Mohamad Assi
email: mohamad-assi@hotmail.com

Frédéric Derbré

Luz Lefeuvre-Orfila

Amélie Rébillard
email: amelie.rebillard@univ-rennes2.fr

D Thesis-

Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Antioxidant supplementation accelerates cachexia development by promoting tumor growth in C26 tumor-bearing mice

Keywords: Muscle wasting, TNF-α, Myostatin, Tumor growth, Oxidative stress, Antioxidants Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling

. Physically active patients exhibited also an improvement in overall QOL and physical performance. These findings affirmed the conviction that PA must constitute a part of the primary anticancer treatment. Little is known about the exact molecular mechanisms through which exercise could mediate its protective effects.

). As a result, patients with lower-extremities cancers like liposarcoma, don't benefit from advises regarding the level of PA that could be practiced during the treatment period.

In some cases, the capacity of patients with advanced stages of cancer (cachectic patients) to pursue a hospital-based PA program could be limited owing to anemia, cardiovascular dysfunction and advanced pro-catabolic state (Argiles et al., 2012). Therefore, an alternative strategy to PA could be useful to slow the deterioration of patients' overall health.

) (Mantovani et al., 2002b). This promotes excessive reactive oxygen species (ROS) formation within skeletal muscle and its subsequent degradation (Li et al., 1998). We might underscore that ROS are also produced within tumor and control its biological response (Schieber and Chandel, 2014; Sabharwal and Schumacker, 2014).

) the type/dose of antioxidants, (3) basal antioxidant status of the patient and (4) other factors that may distort evidence-based conclusion Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Chapter I. Reactive Oxygen Species 12 | P a g e Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 change and maintain low physiological concentrations of O2 .-at ~ 2 × 10 -10 M. This was demonstrated by the early observation of Imlay et al. showing that SODs are able to dismutate 10 - 9 or 10 -6 M of O2 .

.

.

). Even though that CAT is not essential under Chapter I. Reactive Oxygen Species 16 | P a g e Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016

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It is also interesting to know that GSH can across the plasma membrane to the extracellular space.

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. In addition to its antioxidant function GSH plays distinct roles at different cellular compartments. At the sarcoplasmic reticulum (SR) Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during

I will start this section by gratefully thanking the jury members for accepting to examine my Ph.D. work. Namely, Professor Marc Francaux form the University Catholique de Louvain at Belgium and Professor Stéphane Servais from the University Francois Rabelais-Tours, for accepting to report this manuscript. Also, I would like to thank Professor Francoise Redini from the University of Nantes, Professor Odile Sergent from the University of Rennes 1 and Doctor Adrien Rossary from the University of Clermont-Ferrand, for accepting to be among the jury members.

Many thanks for the previous director of the laboratory "Movement, Sport and health Sciences" (M2S), Paul Delamarche and for the actual director Benoit Bideau, for receiving me as a member of the M2S family.

Today, if pursuing a career of a researcher is not a dream anymore, it is in a major part due to Amélie Rebillard who believed in my competences and capacities to conduct this work. I would like to sincerely thank Amélie for her in-science passion, patience, support, motivation and positive energy. From you I learned that we can find a solution for every complicated problem; to never give-up or despair when we fail to obtain positive results (recurrent phenomenon among Ph.D. students); to never neglect literature and reading at the expense on performing experiments; and to explore new fields and ideas, where others don't dare, which is the real meaning of research and being a researcher. I can say that in addition to the phenotypic changes that I have undergone during these three years, where my beard started to grow spontaneously, I have also gained in maturity at both scientific and personal plan.

I also appreciated my discussions with Frédéric Derbré, who transmitted to me the passion to train mice on treadmill and to explore muscle physiology. How can I forget the animated talks with Nicolas Pierre on RNA extraction and PCR?! But also the great moments of jokes and laughs that we have shared with the future doctor Kévin Nay. It was a pleasure to share with you my laboratory experiments, meals and after-noon jogging, even if I was unlikely to follow the rhythm of Frédéric and Nicolas when they were decided to do fractioned exercise! Although I did not understand exactly what the "Biomechanics guys" Camille, Pierre (also known as, Plantouille), Sébastien, Rémy and Sean, were trying to do during these three years, I often saw them seated in front of their computers, while I was shuttling between my office, the biochemistry Lab, the histopathology platform and the animal care facility. However, I enjoyed our coffee breaks to discuss golf with Camille, politics with Pierre and English with Sean! I would say that the concept of "foot-chaise" (playing football while seating on the chair in your office) was innovating and funny, apart the pain that I felt the next day of the match. A special thank for the road partner Jordan with whom I passed more than two weeks of daily travel Rennes-Nantes, to perform radiotherapy on mice. I think that I am a fan of your coaching spirit, especially when you try to gently communicate with your mice and motivate them to pursue the exercise till the end.

I would like to thank Dany and Luz for their help and technical advices in animal handling, gavage, injections and sacrifice. I appreciated chemistry discussions with Brice, which were useful in performing my cell culture experiments. I am also thankful for the Master's students Florian, Moussa and Suzanne for their technical help but also for the energy and the positive ambiance that they deployed in the laboratory, it was a pure pleasure for me to work with such motivated students. I also hope a good luck for the new Ph.D. students Charles and Suzanne in their projects and certainly a lot of scientific publications. I address my gratitude to my mother Abla and father Nabil for their unconditional love and support. I have the chance to be born in a family that encourages their children to learn science and provides them with all the means to realize their own ambitions. This work is the culmination of several years of study, research, perseverance and patience; thus, I dedicate it for them as a sign of recognition. Finally, I want to thank my brothers Ali, Hassan and Hussein for being a part of my life and making it more joyful and meaningful.

Figures and Tables Index

XV | P a g e Assi, 2016 The burden of non-communicable diseases including: cardiovascular dysfunction, type 2 diabetes, and certain types of cancer is increasing in developed countries and account for more than 60% of the 56 million deaths that occur annually (WHO, 2015). Cancer is the first cause of mortality in France and the second after, heart diseases, worldwide (INCa, 2015). Cancer care is a major issue at both health and socioeconomic plan. Each year the French government disburses more than 5 milliard euros on cancer treatment and clinical care. In 2014, 155 million euros were allocated for cancer research by institutional organizations and charitable national foundations.

Figures and Tables Index

According to the recent report released, in 2015, by the French national cancer institute (INCa, 2015), 385 000 new cases were diagnosed with cancer at a median age of 67-68 year-old. The most frequent types are lung, prostate and colorectal cancer in men. Breast cancer remains the most recurrent in women, followed by colorectal and lung cancer. In 2015, 149 500 persons (56% men) have succumbed to cancer in France. Among those, lung and breast cancer were the principal cause leading to death in men and women respectively (INCa, 2015).

A shift in mortality toward an increase could be due to a number of health complications associated to cancer that reduce the tolerance to anticancer therapies (Fearon et al., 2011). The two key limiting factors are the alteration of both nutritional and activity status of patients (WHO, 2004). In fact, advanced stages of cancer are often associated with malnutrition and excessive loss of skeletal muscle mass leading to functional impairment and high risk of inactivity (Fearon et al., 2011). Indeed, the "Plan of cancer 2014-2019" launched by the French government, underscored the importance of improving patients' quality of life (QOL) and physical performance in the completion of anticancer treatments. From this perspective, adjuvant therapies like physical activity (PA) and nutritional support could be helpful when used in combination with conventional treatments.

In this light, the crosstalk between tumor and skeletal muscle is a major event occurring during cancer [START_REF] Jones | Exercise modulation of the host-tumor interaction in an orthotopic model of murine prostate cancer[END_REF]. We suppose that two main routes could drive the crosstalk between tumor and skeletal muscle; in this manuscript, we have chosen to term them the direct or FIGURE 1: Disproportionation of O2 .-by the "Ping-Pong" reaction; O2 .-reduces the oxidized metal ion and then oxidizes the reduced metal ion. From [START_REF] Sheng | Superoxide dismutases and superoxide reductases[END_REF] Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 the ratio GSSG/GSH is much higher compared to the cytosol, due to the efficient formation of disulfide bonds in nascent polypeptides, which is an unavoidable step for proteins to acquire functional conformation [START_REF] Cooper | The enzyme-catalysed conversion of anti-benzo[a]pyrene-7,8-diol 9,10-oxide into a glutathione conjugate[END_REF]. In the nucleus, GSH could be also useful for the reduction of ribonucleotides into deoxyribonucleotides and DNA replication [START_REF] Holmgren | The use of thiols by ribonucleotide reductase[END_REF].

Cysteine-based compounds: Maintaining an optimal pool of thiol within the cell is an efficient way of protection against accumulation of oxidative damages. In continuity with the role of GSH, N-acetylcysteine (NAC), α-lipoic acid and carbocysteine are also important in maintaining cellular redox state. Particularly, NAC has been mainly studied as an antioxidant agent that improves respiratory and skeletal muscle performance [START_REF] Reid | N-acetylcysteine inhibits muscle fatigue in humans[END_REF][START_REF] Supinski | N-acetylcysteine administration alters the response to inspiratory loading in oxygen-supplemented rats[END_REF].

Redox molecular analysis revealed that NAC induced its protective effects via increasing the pool of GSH and preventing apoptosis commitment within muscles [START_REF] Supinski | N-acetylcysteine administration alters the response to inspiratory loading in oxygen-supplemented rats[END_REF]. Thanks to their antioxidant virtues, NAC and carbocysteine are today used clinically in patients with chronic obstructive pulmonary disease (COPD) as a mucolytic agent, able to degrade the three-dimensional structure of mucus by reducing the disulfide bonds and, thus, mediating airways clearance [START_REF] Kim | Pharmacological approaches to discovery and development of new mucolytic agents[END_REF]. Additionally, carbocysteine and α-lipoic acid are used to improve overall performance and QOL in conditions evoking high ROS levels including, advanced stages of cancer [START_REF] Maccio | A randomized phase III clinical trial of a combined treatment for cachexia in patients with gynecological cancers: evaluating the impact on metabolic and inflammatory profiles and quality of life[END_REF].

Vitamin C: Hydrosoluble vitamin C in the form of ascorbic acid is also considered as a powerful scavenger of HClO, OH . , ROO . and SH . , given its capacity to donate two electrons. After losing the first electron, ascorbic acid is transformed to ascorbyl radical that could be either further oxidized to produce dehydroascorbate or regenerated by a reductant such as, GSH [START_REF] Kohen | Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification[END_REF]. A link between vitamin C deficiency and oxidative damage was established in Gulo -/-mice for which lipid peroxidation adducts were much elevated in the liver, compared to control [START_REF] Harrison | Low vitamin C and increased oxidative stress and cell death in mice that lack the sodium-dependent vitamin C transporter SVCT2[END_REF]. The direct antioxidant capacity of ascorbic acid was validated in vitro but there is no convincing evidence about the efficacy of its scavenging effects in animals and humans [START_REF] Paolini | The nature of prooxidant activity of vitamin C[END_REF]. The results of clinical trials were disappointing, rising doubts about the safety of "vitamin C overdosing" in the treatment of patients with cancer and cardiovascular diseases [START_REF] Loria | Vitamin C status and mortality in US adults[END_REF]. Supplementation with vitamin C seems beneficial in particular conditions evoking high levels of lipid peroxidation, inflammation and oxidized low-density lipoprotein (LDL), such as endothelial dysfunction, or when serum levels of vitamin C are opposed to vitamin C intake [START_REF] Loria | Vitamin C status and mortality in US adults[END_REF]. Vitamin C may improve endothelial function by increasing NO .

bioactivity (Williams et al., 1994). NO . can be eliminated by reacting with O2 .-radicals to produce peroxynitrite. At this level, ascorbic acid may stop O2 .-production either (1) directly through scavenging O2 .-molecules produced mainly by NOX or (2) indirectly by filling in the "uncoupled" active site of eNOS in the absence of arginine substrates (in the absence of arginine, eNOS produces O2 .-instead of NO . ) (Traber and Stevens, 2011).

Vitamin E: Another antioxidant that has been extensively used in animal and human studies is the liposoluble vitamin E in the form of α-tocopherol (Halliwell, 1999a). In the human body, various β, γ and δ-tocopherols are absorbed and transferred, but only α-tocopherol is maintained in plasma and tissues (Traber, 2007). This long half-life is due the hepatic α-tocopherol transfer protein (α-TTP) and the rapid recirculation between liver and blood, allowing a daily replacement of the entire α-tocopherol pool (Traber et al., 1994). The α-tocopherol interacts with radicals and give rise to α-tocopherolquinone and subsequently to α-tocopherylquinone. Then, the α-tocopherylquinone is recycled to its reduced form by ascorbic acid [START_REF] Herrera | Vitamin E: action, metabolism and perspectives[END_REF].

Given its bioavailability and pharmacokinetics proprieties, α-tocopherol has the most effective role in stopping the lipid peroxidation process. Its antioxidant effects are largely depend on the concentrations of vitamin C. For example, lipid peroxidation and the great disappearance rate of vitamin E, observed in the presence of high ROS amounts, were normalized by vitamin C supplementation [START_REF] Bruno | Faster plasma vitamin E disappearance in smokers is normalized by vitamin C supplementation[END_REF]. This emphasizes the importance of vitamin C as a "quarterback" for vitamin E regeneration-metabolism. Deficiency in vitamin E may cause severe anemia, neurological disorder and negatively affect the immune system function (Traber and Stevens, 2011). Figure 5 illustrates the overlapping cycles of regeneration between different antioxidants, which is crucial to maintain an effective antioxidant defense.

Polyphenols:

The family of polyphenols may hold thousands of compounds, among these we cite: catechins (e.g. green tea extracts), flavonols (e.g. quercetin), stilbens (e.g. resveratrol), and the list is too long. They could be obtained from various alimentary sources like, green/black tea, coffee, fruits, vegetables, olive, red wine and chocolate [START_REF] Perron | A review of the antioxidant mechanisms of polyphenol compounds related to iron binding[END_REF]. Unlike vitamin E, polyphenols have negligible scavenging effects in vivo, and mediate their antioxidant response via the Keap-Nrf2 pathway [START_REF] Erlank | Polyphenols activate Nrf2 in astrocytes via H2O2, semiquinones, and quinones[END_REF]. In the absence of stressor Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 stimuli, the Cul3-Keap ubiquitin ligase, maintains Nrf-2 sequestrated in the cytoplasm, promotes its ubiquitination and subsequent degradation by the proteasome [START_REF] Sekhar | Nrf2 degradation by the ubiquitin proteasome pathway is inhibited by KIAA0132, the human homolog to INrf2[END_REF]. In the presence of ROS, the thiol groups on Keap become oxidized, thus altering the interaction within complex and liberating Nrf-2 [START_REF] Satoh | Nrf2/ARE-mediated antioxidant actions of proelectrophilic drugs[END_REF]. Then, Nrf-2 will translocate into the nucleus and bind the antioxidant responsive element (ARE) to promote the expression of antioxidant genes including, SOD and TRX [START_REF] Itoh | Molecular mechanism activating Nrf2-Keap1 pathway in regulation of adaptive response to electrophiles[END_REF]. Paradoxically, given their cyclic-benzyl chemical structure, polyphenols could undergo spontaneous oxidation to produce moderate levels of ROS and activate the Keap-Nrf2 system [START_REF] Erlank | Polyphenols activate Nrf2 in astrocytes via H2O2, semiquinones, and quinones[END_REF]. In some diseases, like cancer, an increase in the nuclear accumulation of Nrf2 could be observed due to activating mutations on Nrf2 or silencing of Keap [START_REF] Kansanen | The Keap1-Nrf2 pathway: Mechanisms of activation and dysregulation in cancer[END_REF]. However, the health-related benefits from polyphenols in cancer, are not restricted to their antioxidant virtues but expand to modulate pathways involved in senescence, proliferation and epigenetic modifications (Mileo and Miccadei, 2016a).

Oxidative stress and related macromolecules damage

Even though that ROS are essential for body functioning under normal conditions, excessive and sustained production of ROS has been involved in the development or progression of more than 200 clinical disorders [START_REF] Kohen | Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification[END_REF]. In pathological conditions, a shift in the redox balance toward an increase in the pro-oxidant power leads to damage and the development of oxidative stress (OS). OS is defined as a "disruption of the redox balance towards an increase in pro-oxidant over the capacity of antioxidants, leading to a perturbation of redox signaling and control and/or molecular damage" [START_REF] Jones | Redefining oxidative stress[END_REF]. A redox deregulation in the FIGURE 5: Interrelationship between GSH, vitamin C and vitamin E. From (Kohen and Nyska, 2002) opposite side is called reductive stress (antioxidants over pro-oxidants), but this condition has not been studied in depth.

Despite the relative short half-life of ROS, some species can diffuse and reach sensitive locations, where they can interact with essential biological targets (i.e. lipids, proteins and DNA) and cause damage. Indeed, we can distinguish between three different profiles of OS. Acute OS in response to an external physiological stimuli, which could be reduced by an effective antioxidant defense. In the case of ineffective antioxidant response, the cell is exposed to a more prolonged production of ROS, giving way to the development of chronic OS. Contrary to acute OS, chronic OS could have dangerous consequences on health and it is associated with the development of several diseases including, cancer (Sabharwal and Schumacker, 2014). The third OS profile is called "quasistationary", it occurs when ROS levels do not regain the initial steady state; thus, it is somehow a new redefined level of ROS in the cell, usually much higher than the normal range [START_REF] Lushchak | Glutathione homeostasis and functions: potential targets for medical interventions[END_REF]) (Figure 6).

FIGURE 6: OS profiles in biological system. From [START_REF] Lushchak | Glutathione homeostasis and functions: potential targets for medical interventions[END_REF]) 3.1. Lipid peroxidation [START_REF] Cillard | Mécanismes de la peroxydation lipidique et des anti-oxydations. Oilseeds and fats, Crops and Lipids. Tumor-Skeletal Muscle Crosstalk in Cancer[END_REF]Halliwell, 1999a;[START_REF] Meral | Lipid peroxidation and antioxidant status in beta-thalassemia[END_REF] The lipids of cellular membrane are particularly sensitive to oxidation. The interaction of ROS with lipids induces damage through a process composed of three main stages, known as lipid peroxidation. The first stage called initiation, in which the derivatives of oxygen remove an atom of hydrogen from a methylene (-CH2) group adjacent to a double bound (because of the double bound, the interaction between carbon and hydrogen is weakened and it can be removed easily by oxygen radicals). The formed fatty acid radical is stabilized by rearrangement of the structure to form a conjugated diene. Then, it reacts with oxygen giving place to a lipid peroxyl radical (ROO . ).

During the propagation stage, ROO . can abstract the hydrogen of other neighboring lipids and, therefore, produce further fatty acid radicals.

The interaction of ROO . with a new fatty acid produces a lipid hydroperoxide radical (ROOH) that can be further decomposed into aldehydes, hydrocarbons and isoprostanes. The last stage, termination, takes place when ROO . enters in contact with an antioxidant, like vitamin E.

The major end-products of the reaction include malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) (Figure 7). It has been shown that ω-6 polyunsaturated fatty acids and mitochondrial phospholipid cardiolipin are important sources of 4-HNE formation [START_REF] Zhong | Role of lipid peroxidation derived 4-hydroxynonenal (4-HNE) in cancer: focusing on mitochondria[END_REF]. When produced at high levels in the cell, 4-HNE preferentially reacts with histidine, cysteine and lysine residues of intracellular proteins, resulting in structural modifications of "Schiff-base" type or Michael addition (Negre-Salvayre et al, 2008). The formed HNE-protein adducts are more resistant to removal by the proteasome and favor the aggregation of oxidized protein in the cell; such condition is associated with ageing and cancer [START_REF] Friguet | Oxidized protein degradation and repair in ageing and oxidative stress[END_REF]. Moreover, 4-HNE may directly oxidize proteasome subunits and reduce its activity [START_REF] Keller | Decreased levels of proteasome activity and proteasome expression in aging spinal cord[END_REF]. Nucleic acids are also an important target of 4-HNE. For example, 4-HNE-DNA adduct is a known mutagen in cells mainly found at the p53 gene in human cancers [START_REF] Hussain | Increased p53 mutation load in nontumorous human liver of wilson disease and hemochromatosis: oxyradical overload diseases[END_REF]. Even more important, 4-HNE may compromise DNA repair mechanisms such as nucleotide excision repair (NER) within human cells, in response to mutagenic agents or exposure to UV lights [START_REF] Feng | Trans-4-hydroxy-2-nonenal inhibits nucleotide excision repair in human cells: a possible mechanism for lipid peroxidation-induced carcinogenesis[END_REF]. This could be attributed to the ability of 4-HNE to react with essential proteins involved in NER [START_REF] Feng | Trans-4-hydroxy-2-nonenal inhibits nucleotide excision repair in human cells: a possible mechanism for lipid peroxidation-induced carcinogenesis[END_REF].

Protein oxidation and peroxidation

Proteins are also important targets for biological oxidants due to their important concentration and high rate of constant reaction with numerous oxidant [START_REF] Davies | Protein oxidation and peroxidation[END_REF]. For example, amino acids like cysteine can undergo an abstraction of hydrogen from the thiol (SH) group by OH . , resulting in the formation of thiyl radicals (RS . ). These radicals are able to abstract a hydrogen from α (backbone) and 𝛽-carbon (side-chain) at the same or neighboring amino acid, to give carbon-centered radicals (R . ). Carbon-centered radicals undergo peroxidation, fragmentation, dimerization, disproportionation and isomerization, which results in dramatic effects on protein structure and function [START_REF] Fradkin | UV photodegradation of murine growth hormone: chemical analysis and immunogenicity consequences[END_REF][START_REF] Jiskoot | Protein instability and immunogenicity: roadblocks to clinical application of injectable protein delivery systems for sustained release[END_REF]. The predominant reaction is the interaction of R . with O2 that results in the formation of ROO . radicals.

One of the possible pathways is the cross-reaction of two ROO . radicals (ROO-OOR) that decay to yield aldehydes/ketones, through a succession of reactions. These reactions lead to the formation of protein carbonyls, which is a widely used biomarker for protein oxidation [START_REF] Dalle-Donne | Protein carbonylation, cellular dysfunction, and disease progression[END_REF][START_REF] Dalle-Donne | Protein carbonyl groups as biomarkers of oxidative stress[END_REF]. However, more than 70% of initial ROO . are transformed into ROOH, given the abundance of C-H and S-H bonds in proteins. ROOH radicals can react with more susceptible amino acids like, methionine and cysteine, resulting in the inactivation of several enzymes and alteration of energy metabolism as well as many cellular functions (Nagy and Erhardt, 2010) (Figure 8). Interestingly, oxidized cysteine could undergo reparation by TRX and glutaredoxin (GRDX) [START_REF] Holmgren | Thiol redox control via thioredoxin and glutaredoxin systems[END_REF]. Both TRX and GRDX contain two cysteine residues in their redox active site, which allow them to reduce oxidized cysteine on the damaged protein.

Upon reparation of target proteins, TRX and GRDX become oxidized and are then recycled to their reduced form by the TRX reductase and GSH reductase, respectively (Holmgren et al, 2011).

Protein function could be also restored after the reversion of oxidized methionine to its reduced form under the action of methionine sulfoxide reductase enzymes [START_REF] Moskovitz | Methionine sulfoxide reductases: ubiquitous enzymes involved in antioxidant defense, protein regulation, and prevention of aging-associated diseases[END_REF][START_REF] Sreekumar | Methionine sulfoxide reductase A: Structure, function and role in ocular pathology[END_REF]. Additionally, other small chaperone proteins such as heat shock protein (HSP) could serve as cytoplasmic antioxidants, by binding to and hiding the sensitive amino acids on the target protein (Dahl et al, 2015). HSPs may also hold oxidized/denatured proteins until their refolding or degradation in order to prevent accumulation of protein aggregates (Dahl et al, 2015). Nevertheless, some products like carbonylated amino acid derivatives are irreversible and typically undergo degradation by the proteasome 20 and 26 S in the cytoplasm and by the ATP-dependent Lon protease within mitochondrial matrix [START_REF] Shang | Removal of oxidatively damaged proteins from lens cells by the ubiquitin-proteasome pathway[END_REF][START_REF] Bota | Lon protease preferentially degrades oxidized mitochondrial aconitase by an ATP-stimulated mechanism[END_REF].

FIGURE 8:

Example of oxidative modifications that proteins can undergo in response to high ROS production. Adapted from [START_REF] Davies | Protein oxidation and peroxidation[END_REF]

Oxidative DNA damage

Although DNA is a well-protected molecule, some ROS can reach this location to interact with nucleotides and induce damage. Indeed, OH . possesses a high rate constant reaction with DNA bases and ribose sugar (k~ 5-8×10 9 M -1 s -1 ) [START_REF] Cadet | Photoinduced damage to cellular DNA: direct and photosensitized reactions[END_REF]. Hydroxyl radical reacts indistinctly with DNA either by addition or hydrogen abstraction and, then, the formed products can undergo a secondary reaction with O2 or O2 .- [START_REF] Jaruga | DNA damage products (5'R)-and (5'S)-8,5'-cyclo-2'-deoxyadenosines as potential biomarkers in human urine for atherosclerosis[END_REF]. The DNA-OH . interaction gives rise to a large plethora of end-products depending on the: nature of modified bases, type of the initial reaction and the amounts of pro-oxidants [START_REF] Cobley | Exercise improves mitochondrial and redox-regulated stress responses in the elderly: better late than never![END_REF]. For example, the oxidation of guanine yields more than 20 DNA-adducts, with the 8-oxo-7,8-dihydroguanine (8-oxoG) being the most assayed product to study DNA oxidative damage (Sayin et al., 2014). Interestingly, H2O2, NO . and O2 .-are unlikely to induce direct DNA damage (Halliwell, 1999b), but could serve as a source to produce reactive intermediates that can attack DNA. For example, NO . and O2 which is a less efficient mechanism that screens the whole genome to detect secondary DNA products and the chemical structure of helix-distorting lesions [START_REF] Melis | Oxidative DNA damage and nucleotide excision repair[END_REF]. Both NER and BER could be considered as a part of the intracellular antioxidant defense, given their ability to prevent or repair oxidative DNA damage. In fact, the inactivation of key components of the TC-NER or GG-NER system, like cockayne syndrome A and B factors (CSA/B) and XPC, in animals and cells increases their sensitivity to oxidative stressors and causes the accumulation of DNA damages [START_REF] De Waard | Cell type-specific hypersensitivity to oxidative damage in CSB and XPA mice[END_REF][START_REF] Pascucci | The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Tumor-Skeletal Muscle Crosstalk in Cancer-Ph[END_REF][START_REF] Hollander | Deletion of XPC leads to lung tumors in mice and is associated with early events in human lung carcinogenesis[END_REF]. At the mitochondria, it is the BER system that will recognize and remove 8-oxoG adducts via a specific glycosylase called OGG1 [START_REF] Melis | Oxidative DNA damage and nucleotide excision repair[END_REF]. The damaged base is then released, keeping a free unbound site known as an apurinic/apyrimidinic (AP) site. This is cleaved by an endonuclease and the resulting singlestrand break could be ligated with a single-to-ten nucleotides [START_REF] Melis | Oxidative DNA damage and nucleotide excision repair[END_REF]. Additionally, NER provides support to BER function. For example, CSB regulates the expression of OGG1.

Deficiency in CSB causes weak OGG1 transcription and inefficient 8-oxoG repair [START_REF] Javeri | Downregulation of Cockayne syndrome B protein reduces human 8-oxoguanine DNA glycosylase-1 expression and repair of UV radiation-induced 8-oxo-7,8-dihydro-2'-deoxyguanine[END_REF].All together these findings emphasize the importance of BER and NER to contest oxidative DNA damage and, thus, to reduce cancer incidence.

Biological role of ROS

The mechanisms of cellular signaling are intimately controlled by the redox state of the cell. ROS act as a second messenger to regulate the activity of several transcriptional factors and, therefore, the expression of various genes involved in cell cycle regulation, inflammation, cell death, autophagy, DNA damage repair and the list is too long [START_REF] Alcayaga-Miranda | Prostate tumor-induced angiogenesis is blocked by exosomes derived from menstrual stem cells through the inhibition of reactive oxygen species[END_REF]. This indicates that ROS control the fate of the cell, whether the cell will proliferate or not; will undergo repair and live or dead; will maintain its function or not, are largely dependent on ROS levels inside the cell. Accordingly, the half-cell reduction potential (E hc ) seems to correlate with the biological response of the cell [START_REF] Schafer | Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple[END_REF]. From a mathematical point of view, it could be estimated with the Nernst equation {redox environment = Σ Ei × (reduced species)i} where, Ei is the half-cell reduction potential for a given redox pair (i.e. GSH) and (reduced species)i represents the concentration of the reduced species in the redox pair. Indeed, the redox potential of a normal cell is approximately, E hc ≈ -240 mV, and associated with cell proliferation. In response to progressive increase in intracellular ROS levels, the cell will switch into (1) differentiation (E hc ≈ -200 mV), ( 2) then to apoptosis when the redox potential reaches E hc ≈ -170 mV and (3) finally, to necrosis (E hc ≈ -150 mV) [START_REF] Schafer | Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple[END_REF]) (Figure 9). In addition to controlling quiescence, proliferation, differentiation and apoptosis, ROS could be needed for proper cell migration, chemotaxis and structure stabilization [START_REF] Munnamalai | Reactive oxygen species regulate F-actin dynamics in neuronal growth cones and neurite outgrowth[END_REF]. ROS are crucial for the polymerization of F-actin monomers and α/β-tubulin as well as the regulation of microtubules dynamics and vesicles trafficking [START_REF] Wilson | Regulation of cytoskeletal dynamics by redox signaling and oxidative stress: implications for neuronal development and trafficking[END_REF].

Physiological amounts of ROS are essential for a healthy body functioning. For example, a role for mitochondrial O2 .-and H2O2 in the regulation of vascular endothelial diameter in response to stressor stimuli has been previously unveiled [START_REF] Liu | Endothelin-1 activates endothelial cell nitric-oxide synthase via heterotrimeric G-protein betagamma subunit signaling to protein jinase B/Akt[END_REF]. ROS may also act as O2 sensor in response to hypoxia. When hypoxic non-transformed normal cells suffer from a lack of O2, the intracellular levels of ROS increase and induce the activation of hypoxia-inducible factor -1 (HIF-1), resulting in angiogenesis and tissue oxygenation [START_REF] Guzy | Oxygen sensing by mitochondria at complex III: the paradox of increased reactive oxygen species during hypoxia[END_REF]. The so-called oxidative burst reaction is one of the most effective defense system against pathogens [START_REF] Droge | Free radicals in the physiological control of cell function[END_REF]. Phagocytes will mainly produce O2 .-, H2O2 and HClO via the membrane-anchored NOX, leading to direct pathogens destruction and alarming acquired immune cells [START_REF] Droge | Free radicals in the physiological control of cell function[END_REF]. Interestingly, ROS may also intervene in other vital mechanisms, like: neurotransmission, skeletal muscle contraction and redox cell signaling (Halliwell, 2006a, b;Schieber and Chandel, 2014). All these data provide convincing evidence about a fine tuning of main cellular vital processes by ROS, in which a simple deregulation may lead to catastrophic disorder. which are all sensitive to the actions mediated by ROS [START_REF] Luo | Principles of cancer therapy: oncogene and non-oncogene addiction[END_REF] (Figure 10). The levels of oxidative DNA damages (i.e. 8-oxoG), lipid peroxidation adducts (i.e. 4-HNE and MDA) and/or protein carbonyls were increased in blood samples, urine excretion and tumor tissue of patients with breast, colorectal, lung, prostate, esophageal and thyroid cancer [START_REF] Crohns | Impact of radiotherapy and chemotherapy on biomarkers of oxidative DNA damage in lung cancer patients[END_REF][START_REF] Kosova | Possible relations between oxidative damage and apoptosis in benign prostate hyperplasia and prostate cancer patients[END_REF][START_REF] Lagadu | 8-oxo-7,8-dihydro-2'-deoxyguanosine as a biomarker of oxidative damage in oesophageal cancer patients: lack of association with antioxidant vitamins and polymorphism of hOGG1 and GST[END_REF][START_REF] Sheridan | Nuclear oxidative damage correlates with poor survival in colorectal cancer[END_REF][START_REF] Young | Levels of oxidative damage and lipid peroxidation in thyroid neoplasia[END_REF]. Some studies reported that the levels of oxidative damage in cancer patients correlated with poorer survival or even with more advanced stages of cancer [START_REF] Crohns | Impact of radiotherapy and chemotherapy on biomarkers of oxidative DNA damage in lung cancer patients[END_REF][START_REF] Dziaman | 8-Oxo-7,8-dihydroguanine and uric acid as efficient predictors of survival in colon cancer patients[END_REF]. Accordingly, cancerous tissues have been found to produce elevated levels of H2O2, compared to adjacent normal tissues [START_REF] Szatrowski | Production of large amounts of hydrogen peroxide by human tumor cells[END_REF]. In the same way, polymorphism in some antioxidant genes was linked to increase in cancer incidence [START_REF] Freriksen | Genetic polymorphism 609C>T in NAD(P)H:quinone oxidoreductase 1 enhances the risk of proximal colon cancer[END_REF].

FIGURE 10: Oxidative stress as a hallmark of cancer. From [START_REF] Luo | Principles of cancer therapy: oncogene and non-oncogene addiction[END_REF] ROS induce DNA mutations that might lead to tumor initiation and promotion by targeting tumor suppressor and pro-oncogenic genes [START_REF] Higinbotham | GGT to GTT transversions in codon 12 of the K-ras oncogene in rat renal sarcomas induced with nickel subsulfide or nickel subsulfide/iron are consistent with oxidative damage to DNA[END_REF]. For example, the mutation of the tumor suppressor P53, with a G-to-T conversion leads to its inactivation and is commonly found in skin cancer [START_REF] Brash | A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma[END_REF]. The extent of DNA damage caused by ROS could be irreparable by the base excision repair (BER), allowing the progression of cancer (Obtulowicz et al., 2010). In fact, oxidative DNA damage and a defective repair system are widely accepted as important mutagenic factors involved in carcinogenesis [START_REF] Loft | Cancer risk and oxidative DNA damage in man[END_REF].

The sustained presence of ROS is associated with the development of metabolic perturbations that provide cancer cells with amino acids, lipids and nucleotides to further boost their uncontrolled proliferation [START_REF] Sainz | Radical decisions in cancer: redox control of cell growth and death[END_REF]. Even though that ROS are crucial in normalto-malignant cell transformation and cancer progression, their levels should not exceed the toxicity threshold to guarantee cancer cell survival. For this reason, cancer cells have implemented a mechanism through which some derivatives of the glycolysis pathway could be shuttled to other metabolic circuits involved in the synthesis of reducing compounds endowed with an antioxidant potential [START_REF] Dang | Cancer cell metabolism: there is no ROS for the weary[END_REF]. These findings rejoin the early observation of Otto Warburg that cancer cells presented high glycolytic activity and the ulterior research showing that inhibiting glucose metabolism was effective to induce tumor death [START_REF] Li | Mitochondria-derived ROS bursts disturb Ca(2)(+) cycling and induce abnormal automaticity in guinea pig cardiomyocytes: a theoretical study[END_REF]. Accordingly, Lin and colleagues, showed that low copy number of mitochondrial DNA (mDNA), which reflects low mitochondrial content, coincided with low level of oxidative mDNA damage and lung tumor progression [START_REF] Lin | Low copy number and low oxidative damage of mitochondrial DNA are associated with tumor progression in lung cancer tissues after neoadjuvant chemotherapy[END_REF]. This may suggest that reduced mitochondrial ROS production is associated with decreased extent of mDNA damage, allowing tumors cells to evade apoptosis. These compelling evidences highlight the meticulous regulation of ROS levels within cancer cells. On the one hand, ROS can cause genomic instability, DNA mutations, metabolic alterations and cancer progression.

On the other hand, tumor cell buffers the intracellular content of ROS to avoid excessive oxidative damage and death.

Sources of ROS in cancer

ROS are produced by a set of enzymes and mitochondria within cancerous tissue. To better understand their role in the pathogenesis of cancer we will start by describing the main known sources of ROS in tumor and their impact of disease's evolution.

ROS-producing enzymes

XO: Xanthine oxidoreductase (XOR) exists in two inter-convertible forms that are:

xanthine dehydrogenase (XDH) and xanthine oxidase (XO). In several pathological states, the presence of pro-inflammatory cytokines promotes the cleavage of XDH to XO, which instead uses molecular oxygen to catalyze the hydroxylation of hypoxanthine to xanthine and, then, to uric acid, producing ROS mainly O2 .-and H2O2 [START_REF] Meneshian | The physiology of endothelial xanthine oxidase: from urate catabolism to reperfusion injury to inflammatory signal transduction[END_REF]. Studies in humans, demonstrated an increase in blood XO activity in patients with non-small-cell lung carcinoma (NSCLC), small-cell lung carcinoma (SCLC), head and neck carcinoma, colon and liver cancer compared to control patients [START_REF] Kalcioglu | Adenosine deaminase, xanthine oxidase, superoxide dismutase, glutathione peroxidase activities and malondialdehyde levels in the sera of patients with head and neck carcinoma. Kulak burun bogaz ihtisas dergisi[END_REF][START_REF] Kaynar | Glutathione peroxidase, glutathione-S-transferase, catalase, xanthine oxidase, Cu-Zn superoxide dismutase activities, total glutathione, nitric oxide, and malondialdehyde levels in erythrocytes of patients with small cell and nonsmall cell lung cancer[END_REF][START_REF] Lin | B vitamins deficiency and decreased anti-oxidative state in patients with liver cancer[END_REF]. The activity of XO was in most cases correlated with pro-oxidant parameters in blood samples, like lipid peroxidation adducts. According to the recent review of Battelli et al., high XO activity in plasma of cancer patients is common and may reflect blood vessels damage in response to chronic inflammation, but its modulation within tumor tissue seems to differ between cancers [START_REF] Battelli | Xanthine oxidoreductase in cancer: more than a differentiation marker[END_REF]. Early studies have shown that XO activity was higher in brain and laryngeal tumors, lower in bladder cancer tissue and unchanged in prostate cancer comparing to adjacent normal ones [START_REF] Biri | Activities of DNA turnover and free radical metabolizing enzymes in cancerous human prostate tissue[END_REF][START_REF] Durak | Adenosine deaminase, 5' nucleotidase, xanthine oxidase, superoxide dismutase, and catalase activities in cancerous and noncancerous human laryngeal tissues[END_REF][START_REF] Durak | Adenosine deaminase, 5'nucleotidase, xanthine oxidase, superoxide dismutase, and catalase activities in cancerous and noncancerous human bladder tissues[END_REF][START_REF] Kokoglu | Xanthine oxidase levels in human brain tumors[END_REF]. Authors interpreted this decrease or increase in XO activity as linked to the purine metabolism. Since XO will catabolize purines to produce hypoxanthine, the decrease in XO is expected to promote purine production via the "salvage pathway" in cancer cells and, thus, favor their survival. In the same way, an increase in XO activity could be explained as an attempt to foster purine catabolism and produce high ROS levels, which may deprive tumor from energy blocks and trigger apoptosis.

Glioma cells cultured under hypoxic conditions produced higher levels of H2O2 by XO, which in turn activated HIF-1α and angiogenesis, the inhibition of XO with a specific inhibitor allopurinol abrogated this pathway [START_REF] Griguer | Xanthine oxidase-dependent regulation of hypoxia-inducible factor in cancer cells[END_REF]. Contrariwise, other glioma cells namely, D54-MG, were XO-independent under the same conditions [START_REF] Griguer | Xanthine oxidase-dependent regulation of hypoxia-inducible factor in cancer cells[END_REF]. Indicating that the mechanisms underlying tumor progression may differ between tumor subtypes. XO is also responsible for kidney damage in response to some chemotherapeutic agents [START_REF] Yousef | Personalized Medicine in Kidney Cancer: Learning How to Walk Before We Run[END_REF]. On the other hand, ROS originating from XO may have anticancer therapeutic implications. High levels of XO-produced ROS are responsible for inducing mitochondrial damage and apoptosis in carcinoma OEC-M1 cells [START_REF] Huang | Autophagy-Regulated ROS from Xanthine Oxidase Acts as an Early Effector for Triggering Late Mitochondria-Dependent Apoptosis in Cathepsin S-Targeted Tumor Cells[END_REF]. The tumors of sarcoma-bearing mice treated with XO and hypoxanthine, as a source of ROS, presented great amounts of protein carbonyls and lipid peroxidation, which led to apoptosis induction (Thews et al., 2009). Even more interesting, XO conjugated to polyethylene glycol (PEG) has been proposed as a targeted tumor therapy, given the ability of XO-PEG to selectively accumulate into tumor versus normal cells [START_REF] Sawa | Tumor-targeting chemotherapy by a xanthine oxidasepolymer conjugate that generates oxygen-free radicals in tumor tissue[END_REF].

Therefore, XO is well-recognized as an important source of ROS in cancer. However, its ability to promote or inhibit tumor growth reflects the dual role of ROS in cancer.

NOX:

The family of NOX produces both O2 .-and H2O2 [START_REF] Bedard | The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology[END_REF]. Seven isoforms have been identified to produce ROS, among them: NOX-4 produces H2O2 and NOX-1, 2 and 5 generate O2 .- [START_REF] Bedard | The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology[END_REF]. In conditions evoking chronic inflammation, which is the case of cancer, high amounts of ROS originating from NOX could negatively influence gastrointestinal and pancreatic cancer development [START_REF] Roy | NADPH oxidases and cancer[END_REF]. Clinically, the expression of NOX-1 and NOX-4 in tumor was associated with poor survival and cancer relapse [START_REF] Ha | NADPH Oxidase 1 and NADPH Oxidase 4 Have Opposite Prognostic Effects for Patients with Hepatocellular Carcinoma after Hepatectomy[END_REF][START_REF] Singh | Prognostic significance of NADPH oxidase-4 as an indicator of reactive oxygen species stress in human retinoblastoma[END_REF]. Another isoform, NOX-5, was also found to be overexpressed in numerous cancers, including: colon, melanoma, breast, lung and prostate cancer. However, the role of NOX in cancer has not been addressed in depth clinically and further studies are needed to establish its exact role. At the moment, it seems that the expression of NOX within tumor is associated with cancer progression [START_REF] Roy | NADPH oxidases and cancer[END_REF].

Preclinical evidences highly support the clinical observations and most likely suggest a prooncogenic role for NOX in cancer. The proliferation and vascularization of some tumors including Kaposi's sarcoma are largely dependent on ROS produced by the NOX system [START_REF] Ma | A role for virally induced reactive oxygen species in Kaposi's sarcoma herpesvirus tumorigenesis[END_REF].

NOX family members are considered as a major source of ROS production in endothelial cells (Ushio-Fukai and Nakamura, 2008). In response to pro-angiogenic factors that could be produced by tumor, NOX can: (1) promote endothelial cell proliferation, (2) stimulate branching morphogenesis, (3) tether to actin cytoskeleton, (4) alter cell-cell junction and ( 5) favor the formation of tube-like structure [START_REF] Ikeda | IQGAP1 regulates reactive oxygen species-dependent endothelial cell migration through interacting with Nox2[END_REF][START_REF] Kobayashi | Nox1 regulates apoptosis and potentially stimulates branching morphogenesis in sinusoidal endothelial cells[END_REF]. All these events are essential for the migration of endothelial cells and formation of new micro-vessels to irrigate tumor and facilitate its spread. Recent evidences indicate that the stabilization and activation of NOX-1 and NOX-2 promote oncogenic pathways and invasive behavior in colon cancer cells [START_REF] Joo | NADPH Oxidase 1 Activity and ROS Generation Are Regulated by Grb2/Cbl-Mediated Proteasomal Degradation of NoxO1 in Colon Cancer Cells[END_REF]. Similar results were obtained in pancreatic cells for NOX-4 that could be activated by proteins from the extracellular matrix (ECM) like, fibronectin and laminin, resulting in an increase in ROS production and improving cell survival [START_REF] Edderkaoui | Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase[END_REF]. A recent systematic review and meta-analysis, that included the results of 10 in vitro and in vivo studies, found a positive correlation between NOX expression/activity and lung tumorigenesis [START_REF] Han | Association between NADPH oxidase (NOX) and lung cancer: a systematic review and meta-analysis[END_REF].

A convincing body of evidences in the literature supports the fact that NOX-derived ROS control several aspects of cancer progression. This topic has been plenty reviewed, the most recent review comes from Roy and coworkers, in which they concluded that the inflammatory profile associated with cancer is responsible for the activation of NOX and the resulting ROS-mediated tissue injury and DNA damage; thus, contributing to the evolution of pre-malignant conditions into more advanced stages of disease [START_REF] Roy | NADPH oxidases and cancer[END_REF]. Adapted from [START_REF] Vannini | The dual role of iNOS in cancer[END_REF] Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 presence of pro-inflammatory cytokines such as TNF-α, IFN-γ and IL-1 [START_REF] Reid | Role of nitric oxide in skeletal muscle: synthesis, distribution and functional importance[END_REF]. At high concentration, NO . can induce nitrosative stress through reacting with O2 .-and, subsequently, producing elevated levels of peroxynitrite molecules extremely injurious for living cells [START_REF] Kohen | Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification[END_REF]. High NO . levels were found in plasma of patients with gastric cancer comparing to those without cancer [START_REF] Bakan | Nitric oxide levels and lipid peroxidation in plasma of patients with gastric cancer[END_REF]. iNOS was also expressed in human inflamed colon tissues and colon carcinoma comparing to normal tissue [START_REF] Simon | The NF-kappaB p65 and p50 homodimer cooperate with IRF8 to activate iNOS transcription[END_REF]. Additionally, iNOS and enzymes metabolizing arginine were expressed in soft-tissue sarcoma tumors, including LS, and their expression was particularly higher in malignant LS [START_REF] Cunha | Sarcomas often express constitutive nitric oxide synthases (NOS) but infrequently inducible NOS. Applied immunohistochemistry & molecular morphology[END_REF]Yan et al., 2011).

NOS:

Importantly, iNOS expression correlated positively with tumor size, vascularization and aggressiveness, especially in gastric, breast, prostate and colorectal cancer [START_REF] Song | Relationship between the expression of iNOS,VEGF,tumor angiogenesis and gastric cancer[END_REF].

In animals bearing C6 glioma tumor, the knock down of iNOS with an anti-sense RNA, reduced tumor growth and impaired its vascularization [START_REF] Kostourou | The role of tumour-derived iNOS in tumour progression and angiogenesis[END_REF]. Contrary to colon cancer in which iNOS was likely to promote its growth (Tanaka et al., 2015), the modulation of iNOS expression/activity in other cancers like sarcoma stills to be elucidated. However, it was interesting to note that the activation of iNOS in innate immune cells and the subsequent production of high levels of NO . , has been shown to drive an effective anticancer response against sarcoma [START_REF] Xie | Activation of nitric oxide synthase gene for inhibition of cancer metastasis[END_REF][START_REF] Xie | Direct correlation between expression of endogenous inducible nitric oxide synthase and regression of M5076 reticulum cell sarcoma hepatic metastases in mice treated with liposomes containing lipopeptide CGP 31362[END_REF]. Even in prostate and colon cancer cells, the overexpression of iNOS rendered the cells more sensitive to chemotherapeutic agents [START_REF] Adams | Nitric oxide synthase gene therapy enhances the toxicity of cisplatin in cancer cells[END_REF]. In the same way, the delivery of iNOS gene by micro-encapsulation increased apoptosis of colic and ovarian xenografts [START_REF] Xu | NADPH oxidases are essential for macrophage differentiation. The Journal of biological chemistry[END_REF]. It is not surprising that iNOS seems to have a pro/antioncogenic functions. This may largely depends on the level of produced NO . , low-to-moderate levels ranging from 50-to-400 nM were associated with cell cycle progression, inhibition of apoptosis, angiogenesis, cell migration, cell invasion and carcinogenesis [START_REF] Ridnour | Nitric oxide regulates angiogenesis through a functional switch involving thrombospondin-1[END_REF].

High levels of NO . from 500-to-1000 nM were associated with apoptosis, anti-metastatic and cytostatic effects [START_REF] Wink | Chemical biology of nitric oxide: Insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide[END_REF]) (Figure 11). Vannini and collaborators came to the conclusion that even if "iNOS expression is high in a number of tumors and correlates with poor survival" any approach aiming to inhibit iNOS "needs to be cell situation-based" [START_REF] Vannini | The dual role of iNOS in cancer[END_REF]. Mitochondria is an important hub for O2 .-production. The produced molecules of O2 .-leave the mitochondria by two ways: (1) through its conversion to H2O2 by MnSOD or ( 2) via the voltagedependent anion channels (VDAC) to the cytosol, where its transformed to H2O2 by CuZnSOD [START_REF] Han | Voltage-dependent anion channels control the release of the superoxide anion from mitochondria to cytosol[END_REF] [START_REF] Lustgarten | Complex I generated, mitochondrial matrix-directed superoxide is released from the mitochondria through voltage dependent anion channels[END_REF] . Therefore, picomolar to nanomolar amounts of H2O2 are produced inside the cell that may affect the function of intracellular proteins and the dynamics of gene expression [START_REF] Murphy | Mitochondrial thiols in antioxidant protection and redox signaling: distinct roles for glutathionylation and other thiol modifications[END_REF]. The oncogenic activation of Ras and Myc commonly found in cancer, increases the dependency on the Krebs cycle within mitochondria to produce adenosine triphosphate (ATP), ROS, amino acids, nucleotides and NADPH [START_REF] Gao | c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism[END_REF]. Feeding the Krebs cycle with substrates like glutamine and fatty acid β-oxidation products is crucial to increase tumorigenicity [START_REF] Weinberg | Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity[END_REF]. Particularly, glutamine catabolism by the Krebs cycle is essential for tumor cell growth in the presence or absence of glucose [START_REF] Weinberg | Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity[END_REF].

Mitochondrial ROS generation

Glutamine could be converted to glutamate by glutaminase and, then, to α-ketoglutarate by alanine aminotransferase, which in turn enters the Krebs cycle [START_REF] Carracedo | Cancer metabolism: fatty acid oxidation in the limelight[END_REF]. As a direct consequence, ATP and ROS are, respectively, generated by oxidative phosphorylation and the mitochondrial respiratory chain complexes I, II and III (MRC) [START_REF] Weinberg | Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity[END_REF]. Indeed, mitochondrial ROS production could be prevented by silencing alanine aminotransferase with specific ShRNA [START_REF] Weinberg | Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity[END_REF]. Additionally, the premise that cancer cell requires mitochondrial ROS for proliferation, comes from the observations that cells null from mDNA do not produce ROS and fail to grow [START_REF] Weinberg | Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity[END_REF], while mutations in mDNA (i.e.

respiratory complex I) causing high production of ROS enhanced metastatic behavior of the cells [START_REF] Ishikawa | ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis[END_REF]. In the same way, pre-malignant mammary cells were killed after treatment with the antioxidant MitoQ, which acts as an analogue to the endogenous mitochondrial antioxidant coenzyme Q10 [START_REF] Rao | The antioxidant transcription factor Nrf2 negatively regulates autophagy and growth arrest induced by the anticancer redox agent mitoquinone[END_REF] (Figure 12).

In aerobic conditions, glycolysis provides glycolytic derivatives to the pentose phosphate pathway (PPP) to generate essential macromolecules and intracellular antioxidants (to avoid excessive oxidative damage) [START_REF] Panieri | ROS homeostasis and metabolism: a dangerous liason in cancer cells[END_REF]. Whereas, hypoxic conditions may constitute a weakness for cancer cells, as they become largely dependent on glycolysis itself to generate ATP and survive [START_REF] Weinberg | Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity[END_REF]. Thus, the use of anti-diabetic drugs that limit glucose utilization and, therefore, reduce ROS production by MRC could be useful to restrict tumor growth. In this light, strategies targeting complex I and III were effective in decreasing ROS production and cancer proliferation [START_REF] Janzer | Metformin and phenformin deplete tricarboxylic acid cycle and glycolytic intermediates during cell transformation and NTPs in cancer stem cells[END_REF]. Genetically, the depletion of mitochondrial transcription factor A (TFAM), involved in mitochondrial biogenesis, reduced tumorigenicity in mouse bearing KRAS-induced lung adenocarcinoma [START_REF] Weinberg | Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity[END_REF].

These compelling data highlight the role of mitochondria as important source of ROS generation, essential for tumor progression and metastasis.

Antioxidant defense in cancer

Clinically, histological analysis revealed either a decrease in the expression and/or activity of antioxidant enzymes like SOD and CAT in the epithelium neoplasia prostatic tissue, chronic pancreatitis tissue, pancreas cancer, squamous and basal cell carcinoma [START_REF] Bostwick | Antioxidant enzyme expression and reactive oxygen species damage in prostatic intraepithelial neoplasia and cancer[END_REF][START_REF] Cullen | Expression of antioxidant enzymes in diseases of the human pancreas: another link between chronic pancreatitis and pancreatic cancer[END_REF][START_REF] Sander | Oxidative stress in malignant melanoma and nonmelanoma skin cancer[END_REF] or an increase in the expression and/or activity of SOD, CAT and GPx in breast, bladder, melanoma, lung, stomach, esophageal and colon cancer, when compared to respective normal tissues [START_REF] Durak | Adenosine deaminase, 5' nucleotidase, xanthine oxidase, superoxide dismutase, and catalase activities in cancerous and noncancerous human laryngeal tissues[END_REF][START_REF] Janssen | Superoxide dismutases in gastric and esophageal cancer and the prognostic impact in gastric cancer[END_REF][START_REF] Portakal | Coenzyme Q10 concentrations and antioxidant status in tissues of breast cancer patients[END_REF][START_REF] Sander | Oxidative stress in malignant melanoma and nonmelanoma skin cancer[END_REF]. Interestingly, the expression of MnSOD was higher in gastric cancer tissue referring to normal mucosa and a higher ratio of both levels was associated with poor overall FIGURE 12: Alteration of mitochondrial bioenergetics profiles maintains redox homeostasis in tumor cell and promotes its growth (please see abbreviation list). Adapted from [START_REF] Panieri | ROS homeostasis and metabolism: a dangerous liason in cancer cells[END_REF] Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 survival in patients [START_REF] Janssen | Superoxide dismutases in gastric and esophageal cancer and the prognostic impact in gastric cancer[END_REF]. Based on the references cited here, it appears that early stages of cancer or precancerous conditions, like neoplasia and inflamed tissue, are associated with a decrease in antioxidant response, which may allow ROS to induce damage and malignant transformation. Whereas, in later stages, when cancer acquires a more invasive phenotype, like metastatic melanoma, the activity of antioxidant enzymes may increase to limit oxidative damage related to high ROS and maintain tumor cell survival. In both cases antioxidant are modulated in order to promote the growth of tumor.

Paradoxically, antioxidants are as important as ROS, in maintaining cancer survival. Tumor cells have implemented an intelligent system through which some glycolysis by-products could be shifted toward the PPP to increase their stock of NADPH and GSH and, therefore, limit ROSrelated toxicity. Panieri & Santoro have recently reviewed and listed the impact of some agents targeting the tumor antioxidant defense [START_REF] Panieri | ROS homeostasis and metabolism: a dangerous liason in cancer cells[END_REF]. They found that depleting antioxidants like NADPH and GSH and the inactivation of GPx and TRX enhanced the susceptibility of cell to ROS-mediated apoptosis in breast, colon, ovarian and lung cancer [START_REF] Panieri | ROS homeostasis and metabolism: a dangerous liason in cancer cells[END_REF]. Breast cancer cells deficient in the antioxidant enzymes SOD and CAT lost their ability to form tumors when injected into mice [START_REF] Davison | Antioxidant enzymes mediate survival of breast cancer cells deprived of extracellular matrix[END_REF]. The attachment of cancer cells to ECM is essential for their survival, nonetheless, detached cells may also survive through a mechanism known as anchorage-independent growth. CAT and SOD seems to play a critical role in such process, since their overexpression increased cancer cell survival, while their silencing compromised cellular growth [START_REF] Davison | Antioxidant enzymes mediate survival of breast cancer cells deprived of extracellular matrix[END_REF]. Therefore, a cancerous cell deprived from its antioxidant arms is more vulnerable to ROS toxicity and death. The redox homeostasis in tumor cells is intelligibly controlled and its deregulation may need an intervention with multiple drugs targeting both pro and antioxidant systems.

The double face of ROS in cancer

Mechanisms promoting cancer growth and progression

As mentioned throughout the text, it is becoming clear that ROS play a twin role in cancer, on the one hand they have the ability to promote tumorigenesis and vascularization (Wang et al., 2015b).

On the other hand they can induce DNA damage, cell cycle arrest, senescence and apoptosis (Schieber and Chandel, 2014). Hereafter we will start by describing main ROS-dependent mechanisms involved in tumor growth and progression.

Proliferation: Patients with hepatocellular carcinoma exhibited high rate of mortality, mainly, because of the sustained ROS generation and decrease in MnSOD expression (Wang et al., 2016c). ROS can promote tumor initiation and progression, by increasing the expression of proteins involved in cell cycle activation like cyclin D and E [START_REF] Metodiewa | Anticancer potential of N,N-diethylaminoethyl ethers of flavanone oximes: a comparison with mitoxantrone action on rat Yoshida sarcoma cells in vivo[END_REF]. In this light, the abnormal activation of Wnt/β-catenin pathway has been reported in several types of cancer. Recent observations from Kamihara et al. indicate that in multiple myeloma cells, characterized by the presence of high intracellular iron levels, ROS was responsible for the activation of β-catenin signaling and tumor proliferation [START_REF] Kamihara | The iron chelator deferasirox induces apoptosis by targeting oncogenic Pyk2/beta-catenin signaling in human multiple myeloma[END_REF]. Whereas the use of deferasirox to chelate free iron produced the opposite effect.

The phosphoinositide-3 kinase (PI3K)/Akt is one the major routes that maintain cancer growth. In response to growth factors like, epidermal growth factor (EGF), ROS activate the PI3K/Akt pathway resulting in the phosphorylation and inactivation of Forkhead box3a (FOXO3a).

This abolishes the sustained inhibition exerted by FOXO3a on AP-1, leading to its nuclear accumulation (Saxena et al., 2013a). AP-1 is a transcriptional factor for miR- 21 (Talotta et al., 2009) that promotes carcinogenesis by targeting various tumor suppressors [START_REF] Barreiro | Both oxidative and nitrosative stress are associated with muscle wasting in tumour-bearing rats[END_REF].

Indeed, AP-1 up-regulates the expression of miR21, which in turn decreases the levels of the tumor suppressors including: phosphatase TENsin homolog (PTEN) and programmed cell death 4 gene (PDCD4) (Saxena et al., 2013a;Saxena et al., 2013b), well-known to induce apoptosis and control cell invasion and angiogenesis.

Interestingly, cancer cells are able to produce their proper immunoglobulin G (IgG), which can disturb the activity of antioxidant enzymes like peroxiredoxin-1 (PRDX-1), through direct binding. Such mechanism allows the production of ~ 2μM of H2O2 (low doses) to promote ERK activation and cancer cell proliferation [START_REF] Wang | Cancer-derived immunoglobulin G promotes tumor cell growth and proliferation through inducing production of reactive oxygen species[END_REF]. The family of heat shock protein (HSP)

are also important target for cancer treatment. In this light, renal cancer cells showed lower levels of HSP60 that altered the expression of mitochondrial complex I and increased ROS production.

This makes cells more dependent on the Krebs cycle to help them better proliferate and survive (Tang et al., 2016). All together, these data describe some of the main known mechanisms linking the production of low-to-moderate ROS levels to cancer cells proliferation. Vascularization and Hypoxia: Angiogenesis and formation of micro-vessels are important factors that could drive tumor growth and invasion [START_REF] Culy | Bevacizumab: antiangiogenic cancer therapy[END_REF]. Also, it is wellestablished that altered micro-vessels may impair blood perfusion and delivery of oxygen, causing hypoxia [START_REF] Jain | Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy[END_REF]. The resultant activation of HIF-1α is associated with poor prognosis and an aggressive tumor phenotype [START_REF] Jain | Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy[END_REF]. In response to inflammatory stimulators, ROS enhance the expression and stabilization of HIF-1α, which regulates the expression of several proangiogenic factors involved in blood vessels formation and migration [START_REF] Choi | 15-Deoxy-Delta12,14-prostaglandin J2 stabilizes hypoxia inducible factor-1alpha through induction of heme oxygenase-1 and direct modification ofprolyl-4-hydroxylase 2[END_REF].

Accordingly, ROS also increased the density of blood vessels irrigating mECK36 sarcoma tumor via stimulating the expression and release of pro-angiogenic factors like, vascular endothelial growth factor (VEGF) [START_REF] Ma | A role for virally induced reactive oxygen species in Kaposi's sarcoma herpesvirus tumorigenesis[END_REF]. One of the mechanism through which ROS may mediate VEGF production is β-catenin-dependent. Indeed, in response to TNF-α, NOX-4 is activated and produce high levels of ROS which in turn promote the transcriptional activity of β-catenin and subsequent VEGF expression in epithelial cells (Wang et al., 2016b).

Fibroblasts surrounding cancer constitute an important basis for tumor vascularization.

P53-deficient colon cancer cells produce high levels of ROS, which in turn activate fibroblasts to secret enormous amounts of VEGF and promote tumor neo-vascularization [START_REF] Hayashi | p53 functional deficiency in human colon cancer cells promotes fibroblast-mediated angiogenesis and tumor growth[END_REF].

In the same way, mesenchymal stem cells (MSC) secrete exosomes, containing stimuli that may influence tumor vasculature. For example, menstrual stem cells-derived exosomes reduced ROS production, blood vessels density (i.e. CD31 expression), HIF-1α content and VEGF expression in PC3 prostate tumor, whereas bone marrow MSC-derived exosomes enhanced angiogenesis [START_REF] Alcayaga-Miranda | Prostate tumor-induced angiogenesis is blocked by exosomes derived from menstrual stem cells through the inhibition of reactive oxygen species[END_REF]. These findings emphasize the critical role of ROS in the crosstalk engaged between tumor and its neighboring cells to control the intra-tumor vascularization.

Metastasis:

Angiogenesis constitutes an unavoidable step for tumor metastasis.

Metastasis is a multistep process in which tumor cells (1) interact with the extracellular matrix (ECM), ( 2) degrade the matrix components by releasing several proteases (e.g. metalloproteinase;

MMP) and, then, (3) migrate through vessels irrigation tumor to a second organ [START_REF] Stracke | Cell motility, a principal requirement for metastasis[END_REF]. The early observations that ROS could induce the dissociation of laminin and type IV collagen from Engelbreth-Holm-Swarm (EHS) sarcoma matrix, support a possible role for ROS in promoting tumor dissemination and metastasis [START_REF] Riedle | Reactive oxygen species cause direct damage of Engelbreth-Holm-Swarm matrix[END_REF]. ROS are able to decrease the expression of proteins involved in cell adhesion, including αvβ3-integrin and E- cadherin molecules, on cancer cells (Thews et al., 2009). Also, H2O2 promotes cancer cells invasiveness and migration through the activation of ERK, which in turn provokes NF-κB nuclear accumulation and expression of genes encoding MMP-2 and MMP-9 [START_REF] Cao | Curcumin inhibits H2O2-induced invasion and migration of human pancreatic cancer via suppression of the ERK/NF-kappaB pathway[END_REF]. The release of MMPs together with the decrease in the expression of adhesion molecules, cause ECM degradation and help cells to detach from their initial matrix. However, in a second step, cancer cells will need to reattach to another organ and thus re-express adhesion molecules. In this regard, MICAL-1 is a redox-sensitive enzyme that regulates the dynamics of actin polymers, which confers it the ability to control the trafficking of vesicles containing adhesion molecules like, IgCAM, and regulate their expression on cell surface [START_REF] Van Battum | The intracellular redox protein MICAL-1 regulates the development of hippocampal mossy fibre connections[END_REF]. Upon treatment of breast cancer cells with EGF, the intracellular levels of ROS raised probably leading to the activation of MICAL-1 signaling [START_REF] Deng | MICAL1 controls cell invasive phenotype via regulating oxidative stress in breast cancer cells[END_REF]. These events were responsible for the acquisition of an invasive behavior in breast cancer cells, as the inhibition of MICAL-1 abolished cell metastasis. Through their ability to produce ROS and inflammatory stimuli, cancer-associated fibroblasts (CAF) constitute a signaling niche for tumor to control its propensity to metastasize. A new mechanism linking CAF to cancer metastasis has been recently unveiled by Ren and collaborators [START_REF] Ren | Reprogramming carcinoma associated fibroblasts by AC1MMYR2 impedes tumor metastasis and improves chemotherapy efficacy[END_REF]. The authors showed that CAF induced the expression of miR21 in breast cancer tumor. As we mentioned previously, miR21 is well-known to repress the expression of several tumor suppressor genes. In respect with this function, miR21 inhibited the expression of the tumor suppressor Von Hippel-Lindau (VHL), resulting in the dimerization of NF-κB and βcatenin. As a direct result, F-actin fibers undergone a rearrangement, in a manner that promotes cell migration. This was confirmed in mice bearing breast cancer, as the inhibition of miR21 with AC1MMRY2, reduced lung metastasis [START_REF] Ren | Reprogramming carcinoma associated fibroblasts by AC1MMYR2 impedes tumor metastasis and improves chemotherapy efficacy[END_REF].

Mechanisms suppressing cancer growth and progression

Cell death and Apoptosis: C26 colon and Hepa 1-6 liver cancer cells produce high mitochondrial H2O2, at a level close to toxicity [START_REF] Laurent | Controlling tumor growth by modulating endogenous production of reactive oxygen species[END_REF]. The inhibition of H2O2 with NAC increased cancer proliferation, while the treatment with SOD mimetic, which increases the production of H2O2, killed cancer cells [START_REF] Laurent | Controlling tumor growth by modulating endogenous production of reactive oxygen species[END_REF]. In TNF-α-treated SMMC-7721 hepatocellular carcinoma cells, the down-regulation of PRDX increased sensitivity to H2O2mediated cell death, while overexpression triggered the opposite effect [START_REF] Chen | Expression of FOXO6 is Associated With Oxidative Stress Level and Predicts the Prognosis in Hepatocellular Cancer: A Comparative Study[END_REF].

Mitochondrial dysfunction and ROS accumulation are important pro-apoptotic events occurring in cancer cells [START_REF] Wu | Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC)-mediated generation of reactive oxygen species causes cell cycle arrest and induces apoptosis via activation of caspase-3, mitochondria dysfunction and nitric oxide (NO) in human osteogenic sarcoma U-2 OS cells[END_REF], they lead to the expression of the cell cycle inhibitor P53, cell cycle arrest at the G2/M phase, nucleosomal DNA fragmentation and subsequent apoptosis induction [START_REF] Wu | Benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC)-mediated generation of reactive oxygen species causes cell cycle arrest and induces apoptosis via activation of caspase-3, mitochondria dysfunction and nitric oxide (NO) in human osteogenic sarcoma U-2 OS cells[END_REF]. ROS may also induce apoptosis in a P53-independent manner through a sustained activation of P21. Masgras et al. showed that U2-OS and HT1080 sarcoma cells were more sensitive to P21-mediated apoptosis compared to endometrial (i.e. EJ) and prostate (i.e. PC3) cancer cells [START_REF] Masgras | Reactive oxygen species and mitochondrial sensitivity to oxidative stress determine induction of cancer cell death by p21[END_REF]. Early in vivo observations demonstrated that ROS can trigger apoptosis through increasing lipid, protein and DNA oxidation within tumor itself [START_REF] Xie | Activation of nitric oxide synthase gene for inhibition of cancer metastasis[END_REF][START_REF] Xie | Direct correlation between expression of endogenous inducible nitric oxide synthase and regression of M5076 reticulum cell sarcoma hepatic metastases in mice treated with liposomes containing lipopeptide CGP 31362[END_REF]. This was confirmed by recent studies, showing that chemotherapeutic agents, like cyclophosphamide, increased lipid peroxidation and apoptosis in sarcoma-180 tumor tissues [START_REF] Jana | Antitumorigenic potential of linalool is accompanied by modulation of oxidative stress: an in vivo study in sarcoma-180 solid tumor model[END_REF]. Mitochondria-dependent apoptosis was the most studied path. Indeed, ROS-mediated mDNA damages may cause mutations in genes encoding numerous elements of the MRC, which in turn could disturb the function of MRC, resulting in further overproduction of O2 [START_REF] Van Houten | Role of mitochondrial DNA in toxic responses to oxidative stress[END_REF]. Excessive levels of ROS interact with members of the permeability transition pore (PTP), like the mitochondrial channel VDAC, and induce the hyperpolarization of mitochondrial membrane potential (ΔΨm), which become more permeable allowing the release of cytochrome c into the cytoplasm [START_REF] Santamaria | Efficient execution of cell death in non-glycolytic cells requires the generation of ROS controlled by the activity of mitochondrial H+-ATP synthase[END_REF]. Mitochondria-to-cytoplasm release of cytochrome c is done after the oxidation of cardiolipin that will detach from the latter and constitute a docking platform for the formation of tBid/Bax pore at the outer mitochondria membrane, allowing the exit of cytochrome c into the cytoplasm [START_REF] Kagan | Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors[END_REF]. Then, cytochrome c forms jointly with APAF and pro-caspase-9 the "apoptosome", which once activated will cleave executioner caspases 3/7 to promote DNA fragmentation and apoptosis induction [START_REF] Circu | Disruption of pyridine nucleotide redox status during oxidative challenge at normal and low-glucose states: implications for cellular adenosine triphosphate, mitochondrial respiratory activity, and reducing capacity in colon epithelial cells[END_REF]. Indeed, the anticancer activity of alkylating agents, like cisplatin, and X-ray/γ-radiation rely mainly of the production of high intra-tumor amounts of ROS (Borek, 2004a). It is important to note that given the efficacy of H2O2 in inducing apoptosis, a phase-I clinical trial has tested the effectiveness of chemotherapy combined to 0.05, 0.075 or 0.1% of H2O2 added to the perfusate.

Treatment with H2O2 did not result in adverse side-effects but most likely potentiated tumor-killing effects of the administrated treatment [START_REF] Harrison | A phase I trial of thermal sensitization using induced oxidative stress in the context of HIPEC[END_REF]. Preclinical studies have also tested similar approaches in tumor-bearing animals by introducing XO as a source of ROS production in combination with hyperthermia to inflict a largest level of intra-tumor oxidative damage and increase the rate of apoptosis [START_REF] Frank | Enhancement of oxidative cell injury and antitumor effects of localized 44 degrees C hyperthermia upon combination with respiratory hyperoxia and xanthine oxidase[END_REF][START_REF] Lambert | Selected contribution: Hyperthermia-induced intestinal permeability and the role of oxidative and nitrosative stress[END_REF]Thews et al., 2009).

Senescence: Paradoxically, oncogenes like Ras and Myc, are known to induce cellular senescence in certain cancer cells [START_REF] Weyemi | ROS-generating NADPH oxidase NOX4 is a critical mediator in oncogenic H-Ras-induced DNA damage and subsequent senescence[END_REF]. Senescence disables the proliferating capacity of cancer cells without inducing cell death and constitutes another form of tumor regression [START_REF] Acosta | Senescence: a new weapon for cancer therapy[END_REF]. Numerous mechanisms have been described to drive senescence-like growth arrest. The inhibition of protein kinase CKII resulted in the activation of PI3K/Akt signaling cascade and the subsequent phosphorylation of the transcriptional factor FOXO3a [START_REF] Park | Inactivation of the FoxO3a transcription factor is associated with the production of reactive oxygen species during protein kinase CK2 downregulation-mediated senescence in human colon cancer and breast cancer cells[END_REF]. Then, FOXO3a will remain sequestrated in the cytoplasm unable to translocate into the nucleus. As FOXO3a regulates a plethora of antioxidant genes [START_REF] Huang | Dynamic FoxO transcription factors[END_REF], its cytoplasmic accumulation inhibits the expression of those genes and, therefore, increases intracellular ROS levels. Then, ROS are able to induce senescence through the cell cycle inhibitor axis P53-P21, resulting in irreversible cell cycle arrest at phase G1 [START_REF] Colavitti | Reactive oxygen species as mediators of cellular senescence[END_REF][START_REF] Zdanov | Identification of p38MAPKdependent genes with changed transcript abundance in H2O2-induced premature senescence of IMR-90 hTERT human fibroblasts[END_REF]. In turn, activated P21 may further enhance ROS production in senescent cancer cells [START_REF] Kim | Pharmacological approaches to discovery and development of new mucolytic agents[END_REF]. An increase in ROS production, to a certain level, induces DNA damage, reduces the fork of DNA replication and causes senescence [START_REF] Maya-Mendoza | Myc and Ras oncogenes engage different energy metabolism programs and evoke distinct patterns of oxidative and DNA replication stress[END_REF]; thus, a kind of "ROS-P21-ROS" loop exists to drive and maintain senescence.

Senescent cells produce specific mediators, a phenomenon known as senescence-associated secretory phenotype (SASP) consisting mainly of cytokines like IL-6 and IL-8 and TGF-β (Munoz-Espin and Serrano, 2014). In response to Ras activation, ROS are able to induce the activation of NF-κB and subsequent release of IL-6/IL-8, which is essential to maintain cancer cells in a senescent state [START_REF] Wang | Protein kinase D1 is essential for Ras-induced senescence and tumor suppression by regulating senescence-associated inflammation[END_REF]. We cannot describe main tumor suppressive mechanisms without mentioning the important role of P38 MAPK in driving senescence. Han & Sun have illustrated the fruits of several research and proposed a mechanism for the activation of P38 and P38-induced cellular senescence [START_REF] Han | The pathways to tumor suppression via route p38[END_REF]. Indeed, the increase in ROS accompanying the oncogenic transformation, induces activation of MKK3/6 an upstream of P38 MAPK, which once activated, will phosphorylate P53 at three different serine residues Ser33, 37 and 46. Activated P53 then promotes the expression and stabilization of P16 INK4α , P14/P19 ARF and P21 to induce replicative senescence [START_REF] Collado | The power and the promise of oncogene-induced senescence markers[END_REF][START_REF] Deng | High intensity ras signaling induces premature senescence by activating p38 pathway in primary human fibroblasts[END_REF][START_REF] Serrano | Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a[END_REF]. However, P38 may also induce P21 stabilization in a P53-independent manner [START_REF] Lafarga | p38 Mitogen-activated protein kinase-and HuR-dependent stabilization of p21(Cip1) mRNA mediates the G(1)/S checkpoint[END_REF]. To date, research in this field indicates that PI3K/Akt and P38 MAPK are the two main ROS-dependent pathways that trigger senescence [START_REF] Xu | Suppression of tumor growth and metastasis by dietary fish oil combined with vitamins E and C and cisplatin[END_REF].

Innate immune response:

The role of ROS in mediating immune anti-tumor response is well-known [START_REF] Corzo | Mechanism regulating reactive oxygen species in tumorinduced myeloid-derived suppressor cells[END_REF]. Tumor-associated macrophages could be divided into M1 and M2 clones, which exhibit pro and anti-inflammatory secretory profile respectively [START_REF] Mills | M1 and M2 Macrophages: Oracles of Health and Disease[END_REF].

The M2 macrophages may favor tumor immune escape as they are unlikely to produce NO . and ROS; Interestingly, their treatment with specific mediators triggering polarization toward a M1like profile, increased iNOS/arginine metabolism, NOX expression and the subsequent release of NO . and ROS [START_REF] Simoes | Lipoxin A4 selectively programs the profile of m2 tumor-associated macrophages which favour control of tumor progression[END_REF]. This could lead to apoptosis induction and the reduction of intra-tumor angiogenesis. The differentiation of innate immune cells is an important step to mediate tumor suppression. Accordingly, accumulation of immature immune cells in tumor microenvironment protects the tumor through a mechanism known as immune suppression [START_REF] Xiao | Interleukin 33 in tumor microenvironment is crucial for the accumulation and function of myeloid-derived suppressor cells[END_REF]. In this light, NOX-1 and NOX-2 play a pivotal role in macrophages differentiation, as NOX-1 or NOX-2 null macrophages failed to fully differentiate and produce ROS, thus delaying the anti-tumor response (Xu et al., 2016). Convincing pieces of evidence suggest that immunemodulatory agents, such as methylglyoxal, mediate their anticancer functions via increasing ROS production. Interestingly, an important number of these studies was performed on sarcoma-180 tumors implanted either subcutaneously or intramuscularly [START_REF] Kong | Anti-tumor and macrophage activation induced by alkali-extracted polysaccharide from Pleurotus ostreatus[END_REF][START_REF] Sanders | Cancer resistance of SR/CR mice in the genetic knockout backgrounds of leukocyte effector mechanisms: determinations for functional requirements[END_REF]Tomimori et al., 2012). The results were reproducible between studies, indicating that tumorinfiltrating macrophages and neutrophils suppressed sarcoma growth by producing high amounts of ROS, particularly NO . radicals.

►Key Messages from Chapter II -ROS are mainly produced by XO, NOX, iNOS and mitochondria in cancer cells. ROS play a dualistic role in cancer depending on the produced doses inside the cell and its microenvironment. For example, doses ranging from 50-to-400 nM for NO .

or 2-to-1000 μM for H2O2 are most likely associated with cancer proliferation, invasion and angiogenesis, whereas doses exceeding these intervals cause apoptosis or senescence. The amounts of ROS are controlled by the antioxidant system, which decreases its activity during the precancerous stage allowing low-to-moderate ROS levels to produce DNA damage and malignant transformation; but increases its antioxidant activity during cancer progression to protect cells from

Defining cancer cachexia

Approximately, 50% of patients with advanced stages of cancer experience cachexia and more than the third die within six months following the manifestation of first cachexia symptoms [START_REF] Wigmore | Contribution of anorexia and hypermetabolism to weight loss in anicteric patients with pancreatic cancer[END_REF]. Cancer cachexia is a multifactorial syndrome characterized by an ongoing loss of skeletal muscle mass, with or without depletion of adipose tissue, that cannot be reversed by conventional nutritional intervention (Fearon et al., 2011). The prevalence of cachexia depends on tumor type, size and localization. Approximately, 80% of patients with gastric, pancreatic and esophageal cancer and 50-60% with colorectal, lung and prostate cancer have substantial weight loss at the time of diagnosis [START_REF] Laviano | Therapy insight: Cancer anorexia-cachexia syndrome--when all you can eat is yourself[END_REF][START_REF] Bruera | ABC of palliative care. Anorexia, cachexia, and nutrition[END_REF]. Excessive involuntary weight loss and cachexia related-death are frequently found in patients with colon, pancreatic and lung cancer, whereas those with sarcomas, non-Hodgkin's lymphoma and breast cancer are spared. Death usually occurs, when patients loose more than 30% of their historic stable body weight, which corresponds to a loss of 75% of their skeletal muscle mass [START_REF] Wigmore | Contribution of anorexia and hypermetabolism to weight loss in anicteric patients with pancreatic cancer[END_REF].

Cancer cachexia could be considered as a public health problem [START_REF] Farkas | Cachexia as a major public health problem: frequent, costly, and deadly[END_REF] and, therefore, the management of cachexia represents a real challenge for researchers and clinicians in the biomedical field. During the time course of cachexia, alteration within metabolism and eventual depletion of adipose tissue manifest concomitantly and characterize the early stage of the disease.

These events constitute a favorable environment for the development of muscle wasting in a later stage. Once the muscle compartment is affected, the evolution of cachexia becomes poorly controlled, even irreversible, because the currently used anti-cachexia treatments do not offer the possibility to effectively reverse and abolish muscle wasting.

Pharmacological and nutritional agents proposed to treat cachexia are mainly appetite stimulants (e.g. ghrelin, megestrol acetate and corticosteroids) to reduce anorexia [START_REF] Loprinzi | Phase III evaluation of 4 doses of megestrol acetate as therapy for patients with cancer anorexia and/or cachexia[END_REF];

androgens and anabolic steroids (e.g. testosterone, growth hormone and insulin growth factor) to improve muscle mass and promote protein synthesis [START_REF] Waters | Does growth hormone drive breast and other cancers?[END_REF]; Omega-3-derived fatty acids (e.g. eicosapentaenoic acid and docosahexaenoic acid) to increase total body mass and reduce chemotherapy-related toxicity [START_REF] Ries | A systematic review on the role of fish oil for the treatment of cachexia in advanced cancer: an EPCRC cachexia guidelines project[END_REF]; pro-inflammatory cytokines inhibitors and no-steroidal anti-inflammatory drugs (NSAID) (e.g. TNF-α inhibitors) to slow lipolysis and muscle atrophy [START_REF] Saraceno | Effect of anti-tumor necrosis factor-alpha therapies on body mass index in patients with psoriasis[END_REF]. Unfortunately, all these treatments given alone or in combination, mainly resulted in an increase in fat body mass but failed to restore lean body mass (Gullett et al., 2011). However, a new window of hope could be opened with the introduction of anamorelin as a pharmacological option to treat cachexia. Indeed, two randomized phase III trials, ROMANA 1 and ROMANA 2, have shown that anamorelin was effective to increase total body weight mainly by improving lean body mass [START_REF] Schafer | Ataxin-10 is part of a cachexokine cocktail triggering cardiac metabolic dysfunction in cancer cachexia[END_REF].

Muscle wasting: key feature of cachexia

At the beginning of this paragraph we might underscore that cancer cachexia could affect the function of many organs, as illustrated in Figure 15. However, herein we will focus on the most common symptom, muscle atrophy, since it is the component of cancer cachexia that has the greatest negative impact on patients' QOL and anticancer treatment efficiency [START_REF] Bachmann | Cachexia worsens prognosis in patients with resectable pancreatic cancer[END_REF]. Clinically, muscle atrophy is a serious debilitating condition characterized by the presence of fatigue, weakness and pain, leading to functional impairment (Fearon et al, 2011). Patients with advanced cancer often report a decrease in their autonomy, spontaneous physical activity and FIGURE 15: The impact of tumor derived-factors on multiple organs during cancer cachexia (Argilés et al., 2015;Tisdale, 2009) Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 ability to perform daily tasks [START_REF] Fouladiun | Daily physical-rest activities in relation to nutritional state, metabolism, and quality of life in cancer patients with progressive cachexia[END_REF]. They are aware of the severity of functional symptoms that become more accentuated during the time course of cachexia (Leblanc et al, 2015).

A number of epidemiological studies have conducted correlation analysis in order to determine factors that affect the QOL in cancer cachectic patients. Their results indicate that fatigue, reduced physical activity and non-capacity to perform exercise were the most significant predictors of QOL despite the type and location of cancer (Cramarossa et al, 2014;Kawaguchi et al, 2012). As muscle wasting evolves, cancer patients become more vulnerable to the toxic effect of chemotherapy, requiring doses reduction or treatment delay [START_REF] Prado | Sarcopenia as a determinant of chemotherapy toxicity and time to tumor progression in metastatic breast cancer patients receiving capecitabine treatment[END_REF]. Given the critical situation of the patient, a complete resection of tumor by chirurgical intervention is also highly risked [START_REF] Muscaritoli | Prevention and treatment of cancer cachexia: new insights into an old problem[END_REF]; thus, the reduced efficiency of anticancer treatment reflects the poor performance status in patients rather than alteration of the therapy itself. Therefore, preservation of overall muscle mass and function is essential to achieve successful treatment and reduce the likelihood of mortality. From this perspective, it is crucial to understand, in a first step, the underpinning molecular mechanisms responsible for the development of muscle wasting, in an attempt to identify potential therapeutic targets.

Tumor-muscle crosstalk: a principle axis for ROS production and muscle wasting

Pro-inflammatory cytokines, TGF-β family ligands (e.g. myostatin and activin) and other mediators like proteolysis-inducing factor (PIF) could be expressed and released continuously by tumor cells (Tisdale, 2002). Elevated levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) in biological fluids (e.g. blood and urine) of patients experiencing advanced stages of cancer [START_REF] Cariuk | Induction of cachexia in mice by a product isolated from the urine of cachectic cancer patients[END_REF][START_REF] Fortunati | Pro-inflammatory cytokines and oxidative stress/antioxidant parameters characterize the bio-humoral profile of early cachexia in lung cancer patients[END_REF]Puig-Vilanova et al., 2015;Tisdale, 2009). However, whether PIF is expressed in humans and/or plays a significant role in the development of cancer cachexia, remains a subject of debate, as results have been contradictory between studies performed by different research teams [START_REF] Wieland | Is there a human homologue to the murine proteolysis-inducing factor? Clinical cancer research : an official journal of the[END_REF]Russel 2010a). Mice bearing colon C26 tumor exhibited high circulating levels of TNF-α, IL-1β and IL-6, comparing to control (Assi et al., 2016;Zhou et al., 2010).

Once in bloodstream, these mediators can act in an autocrine and paracrine manner to promote ROS formation in both tumor and skeletal muscle, respectively [START_REF] Arthur | Oxidative stress as a therapeutic target during muscle wasting: considering the complex interactions[END_REF].

Indeed, ROS control cell cycle entry-withdrawal gate within tumor (Schieber and Chandel, 2014) as well as anabolism-catabolism balance in skeletal muscle (Baracos, 2001). With the progression of tumor, skeletal muscle becomes more exposed to inflammatory stimuli and, as a direct result, important amounts of ROS are generated within muscle (Figure 16). In this condition, ROS promote the activation of catabolic pathways over those involved in protein synthesis (Tisdale, 2009), leading to the development of oxidative damage and muscle wasting. Indeed, treatment of muscle cells with the culture medium of different cancer cells (rich in tumor-specific factors)

induced protein oxidation and atrophy [START_REF] Mu | Notch Signaling Mediates Skeletal Muscle Atrophy in Cancer Cachexia Caused by Osteosarcoma[END_REF].

In fact, evidences from human and animal studies clearly indicate that skeletal muscle is greatly affected during cancer cachexia. Oxidative damage markers were increased in the skeletal muscle of cancer patients. Patients with lung cancer exhibited an increase in the levels of protein carbonyls in vastus lateralis, which correlated positively with muscle proteolysis (Puig-Vilanova et al., 2015). The interesting study from Buck's team showed that lipid peroxidation adducts, MDA, were elevated within skeletal muscle (i.e. vastus lateralis) of patients with colon, lung and esophageal cancer comparing to control subjects [START_REF] Ramamoorthy | Decreased Jun-D and myogenin expression in muscle wasting of human cachexia[END_REF]. OS was also reported in skeletal muscle and other tissues of cachectic animals. For example, protein carbonylation and lipid peroxidation adducts namely 4-HNE and MDA were increased in the gastrocnemius (Gas) muscle of rodents bearing different types of tumors [START_REF] Barreiro | Both oxidative and nitrosative stress are associated with muscle wasting in tumour-bearing rats[END_REF][START_REF] Guarnier | Time course of skeletal muscle loss and oxidative stress in rats with Walker 256 solid tumor[END_REF][START_REF] Mastrocola | Muscle wasting in diabetic and in tumor-bearing rats: role of oxidative stress[END_REF]. These observations emphasize a key role of tumor-muscle crosstalk in promoting ROS formation within muscle and the subsequent development of muscle atrophy.

Sources of ROS in cancer-related muscle wasting

ROS-producing enzymes

XO:

Little is known about the regulation of XO in the skeletal muscle compartment during cancer. In numerous models of tumor-bearing animals, the activity of XO was elevated in skeletal muscle and correlated with an increase in muscle oxidative damage [START_REF] Hinch | Disruption of pro-oxidant and antioxidant systems with elevated expression of the ubiquitin proteosome system in the cachectic heart muscle of nude mice[END_REF][START_REF] Konishi | Febuxostat improves outcome in a rat model of cancer cachexia[END_REF][START_REF] Springer | Inhibition of xanthine oxidase reduces wasting and improves outcome in a rat model of cancer cachexia. International journal of cancer[END_REF][START_REF] Springer | The xanthine oxidase inhibitor oxypurinol reduces cancer cachexia-induced cardiomyopathy[END_REF]. Although XO is not usually present at high levels within skeletal muscle, the hyper-activation of XO during cachexia could be explained by an increase in the cleavage of XDH to XO [START_REF] Springer | Inhibition of xanthine oxidase reduces wasting and improves outcome in a rat model of cancer cachexia. International journal of cancer[END_REF]. The few number of studies who addressed the role of XO in cancer-induced muscle wasting, demonstrated that targeting XO with selective inhibitors such as allopurinol (4 and 40 mg/kg/day), oxypurinol (4 and 40 mg/kg/day) and febuxostat (5 mg/kg/day) can reduce body weight loss and skeletal muscle atrophy [START_REF] Konishi | Febuxostat improves outcome in a rat model of cancer cachexia[END_REF][START_REF] Springer | Inhibition of xanthine oxidase reduces wasting and improves outcome in a rat model of cancer cachexia. International journal of cancer[END_REF][START_REF] Springer | The xanthine oxidase inhibitor oxypurinol reduces cancer cachexia-induced cardiomyopathy[END_REF]. Only one study from Vaughan et al. showed that oxypurinol (1mmol/L of water) was likely to aggravate weight loss in C26 mice, emphasizing a possible protective role for XO [START_REF] Vaughan | Eicosapentaenoic acid and oxypurinol in the treatment of muscle wasting in a mouse model of cancer cachexia[END_REF]. Accordingly, preliminary results from our laboratory indicate that allopurinol (50/mg/kg/day) failed to improve cancer cachexia symptoms (Assi and Rebillard, 2016). Similarities between our findings and that of Vaughan's study could be attributed to the fact that protein carbonyls, 4-HNE and/or 8-oxoG content, were absent in skeletal muscle. While in the study of Springer et al. showing improvement of muscle mass after allopurinol administration, the content of protein carbonyls was greater within wasted muscles and significantly decreased in response to allopurinol [START_REF] Springer | Inhibition of xanthine oxidase reduces wasting and improves outcome in a rat model of cancer cachexia. International journal of cancer[END_REF]. Additionally, allopurinol failed to attenuate systemic oxidative damage in our model of C26 mice. This may suggest that XO is not a primary actor in the pathogenesis of muscle wasting related to C26 tumor. NOX: TNF-α, IFN-γ, PIF and Angiotensin-II (Ang-II) are known to induce ROS production via the activation of NOX [START_REF] Hubackova | IFNgamma induces oxidative stress, DNA damage and tumor cell senescence via TGFbeta/SMAD signaling-dependent induction of Nox4 and suppression of ANT2[END_REF][START_REF] Sukhanov | Angiotensin II, oxidative stress and skeletal muscle wasting[END_REF]. Russell et al. showed that treatment of C2C12 myotubes cells with PIF and Ang-II induced atrophy through the NOX system [START_REF] Russell | Role of reactive oxygen species in protein degradation in murine myotubes induced by proteolysis-inducing factor and angiotensin II[END_REF]. NOX-induced excessive O2 .-production was also observed in rat L6 myotubes treated with Ang-II [START_REF] Wei | Angiotensin II-induced NADPH oxidase activation impairs insulin signaling in skeletal muscle cells[END_REF]. In a model of Ang-II-infused mice, the high formation of O2 .-levels within muscles promoted degradation of sarcomeric proteins [START_REF] Sukhanov | Angiotensin II, oxidative stress and skeletal muscle wasting[END_REF]. This elevated production of O2 .-was NOX-dependent, since its blockade with a specific inhibitor, apocynin, partially prevented atrophy. Contrariwise, it is thought that the enhanced O2 .-formation within skeletal muscle of cachectic mice bearing colon MAC 16 adenocarcinoma, was due to an aberrant antioxidant response rather than an increase in NOX activity [START_REF] Sullivan-Gunn | Decreased NADPH oxidase expression and antioxidant activity in cachectic skeletal muscle[END_REF]. PIF was able to promote O2 .-production via NOX and the subsequent activation of proteolysis pathway in muscle cells [START_REF] Russell | Role of reactive oxygen species in protein degradation in murine myotubes induced by proteolysis-inducing factor and angiotensin II[END_REF]. Since NOX controls the activation of various downstream kinases that play an essential role in proliferation, differentiation and inflammation, the silencing of NOX isoforms could provide a particular therapeutic interest to limit cancer cells proliferation and reduce the magnitude of muscle degradation.

NOS: Today, it is admitted that the arginine metabolism is altered in cancer cachectic patients and responsible for the inhibition of protein synthesis and activation of proteolysis [START_REF] Buijs | The role of a disturbed arginine/NO metabolism in the onset of cancer cachexia: a working hypothesis[END_REF]. Accordingly, cachectic patients with advanced stages of cancer presented a greater NO . production, nitrotyrosine content and iNOS expression in skeletal muscle tissues, comparing to non-cachectic subjects [START_REF] Ramamoorthy | Decreased Jun-D and myogenin expression in muscle wasting of human cachexia[END_REF][START_REF] Reid | Role of nitric oxide in skeletal muscle: synthesis, distribution and functional importance[END_REF]. TNF-α is a potent activator of iNOS signaling and the subsequent alteration of muscle cell differentiation [START_REF] Buck | Muscle wasting and dedifferentiation induced by oxidative stress in a murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and antioxidants[END_REF]. During differentiation, myogenic factors including MyoD and myogenin bind to a DNA consensus sequence called E-box, which is present in the promoter of many muscle-specific genes, like troponin and myosin creatine kinase (MCK) [START_REF] Yutzey | Different E-box regulatory sequences are functionally distinct when placed within the context of the troponin I enhancer[END_REF]. In cachectic nude mice overexpressing TNF-α gene, the binding of D-Jun/myogenin complex to MCK-specific Ebox region was disrupted by the activation of NOS, leading to muscle atrophy and dedifferentiation [START_REF] Buck | Muscle wasting and dedifferentiation induced by oxidative stress in a murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and antioxidants[END_REF] (see Box 1 for more information about the role of CK in skeletal muscle).

The inhibition of NOS, by nitro-L-arginine, prevented weight loss and muscle wasting in TNF-α-treated animals [START_REF] Buck | Muscle wasting and dedifferentiation induced by oxidative stress in a murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and antioxidants[END_REF]. Apoptosis is one of the mechanisms that could be involved in muscle atrophy [START_REF] Costelli | Ca(2+)-dependent proteolysis in muscle wasting[END_REF]. Interestingly, a link between iNOS and apoptosis activation has been suggested, since the administration of IL-15 to cachectic rats inhibited apoptosis by disturbing TNF-α signaling and the resulting NO . formation [START_REF] Figueras | Interleukin-15 is able to suppress the increased DNA fragmentation associated with muscle wasting in tumour-bearing rats[END_REF]. In C2C12 cells, TNF-α and IFN-γ were able to induce the activation of NF-κB and its FIGURE 17: The identified pathways for iNOS/NO . in muscle atrophy and dedifferentiation. [START_REF] Buck | Muscle wasting and dedifferentiation induced by oxidative stress in a murine model of cachexia is prevented by inhibitors of nitric oxide synthesis and antioxidants[END_REF][START_REF] Marco | NF-kappa B-mediated MyoD decay during muscle wasting requires nitric oxide synthase mRNA stabilization, HuR protein, and nitric oxide release[END_REF]) downstream target iNOS (Di [START_REF] Marco | NF-kappa B-mediated MyoD decay during muscle wasting requires nitric oxide synthase mRNA stabilization, HuR protein, and nitric oxide release[END_REF]. The activation of TNF-α/NF-κB/iNOS pathway was efficient to promote the degeneration of muscle via stimulating the loss of proteins playing a key role in muscle cell proliferation and differentiation such as MyoD [START_REF] Marco | NF-kappa B-mediated MyoD decay during muscle wasting requires nitric oxide synthase mRNA stabilization, HuR protein, and nitric oxide release[END_REF].

These compelling evidences indicate that selective inhibition of iNOS could decelerate cachexia progression in cancer (Figure 17).

Mitochondrial dysfunction

Mitochondria are main organelles producing energy molecules in the form of ATP, mainly through glycolysis and oxidative phosphorylation [START_REF] Ernster | Mitochondria: a historical review[END_REF]. In respect with this function, skeletal muscle constitutes a "niche" of mitochondria, since it is one of the main organs continuously producing energy and exerting thermoregulation function in the body [START_REF] Hocquette | Lipoprotein lipase activity and mRNA levels in bovine tissues. Comparative biochemistry and physiology[END_REF]. A scarce number of studies have addressed the mitochondrial events that occur within skeletal muscle during cancer. Mitochondrial dysfunction and altered mitochondrial plasticity are a primary source of ROS generation in cancer. ROS exert direct deleterious effects on MRC (e.i.

complexes I, II and IV) by decreasing their activities in skeletal and respiratory muscles of cachectic mice [START_REF] Fermoselle | Mitochondrial dysfunction and therapeutic approaches in respiratory and limb muscles of cancer cachectic mice[END_REF]. Thus, it makes sense that ROS-mediated MRC dysfunction could lead to impaired oxidative phosphorylation and low ATP synthesis. In numerous animal models of cancer-related muscle wasting, skeletal mass degradation was associated with a decrease in respiratory chain activity and low ability of wasted muscles to synthetize the required ATP [START_REF] Antunes | Molecular insights into mitochondrial dysfunction in cancer-related muscle wasting[END_REF][START_REF] Constantinou | Nuclear magnetic resonance in conjunction with functional genomics suggests mitochondrial dysfunction in a murine model of cancer cachexia[END_REF]. Indeed, treatment of C2C12 muscle cells with Lewis lung carcinoma conditioned culture medium (rich in pro-inflammatory mediators) increased ROS production and reduced ATP rates [START_REF] Mclean | Mitochondria dysfunction in lung cancer-induced muscle wasting in C2C12 myotubes[END_REF]. These disruptions in respiratory chain function were mainly due to mitochondrial loss (i.e. mitophagy) and structural abnormalities (i.e. giant mitochondria) (Tzika et al., 2013;White et al., 2011).

As depicted in Figure 18, a weak ATP production leads to a low mitochondrial transmembrane potential [START_REF] Loschen | Proceedings: Formation of oxygen radicals and hydrogen peroxide in mitochondrial membranes[END_REF], allowing mitochondria to generate excessive amounts of ROS potentially damaging for mitochondria membrane and muscle. Thus, there is a ROS-ATP-ROS loop during cachexia. ROS primarily produced in response to inflammatory stimuli disturb the MRC function within muscle, leading to a decreased ATP formation [START_REF] Mclean | Mitochondria dysfunction in lung cancer-induced muscle wasting in C2C12 myotubes[END_REF]. This poor ATP level is a favorable condition for high mitochondrial ROS production [START_REF] Loschen | Proceedings: Formation of oxygen radicals and hydrogen peroxide in mitochondrial membranes[END_REF], thereby maintaining the vicious circle. The mitochondrial energetic inefficiency and the subsequent accumulation of oxidative insults, may impede the capacity of muscle to generate sufficient force and ensure basic physical needs [START_REF] Argiles | Muscle wasting in cancer: the role of mitochondria[END_REF]. This ROS-dependent mechanism observed in skeletal, cardiac and respiratory muscles may in part explain the increased fatigue and decreased autonomy observed in cachectic individuals with advanced stages of cancer.

Defective antioxidant response

In addition to the above-mentioned sources of ROS, the loss of antioxidant counterbalance and control can exacerbate OS in cancer. At the systemic level, SOD activity was up-regulated in patients, with stage II to stage IV colon cancer, presenting a good performance status. While SOD activity decreased along with GPx activity in patients with compromised physical performance at the stage IV (Mantovani et al., 2002a). This may indicate that high grades of cancer and poor muscle strength are, most likely, associated with a weak enzymatic antioxidant activity. Patients with soft-tissue sarcoma exhibited low blood SOD and CAT levels [START_REF] Nathan | Oxidative stress and antioxidant status in primary bone and soft tissue sarcoma[END_REF], especially in those poor responding to treatment [START_REF] Nathan | Oxidative stress and antioxidant status in primary bone and soft tissue sarcoma[END_REF]. Furthermore, patients bearing colon cancer displayed a low blood level of GSH [START_REF] Navarro | Changes in glutathione status and the antioxidant system in blood and in cancer cells associate with tumour growth in vivo[END_REF]. The decrease in GSH content may be due to a decrease in the available substrates needed for GSH synthesis. In fact, glucose plays a pivotal role in the synthesis of compounds with high reducing potential, like NADPH, through the PPP (Figure 19). NADPH is required for ( 1) the reduction of GSSG to GSH, by the GSH reductase, and ( 2) formation of active catalase tetramers [START_REF] Kashiwagi | Pyruvate improves deleterious effects of high glucose on activation of pentose phosphate pathway and glutathione redox cycle in endothelial cells[END_REF]. The perturbations in glucose metabolism and reduced nutrients supply, due to symptoms such as anorexia and vomiting, can lead to an inadequate synthesis of reducing compounds and, therefore, may explain the GSH deficiency observed in cancer cachectic individuals [START_REF] Maccio | Interleukin-6 and leptin as markers of energy metabolic changes in advanced ovarian cancer patients[END_REF].

Skeletal muscle biopsies from lung cancer patients showed an increase in both SOD expression and activity, which coincided with high content of intramuscular protein carbonyls (Puig-Vilanova et al., 2015). Accordingly, experimental studies found that the expression of SOD was up-regulated within atrophied skeletal muscles [START_REF] Springer | Inhibition of xanthine oxidase reduces wasting and improves outcome in a rat model of cancer cachexia. International journal of cancer[END_REF]. We have reported an increase in catalase, but not in CuZnSOD and MnSOD, expression within skeletal muscle of cachectic C26 tumor-bearing mice (Assi et al., 2016). Contrariwise, others reported that both expression and activity of SOD and GPx were decreased in the skeletal muscle of cancer cachectic mice [START_REF] Hinch | Disruption of pro-oxidant and antioxidant systems with elevated expression of the ubiquitin proteosome system in the cachectic heart muscle of nude mice[END_REF][START_REF] Mastrocola | Muscle wasting in diabetic and in tumor-bearing rats: role of oxidative stress[END_REF][START_REF] Sullivan-Gunn | Decreased NADPH oxidase expression and antioxidant activity in cachectic skeletal muscle[END_REF]. Nonetheless, studies who denoted an increase in SOD activity have also demonstrated an increase in OS profiles, suggesting that SOD activation was inefficient and insufficient to antagonize muscular and systemic OS. An accumulation of high H2O2 rates due to the elevated SOD activity, might explain this paradox. However, data available from the literature strongly suggest that the decrease of muscle and blood GSH content, GSH/GSSG ratio and GPx activity occur during cancer-related muscle wasting.

ROS-dependent mechanisms in cancer-related muscle wasting

It is becoming clear from the pieces of conviction cited previously that ROS are mainly responsible for the atrophic events that occur in skeletal muscle during the progression of cancer.

In this way, Mantovani and co-workers established a direct association between ROS levels and the performance status of cancer cachectic patients. They found that the high blood levels of ROS were somehow associated with increased fatigue, decreased autonomy and elevated concentrations of pro-inflammatory cytokines (Mantovani et al., 2002a). As mentioned previously, oxidative damage were also reported in skeletal muscle biopsies of cancer patients (Paragraph 3). These findings highlight a possible link between ROS and the functional impairment observed in cancer patients. Indeed, the main mechanisms through which ROS provoke muscle wasting have been mainly unveiled in animal models and cell culture studies.

Anabolic pathways

Inhibition of IGF-1/Akt pathway and enhancement of PKR activation:

Muscle atrophy is mainly due to an increase in protein catabolism, by cytokine-dependent hyperactivation of various pathways, associated with hypo-anabolism (Baracos, 2001). The anabolic response in skeletal muscle is mainly derived by the insulin-like growth factor (IGF-1)/Akt pathway [START_REF] Lai | Conditional activation of akt in adult skeletal muscle induces rapid hypertrophy[END_REF]. IGF-1 signals through its specific receptor IGF1R to activate Akt, which in turn promotes the induction of mTOR. Then mTOR phosphorylates its downstream targets, p70S6K and 4E-binding protein/eukaryotic translation initiation factor 4E (4E-BP/eIF4E) inactive complex. This results in the release of the translational factor eIF4E, which is then able to associate with other factors and recruit ribosome subunits (Figure 20) (Kim et al., 2002). The inhibition of IGF-1/Akt pathway in cultured muscle cells and skeletal muscle tissues of cachectic rodent bearing tumor is well-described [START_REF] Morissette | Myostatin inhibits IGF-I-induced myotube hypertrophy through Akt[END_REF][START_REF] Zhuang | Reversal of muscle atrophy by Zhimu and Huangbai herb pair via activation of IGF-1/Akt and autophagy signal in cancer cachexia[END_REF]. Accordingly, the injection of low-doses IGF-1 reduced mortality and improved muscle performance in cachectic rats bearing Yoshida tumor [START_REF] Schmidt | IGF-1 treatment reduces weight loss and improves outcome in a rat model of cancer cachexia[END_REF]. However, the ability of ROS to enhance or inhibit Akt signaling in muscle cells, has previously aroused much controversy. A logical explanation seems to be provided by the recently published work of Tan et al. in which they demonstrated that Akt, but not its negative regulator PTEN, was sensitive to chronic OS in C2C12 cells treated with low-doses of H2O2 (Tan et al., 2015). On the other hand, very high doses of H2O2 increased Akt phosphorylation through oxidizing and perturbing the activity of PTEN.

Interestingly, the treatment of C2C12 cells with catalase reduced the levels of H2O2 in culture media and consequently increased Akt phosphorylation (Tan et al., 2015). Therefore, the sustained OS and defective antioxidant response present in cancer cachexia could be responsible for the inhibition of Akt phosphorylation in wasted muscles and the subsequent reduced protein synthesis.

In addition to the disruption of IGF-1/Akt signaling, the activation of dsRNA-dependent protein kinase (PKR) depresses protein synthesis by phosphorylating eIF2 on the α-subunit, thus preventing the assembly of the ribosome 43S complex and initiation of mRNA translation [START_REF] Rowlands | The catalytic mechanism of guanine nucleotide exchange factor action and competitive inhibition by phosphorylated eukaryotic initiation factor 2[END_REF]. Activation of PKR and phosphorylation of eIF2-α were elevated in atrophied muscles of cachectic patients with esophagogastric cancer and animals bearing MAC 16

tumor (Eley et al., 2008;Eley and Tisdale, 2007). Furthermore, tumor-derived factors such as PIF, isolated from cancer cachectic patients, was able to block protein synthesis in cultured muscle cells via the activation of PKR (Russell et al., 2010a). These results provide compelling evidence for the involvement of inflammation and ROS in the development of muscle atrophy by inhibiting protein synthesis. Nonetheless, ROS are also responsible for the activation of various proteolysis pathways.

Catabolic pathways

Ubiquitin-Proteasome Pathway (UPP): Three principal pathways are responsible for protein catabolism during cachexia: the ubiquitin-proteasome system (UPS), autophagy lysosome pathway (ALP) and calpain-dependent pathway. UPS is the predominantly studied and decorticated proteolytic framework and is the main culpable of sarcomeric protein breakdown in cachexia. Indeed, substrates destined for degradation, by the proteasome, are labeled with polyubiquitin chain thanks to the activity of two muscle-specific E3 ubiquitin-ligases, known as, muscle Ring finger protein-1 (MuRF-1) and muscle atrophy F-box (MAFbx) (Llovera et al., 1998).

Then, the ubiquitin-tagged proteins enter the proteasome core where they undergo enzymatic degradation into small peptide fragments. The expression of the UPS components was increased in the skeletal muscle of cachectic patients and tumor-bearing animals as well as in cultured myotube cells [START_REF] Bedard | Inactivation of the ubiquitin-specific protease 19 deubiquitinating enzyme protects against muscle wasting[END_REF][START_REF] Costelli | Anticytokine treatment prevents the increase in the activity of ATP-ubiquitin-and Ca(2+)-dependent proteolytic systems in the muscle of tumour-bearing rats[END_REF]Khal et al., 2005). It has been demonstrated that ROS can up-regulate the expression of MuRF-1/MAFbx and the proteasome subunits both in vitro and in vivo (Gomes-Marcondes and Tisdale, 2002;Tisdale, 2005). Basically, four upstream cascades in which ROS act as a signal transducer, allow the induction of the UPS during cachexia:

(1) TNF-PIF/ nuclear factor-κB (NF-κB) pathway, (2) TGF-β family ligands /Smad/FOXO pathway, (3) MAPK pathway and ( 4) IL-6/Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) pathway.

ROS are potent activators of the NF-κB signaling cascades, they activate IκB Kinase (IKK) leading to the phosphorylation and degradation of the NF-κB inhibitor, also known as, IκB [START_REF] Schmidt | The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-kappa B[END_REF]. NF-κB, was rapidly activated by O2 .-and H2O2 produced by NOX, following the treatment of C2C12 cells with TNF-α or PIF (Li and Reid, 2000;Li et al., 1998;[START_REF] Wyke | NF-kappaB mediates proteolysis-inducing factor induced protein degradation and expression of the ubiquitin-proteasome system in skeletal muscle[END_REF]. In the study of Cai et al, the use of mice expressing a constitutively active form of IKK, provoked the nuclear translocation of NF-κB leading to the transcriptional activation of MuRF-1 but not MAFbx [START_REF] Cai | IKKbeta/NF-kappaB activation causes severe muscle wasting in mice[END_REF]. Newly emerging data provide the evidence that during muscle atrophy, NF-κB selectively activated MuRF-1 by specific binding to its promoter region [START_REF] Wu | NF-kappaB but not FoxO sites in the MuRF1 promoter are required for transcriptional activation in disuse muscle atrophy[END_REF]. In the same study authors demonstrated that MuRF-1 promoter activation requires NF-κB sites but not FOXO sites.

Mstn plays an important role in the establishment of muscle atrophy during cancer and its inhibition could rescue skeletal muscle mass (Benny [START_REF] Klimek | Acute inhibition of myostatin-family proteins preserves skeletal muscle in mouse models of cancer cachexia[END_REF]. In response to Mstn, SMAD2/3 transducers remove the sustained inhibition of FOXO1/3 exerted by Akt and, therefore, allow its translocation into the nucleus to promote MAFbx expression (Trendelenburg et al., 2009).

Mstn promotes ROS production [START_REF] Sriram | Myostatin augments muscle-specific ring finger protein-1 expression through an NF-kB independent mechanism in SMAD3 null muscle[END_REF], which increase the transcriptional activity of NF-κB and FOXO, resulting in the up-regulation of the essential UPS components and proinflammatory cytokines, like IL-6 [START_REF] Essers | Functional interaction between beta-catenin and FOXO in oxidative stress signaling[END_REF][START_REF] Klotz | Redox regulation of FoxO transcription factors[END_REF][START_REF] Libermann | Activation of interleukin-6 gene expression through the NF-kappa B transcription factor[END_REF][START_REF] Manolopoulos | Linking Alzheimer's disease to insulin resistance: the FoxO response to oxidative stress[END_REF]. Previous studies have shown that high circulating IL-6 levels were associated with severe muscle wasting in Apc Min/+ mice [START_REF] Goodman | Interleukin-6 induces skeletal muscle protein breakdown in rats[END_REF]. Indeed, the levels of IL-6-downstream effector, Phospho-STAT3 (active form) were increased in the wasted muscles of cachectic Apc Min/+ mice [START_REF] Velazquez | Quercetin supplementation attenuates the progression of cancer cachexia in ApcMin/+ mice[END_REF]. In a model of IL-6-treated C2C12 cells, the pharmacological inhibition of STAT3 and JAK abolished the activation of the UPS [START_REF] Bonetto | JAK/STAT3 pathway inhibition blocks skeletal muscle wasting downstream of IL-6 and in experimental cancer cachexia[END_REF], indicating that the IL-6/JAK-STAT3 pathway is a primary actor in muscle wasting during cachexia. All these data indicate that inflammatory stimuli promote ROS production through the activation of various redox responsive pathways (Figure 21). Autophagy Lysosome Pathway (ALP): Autophagy is a highly conserved and selective mechanism that promotes the removal and degradation of damaged cellular organelles and long-lived proteins by the lysosomal machinery [START_REF] Mizushima | Autophagy in protein and organelle turnover[END_REF]. The main lysosomal proteases are cathepsins B, D, H and L [START_REF] Taillandier | Coordinate activation of lysosomal, Ca 2+activated and ATP-ubiquitin-dependent proteinases in the unweighted rat soleus muscle[END_REF]. An excessive activation of the autophagy-lysosome system provokes an exaggerated degradation of intracellular proteins and organelles, leading to muscle dysfunction and atrophy [START_REF] Masiero | Autophagy is required to maintain muscle mass[END_REF]. Indeed, the expression and activity of cathepsins, as well as autophagy-related markers content such as, GABARAPL1,

Beclin-1 and Light Chain-3 (LC3), raised in muscles of cachectic patients and animals [START_REF] Stephens | Evaluating potential biomarkers of cachexia and survival in skeletal muscle of upper gastrointestinal cancer patients[END_REF]Tardif et al., 2013). The study of McClung and coworkers elegantly, showed that P38 activation and OS markers were increased in the skeletal muscle of cachectic mice, concomitantly with the up-regulation of autophagy-related genes [START_REF] Mcclung | p38 MAPK links oxidative stress to autophagyrelated gene expression in cachectic muscle wasting[END_REF]. The matter that ROS can induce autophagy via the activation of P38, was evidenced by the treatment of differentiated myotubes cells with H2O2, which resulted in P38 phosphorylation and autophagy mobilization [START_REF] Mcclung | p38 MAPK links oxidative stress to autophagyrelated gene expression in cachectic muscle wasting[END_REF]. These findings were corroborated by the work of Barreiro's team showing that the inhibition of NF-κB and MAPK pathways partly prevented atrophy by decreasing the LC3II/LC3I ratio and oxidative damage in skeletal and respiratory muscles of lung tumor-bearing mice [START_REF] Chacon-Cabrera | Pharmacological strategies in lung cancer-induced cachexia: effects on muscle proteolysis, autophagy, structure, and weakness[END_REF]. In response to catabolic stimuli, FOXO1/3 levels, raised in wasted muscles, both in cell culture and animal studies [START_REF] Chacon-Cabrera | Pharmacological strategies in lung cancer-induced cachexia: effects on muscle proteolysis, autophagy, structure, and weakness[END_REF][START_REF] Zhao | FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells[END_REF]. As we said previously, ROS up-regulate the transcriptional activity of FOXO family, furthermore FOXO is a master regulator of a plethora of genes involved in autophagosome biogenesis, autophagosome-lysosome fusion and mitophagy [START_REF] Sandri | Misregulation of autophagy and protein degradation systems in myopathies and muscular dystrophies[END_REF]. Thus, it makes sense that increased muscular OS could promote autophagy by activating nuclear FOXO translocation. However, the preferential activation of the NF-κB pathway, for example, over the smad/FOXO one's, depends on the type/localisation of tumor and consequently the nature of circulating humoral factors that regulate the resulting catabolic response. A Japanese study demonstrated that transgenic mice overexpressing IL-6 suffered from excessive muscle atrophy which coincided with an increase in the activities and expression of cathepsins B and L within atrophied skeletal muscle [START_REF] Fujita | Role of interleukin-6 in skeletal muscle protein breakdown and cathepsin activity in vivo[END_REF]. Few months later the same research group, published another study in which they showed that the blockade of IL-6 Receptor, with a specific antibody, improved muscle mass and decreased the expression of cathepsins B and L in C26-tumor bearing mice [START_REF] Fujita | Role of interleukin-6 in skeletal muscle protein breakdown and cathepsin activity in vivo[END_REF], emphasizing a possible involvement of the redox-sensitive JAK-STAT3 pathway, through which IL-6 exerts its major atrophic effects.

Calpain-dependent Pathway and apoptosis:

Calpains are Calcium (Ca 2+ )dependent, non-lysosomal cysteine-proteases involved in the cleavage of cytoskeletal proteins such as spectrin, desmin, titin and nebulin that anchor and stabilize the essential myofibrillar contractile elements [START_REF] Goll | The calpain system[END_REF]. These cytoskeletal proteins are encoded by a panel of genes called easily releasable myofilaments (ERMs) whose expression could be up-regulated within atrophied muscles in cancer cachexia, due to the high demand of myofibrillar components replacement [START_REF] Fontes-Oliveira | A differential pattern of gene expression in skeletal muscle of tumor-bearing rats reveals dysregulation of excitation-contraction coupling together with additional muscle alterations[END_REF]. Caspase-3 may also play a role in sarcomeric protein degradation, especially that its activity was found to be increased in atrophied muscles and coincided with high rate of myonuclear apoptosis [START_REF] Dupont-Versteegden | Apoptosis in skeletal muscle and its relevance to atrophy[END_REF]. However, whether myonuclear apoptosis is crucial in the catabolic process remains to be elucidated. Calpains jointly with caspase-3 mediate the disintegration of sarcomere structure and dissociation of actomyosin complexes, making actin and myosin filaments accessible and degradable by the UPS [START_REF] Costelli | Ca(2+)-dependent proteolysis in muscle wasting[END_REF].

More than ten calpain isoforms exist, the two best-characterized are named µ-calpain (cap-1) and m-calpain (cap-2), referring to micromolar and millimolar amounts of Ca 2+ required to activate each isoform, respectively [START_REF] Goll | The calpain system[END_REF]. Sarcoplasmic Ca 2+ is primordial for the regulation of calpain system but a sustained elevation in free Ca 2+ may increase calpain activity. Indeed, several scenarios have been proposed as main contributors to sarcoplasmic Ca 2+ overload: (1) oxidative damages (e.i. 4-HNE) promoted cytosolic Ca 2+ accumulation by inhibiting its removal through the Ca 2+ -ATPase pumps, located at the membrane of muscle cells [START_REF] Siems | Intracellular metabolism of 4-hydroxynonenal[END_REF]) [START_REF] Costelli | Activation of Ca(2+)-dependent proteolysis in skeletal muscle and heart in cancer cachexia[END_REF], ( 2) mitochondrial ROS directly induced Ca 2+ release into the cytosol by activating the Ca 2+ -sensitive non-specific channels located at the sarcoplasmic reticulum (SR) membrane [START_REF] Delmotte | Interaction between endoplasmic/sarcoplasmic reticulum stress (ER/SR stress), mitochondrial signaling and Ca(2+) regulation in airway smooth muscle (ASM)[END_REF][START_REF] Li | Mitochondria-derived ROS bursts disturb Ca(2)(+) cycling and induce abnormal automaticity in guinea pig cardiomyocytes: a theoretical study[END_REF] and ( 3) the expression of genes encoding proteins involved in the binding of the intracellular Ca 2+ (e.g. calmodulin) was down-regulated in skeletal muscle of tumor-bearing rodents (Fontes-Oliveira et al., 2014) (Figure 22). Interestingly, a number of studies have unveiled a direct link between ROS generation and Ca 2+ -mediated calpain activation. Smuder et al. showed that exposure of fibers isolated from rat skeletal muscle to H2O2

and Fe 2+ accelerated the proteolysis of oxidized myofibrillar components by cap-1 and cap-2 [START_REF] Smuder | Oxidation enhances myofibrillar protein degradation via calpain and caspase-3[END_REF]. H2O2 seemed to mediate calpain activation by inducing SR stress and the subsequent Ca 2+ leakage [START_REF] Mcclung | Calpain-1 is required for hydrogen peroxide-induced myotube atrophy[END_REF][START_REF] Pierre | Activation of ER stress by hydrogen peroxide in C2C12 myotubes[END_REF]. Furthermore, NO . induced myotubes atrophy by activating calpain-dependent cleavage of αII-spectrin and talin [START_REF] Soltow | Cessation of cyclic stretch induces atrophy of C2C12 myotubes[END_REF]. OS profiles, together, with the calpain activity raised in skeletal muscles of tumor-bearing animals, whereas the activity of calpastatin, the natural calpain inhibitor, was decreased [START_REF] Borges | Oxidative and proteolytic profiles of the right and left heart in a model of cancer-induced cardiac cachexia[END_REF][START_REF] Costelli | Anticytokine treatment prevents the increase in the activity of ATP-ubiquitin-and Ca(2+)-dependent proteolytic systems in the muscle of tumour-bearing rats[END_REF]. On the other hand and contrary to the above-mentioned calpains, cap-3 could play a counter-regulatory role in muscle proteolysis, since its expression was downregulated in wasted muscles of cachectic animals with malignancy [START_REF] Busquets | Calpain-3 gene expression is decreased during experimental cancer cachexia[END_REF][START_REF] Fujita | Role of interleukin-6 in skeletal muscle protein breakdown and cathepsin activity in vivo[END_REF].

Tumor-muscle crosstalk: what about adjuvant therapies?

The loss of skeletal muscle, caused by tumor-mediated inflammation and subsequent intramuscular ROS generation, is usually associated with poor prognosis. Patients become weak, less active and unlikely to support heavy anticancer treatments, like chemotherapy or surgery [START_REF] Muscaritoli | Prevention and treatment of cancer cachexia: new insights into an old problem[END_REF][START_REF] Prado | Sarcopenia as a determinant of chemotherapy toxicity and time to tumor progression in metastatic breast cancer patients receiving capecitabine treatment[END_REF]. This obligates physicians to prolong the period of treatment and/or reduce intervention doses; thus, making complete tumor eradication an unachievable aim. To slow further deterioration or avoid arriving at this critical stage, additional strategies must be combined to conventional ones. These strategies must seek, in the first place, to improve muscle function and preserve its mass.

In this light, adjuvant therapies like, physical activity (PA) and diet, could constitute a part of the global solution. First of all, repetitive PA is well-known to drive antioxidant and metabolic adaptations, improve performance, decrease fatigue and promote anabolism [START_REF] Bishop | Can we optimise the exercise training prescription to maximise improvements in mitochondria function and content?[END_REF][START_REF] Gomez-Cabrera | Moderate exercise is an antioxidant: upregulation of antioxidant genes by training[END_REF]. Even more, benefits in term of cancer progression, recurrence and related mortality have been also denoted in physically active persons (Friedenreich et al., 2016;Meyerhardt et al., 2006a;Meyerhardt et al., 2006b). Collectively, a regular PA may slow the degeneration of skeletal muscle, allow patients to complete their treatment and install a reciprocal muscle-tumor crosstalk to counteract and limit the detrimental effects of tumor on muscle.

Secondly, in some advanced stages of cancers (e.g. colon, pancreas), ROS may have a more important role to play in muscle wasting and tumor growth (Assi et al., 2016;[START_REF] Von Grabowiecki | Regulation of a Notch3-Hes1 pathway and protective effect by a tocopherol-omega alkanol chain derivative in muscle atrophy[END_REF]. Indeed, nutritional supplements endowed with antioxidant potential may be more effective than PA to antagonize these ROS-dependent pathways and slow the time course of disease. However, the ambivalence of ROS in both tumor and skeletal muscle, may render the intervention with such strategies more complicated. For example, inhibition of ROS could slow muscle wasting but at the same time it may prevent apoptosis induction in tumor. In the two coming chapters we will tackle the impact of PA and antioxidants on cancer and related muscle wasting.

►Key Messages from Chapter III -Crosstalk between tumor and skeletal muscle is responsible for the development of muscle wasting during cancer. As illustrated in the Figure 23

Cancer progression, muscle wasting and related physical inactivity

After what has been said previously, it is becoming clear that advanced stages of cancer are often associated with excessive loss of skeletal muscle mass, leading to functional impairment and high risk of inactivity (Fearon et al., 2011;[START_REF] Fouladiun | Daily physical-rest activities in relation to nutritional state, metabolism, and quality of life in cancer patients with progressive cachexia[END_REF]. In turn, physical inactivity is an aggravating factor in both healthy and ill individuals, as it negatively impacts skeletal muscle mass and function [START_REF] Pierre | From physical inactivity to immobilization: Dissecting the role of oxidative stress in skeletal muscle insulin resistance and atrophy[END_REF]; thus, a kind of loop between "cancer, muscle wasting and physical inactivity" may take place in cancer patients (Figure 24). A previous study demonstrated that physical inactivity increased colon cancer-related mortality [START_REF] Morrison | Behavioural and metabolic risk factors for mortality from colon and rectum cancer: analysis of data from the Asia-Pacific Cohort Studies Collaboration[END_REF]. Furthermore, in 2009, Pedersen proposed the hypothesis of "the diseasome of physical inactivity", in which the author hypothesized that physical inactivity promotes chronic systemic inflammation, a condition that favor the progression of cancer and associated disorders (Pedersen, 2009). This indicates that being inactive, especially when diagnosed with a cancer, may promote further inflammation, muscle weakness, fatigue and, therefore, aggravate the time course of the disease [START_REF] Chambers | Physical inactivity and muscle weakness in the critically ill[END_REF]. Add to this, in particular cases, cancer could be detected within lower-extremities, constituting an awkward mass that impede the capacity of muscle to produce sufficient force and ensure basic physical needs [START_REF] Wampler | Physical activity among adult survivors of childhood lower-extremity sarcoma[END_REF]. Indeed, survivors of lower-extremity soft tissue sarcoma were at risk of inactivity and less likely to meet recommended guidelines of physical activity [START_REF] Wampler | Physical activity among adult survivors of childhood lower-extremity sarcoma[END_REF]. Predictors of inactivity were female sex, heavy surgical intervention and chemotherapy [START_REF] Ness | More than physical function: HRQOL among children and adolescents with lower extremity bone sarcoma[END_REF][START_REF] Wampler | Physical activity among adult survivors of childhood lower-extremity sarcoma[END_REF]. Therefore, sarcoma survivors may face a high risk of functional abnormalities as a result of sarcoma itself or its treatment [START_REF] Meacham | Cardiovascular risk factors in adult survivors of pediatric cancer--a report from the childhood cancer survivor study. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF]. Collectively, the function of skeletal muscle is highly altered in cancer patients, exposing them to further health complications that may delay complete tumor eradication and cure [START_REF] Prado | Sarcopenia as a determinant of chemotherapy toxicity and time to tumor progression in metastatic breast cancer patients receiving capecitabine treatment[END_REF]. In this regard, PA is an important health behavior that could help to better manage the complications related to inactivity, for which cancer patients are at high risk.

FIGURE 24: The vicious circle between physical inactivity, tumor development and muscle wasting

Molecular mechanisms modulated by PA in healthy conditions

PA is a bodily movement that solicits skeletal muscle, resulting in cardiorespiratory adaptations, musculoskeletal fitness and energy expenditure [START_REF] Shephard | Physical activity, fitness, and health: international proceedings and consensus statement[END_REF]. The intensity of PA is evaluated using metabolic equivalent task (MET), with 1 MET corresponds to the energy expended at rest.

The WHO recommends 150 min of moderate-intensity PA (3-5.9 MET) or 75 min high intensity PA (≥ 6 MET) throughout the week (WHO, 2010). The health benefits of regular PA are driven by a number of molecular mechanisms, hereafter we will describe the most important.

Antioxidant response, mitochondrial biogenesis and angiogenesis

Antioxidant response: Recently, the systematic review by de Sousa et al. incorporated the results of 30 intervention trials with exercise and concluded that regardless the intensity, frequency, type of exercise and the studied population, it seems that exercise increased antioxidant parameters and decreased pro-oxidants markers (de Sousa et al., 2016). It is thought that in response to steady exercise, the skeletal muscle is repetitively exposed to low doses of ROS, produced locally, provoking a so-called "hormetic-like adaptations". Indeed, hormesis is a biological phenomenon in which living cells are regularly exposed to low doses of "toxins" leading to longterm beneficial adaptations. A substance called hormetic must fulfill three main characteristics: (1) biphasic dose-response with low doses induce the opposite effects to high doses, (2) low doses should be measurable and ( 3) present naturally in the biological micro-environment [START_REF] Jargin | Hormesis and homeopathy: The artificial twins[END_REF].

The fact that exercise induces hormetic-like responses, comes from the early observation that wild animals exhibited stronger antioxidant defense compared to domestic animals, due to their higher muscular activity [START_REF] Jenkins | Effects of nonesterified fatty acid availability on tissue-specific glucose utilization in rats in vivo[END_REF]. Based on these observations, it has been suggested that regular PA promotes low ROS generation within skeletal muscle and, as an adaptive response, the expression of antioxidant genes is up-regulated to allow a better resistance in the presence of stressful conditions (Figure 25) [START_REF] Higuchi | Superoxide dismutase and catalase in skeletal muscle: adaptive response to exercise[END_REF]. Although both endurance (aerobic) and resistance (anaerobic) exercise may induce an antioxidant/metabolic response [START_REF] Park | Impact of aerobic and anaerobic exercise training on oxidative stress and antioxidant defense in athletes[END_REF], the involved signaling pathways could be much different and complex. For example, muscle contraction can activate various redox sensitive kinases, leading to signal transduction and skeletal muscle adaptation. In most studies cited here, endurance moderate-intensity exercise was the most chosen modality of PA. The activation of P38 and ERK1/2 MAPKs by H2O2 originating from XO and NOX, has been reported in exercised skeletal muscles [START_REF] Henriquez-Olguin | NOX2 Inhibition Impairs Early Muscle Gene Expression Induced by a Single Exercise Bout[END_REF]Kang et al., 2009). Activated P38 and ERK1/2 provoked the nuclear accumulation of NF-κB (p65) subunit and the transcription of SOD2 and GPx genes [START_REF] Kang | Exercise activation of muscle peroxisome proliferator-activated receptor-gamma coactivator-1alpha signaling is redox sensitive[END_REF]. Mitochondrial biogenesis and angiogenesis: Mitochondrial biogenesis is stimulated by exercise, allowing an effective production of ATP and protection against oxidative insults [START_REF] Davies | Exercise bioenergetics following sprint training[END_REF]. Accordingly, mitochondria from trained individuals were more resistant to OS than those from sedentary ones [START_REF] Sen | Skeletal muscle and liver glutathione homeostasis in response to training, exercise, and immobilization[END_REF]. The AMP-activated protein kinase (AMPK) is particularly activated in response to endurance exercise and could be considered as a marker of physical exercise in muscles. Indeed, O2 .-and H2O2 produced by NOX and XO can increase AMPK activity through phosphorylating the Thr 172 residue, by three main mechanisms:

(1) increase Ca 2+ leakage from the sarcoplasmic reticulum, which in turn promotes the autophosphorylation of calmodulin-dependent kinase II (CaMKII) [START_REF] Hudmon | Structure-function of the multifunctional Ca2+/calmodulin-dependent protein kinase II[END_REF], (2) increase AMP:ATP ratio [START_REF] Choi | The regulation of AMP-activated protein kinase by H(2)O(2)[END_REF]) and ( 3) activate Sestrin-2 [START_REF] Morrison | Sestrin2 promotes LKB1-mediated AMPK activation in the ischemic heart[END_REF]. All these events lead to the activation and stabilization of LKB1 kinase that phosphorylates AMPK on the Thr 172 site of the α-subunit. Activation of AMPK as well as ERK1/2 and P38 MAPKs are important events related to exercise, as they increase the mRNA expression of PGC-1α [START_REF] Puigserver | Cytokine stimulation of energy expenditure through p38 MAP kinase activation of PPARgamma coactivator-1[END_REF] and can also directly phosphorylate PGC-1α and stabilize its expression in the nucleus [START_REF] Puigserver | Cytokine stimulation of energy expenditure through p38 MAP kinase activation of PPARgamma coactivator-1[END_REF]. Accumulation of PGC-1α proteins was reported in the skeletal muscle of exercised animals and was associated with increased ( 1) nuclear respiratory factor-1 (NRF-1) and TFAM content, two markers of mitochondrial biogenesis, (2) angiogenic genes expression, like VEGF, and (3) slow-to fast twitch muscle fiber transformation [START_REF] Kang | Exercise activation of muscle peroxisome proliferator-activated receptor-gamma coactivator-1alpha signaling is redox sensitive[END_REF][START_REF] Lin | Transcriptional co-activator PGC-1 alpha drives the formation of slow-twitch muscle fibres[END_REF] (Figure 26). Also, NF-κB is a transcriptional factor that regulates various physiological changes after endurance and resistance exercise, such as blood flow and vasodilatation of the vessels, through inducing the expression of iNOS and eNOS genes [START_REF] Hollander | Superoxide dismutase gene expression is activated by a single bout of exercise in rat skeletal muscle[END_REF]Li et al., 2005;[START_REF] Reid | The effect of moderate aerobic exercise and relaxation on secretory immunoglobulin A[END_REF][START_REF] Vella | Resistance exercise increases NF-kappaB activity in human skeletal muscle[END_REF].

Insulin sensitivity, hormonal changes and muscle-related secretory phenotype

Insulin sensitivity: PA is a high determinant of insulin sensitivity. A recent study investigated how the fluctuation in PA may influence insulin responsiveness; healthy active men enrolled in the study were submitted to one month of detraining, that is suppression of all kind of exercises and reduction of spontaneous daily activity [START_REF] Gastebois | Transition from physical activity to inactivity increases skeletal muscle miR-148b content and triggers insulin resistance[END_REF]. Scientists found an increase in miR148b within inactive muscle biopsies. In turn, miR148b was responsible for the down-regulation of ROCK1 and NRAS involved in glucose uptake and insulin signaling (Gastebois , 2016). Additionally, in healthy children aged between 8-12 years-old, the levels of C-peptide (reflecting circulating pro-insulin) were decreased in boys compared to girls, because of their higher level of PA [START_REF] Huus | Physical Activity, Blood Glucose and C-Peptide in Healthy School-Children, a Longitudinal Study[END_REF]. A longitudinal follow-up showed that a drop in PA reinforced the circulating C-peptide levels and the load of insulin-producing β-cells islets [START_REF] Huus | Physical Activity, Blood Glucose and C-Peptide in Healthy School-Children, a Longitudinal Study[END_REF]. Similar data showing greater circulating glucose and insulin levels in inactive adults were also reported [START_REF] Jung | Obesity and associated lifestyles modify the effect of glucose metabolism-related genetic variants on impaired glucose homeostasis among postmenopausal women[END_REF]. These studies highlighted that the switch from active-toinactive behavior is associated with high circulating insulin, reduced efficiency of insulin cascade and glucose uptake in muscle, which could evolve into insulin resistance. It has been postulated

that the impairment of insulin responsiveness could be due to an increase in circulating fatty acids and accumulation of long-chain fatty acids within skeletal muscle (the primary site of insulin resistance) [START_REF] Jenkins | Effects of nonesterified fatty acid availability on tissue-specific glucose utilization in rats in vivo[END_REF]. The condensation of bioactive lipid metabolites, alters insulin signaling by interfering with the translocation of glucose transporter-4 (GLUT-4) and glucose transport [START_REF] Chavez | Acid ceramidase overexpression prevents the inhibitory effects of saturated fatty acids on insulin signaling[END_REF]. Using liquid chromatography coupled to mass spectrometry, Xiang and colleagues, identified 24 metabolites modulated by exercise in the plasma of diabetic db/db mice [START_REF] Xiang | Plasma metabolic signatures reveal the regulatory effect of exercise training in db/db mice[END_REF]. The most significant finding was the increase in pantothenic acid and palmitoylcarnitine two enhancers of fatty acid β-oxidation [START_REF] Xiang | Plasma metabolic signatures reveal the regulatory effect of exercise training in db/db mice[END_REF]. This corroborates with the early findings of Goodpaster et al. showing that physical exercise may improve insulin sensitivity and glucose tolerance, by enhancing fatty acid oxidation in muscles [START_REF] Goodpaster | Enhanced fat oxidation through physical activity is associated with improvements in insulin sensitivity in obesity[END_REF]. Such mechanism seems to be dependent on the activation of PGC-1α which up-regulates the expression of genes involved in fatty acid oxidation and oxidative phosphorylation [START_REF] Handschin | Peroxisome proliferator-activated receptor gamma coactivator 1 coactivators, energy homeostasis, and metabolism[END_REF][START_REF] Liepinsh | Decreased acylcarnitine content improves insulin sensitivity in experimental mice models of insulin resistance[END_REF]. Additionally, in L6 and C2C12 muscle overexpressing PGC-1α, the insulin-mediated glucose transport was largely increased via a greater GLUT-4 expression. The authors showed that PGC-1α was able to bind the promoter of GLUT-4

to increase its expression [START_REF] Michael | Restoration of insulin-sensitive glucose transporter (GLUT4) gene expression in muscle cells by the transcriptional coactivator PGC-1[END_REF]. These findings forge the notion that exercisemediated PGC-1α activation have insulin-sensitizing effects (Figure 26).

Hormonal changes and myokines:

As mentioned previously, PGC-1α could be activated by ROS-sensitive cascades (AMPK and P38 MAPK) but also by other pathways depending on hormones like epinephrine and norepinephrine. The levels of circulating epinephrine rise after training and contribute to cAMP signaling through β-adrenergic receptor (β-AR) [START_REF] Hartley | Multiple hormonal responses to prolonged exercise in relation to physical training[END_REF]. The binding of epinephrine to β-AR activates adenylyl cyclase (AC), which converts ATP into cAMP [START_REF] Hartley | Multiple hormonal responses to prolonged exercise in relation to physical training[END_REF]. The major effector of cAMP is PKA, a tetramer kinase composed of two dimers: regulatory and catalytic subunits. The binding of cAMP to PKA, provokes the release of catalytic subunits, which become free to phosphorylate several proteins including the transcriptional factor CREB [START_REF] Gonzalez | Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133[END_REF]. Activated CREB is known to promote the expression of PGC-1α and the transcriptional activation of corresponding genes, including interleukins like, IL-6 and IL-15 [START_REF] Shin | The effect of exercise on expression of myokine and angiogenesis mRNA in skeletal muscle of high fat diet induced obese rat[END_REF] (Figure 26).

The capacity of muscle to up-regulate the expression of cytokines and interleukins, conferred it the reputation of being a secretory organ [START_REF] Febbraio | Contraction-induced myokine production and release: is skeletal muscle an endocrine organ?[END_REF]. In response to exercise, skeletal muscle releases factors known as "myokines", which could act in an autocrine or paracrine manner to activate several signaling pathways, as a part of the adaptive response of skeletal muscle to exercise [START_REF] Pedersen | Muscle-derived interleukin-6: lipolytic, anti-inflammatory and immune regulatory effects[END_REF]. These factors regroup mainly pro-inflammatory cytokines like, the IL-6 family members: IL-11, oncostatin M (OSM) and leukemia inhibitory factor (LIF); as well as anti-inflammatory cytokines such as, IL-4, IL-10 and IL-15 [START_REF] Petersen | The anti-inflammatory effect of exercise[END_REF]. Indeed, the identification of exercise-derived factors was mainly realized in plasma and muscle biopsies of exercised human and animals or in cultured muscle cells after electrical pulse stimulation (an elegant approach used to mimic the effect of exercise in vitro by applying regular and repetitive electrical pulses) [START_REF] Ost | Regulation of myokine expression: Role of exercise and cellular stress[END_REF]. These cytokines are produced in response to stress stimuli such as exercise-induced ROS generation but are not specific for muscle cells and can be produced by adipose tissue for example. However, other myokines like irisin have been identified by the group of Bruce Spiegelman as produced mainly by exercised muscles [START_REF] Jedrychowski | Detection and Quantitation of Circulating Human Irisin by Tandem Mass Spectrometry[END_REF]. Spiegelman's group proved by mass spectrometry that irisin is produced at low levels reaching 3.6 ng/ml at rest and increase to 4.3 ng/ml after exercise [START_REF] Jedrychowski | Detection and Quantitation of Circulating Human Irisin by Tandem Mass Spectrometry[END_REF]. Whether this slight change in irisin has a meaningful physiological significance or not remain to be elucidated. Other mediators can be added to the list of myokines produced by trained skeletal muscle, including: apelin, (Besse-Patin et al., 2014), decorin [START_REF] Heinemeier | Expression of extracellular matrix components and related growth factors in human tendon and muscle after acute exercise[END_REF], musclin and secreted protein acidic and rich in cysteine (SPARC) [START_REF] Bradshaw | Diverse biological functions of the SPARC family of proteins[END_REF] but their exact role and mechanism of actions in health and disease still poorly understood and need further investigations [START_REF] Ost | Regulation of myokine expression: Role of exercise and cellular stress[END_REF].

In summary, it is becoming clear that endurance and resistance exercise are associated with production of O2 A body of evidence indicates that PA reduces about 25% the risk of colon and breast cancer, with possible evidence in prostate, lung and ovary cancer (Friedenreich, 2011). In addition to the potential beneficial role of PA in cancer prevention, it may also delay disease recurrence, highlighting a possible role in the suppression of tumor growth [START_REF] Ballard-Barbash | Physical activity, biomarkers, and disease outcomes in cancer survivors: a systematic review[END_REF]Meyerhardt et al., 2006b). Epidemiologists and exercise oncologists believe that PA could be prescribed for cancer patients at three different levels throughout the treatment protocol (Friedenreich, 2011). Before starting treatment, PA may help patients to (1) manage symptoms and ( 2) maintain a certain level of physical function to increase treatment tolerance. During the treatment, to (1) reduce adverse side-effects of anticancer therapies, (2) preserve muscle strength,

Exercise-oncology: the impact of PA on cancer progression

(3) prevent skeletal muscle loss, ( 4) reduce anxiety and ( 5) allow hard treatment to go forward.

After the treatment, PA could (1) optimize recovery, ( 2) reduce cancer relapse and ( 3) prevent the appearance of late chronic complications, for which cancer survivors are at an increased risk (i.e. cardiovascular diseases) (Friedenreich, 2011).

Physically active cancer patients are more confident concerning their health status and express less fear of cancer recurrence comparing to less active or sedentary patients [START_REF] Fisher | Health behaviours and fear of cancer recurrence in 10 969 colorectal cancer (CRC) patients[END_REF]. This could be due to the increased awareness and accumulating evidence about the beneficial effects of PA in reducing cancer progression and recurrence. In this light, Lahart and colleagues performed a systematic review and meta-analysis in which they included 22 prospective cohort study addressing the effects of PA on breast cancer [START_REF] Lahart | Physical activity, risk of death and recurrence in breast cancer survivors: A systematic review and meta-analysis of epidemiological studies[END_REF]. They found reduced risk of breast cancer progression in both recurrence and new primaries [START_REF] Lahart | Physical activity, risk of death and recurrence in breast cancer survivors: A systematic review and meta-analysis of epidemiological studies[END_REF].

Similarly, findings were obtained from Richman et al. in patients with diagnosed prostate cancer,

showing that brisk walking reduced the risk of cancer progression [START_REF] Richman | Physical activity after diagnosis and risk of prostate cancer progression: data from the cancer of the prostate strategic urologic research endeavor[END_REF].

Interestingly, the delay in prostate cancer progression was seen in patients who practiced brisk walking for 3h/week or more, comparing to those who were engaged in less than 3h/week with an easy pace [START_REF] Richman | Physical activity after diagnosis and risk of prostate cancer progression: data from the cancer of the prostate strategic urologic research endeavor[END_REF]. Patients with colon cancer practicing 18-to-27 MET-hours/week or more exhibited an increase in survival-free recurrence (Meyerhardt et al., 2006a). More interesting, even in patients with diagnosed stage I and II colon cancer or with recurrent stage III colon cancer, 18-MET-hours/week was sufficient to prolong survival [START_REF] Jeon | Impact of physical activity after cancer diagnosis on survival in patients with recurrent colon cancer: Findings from CALGB 89803/Alliance[END_REF]Meyerhardt et al., 2006b). Intervention with PA was feasible and safe in lung cancer patients (Friedenreich, 2011). Recent studies showed that higher levels of PA were associated with lower cancer-related mortality in post-menopausal women with NSCLC and lung adenocarcinoma (Wang et al., 2016a). Other studies still ongoing to investigate the impact of 24-week exercise intervention on the quality of life and physical performance of lung cancer patients (Wiskemann et al., 2016). The impact of PA on hematological cancer patients was mainly addressed in those receiving bone marrow transplantation (Friedenreich, 2011). Battaglini et al. noted that combined resistance and aerobic exercise, in leukemia patients undergoing chemotherapy, significantly improved cardiorespiratory endurance, muscle strength, lean body mass, fatigue and depression [START_REF] Battaglini | The effects of an exercise program in leukemia patients[END_REF]. Interestingly, PA seems to affect cancer patients differently according to the molecular subgroups. For example, patients with tumors expressing (high versus low levels) of the cell cycle inhibitors P21 or P27; or with low/negative status of insulin receptor substrate-1

(IRS-1), benefit the most from PA in term of cancer-related mortality [START_REF] Hanyuda | Survival Benefit of Exercise Differs by Tumor IRS1 Expression Status in Colorectal Cancer[END_REF][START_REF] Meyerhardt | Interaction of molecular markers and physical activity on mortality in patients with colon cancer[END_REF]. In this light, several preclinical studies have been launched to further understand the interactions between PA and cancer at the molecular level. The most commonly studied factors include: OS, insulin metabolism, inflammation, immune system and sex hormones (McTiernan, 2008).

OS and Inflammation:

A 10-week exercise program consisting of endurance and resistance training increased total antioxidant capacity, reduced levels of protein carbonyls and improved muscle strength in patients with lymphoma, ovarian, pancreatic and breast cancer [START_REF] Repka | Oxidative Stress and Fitness Changes in Cancer Patients after Exercise Training[END_REF]. In the same way, PA decreased H2O2 rates in prostate but not in breast cancer patients; interestingly, only prostate cancer patients displayed higher H2O2 levels compared to healthy men, at the baseline [START_REF] Knop | Sport and oxidative stress in oncological patients[END_REF]. A 12-week of endurance treadmill training decreased circulating levels of lipid peroxidation markers, IL-1β, IL-6, TNF-α and IFN-γ, while its increased IL-10 in women with chronic inflammation [START_REF] Farinha | Response of oxidative stress and inflammatory biomarkers to a 12-week aerobic exercise training in women with metabolic syndrome[END_REF]. Accordingly, the circulating levels of pro-inflammatory cytokines, IL-6 and IL-1ra, were attenuated in prostate and breast cancer patients in response to resistance and/or aerobic exercise [START_REF] Galvao | Combined resistance and aerobic exercise program reverses muscle loss in men undergoing androgen suppression therapy for prostate cancer without bone metastases: a randomized controlled trial[END_REF][START_REF] Schmidt | Resistance Exercise and Inflammation in Breast Cancer Patients Undergoing Adjuvant Radiation Therapy: Mediation Analysis From a Randomized, Controlled Intervention Trial[END_REF]. Therefore, it seems that improvement in systemic antioxidant and antiinflammatory status could be more pronounced in patients with chronic inflammation and OS biomarkers at the baseline, before starting exercise.

The increase in blood antioxidant activity could be beneficial to narrow damage for proteins, lipoproteins and lipids and avoid further disorders, for example, reduce the oxidation of LDL and related cardiovascular complications [START_REF] Loria | Vitamin C status and mortality in US adults[END_REF]. However, it has been postulated, (Gueritat et al., 2014). Mice bearing orthotopic T269 glioma exhibited an important reduction in tumor volume when combining endurance exercise on treadmill with the chemotherapeutic agent, temozolomide (TMZ) [START_REF] Lemke | Slowing down glioblastoma progression in mice by running or the anti-malarial drug dihydroartemisinin? Induction of oxidative stress in murine glioblastoma therapy[END_REF]. Contrary to the former study, physical exercise inhibited tumor growth through the induction of ROS, as the use of NAC partially blunted these beneficial effects [START_REF] Lemke | Slowing down glioblastoma progression in mice by running or the anti-malarial drug dihydroartemisinin? Induction of oxidative stress in murine glioblastoma therapy[END_REF]. Similar results were obtained from our laboratory showing that combination of radiotherapy with physical exercise was effective to reduce PPC-1 prostate tumor growth via increasing tumor oxidative damage (unpublished data).

Seemingly, combining moderate exercise with anticancer therapies induces tumor-killing effects by increasing ROS production and causing oxidative damage within tumor; while moderate intensity exercise alone may rather induce an increase in antioxidant defense and reduce OS.

Therefore, the modality of exercise (intensity, type and frequency) must be individualized according to tumor type and clinical case/treatment of each patient. Low-to-moderate endurance exercise could to be more suitable and effective for patients undergoing chemo/radiotherapy

Insulin, IGF and sex hormones: Insulin resistance which is reflected by the presence of high circulating levels of insulin, is linked to higher cancer incidence and mortality [START_REF] Kaaks | Energy balance and cancer: the role of insulin and insulin-like growth factor-I[END_REF]. This notion is supported by the observation that type 2 diabetes patients displaying high insulin levels, chronic inflammation and greater androgen/estrogen secretion, are at a higher risk of cancer development [START_REF] Joung | The association between type 2 diabetes mellitus and women cancer: the epidemiological evidences and putative mechanisms[END_REF]. Accordingly, breast cancer-related mortality showed 16% increase in women with type II diabetes [START_REF] Campbell | Feasibility of a lifestyle intervention on body weight and serum biomarkers in breast cancer survivors with overweight and obesity[END_REF]. In fact, as the development of estrogen-dependent breast cancer is highly influenced by estrogen levels, insulin may drive its pro-oncogenic effects through its ability to increase the expression of estrogen receptor and binding potential [START_REF] Panno | Effect of oestradiol and insulin on the proliferative pattern and on oestrogen and progesterone receptor contents in MCF-7 cells[END_REF]. In turn, activated estrogen signaling will further increase insulin mitogenicity by promoting IRS-1 expression and PI3K/Akt signaling [START_REF] Mauro | Estradiol increases IRS-1 gene expression and insulin signaling in breast cancer cells[END_REF]. PA is known to reduce the levels of insulin in humans with hyperinsulinaemia and this can occur independently of body weight and fatness (McTiernan, 2008). However, the impact of exercise on insulin-like factors axis in cancer is less convincing, as circulating IGFs and IGF binding proteins (IGFBPs) levels fluctuated between breast, colon and prostate cancer as well as according to the type of exercise; thus, preventing the generalizability of such findings [START_REF] Devin | The Influence of Exercise on the Insulinlike Growth Factor Axis in Oncology: Physiological Basis, Current, and Future Perspectives. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF]. However, Meneses-Echavez and collaborators were more optimist and reported in their meta-analysis that PA was associated with reduced insulin and IGF-1 levels in breast cancer survivors [START_REF] Meneses-Echavez | The insulin-like growth factor system is modulated by exercise in breast cancer survivors: a systematic review and meta-analysis[END_REF]. In the same way, authors also reported an increase in IGFBP-1 and IGFBP-3 levels which could bind IGF-1 and IGF-3 to limit their mitotic signaling in cancer [START_REF] Meneses-Echavez | The insulin-like growth factor system is modulated by exercise in breast cancer survivors: a systematic review and meta-analysis[END_REF]. Interestingly, Orsatti et al. and Nishida et al. demonstrated that PA decreased IGF-1 in patients exhibiting high levels at the baseline, but it increased IGF-1 when low levels were reported pre-intervention [START_REF] Nishida | Effect of low-intensity aerobic exercise on insulin-like growth factor-I and insulin-like growth factor-binding proteins in healthy men[END_REF][START_REF] Orsatti | Plasma hormones, muscle mass and strength in resistance-trained postmenopausal women[END_REF]. This has been interpreted later by Devin and coworkers, as a corrective response of PA to maintain optimal IGF levels [START_REF] Devin | The Influence of Exercise on the Insulinlike Growth Factor Axis in Oncology: Physiological Basis, Current, and Future Perspectives. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF]. In fact, high levels of IGF-1 may increase carcinogenesis, while low levels may promote catabolism; thus, this physiological dichotomy of IGFs must be taken into account before PA intervention and data interpretation. With the same logic, although insulin has been mainly described as a hormone driving mitotic and anti-apoptotic effects in cancer cells [START_REF] Kaaks | Energy balance and cancer: the role of insulin and insulin-like growth factor-I[END_REF], we must recall that this could be linked to conditions evoking hyperinsulinaemia. Contrariwise, at levels close to the normal range, insulin may exert several functions including the arrest of cell cycle/proliferation to induce differentiation in skeletal muscle cells and pre-adipocytes [START_REF] Zetser | p38 mitogen-activated protein kinase pathway promotes skeletal muscle differentiation. Participation of the Mef2c transcription factor[END_REF], this may shed the light on a forgotten side of insulin actions.

Angiogenesis and immune system:

Angiogenesis is an important process for tumor to gain O2 and nutrients. High levels of circulating pro-angiogenic factors like VEGF (> 314.2 pg/mL) are associated with high risk of cancer and poor prognosis [START_REF] Reeves | Vascular endothelial growth factor and breast cancer risk[END_REF]. Tumor of prostate cancer patients, engaged in vigorous PA, exhibited regular shaped blood vessels with larger diameter when compared to those who practiced non-vigorous activity [START_REF] Van Blarigan | Physical Activity and Prostate Tumor Vessel Morphology: Data from the Health Professionals Follow-up Study[END_REF]. Indeed, intra-tumor vessels are usually irregular and poorly functioning, giving way to the development of hypoxic domains within tumor and activation of HIF-1α, which will further promote angiogenic genes expression and transformation of tumor into a more aggressive phenotype [START_REF] Jain | Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy[END_REF]. PA-mediated improve in the morphology of blood vessels will possibly increase tumor oxygenation and reduce hypoxia. Hypoxic tumors are usually refractory to chemotherapy, as agents are not efficiently delivered to the tumor given the altered quality of irrigating vessels and the ability of HIF-1α to increase the expression of ATP-dependent drug efflux pumps [START_REF] Comerford | Hypoxia-inducible factor-1-dependent regulation of the multidrug resistance (MDR1) gene[END_REF] bearing breast tumor, increased blood vessels density, maturity, blood perfusion but reduced tumor hypoxia. As a direct result, the delivery of the chemotherapeutic agent, cyclophosphamide, into tumor was improved, leading to further tumor suppression [START_REF] Betof | Modulation of murine breast tumor vascularity, hypoxia and chemotherapeutic response by exercise[END_REF]. Therefore, in a first step an increase in blood vessels maturity and repair is essential to reduce hypoxia and "normalize" the microenvironment of the tumor, facilitating the access of toxic drugs into tumor. These findings were consolidated more recently by a study demonstrating an increase in VEGF expression and blood vessels density and decreased tumor growth in trained rats bearing mammary cancer [START_REF] Faustino-Rocha | Long-term exercise training as a modulator of mammary cancer vascularization[END_REF]. However, studies reporting a decrease in VEGF, HIF-1α and blood vessels density markers in tumors of trained breast tumor-bearing mice [START_REF] Isanejad | MicroRNA-206, let-7a and microRNA-21 pathways involved in the antiangiogenesis effects of the interval exercise training and hormone therapy in breast cancer[END_REF] [START_REF] Jones | Modulation of circulating angiogenic factors and tumor biology by aerobic training in breast cancer patients receiving neoadjuvant chemotherapy[END_REF].

The modulation of immune system by physical exercise remains largely untested and evidence on moderate-intensity PA from randomized controlled trials is inconclusive (McTiernan, 2008). However, the most plausible hypothesis is that PA could increase the activity of natural killer cells (NK), which are involved in tumor suppression [START_REF] Wetmore | An angiotensin converting enzyme haplotype predicts survival in patients with end stage renal disease[END_REF]. In this regard, the team of Pernille Hojman recently showed that voluntary running on wheels decreased lung, liver and melanoma tumor incidence or growth in mice through a mechanism involving a mobilization and redistribution of IL6-dependent NK cells within tumor [START_REF] Pedersen | Voluntary Running Suppresses Tumor Growth through Epinephrine-and IL-6-Dependent NK Cell Mobilization and Redistribution[END_REF].

Interestingly, Zimmer et al. demonstrated that the expression of NKG2D gene encoding, the tumorcell recognition receptor increased in the NK cells of cancer patients after endurance run. This was due to the acetylation of NKG2D gene, supporting a role for exercise in enhancing NK antitumor activity through the modification of epigenetic profiles [START_REF] Zimmer | Exercise-induced Natural Killer Cell Activation is Driven by Epigenetic Modifications[END_REF]. In addition to NK, acute swimming exercise increased the phagocytic capacity of circulating macrophages and their ability to produce NO . and H2O2 via the activation of NF-κB-iNOS pathway [START_REF] Silveira | Acute exercise stimulates macrophage function: possible role of NF-kappaB pathways[END_REF].

However, acute exercise may not be the ideal type of exercise to prescribe for cancer patients.

Experimental designs of low-to-moderate-intensity exercise, seems to be the more close and representative to the clinical context. Together these data highlight the beneficial effects of moderate-intensity PA in slowing the progression of cancer through affecting several pathways linked to inflammation, redox state, metabolic hormones, angiogenesis and immunity (Figure 27). Our understanding in the exerciseoncology field is increasing and the currently available knowledge clearly supports PA as a protective strategy for cancer patients. However, we must expand our understanding to other unexplored areas in this field. In this regard, the impact of PA on intramuscular tumors has been largely underestimated and untested.

Paucity of data: the impact of PA on intramuscular cancers, taking liposarcoma as an example

Despite the benefits of PA in several types of cancer, its impact on tumors located within skeletal muscle itself could be quite different. In this particular case, tumors are directly exposed to mechanical and chemical muscular stimuli, what we called here "direct tumor-muscle crosstalk". Indeed, skeletal muscle is a hub for high rate ATP and ROS production, which could be a favorable context for cancer progression. The cells are multinucleated and, thus, may allow efficient synthesis of pro-oncogenic proteins. On the other hand, we can hypothesize that practicing PA program in patients with intramuscular sarcoma before, during or after chirurgical operation could negatively affect tumor growth and reduce undesirable side-effects related to treatment. However, these ideas remain speculative and need to be confirmed Liposarcoma (LS) is the most frequent type of soft tissue sarcoma that arises from adipocytes, fibroblasts and vascular endothelial cells located within deep soft tissue [START_REF] Sim | Soft-Tissue Tumors: Diagnosis, Evaluation, and Management[END_REF]. Among the 10 000 identified sarcomas by the French sarcoma network, from 2010 till 2013, 20-25% of cases were diagnosed with LS [START_REF] Stock | Adipocytic tumors[END_REF]. It is most commonly found within tights of patients (Peterson et al., 2003). This particular localization inside skeletal muscle constitutes an awkward mass that may impede the capacity of muscle to produce sufficient force and ensure basic physical needs. Indeed, survivors of lower-extremities sarcoma exhibit physical quality deterioration and are unlikely to meet guidelines of PA [START_REF] Wampler | Physical activity among adult survivors of childhood lower-extremity sarcoma[END_REF]. However, the impact of PA on intramuscular tumors and related muscle dysfunction still poorly understood; thus, there is no recommendation about the levels of PA to advise for patients with lower-extremities sarcomas.

No studies were found when typing "physical activity" and "liposarcoma" on PubMed. We focused our research on soft-tissue sarcoma with preference to those located in the lowerextremities, which could be the closest condition to LS. We searched with a combination of the following items: "physical activity" "exercise" "quality of life" and "sarcoma". We identified eight studies that addressed the effect of sarcoma or its treatment on physical QOL and two studies that assessed the effect of short-term rehabilitation on physical function (Table 4). All these studies were performed on childhood sarcoma survivors and most of them (6/8) were performed on a population of patients with different cancer types, including sarcomas. The findings of these studies were quite similar. They reported that patients, especially those with lower-extremities sarcoma, exhibited: (1) reduced PA, (2) deteriorated QOL, (3) low muscle strength, (4) limited performance and ( 5) high risk of cardiac dysfunction. These adverse effects are mostly related to physiologic impairment due to the heavy intervention with surgery and chemotherapy or to the presence of tumor itself within the musculoskeletal apparatus. In patients with lower-extremities osteosarcoma, the intervention with strength, endurance, coordination and flexibility exercise, during inpatient stay, was feasible and improved the capacity of patients to perform PA during home stays [START_REF] Winter | The assessment of physical activity in children undergoing cancer treatment[END_REF]. Interestingly, the authors of this study recommended that exercise must be individually adapted to patient's health status and that PA should outlast the treatment period and be carried on patients showed an improvement in PA levels after six months of follow-up [START_REF] Muller | Physical activity and health-related quality of life in pediatric cancer patients following a 4-week inpatient rehabilitation program. Supportive care in cancer[END_REF].

Moreover, the comprehensive cancer center from the University of Michigan at the USA, has launched the "Sarcoma Survivorship Program" aiming to minimize the risk of heart diseases, obesity and diabetes in sarcoma patient survivors with poor physical function, by modifying lifestyle factors, like diet and exercise. This reflects the awareness that patients with lowerextremities sarcoma are at high risk of functional impairment and must be adequately supported.

The few available data point the importance of PA in ( 1) improving the performance of lowerextremities sarcoma patients, ( 2) help them to reintegrate social life and regain school/work as well as ( 3) perform other usual activities. However, the timing, intensity, frequency, duration and type of PA required to slow sarcoma development/growth still to be determined.

Limited application of PA in cancer cachectic patients: need for an alternative solution

Cachectic patients with advanced stages of colon, lung and pancreatic cancer can develop anemia in ~ 40% of cases [START_REF] Bruera | Cachexia and asthenia in cancer patients[END_REF]. Due to the pro-advanced catabolic state, cardiomyocytes and ventricular cells could undergo degradation [START_REF] Schafer | Ataxin-10 is part of a cachexokine cocktail triggering cardiac metabolic dysfunction in cancer cachexia[END_REF] and this is associated in ~ 48% with respiratory muscle atrophy (i.e. diaphragm) [START_REF] Houten | An investigation of the cause of death from cancer[END_REF].

These events are health-threating, as they lead to the development of cardiovascular dysfunction, respiratory troubles and favor further skeletal muscle atrophy [START_REF] Bruera | Cachexia and asthenia in cancer patients[END_REF]. In such condition, the ability of patients to follow a moderate-intensity exercise could be compromised and replaced with other easy pace activities such as, slow walking [START_REF] Stewart | Cancer cachexia and fatigue[END_REF]. It is important to know that there is a kind of PA level threshold that should be attained to reap PA-related health benefits [START_REF] Richman | Physical activity after diagnosis and risk of prostate cancer progression: data from the cancer of the prostate strategic urologic research endeavor[END_REF][START_REF] Van Blarigan | Physical Activity and Prostate Tumor Vessel Morphology: Data from the Health Professionals Follow-up Study[END_REF]. In this regard, non-vigorous activities may improve fatigue and avoid immobilization-induced atrophy, but are not sufficient to potentiate the anticancer activity of the treatment or even significantly improve QOL. Accumulating preclinical studies indicate that C26 colon tumor-bearing mice were particularly intolerant to lowto-moderate intensity endurance exercise. The presence of anemia in C26 mice exacerbated the loss of total body weight and skeletal muscle mass after exercising on treadmill (Argiles et al., 2012). Using a similar protocol, Pin et al. demonstrated that muscle atrophy was worsened in C26 mice after 2-weeks of exercise, but not in mice bearing LLC tumor [START_REF] Pin | Combination of exercise training and erythropoietin prevents cancer-induced muscle alterations[END_REF]. Ladder climbing resistance exercise also enhanced total body weight waste in C26 mice and increased the expression of genes related to damage and repair within skeletal muscle, compared to no trained C26 mice [START_REF] Khamoui | Aerobic and resistance training dependent skeletal muscle plasticity in the colon-26 murine model of cancer cachexia[END_REF]. These data may underscore the particular susceptibility of cachectic colon cancer-bearing host to endurance and resistance exercise. Since the antioxidant virtues of PA are believed to be one of the most effective arms of exercise against ROS in cancer [START_REF] Rebillard | Prostate cancer and physical activity: adaptive response to oxidative stress[END_REF], a supplementation with antioxidant complements could be a plausible alternative strategy. Add to this, such compounds are highly used among cancer patients especially those with colon, lung and prostate cancer, even without medical counseling [START_REF] Sandler | Use of vitamins, minerals, and nutritional supplements by participants in a chemoprevention trial[END_REF].

Given the inconsistency of conclusions regarding the usefulness of such practice in cancer patients [START_REF] Hardy | Dietary supplement use in cancer care: help or harm[END_REF], further studies are warranted. may not be able to perform vigorous PA, which means that they must rely on other alternative strategies to improve their overall health condition.

The use of antioxidants in cancer and related cachexia

Nutritional supplements in form of capsules, tablets, powder, drops and liquid are usually taken orally and used to improve human nutrition (Mut-Salud et al., 2016b). It has been suggested

since 1968 by the double-Noble prize, Linus Pauling, that megavitamin therapy is the better way to live longer and healthier. Indeed, Pauling showed the example by taking daily mega-doses of vitamin C going from 6 to18 grams, corresponding to 200 glasses of orange jus. Before him, Denham Harman proposed the free radical theory of ageing, in which he hypothesized that ageing is a natural process resulting from the accumulation of oxidative insults that could be avoided by taking antioxidants as a source of "youth-renewal" [START_REF] Harman | Aging: a theory based on free radical and radiation chemistry[END_REF]. Unfortunately, these ideas contributed to the fact that today antioxidant supplements are increasingly used among young and old people, especially in those with health problems, including cancer.

The use of antioxidants in cancer remains questionable and was a subject of debate for several years (Harvie, 2014). However, our understanding on the topic is largely increasing but further in-depth studies still be needed to establish clear guidelines for the use of antioxidants in cancer. A search on PubMed with "antioxidants" and "cancer" as keywords, revealed that the highest record of publications, from 1945, was attained between 2012 and 2015 (Figure 28). This attests that the use of antioxidants in cancer constitutes a contemporary dilemma. Antioxidants may open a window of hope to ameliorate the QOL in cachectic patients with advanced stages of cancer, for whom the application of other adjuvant strategies, like exercise, is limited (Argiles et al., 2012).

Another reason that could motivate more studies in this field, is the behavior of patients toward antioxidant supplementation and their perception of supplements as potential anticancer compounds (Harvie, 2014). Cancer patients are highly interested in vitamins and other antioxidant supplements, as they believe that these compounds are natural and beneficial for health (Harvie, 2014). The prevalence of supplements use is approximately, 60% in lung, 49% in colon and 35%

in prostate cancer patients [START_REF] Hall | Motivations and influences on the use of complementary medicine in patients with localized prostate cancer treated with curative intent: results of a pilot study[END_REF][START_REF] Jatoi | Is voluntary vitamin and mineral supplementation associated with better outcome in non-small cell lung cancer patients? Results from the Mayo Clinic lung cancer cohort[END_REF][START_REF] Sandler | Use of vitamins, minerals, and nutritional supplements by participants in a chemoprevention trial[END_REF]. Previous studies have shown that the use of alternative medicine was associated with higher education, fear of cancer recurrence, influence of family members and participation to social groups [START_REF] Hall | Motivations and influences on the use of complementary medicine in patients with localized prostate cancer treated with curative intent: results of a pilot study[END_REF][START_REF] Lee | Complementary and alternative medicine use among men with prostate cancer in 4 ethnic populations[END_REF]. Approximately, 50% of patients taking antioxidants or multivitamins do not inform their treating physician [START_REF] Hall | Motivations and influences on the use of complementary medicine in patients with localized prostate cancer treated with curative intent: results of a pilot study[END_REF]. Such random and inappropriate use could be lifethreating and deleterious for cancer patients [START_REF] Bjelakovic | Antioxidant supplements for prevention of gastrointestinal cancers: a systematic review and meta-analysis[END_REF]. Practitioners usually prohibit the use of supplements during chemotherapy or radiotherapy, as a preventive strategy against an unproven product that could be deleterious for patients' health (D'Andrea, 2005).

Antioxidants were initially given for cancer patients to offer an effective nutritional care and alleviate secondary symptoms, since it is though that these compounds could be depleted in response to the disease and/or the used anticancer therapies [START_REF] Fortunati | Pro-inflammatory cytokines and oxidative stress/antioxidant parameters characterize the bio-humoral profile of early cachexia in lung cancer patients[END_REF]Santiago-Arteche et al., 2012). Certainly, ROS and OS contribute to the pathogenesis of cancer and related muscle wasting, as they control pathways involved in tumor growth and muscle catabolism (Baracos, 2001;Schieber and Chandel, 2014). We have recently reviewed the role of ROS in cancer cachexia and found that ROS are not limited to tumor and skeletal muscle but could also expand to blood, heart, liver and adipose tissue (Assi and Rebillard, 2016). This multi-organ presence of ROS confers cancer cachexia an overelaborate nature that renders the intervention with antioxidants more complicated. For example, inhibition of ROS could be beneficial in skeletal muscle to reduce atrophy but deleterious in tumor as this may accelerate its growth (Assi et al., 29). In the literature, there is a huge number of research articles, reviews, perspectives and opinion papers trying to provide potential explanations and solutions. An example of these efforts, the recently published review by Mut-Salud et al. in which they concluded that "at the moment it cannot be possible to give a general recommendation on whether or not to take antioxidants" (Mut-Salud et al., 2016b). Accordingly, emerging data suggest that if antioxidant supplementation cannot be "generalized" it could be more likely "individualized". In the coming lines we will describe the impact of mostly used antioxidants in cancer/cancer cachexia and tackle main identified molecular mechanisms.

Antioxidant vitamins

Most intervention studies with antioxidant vitamins realized on cancer patients did not explore the concept of cachexia or take into account cachexia staging criteria to select patients.

However, as mentioned above, tumor occupies a central role in the development of cachexia, thus, in a first step it could be helpful to draw a global view about the impact of vitamins on cancer itself in the aim to better use these products in cancer cachexia. Data available from clinical studies suggest a lack of convincing evidence concerning the beneficial effects of vitamin supplementation in cancer patients (Mut-Salud et al., 2016b). The systematic review and meta-analysis of Bjelakovic et al incorporated the results of 14 randomized trials and concluded that high-doses of vitamin A/E and β-carotene were associated with increased mortality in patients with gastrointestinal cancer (Bjelakovic and Gluud, 2007;[START_REF] Bjelakovic | Antioxidant supplements for prevention of gastrointestinal cancers: a systematic review and meta-analysis[END_REF]. Accordingly, the meta-analysis from Pais and Dumitraşcu indicated that the combination of β-carotene with vitamin E could increase mortality in patients with colorectal cancer [START_REF] Pais | Do antioxidants prevent colorectal cancer? A meta-analysis[END_REF]. Interestingly, this increase in mortality seems to be more pronounced when doses of vitamin E exceeded 134 mg/day [START_REF] Miller | Metaanalysis: high-dosage vitamin E supplementation may increase all-cause mortality[END_REF]. Thus, if antioxidant vitamins are problematical in some cases, how should we improve the use of such compounds? In other words, what is the factor that will provide eligibility for a cancer patient to undergo antioxidant supplementation? The response appears to be provided by the randomized double-blinded trial "SUVIMAX". At the baseline, healthy men enrolled in the study exhibited a low blood antioxidant status compared to healthy women, because of the reduced intake of fruits/vegetables often observed in men's alimentary habits [START_REF] Hercberg | The SU.VI.MAX Study: a randomized, placebo-controlled trial of the health effects of antioxidant vitamins and minerals[END_REF]. After eight years of daily supplementation with complements including vitamin C/E and β-carotene at nutritional doses, men presented a reduced risk of 31% to prostate cancer, while women with adequate antioxidant status at the baseline developed an increased risk of 67% to skin cancer [START_REF] Hercberg | The SU.VI.MAX Study: a randomized, placebo-controlled trial of the health effects of antioxidant vitamins and minerals[END_REF]. This suggests that only individuals with particular antioxidant deficiencies will benefit from supplementation in term of cancer prevention. This conclusion, although obtained in disease-free subjects, could be logically transposable to cancer cachectic patients.

Intriguingly, a previous study has shown that patients with lung cancer exhibited low blood levels of vitamin E comparing to controls, but the depletion of vitamin E was more pronounced in cachectic patients [START_REF] Fortunati | Pro-inflammatory cytokines and oxidative stress/antioxidant parameters characterize the bio-humoral profile of early cachexia in lung cancer patients[END_REF]. This may indicate that even in the same type of cancer the doses of vitamins must be adapted taking into account the presence or absence of cachexia. The few number of intervention studies with antioxidants conducted on cachectic patients with colorectal, lung and breast cancer, support the evidence that vitamins in combination to other antioxidants could be beneficial in patients with weak blood antioxidant activity and high ROS levels (Mantovani et al., 2012). Recently, the French speaking society of clinical nutrition and metabolism (SFNEP) has discouraged the use of α-tocopherol and β-carotene for patients with esophageal and head and neck cancer without diagnosed deficiency (French Speaking Society of [START_REF] Clinical | Clinical nutrition guidelines of the French Speaking Society of Clinical Nutrition and Metabolism (SFNEP): Summary of recommendations for adults undergoing non-surgical anticancer treatment[END_REF]. The SFNEP has also stressed out the negative impact of a highdose and long-term antioxidant vitamins administration on the effectiveness of radio/chemotherapy (French Speaking Society of [START_REF] Clinical | Clinical nutrition guidelines of the French Speaking Society of Clinical Nutrition and Metabolism (SFNEP): Summary of recommendations for adults undergoing non-surgical anticancer treatment[END_REF]. Accordingly, supplementation with vitamin E and β-carotene increased cancer recurrence and overall mortality in head and neck cancer patients undergoing radiotherapy [START_REF] Bairati | Randomized trial of antioxidant vitamins to prevent acute adverse effects of radiation therapy in head and neck cancer patients[END_REF]. In fact, the use of these compounds without restriction is risked, as a given antioxidant could synergize or counteract the anticancer activity of a given drug depending on its type and mechanism of action (Mut-Salud et al., 2016b). According to the review of Harvie, the administration of antioxidant vitamins during radiotherapy was associated with a reduction of its anticancer potential (Harvie, 2014). However, there is a lack of evidence concerning the combination of vitamins with chemotherapy. At the moment, the best way to provide an effective nutritional support for cachectic cancer patients is to determine and adapt vitamins doses on a patient-by-patient basis. The supplementation must target cachectic patients exhibiting reduced blood levels of vitamin A, C and E, β-carotene and lycopene [START_REF] Crespo-Sanjuan | Early detection of high oxidative activity in patients with adenomatous intestinal polyps and colorectal adenocarcinoma: myeloperoxidase and oxidized low-density lipoprotein in serum as new markers of oxidative stress in colorectal cancer[END_REF][START_REF] Mayne | Low plasma lycopene concentration is associated with increased mortality in a cohort of patients with prior oral, pharynx or larynx cancers[END_REF].

Experimental models of cancer permitted us to understand some of the mechanisms borrowed by vitamins to induce their deleterious effects. The principal and commonly described Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 mechanism was via lessening oxidative damages and ROS-induced apoptosis in tumor. In preclinical studies vitamin E, in the form of α-tocopherol, was the most used antioxidant vitamin giving its kinetic ability to scavenge certain free radicals (k~10 5 -10 6 M -1 s -1 ) [START_REF] Forman | How do nutritional antioxidants really work: nucleophilic tone and para-hormesis versus free radical scavenging in vivo[END_REF].

Vitamin E (100-500 mg/kg) accelerated lung cancer progression in mice through decreasing ROS production and oxidative damage to DNA (i.e. 8-oxoguanine) within tumor (Sayin et al., 2014). Moreover, vitamin E enhanced the proliferation of lung cancer cells by reducing the expression of the redox-dependent protein P53, which is responsible for cell cycle arrest and apoptosis induction (Sayin et al., 2014).

Le Gal et al. demonstrated that vitamin E was also able to enhance metastasis in melanoma without affecting proliferation. Vitamin E mediated its effects by increasing the GSH synthesis and GSH/GSSG ratio only in metastatic nodules but not in primary tumors (Le Gal et al., 2015). The capacity of metastatic cells to migrate after leaving the primary site could be limited owing to OS; thus, an increase in GSH content may help them to overcome such obstacle. Mechanistically, vitamin E induced its effect by activating the redox-sensitive GTP-RhoA protein, responsible for cytoskeletal changes during cell invasion and migration (Le Gal et al., 2015). Vitamin C (8 mg/kg) and E (40 mg/kg) were also capable to attenuate the anticancer activity of cisplatin combined to an omega-3 enriched diet, by decreasing lipid peroxidation in lung tumor tissue [START_REF] Yam | Suppression of tumor growth and metastasis by dietary fish oil combined with vitamins E and C and cisplatin[END_REF].

Since muscle wasting is a key feature of cancer cachexia, most experimental studies have attempted to use antioxidants in the aim to prevent OS in muscle but didn't take into account the redox status of tumor. Although vitamin E was able to attenuate skeletal muscle proteolysis in unloaded mice by reducing muscular OS (Servais et al., 2007), the use of a mixture containing nutritional doses of vitamin A (0.06 mg/kg), C (11.53 mg/kg) and E (1.73 mg/kg) selectively reduced oxidative damages in C26 tumor and promoted its growth but exacerbated OS within skeletal muscle (Assi et al., 2016). Remarkably, these findings may indicate that the use of antioxidant vitamins is more complicated in cachexia-related muscle wasting due to the ambivalence of OS between skeletal muscle and tumor (Figure 29).

Polyphenols

A short-term treatment of prostate cancer patients with green tea extracts reduced the circulating levels of prostate-specific antigen (PSA) and VEGF, supporting a potential positive role for polyphenols in cancer prevention and treatment [START_REF] Mclarty | Tea polyphenols decrease serum levels of prostate-specific antigen, hepatocyte growth factor, and vascular endothelial growth factor in prostate cancer patients and inhibit production of hepatocyte growth factor and vascular endothelial growth factor in vitro[END_REF]. Green tea polyphenols (474 mg/day) also attenuated ROS levels in plasma of patients with liver cancer undergoing arterial infusion chemotherapy [START_REF] Baba | Reduction of oxidative stress in liver cancer patients by oral green tea polyphenol tablets during hepatic arterial infusion chemotherapy[END_REF]. Cachectic patients with colon, head and neck and lung cancer displayed higher ROS levels and low enzymatic antioxidant activity in the blood compared to healthy individuals (Mantovani et al., 2003a). Their supplementation with an antioxidant formula containing polyphenols (300 mg/kg) partially reduced systemic OS and improved performance status [START_REF] Mantovani | A phase II study with antioxidants, both in the diet and supplemented, pharmaconutritional support, progestagen, and anti-cyclooxygenase-2 showing efficacy and safety in patients with cancer-related anorexia/cachexia and oxidative stress. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF].

Additionally, polyphenols have been shown to reduce cancer growth and muscle atrophy in animal models of cancer cachexia, through controlling the activity of numerous transcriptional factors. Epigallocatechin-3-gallate (EGCG) and theaflavin-3,3 ' -digallate, found in green and black tea respectively, were effective to reduce skeletal muscle atrophy, through inhibiting TNF-αmediated activation of NF-κB system [START_REF] Liang | Suppression of extracellular signals and cell proliferation by the black tea polyphenol, theaflavin-3,3'-digallate[END_REF]Wang et al., 2011). Rats bearing Walker 256 tumor receiving daily intra-peritoneal (I.P.) quercetin injections (10 mg/kg), presented tumor regression and prolonged survival (Camargo et al., 2011). These beneficial effects of quercetin on tumor growth could be attributed to its anti-angiogenic properties, as evidenced by the inhibition of VEGF production in liver extracts (Camargo et al., 2011). Furthermore, oral quercetin supplementation (25 mg/kg) improved the musculoskeletal function and altered IL-6 production in Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 Apc Min/+ mice (develop spontaneous colon cancer), independently of tumor burden [START_REF] Velazquez | Quercetin supplementation attenuates the progression of cancer cachexia in ApcMin/+ mice[END_REF]. Accordingly, the phosphorylated levels of STAT3 (downstream effector of IL-6) were decreased in skeletal muscle of Apc Min/+ mice supplemented with quercetin, whilst the phosphorylation status of NF-κB remained unchanged [START_REF] Velazquez | Quercetin supplementation attenuates the progression of cancer cachexia in ApcMin/+ mice[END_REF]. Resveratrol, abundantly found in the skin of grapes, peanuts and pines, seems to exert its anti-wasting effects in vivo largely depending on tumor type and the route of administration. Oral resveratrol (200 mg/kg) therapy reduced muscle loss through impairing the DNA binding activity of NF-κB (p65) subunit in both skeletal and cardiac muscles of mice bearing C26 tumor, without influencing tumor growth [START_REF] Shadfar | Oral resveratrol therapy inhibits cancer-induced skeletal muscle and cardiac atrophy in vivo[END_REF]. Whereas, I.P. resveratrol injection failed to ameliorate muscle wasting in mice bearing LLC (1mg/kg) or Yoshida AH-130 (5 and 25 mg/kg) tumor (Busquets et al., 2007).

Although most of these studies found that polyphenols positively affected muscle mass and function, there is a lack of evidence concerning their effects on tumor growth. Globally, tumor weight was the sole parameter used to demonstrate tumor regression, this data must be consolidated by performing direct analysis on tumor proliferation (e.g. Ki-67, mitotic index), apoptosis, OS and local inflammation. We must underscore that in vitro and ex vivo studies on colon cancer cells reported that polyphenols or their derived-metabolites were able to reduce cell proliferation, induce cell cycle arrest, promote Nrf2-dependent antioxidant response without affecting the efficacy of chemotherapeutic agents [START_REF] Haraguchi | Cecal succinate elevated by some dietary polyphenols may inhibit colon cancer cell proliferation and angiogenesis[END_REF][START_REF] Signorelli | Natural grape extracts regulate colon cancer cells malignancy[END_REF]. However, the use of antioxidants in vitro is considered unreliable, since these compounds could react with the constituents of culture media to produce H2O2 and other secondary products that interact with the cells and modify their behavior [START_REF] Halliwell | Cell culture, oxidative stress, and antioxidants: avoiding pitfalls[END_REF]. Thus, these in vitro findings cannot be extrapolated to living animals and humans, as polyphenols and other antioxidants are expected to react differently and produce distinct outcomes.

Interestingly, polyphenols may restrict tumor growth by exerting pro-senescence and epigenomic modifications within tumor cells. For example, mice bearing gastric cancer and supplemented with resveratrol orally (40 mg/kg) exhibited a senescence-like growth arrest through the activation of cell cycle inhibitors, p21 and p16, decreasing Ki-67 staining (proliferation marker) and increasing β-Galactosidase staining (marker of senescence) [START_REF] Yang | Resveratrol inhibits the growth of gastric cancer by inducing G1 phase arrest and senescence in a Sirt1-dependent manner[END_REF]. Epigenetic modifications, like histone acetylation and DNA methylation, are produced by a set of enzymes including histones deacetylase (HDAC), histone acetyltransferase (HAT) and DNA methyltransferase (DNMT). These modifications, sensitive to intracellular ROS, permit a reversible activation or repression of genes expression involved in the processes of carcinogenesis [START_REF] Sarkar | Cancer development, progression, and therapy: an epigenetic overview[END_REF]. Accordingly, it has been suggested that polyphenols-induced Nrf2 activation may reduce ROS levels and contribute to change in epigenetic profiles (Mileo and Miccadei, 2016b). For example, EGCG reversed the hyper-methylation of several anti-oncogene genes (i.e.

p16 INK4α , p15) and thus reactivated their expression [START_REF] Fang | Tea polyphenol (-)epigallocatechin-3-gallate inhibits DNA methyltransferase and reactivates methylation-silenced genes in cancer cell lines[END_REF]. These evidence may unveil a new function for polyphenols as an epigenetic-corrector and, therefore, as a direct modulator of tumor growth

Today, nanoparticles are used as a drug carriers in targeted therapies, including those with anticancer potential [START_REF] Lin | HSP90 Inhibitor Encapsulated Photo-Theranostic Nanoparticles for Synergistic Combination Cancer Therapy[END_REF][START_REF] Mccarthy | Multifunctional magnetic nanoparticles for targeted imaging and therapy[END_REF]. Interestingly, the uptake of these particles by human colon cancer cells and glioma cells in vivo and/or in vitro was enhanced when coated with green tea or EGCG [START_REF] Zimmer | Exercise-induced Natural Killer Cell Activation is Driven by Epigenetic Modifications[END_REF] [START_REF] Lu | Augmented cellular uptake of nanoparticles using tea catechins: effect of surface modification on nanoparticle-cell interaction[END_REF][START_REF] Xiao | Enhanced in vitro and in vivo cellular imaging with green tea coated water-soluble iron oxide nanocrystals[END_REF].

Structural analyzes revealed that EGCG possesses a hydrophobic characteristics that facilitate its interaction with lipid rafts of the plasma membrane to promote endocytosis (Nakayama et al., . Additionally, EGCG may induce internalization by interaction with specific membrane receptors [START_REF] Hsu | The anti-cancer effects of (-)-epigallocatechin-3-gallate on the signaling pathways associated with membrane receptors in MCF-7 cells[END_REF].

The affinity of EGCG was particularly higher to cancerous cells compared to normal cells [START_REF] Lu | Augmented cellular uptake of nanoparticles using tea catechins: effect of surface modification on nanoparticle-cell interaction[END_REF]. This may indicate that tumor cells could express specific receptors and present different plasma membrane characteristics that favor the physical interaction of EGCG with malignant cells.

This selectivity of EGCG is very promising and must be exploited in the delivery of anticancer agents encapsulated in EGCG-coated nanoparticles (Figure 30).

Oligoelements: zinc and selenium

Bjelakovic et al. found that selenium could be related to a decrease in gastrointestinal cancer incidence [START_REF] Bjelakovic | Antioxidant supplements for prevention of gastrointestinal cancers: a systematic review and meta-analysis[END_REF]. Accordingly, selenium (200 μg) and zinc (21 mg) were both effective to improve overall clinical condition of patients with gut cancer [START_REF] Federico | Effects of selenium and zinc supplementation on nutritional status in patients with cancer of digestive tract[END_REF].

The nutritional prevention of cancer trial (NPCT) showed that supplementation with 200 μg of selenium during ten years decreased the incidence of prostate cancer in men, only if the plasmatic value was < 121.6 μg/L [START_REF] Duffield-Lillico | Baseline characteristics and the effect of selenium supplementation on cancer incidence in a randomized clinical trial: a summary report of the Nutritional Prevention of Cancer Trial. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF]. In fact, the development of different types of leukemia, lymphoma and solid cancers could be associated with reduced circulating levels of zinc and selenium when compared to control [START_REF] Baltaci | Changes in the Serum Levels of Trace Elements Before and After the Operation in Thyroid Cancer Patients[END_REF][START_REF] Karaman | Selenium status in blood, urine, and hair samples of newly diagnosed pediatric cancer patients[END_REF].

Deficiency in zinc and selenium seems to be caused by the presence of tumor. This interpretation is consistent with recent findings in thyroid cancer patients [START_REF] Baltaci | Changes in the Serum Levels of Trace Elements Before and After the Operation in Thyroid Cancer Patients[END_REF], showing that the levels of circulating zinc and selenium were decreased compared to control, during the preoperative period, whereas they returned to normal level 15 days after operation. More intriguing, when examining cancerous and normal thyroid tissues, they found ~ two fold more selenium and zinc in tumors [START_REF] Baltaci | Changes in the Serum Levels of Trace Elements Before and After the Operation in Thyroid Cancer Patients[END_REF]. Accordingly, in prostate tumor tissues the levels selenium binding protein-1 (SBP-1), which binds selenium and reduces its bioavailability, were inversely correlated with tumor grade. Tumors expressing lower levels of SBP-1 were also twice more likely to recur [START_REF] Ansong | Evidence that selenium binding protein 1 is a tumor suppressor in prostate cancer[END_REF]. It is interesting to know that in 1987, Larsson and colleagues reported similar findings in cachectic rats bearing sarcoma tumor. Authors demonstrated that selenium was well detectable in tumor but not in muscle and that selenium load increased within the progression of the disease [START_REF] Larsson | Trace element changes in serum and skeletal muscle compared to tumour tissue in sarcoma-bearing rats[END_REF]. Contrary to selenium, zinc was highly found in skeletal muscle and increase with the progression of cancer [START_REF] Larsson | Trace element changes in serum and skeletal muscle compared to tumour tissue in sarcoma-bearing rats[END_REF]. Based on these findings, it seems that circulating selenium and zinc undergo redistribution during cancer, with selenium preferentially shuttled to the tumor and zinc to skeletal muscle (Figure 31).

Siren et al. showed that the concentration of zinc within skeletal muscle of cancer cachectic patients was the double when comparing to control [START_REF] Siren | Systemic zinc redistribution and dyshomeostasis in cancer cachexia[END_REF]. Similar findings were obtained in cachectic animals bearing MAC 16 and LLC tumors [START_REF] Siren | Systemic zinc redistribution and dyshomeostasis in cancer cachexia[END_REF]. In the study of Russell and coworkers, the levels of zinc in plasma remained unchanged between tumorbearing MAC 16 mice and non-tumor mice but were 3 times higher in skeletal muscle of tumor group [START_REF] Russell | Attenuation of skeletal muscle atrophy in cancer cachexia by D-myo-inositol 1,2,6-triphosphate[END_REF]. In this case, the supplementation with zinc further increased its concentration in plasma and skeletal muscle, whereas zinc chelation with alpha trinositol reduced its accumulation and rescued muscle atrophy [START_REF] Russell | Attenuation of skeletal muscle atrophy in cancer cachexia by D-myo-inositol 1,2,6-triphosphate[END_REF]. This indicates that the accumulation of zinc in wasted muscle during cancer cachexia is rather a cause of muscle wasting than a consequence. In a second study, Russell et al. demonstrated that zinc was able to activate NF-κB (p65) subunits well-known to activate MuRF-1 expression and drive muscle catabolism. Zinc was also able to activate PKR, induce eIF-2 phosphorylation and maintain 4E-BP1-eIF-4E complex in a hypo-phosphorylated state, leading to the depression of protein synthesis (Russell et al., 2010b). Also, the presence of inflammatory factors like IL-6 during cachexia could up-regulate the expression of zinc influx transporters, which play a central role in mobilization and redistribution of zinc during cachexia [START_REF] Liuzzi | Interleukin-6 regulates the zinc transporter Zip14 in liver and contributes to the hypozincemia of the acute-phase response[END_REF]. In addition to muscle wasting, the accumulation of zinc within some tissues while depriving others, results in zinc dyshomeostasis and the subsequent development of anorexia, hypogonadism, insulin resistance and taste dysfunction [START_REF] Siren | Systemic zinc redistribution and dyshomeostasis in cancer cachexia[END_REF].

Selenium is essential for the functioning of several antioxidant enzymes including GPx [START_REF] Lubos | Glutathione peroxidase-1 in health and disease: from molecular mechanisms to therapeutic opportunities[END_REF]. Since selenium was accumulated in the tumor during cancer cachexia we can suppose that this could enhance the antioxidant defense of the tumor and protect it against oxidative damage; thus, favoring tumor progression. However, practitioners usually recommend to measure the levels of circulating selenium and to supplement only selenium-deficient patients [START_REF] Muecke | Selenium supplementation in radiotherapy patients: do we need to measure selenium levels in serum or blood regularly prior radiotherapy? Radiation oncology[END_REF]. Animal studies have shown that treatment of tumor-bearing mice with cisplatin reduced 40% of tumor mass but did not improve, even worsened, muscle atrophy due to treatment-related toxicity (Wang et al., 2015a). Supplementation with low doses of selenium (0.69 μg) in combination with chemotherapy reduced muscle wasting as evidenced by the repression of MAFbx, calpains and cathepsin L expression within skeletal muscle and the overall increase in muscle mass (Wang et al., 2015a). Additionally, nanoparticles containing selenium were effective to reduce chemotherapy-related lung damage in Swiss albino mice [START_REF] Bhattacharjee | Nano-Se attenuates cyclophosphamideinduced pulmonary injury through modulation of oxidative stress and DNA damage in Swiss albino mice[END_REF].

Ultimately, zinc and selenium could be depleted from circulation and redirected into skeletal muscle and tumor respectively. Muscle becomes deprived from selenium but highly loaded with zinc rendering it more vulnerable to oxidative insults and wasting [START_REF] Larsson | Trace element changes in serum and skeletal muscle compared to tumour tissue in sarcoma-bearing rats[END_REF][START_REF] Russell | Attenuation of skeletal muscle atrophy in cancer cachexia by D-myo-inositol 1,2,6-triphosphate[END_REF]. In the same way, accumulation of selenium within tumor may fortify it against oxidative damages and promote its growth. The intervention with chemotherapy or radiotherapy will shrink tumor and break this vicious circle, but could be not sufficient to "renormalize" the micronutrients homeostasis within the body. Therefore, intervention with low doses of selenium or zinc may attenuate toxicity related to treatment, recover muscle mass and improve the functioning of other organs. Patients with circulating selenium and zinc deficiency, probably, reflect a simple depletion of these compounds and/or a severe change in micronutrients redistribution that favors tumor progression and muscle wasting; thus, for safety concerns, they could undergo supplementation, preferentially and the end or after completion of anticancer therapies.

Antioxidant mixture and pharmacological agents

Since the etiology of cancer cachexia is multifactorial, treatments should be multidimensional to manage cachexia symptoms and alleviate cachexia-related sufferance. In this paragraph, we will discuss studies that have integrated antioxidants in their treatment arms against cancer cachexia (Table 5). In a randomized phase III study, treatment of gynecological cancer patients with antioxidants, namely α-lipoic acid and carbocysteine, combined to megestrol acetate (MA; appetite stimulant) and L-carnitin (antioxidant properties) decreased fatigue, circulating TNF-α concentrations and ROS blood levels, whereas MA alone failed to induce any significant changes in all these parameters [START_REF] Maccio | A randomized phase III clinical trial of a combined treatment for cachexia in patients with gynecological cancers: evaluating the impact on metabolic and inflammatory profiles and quality of life[END_REF]. Decidedly, the pioneer work of Mantovani's team clearly indicates that the supplementation of cancer cachectic patients with a cocktail of antioxidants, including: polyphenols, vitamins and cysteine-containing compounds, alone or associated to drugs like MA, L-carnitine and thalidomide (immune-modulatory function), increased the activity of GPx and reduced ROS levels in blood [START_REF] Mantovani | A phase II study with antioxidants, both in the diet and supplemented, pharmaconutritional support, progestagen, and anti-cyclooxygenase-2 showing efficacy and safety in patients with cancer-related anorexia/cachexia and oxidative stress. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF]Mantovani et al., 2002b). Additionally, this combination regimen can effectively ameliorate lean body mass and the performance status in cachectic patients, as assessed by the European cooperative oncology group (ECOG) scale. These clinical positive outcomes could be attributed to the presence of high ROS amounts and the low activity of antioxidant enzymes in blood samples at the baseline.

Consistent with this interpretation, the study of Block et al. showed that supplementation with highdoses vitamin C (1000 mg) and E (800 UI) during two months reduced the plasmatic levels of isoprostanes (marker of lipid peroxidation), only if it was superior to 50 µg/ml [START_REF] Block | The effect of vitamins C and E on biomarkers of oxidative stress depends on baseline level[END_REF]. This indicates the existence of plasmatic critical threshold values for antioxidants and OS biomarkers. When the plasmatic values of antioxidants are inferior to the normal or when blood ROS levels are much higher than healthy control, then antioxidant supplementation will be potentially positive. While, when plasmatic values of a given antioxidant are superior to the normal, then antioxidant restriction will be a valuable measure (Figure 32). Thence the importance to perform laboratory blood tests in order to determine the antioxidant status before starting intervention. Another interesting detail that may explain the beneficial effects in these trials was, probably, the short duration of treatment going from ten days to four months. Supplementation for a short period seems to be beneficial even when high-doses of antioxidants will be used.

Furthermore, short-term supplementation was likely to reduce chemotherapy-related toxicity and side-effects in cachectic patients, without affecting its anticancer potential [START_REF] Argyriou | A randomized controlled trial evaluating the efficacy and safety of vitamin E supplementation for protection against cisplatin-induced peripheral neuropathy: final results. Supportive care in cancer[END_REF].

►Key Messages from Chapter V -Antioxidants are exponentially used among cancer patients. In some cases, it may constitute the main adjuvant strategy to provide adequate nutritional support and improve overall QOL. Globally, supplementation in patients with systemic antioxidant deficiencies is expected to be beneficial and not risky. The use of vitamins seems to be complicated in cancer especially after the establishment of muscle atrophy, due to the ambivalence and dualistic role of ROS between tumor and skeletal muscle. Alternatively, polyphenols appear to be promising compounds given their high affinity for tumor cells and stunning capacity to modulate cellular senescence and epigenetic profiles. Essential micronutrients undergo redistribution between tumor and skeletal muscle, in a way, triggering the evolution of tumor growth and muscle wasting.

Supplementation of deficient patients undergoing chemo/radiotherapy could help to reestablish homeostasis and overcome cachexia symptoms. At the moment, referring to serum or plasma antioxidant status could be the optimal way to individualize antioxidant intervention, clinically, and maximize gains. In this light, self-prescribe of antioxidants by cancer patients must be firmly controlled by clinicians to avoid undesirable side-effects. Further preclinical studies could be interesting to in-depth identify molecular mechanisms of action of antioxidants and try to improve their use in cancer. Tumor mainly induces muscle wasting through releasing inflammatory mediators, which in turn enhance ROS production within muscle and its subsequent catabolism (Tisdale, 2009).

Given the importance of skeletal muscle in movement production and maintaining posture as well as articulations stability, its wastage seriously alters the physical function and, thus, decreases the probability of anticancer treatment completion [START_REF] Fouladiun | Daily physical-rest activities in relation to nutritional state, metabolism, and quality of life in cancer patients with progressive cachexia[END_REF]. It has been suggested that PA could constitute a part of the primary cancer care to better control tumor evolution and muscle wasting/weakness/ fatigue as well as allow hard therapies to go forward (Friedenreich, 2011). A body of evidence indicates that PA is beneficial in reducing cancer progression, recurrence and related mortality, notably, in breast, colon and prostate cancer (Friedenreich et al., 2016). The main mechanisms through which PA can slow cancer progression are mainly: the modulation of tumor oxidative damage profiles and the reduction of systemic inflammation, circulating insulin levels as well as tumor hypoxia/angiogenesis (Gueritat et al., 2014;[START_REF] Knop | Sport and oxidative stress in oncological patients[END_REF][START_REF] Lemke | Slowing down glioblastoma progression in mice by running or the anti-malarial drug dihydroartemisinin? Induction of oxidative stress in murine glioblastoma therapy[END_REF]McTiernan, 2008). Even some scientists believe that exercised muscles may release myokines able to induce anticancer effects [START_REF] Aoi | A novel myokine, secreted protein acidic and rich in cysteine (SPARC), suppresses colon tumorigenesis via regular exercise[END_REF][START_REF] Hojman | Exercise-induced muscle-derived cytokines inhibit mammary cancer cell growth[END_REF]. When we know that muscle is firstly solicited during exercise and that PA is likely to be beneficial in cancer, it gets intriguing that no previous studies have assessed that impact of PA on intramuscular tumors. Especially that these particular forms of malignancy are highly aggressive and directly affect the musculoskeletal apparatus. We can suppose that the unique cellular architecture, organization and contractibility as well as the high level of energy/ROS production and changes in pH acidity [START_REF] Bangsbo | Elevated muscle acidity and energy production during exhaustive exercise in humans[END_REF], make muscle a sanctuary in which tumor can behave differently in response to exercise. Based on these questioning, the first purpose of this thesis was to investigate the impact of regular PA on tumor growth and skeletal muscle dysfunction in an orthotopic model of intramuscular tumor-bearing mice.

In particular cases the ability of cancer patients with cachexia to move and practice exercise could be limited owing to anemia and cardiovascular dysfunction (Argiles et al., 2012); thus, alternative strategies are needed. Antioxidants have been proposed to counteract the role of ROS in both tumorigenesis and muscle wasting and to provide deficient patients with adequate nutritional support (Mantovani et al., 2012). However, results from the literature are inconclusive, most likely because previous studies have tried to standardize the same treatment to a heterogeneous population of patients exhibiting dissimilar clinical status and antioxidant needs (Mut-Salud et al., 2016a). To date, it is becoming clear that the best way to use such products is to individualize the treatment according to patient's need (Assi and Rebillard, 2016). Even if physicians prohibit supplementation with antioxidants during treatment (D'Andrea, 2005), it is not a secret that patients especially those with colon and lung cancer take antioxidants randomly [START_REF] Sandler | Use of vitamins, minerals, and nutritional supplements by participants in a chemoprevention trial[END_REF]. This practice can be detrimental for patients' health (Bjelakovic and Gluud, 2007) and could be the main motivation behind the exponential increase in research on this topic, these few last years. The use of antioxidants in cancer is a contemporary problem that requires further investigations. In this regard, the second objective was to determine the effect of commercialized antioxidants on tumor growth and severe muscle wasting/dysfunction in a model of cachectic mice bearing subcutaneous tumor.

Based on what has been said here and that research must explore new ideas and evolve in harmony with the clinical context nowadays, we decided to test the efficacy of two adjuvant strategies in two distinct animal models of cancer with significant translational relevance.

INTRODUCTION

Cachexia is a multifactorial syndrome and a complex metabolic disorder that manifests mainly in patients with diabetes, acquired immune deficiency syndrome (AIDS) and cancer. It is characterized by a continuous loss of muscle mass with or without depletion of adipose tissue [1].

Contrary to anorexia, in which adipose tissue is mainly affected but rarely the muscle compartment, conventional and single-nutritional intervention failed to prevent total weight loss and/or restore lean body mass in cancer cachexia [2]. Patients with advanced stages of colon cancer are mainly affected by cachexia, which accounts for more than 20% of mortality in total cancer patients [3].

Muscle atrophy is one of the most significant clinical events in cancer cachexia that negatively impact patient's quality of life and strongly reduce anti-cancer treatment efficiency [4]. Cachexiainduced muscle atrophy (CIMA) is often associated with psychosocial effects due to functional impairment and a dramatically reduced physical activity in cachectic patients [5]. Therefore, the management of cancer cachexia, notably muscle atrophy, represents a real challenge and a major issue for researchers and clinicians in the biomedical field.

Disease-related muscle atrophy have received much attention during the past decade and several studies have been devoted to understand the pathophysiology and explore the underlying molecular mechanisms involved in the wasting process. Muscle atrophy is mainly due to an increase in protein catabolism associated with hypo-anabolism, resulting in a net decrease in overall muscle mass [6].

High circulating levels of tumor-derived factors (e.g. Tumor Necrosis Factor-α and Interleukin-6)

and some Transforming Growth Factor-beta (TGF-β) family ligands (e.g. myostatin and activin), are responsible for the inhibition of the anabolic/hypertrophic, Phosphoinositide 3-kinase (PI3K)-Akt pathway and subsequently the depression in protein synthesis rate [7,8]. On the other hand, these cytokines, trigger the activation of catabolic/atrophic upstream signaling cascades (e.g.

Nuclear Factor-κB /AP-1), leading to the transcriptional activation of two muscle-specific E3 ubiquitin-ligases, known as, Muscle Ring finger protein-1 (MuRF-1) and Muscle Atrophy F-box (MAFbx) [9,10]. Sarcomeric proteins destined to degradation are tagged with polyubiquitin chain, thanks to MuRF-1 and MAFbx, prior the entry into the proteasome core, where they undergo degradation into small peptide fragments. Hyper-activation of the ubiquitin-proteasome proteolytic system is common in CIMA and is mainly responsible for the high breakdown of myofibrillar proteins [11]. Currently, it is well established and recognized that muscle wasting is intimately linked to oxidative stress (OS) in various conditions including disuse, ageing and spinal cord injury [12,13]. It seems that OS is also implicated in cachexia pathogenesis, since oxidative damage markers (e.i. carbonyls proteins) were increased in the skeletal muscle of cachectic patients with lung cancer and were positively correlated with muscle proteolysis [14]. Furthermore, Reactive Oxygen Species (ROS)

have been proposed to act as a second messenger to activate the ubiquitin-proteasome system in an in vitro model of muscle atrophy [15]. Actually, there is growing evidence that sarcopenia or disuse-induced catabolic state in the muscles of rodents models, could be prevented or delayed by the use of different types and doses of antioxidants such as Vitamin E, Resveratrol, Cystein-based antioxidants and Allopurinol [16][START_REF] Jackson | Long-term supplementation with resveratrol alleviates oxidative stress but does not attenuate sarcopenia in aged mice. The journals of gerontology[END_REF][START_REF] Derbre | Inhibition of xanthine oxidase by allopurinol prevents skeletal muscle atrophy: role of p38 MAPKinase and E3 ubiquitin ligases[END_REF][START_REF] Sinha-Hikim | Long-term supplementation with a cystine-based antioxidant delays loss of muscle mass in aging. The journals of gerontology[END_REF]. Since a broad range of ROS is involved in cachexia-related muscle atrophy pathogenesis, we suppose that the use of a combination of antioxidants could be more valuable to target a large spectrum of these species and limit their detrimental activities.

Herein, we tested our hypothesis in a model of colon 26 (C26) tumor-bearing mice supplemented

or not with a mixture of antioxidants containing mainly, catechins, curcumin, quercetin and vitamin C.

MATERIALS AND METHODS

Animals care and protocol

All experiments were approved by the Regional Ethics Committee for Animal Experimentation of or fixed in 4%-paraformaldehyde (PFA). Venous blood was collected into EDTA tubes and centrifuged (1500 g, 10 min, 4°C) for plasma collection.

Culture of C26 cell line and inoculation

Frozen C26 colon cancer cells were obtained from Cell Lines Service (CLS, Eppelheim, Germany).

The murine tumor cells were maintained in a humidified atmosphere at 37°C and 5% CO2 and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM Lglutamine (Life technologies, Saint Aubin, France). Cells were collected using 0.25% trypsin-EDTA. Before the injection of C26 cells into mice (day 1), cells were counted using a hemocytometer, pelleted via centrifugation (500 g, 5 min, 25°C) and resuspended at 1x10 6 C26 cells/ml of sterilized PBS. Mice were shaved on the dorsal side and given a subcutaneous injection of either 1x10 6 C26 cells suspended in 100 µl of sterilized PBS or 100 µl of sterilized PBS only (control) [START_REF] Tanaka | Experimental cancer cachexia induced by transplantable colon 26 adenocarcinoma in mice[END_REF][START_REF] Fujimoto-Ouchi | Establishment and characterization of cachexia-inducing and -non-inducing clones of murine colon 26 carcinoma. International journal of cancer[END_REF]. Body mass was monitored daily after inoculation.

Determination of cachexia symptoms severity

Mice were classified as having mild, moderate, or severe cachexia based on their body mass and gastrocnemius muscle mass. C26 tumor-bearing mice were categorized as having mild cachexia, if both variables were within 1 SD (Standard Deviation) of the mean of control mice. Mice with severe cachexia were > 2 SDs away from the mean of control mice [START_REF] White | Muscle oxidative capacity during IL-6-dependent cancer cachexia[END_REF].

Wire hang test

At the beginning of the protocol, 10 days and 22 days after inoculation, a wire test was used to assess whole body force. Mice were placed on top of a wire cage lid that was shaken gently 3 times, causing the mouse to grip the wire. Then, the wire cage lid was inverted. Three trials per animal were performed for each evaluation and the best time of latency was retained to calculate the holding impulse [START_REF] Ray | Development of a mouse model for assessing fatigue during chemotherapy[END_REF].

Histological analysis

EDL muscle or tumor samples were 24h PFA-fixed and paraffin-embedded in blocks. Serial transverse sections of 4 μm were obtained from each sample using a LEICA microtome and were mounted on glass slides, three cuts in each. The cuts were performed in the wider part of the tissues.

Subsequently, EDL sections were stained for reticulin using the Gomori method to measure muscle fibers diameter and tumor sections were stained for Ki-67 in order to determine the percentage of tumor proliferating cells. All the histological sections were scanned (20× objective and 40× magnification). The minimal Feret's diameter of muscle fiber was determined using the Image J software. Mean fiber diameter for each muscle was determined from at least 150 fibers per muscle. For ki-67 quantification four images were taken per section and the number of ki-67 positive nuclei was counted using NDP image software.

RNA isolation and RT-qPCR

Total RNA were extracted from Gas muscles and tumor tissues using Trizol® reagent according to the manufacture's protocol (Sigma Aldrich). RNA amount and integrity was determined and analyzed using RNA stdsens chips (700-7153) and Experion automated electrophoresis station (Bio-Rad, France). All samples were of high RNA quality after electrophoresis on agarose The expression of target genes (MuRF-1, MAFbx, TNF-α and IL-6) was normalized to the reference genes 18S or HPRT1 and the relative expression was calculated using the ΔΔCt method.

Primer sequences are listed in Table 2. µM sodium orthovanadate and proteases inhibitors cocktail (Sigma P8340, 5 µl/ml). The samples were homogenized using a Polytron homogenizer at 4°C. Each sample was then incubated on ice Densitometry analysis of the bands was conducted using GS-800 Imaging densitometer and QuantityOne software. Phospho-specific signal was normalized to the total signal to estimate the ratio of activation.

Enzyme Linked Immunosorbent Assay (ELISA)

Plasma myostatin levels were detected using Quantikine® ELISA kit purchased from R&D systems (DGDF80, R&D systems Europe, Lille, France). Briefly, 20µl of mouse plasma samples were activated by adding 10 µl of 1N Hcl and neutralized with 10 µl of 1.2 N NaOH/0.5M HEPES. Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 Then the pH of neutralized samples was measured to ensure that it is within 7.2 and 7.6. Plasma samples were added to a 96 wells plate pre-coated with capture monoclonal antibody specific to myostatin. A detection antibody linked to a peroxidase enzyme was next added to each well.

Finally, a substrate solution was added and color developed in proportion with the amount of myostatin bound in the initial step. Color change was measured at 450 nm using a microplate reader. Plasma TNF-α concentrations were determined using a mouse TNF-α detection kit according to the manufacture's protocol (430907, LEGEND MAX TM , BioLegend)

Oxygen Radical Absorbance Capacity assay (ORAC)

Total antioxidant capacity was measured by spectro-fluorometry. A mixture containing: 0.5 M perchloric acid (Prolabo), 100 mM PBS and 100 µL of plasma was centrifuged at 3000g for 5 min at 4°C. The pH of the obtained supernatant was adjusted to ~7 with a solution of 5 M NaOH and 50µl of supernatant were added into each well in a 96-well plate. Then, 14 µM of fluorescein (Fluka) and 17.2 mg/ml of 2,2'-azobis (2-aminopropane) dihydrochloride (AAPH) were dispensed, into each well. Fluorescence was recorded for 60 min at excitation and emission wavelengths of 485 and 530 nm, respectively, using a microplate reader. Calibration solutions of Trolox were also tested to establish a standard curve.

Protein carbonylation

Analysis of protein carbonyl levels was performed using an oxyblot kit according to the manufacture's protocol. (S7150; Millipore, Île-de-France, France). Protein extracts (10 µg) were derivatized with 1% DNPH for 15 min at RT and then separated on 12.5% SDS-PAGE. After transfer, membranes were blocked with 1%BSA in PBST (Phosphate Buffered Saline/0.05% Tween-20) and then incubated overnight at 4°C with anti-DNP primary antibody (1:100) supplied with OxyBlot kit. After washes, secondary IgG goat anti-rabbit antibody (IRDye 680; LI-COR, Lincoln, NE, USA) was added for one hour in the dark at RT. Membranes were scanned using Odyssey Infrared Imaging System.

4-Hydroxy-2-nonenal analysis

Detection of 4-hydroxy-2-nonenal (4-HNE) adducts, was performed as described previously [START_REF] Gueritat | Exercise training combined with antioxidant supplementation prevents the antiproliferative activity of their single treatment in prostate cancer through inhibition of redox adaptation[END_REF].

Briefly, membranes were incubated in a solution of 250 mM sodium borohydride in 100 mM MOPS, for 15 min at RT, immediately after transfer. Then, were incubated overnight at 4°C with Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 primary antibody, specific for 4-HNE, which was kindly provided by Pr. Luke Szweda (OMRF, Oklahoma City, OK, USA). Membranes were washed with PBST and incubated for one hour at RT with secondary IgG goat anti-rabbit antibody (IRDye 680; LI-COR, Lincoln, NE, USA). After washing, membranes were scanned using the Odyssey Imaging System.

Statistical analysis

Median survival was determined by the Kaplan-Meier method and analyzed by the log-rank test.

Other results are presented as means ± Standard Error Mean (SEM). Significance of intergroup differences was examined using Student t-test for tumor comparisons or One-way analysis of variance (ANOVA) for muscle and plasma comparisons. Normality and equal variance were checked before the statistical analysis was conducted. Significant main effects or interactions were further analyzed by the Fischer LSD post hoc test. If normality of the data was not present, the data were analyzed using an ANOVA on ranks, and further analyzed by the Dunn's test. For all statistical analyzes, the level of significance was set at P < 0.05.

RESULTS

Antioxidant supplementation enhanced colon cancer cachexia development and caused premature death

To assess the effect of antioxidant supplementation on cancer cachexia development, we supplemented C26-mice, an experimental model widely used to study cancer cachexia, with a mixture of antioxidants or PBS, as described above. Surprisingly, we found that body weight of supplemented animals (C26-Antiox) started to decrease at 10 th day post injection, whereas C26-PBS mice presented significant body mass reduction only after 20 days post injection (Figure 1A).

When mortality started to occur in C26-mice groups, body weight measures were no more taken into account in data interpretation, which explains why body weight measures were stopped at 13 th and 20 th days for C26-Antiox and C26-PBS mice, respectively. Here, we showed that antioxidant supplementation caused 50% and 100% of C26-mice deaths at 18 days and 22 days post injection, respectively. In comparison, only 20% of C26-PBS mice died after 22 days (Figure 1B).

Furthermore, neither C26-PBS nor C26-Antiox mice developed anorexia since no change in food intake was observed in both groups compared to control group (Figure 1C). Based on various criteria of cancer cachexia development (see Materials and Methods), we found that all supplemented mice developed severe cachexia, while C26-PBS mice were distributed between mild, moderate and severe cachexia (Figure 1D). These results demonstrated that natural antioxidants enhanced colon cancer cachexia development and caused premature death in C26mice.

Cachexia-induced muscle wasting was not prevented with antioxidants

Next, we investigated whether muscle wasting was modulated with antioxidants. As depicted in vs. 8 ± 1.4 and 94.5 ± 11.3 mg, P < 0.001, Figure 2C). Figure 2A shows EDL muscle sections stained for reticulin with the Gomori method. We observed that fibers diameter of both C26-PBS and C26-Antiox-mice were lower than control group (29.8 ± 5.8 and 28.9 ± 3.1

Antioxidants suppressed Akt activation but failed to block ubiquitin-proteasome system induction

Tumor Necrosis Factor-α (TNF-α) and Myostatin (Mstn) are two mediators known to play a key role in the wasting process. We found that circulating TNF-α levels were up-regulated in both C26-PBS and C26-Antiox groups (10.52 ± 2.63 and 7.5 ± 2.23 pg/mL respectively, P < 0.001) compared to control (2.17 ± 0.34 pg/mL; Figure 3A). Whereas the relative expression of TNF-α and IL-6 transcripts in Gas remained unchanged between all three experimental groups (Figure S1).

Counterintuitively, plasma Mstn levels were drastically decreased in C26-PBS compared to control (16819 ± 4283 vs 41464 ± 5418 pg/mL). This decrease was more pronounced in C26-Antiox (10603 ± 2523 pg/mL; Figure 3B). Furthermore, we found that Mstn levels were positively correlated with Gas muscle mass (r = 0.91, P < 0.05). Gas muscles were used to perform western blotting and RT-qPCR analysis. Interestingly, Akt activation (ratio phospho-Akt/Akt total), a marker of protein synthesis, was down-regulated only in antioxidants-supplemented mice comparing to C26 and control (-77%, P < 0.05, Figure 3D). The phosphorylation of Nuclear

Factor-κB (NF-κB) on the p65 subunit was up-regulated in C26-PBS and C26-Antiox mice (+141% and +54% respectively, P < 0.001; Figure 3C). Although, p65 was phosphorylated to a lesser extend in supplemented comparing no-supplemented mice, the difference between both groups was not statistically significant. At the mRNA level the relative expression of MAFbx and MuRF-1, was flagrantly increased in both C26-PBS (7.6 and 4.7 fold, P < 0.001) and C26-Antiox (7.5 and 4.5 fold, P < 0.001) comparing to control (Figure 3E). Accordingly, protein ubiquitination was increased in both groups of C26 (+29% and +26%, respectively, P < 0.05; Figure 3F). Taken together, these data suggest that antioxidants decreased plasma Mstn levels, depressed Phosphotogether, these data suggest that antioxidants decreased plasma Mstn levels, depressed Phospho-Akt expression and failed to prevent ubiquitin-proteasome system activation.

Antioxidants exerted a pro-oxidant effect in skeletal muscle of C26 mice

As shown in Figure 4A, we observed that C26-Antiox mice exhibited higher carbonylated proteins levels compared to control animals (+63%; P < 0.001). Interestingly, no change was observed in C26-PBS mice when compared to control. Contrary to carbonylated proteins, no change in the protein content of 4-HNE, a marker of lipid peroxidation, was detected between the three experimental groups (Figure 4B). Expression of the antioxidant enzymes, magnesium superoxide dismutase (MnSOD) and copper-zinc superoxide dismutase (CuZnSOD) remained unchanged between all groups of mice (Figure 4C). While, catalase expression was significantly higher in C26-PBS and C26-Antiox (+70% and + 71% respectively; P < 0.01;

C26-Antiox mice exhibited a paradoxical reduction in systemic antioxidant capacities

As patients with colorectal cancer exhibit systemic OS [START_REF] Bhagat | Lipid peroxidation and antioxidant vitamin status in colorectal cancer patients[END_REF][START_REF] Santiago-Arteche | Cancer chemotherapy reduces plasma total polyphenols and total antioxidants capacity in colorectal cancer patients[END_REF], we hypothesized that antioxidant supplementation in our experimental model of colon cancer could reduce systemic oxidative damages. We observed that C26-PBS and C26-Antiox mice exhibited systemic OS (Figure 5B and C), as evidenced by the increase of plasma carbonylated proteins content (+85 and +65% vs. control mice respectively; P < 0.01, Figure 5B) and 4-HNE (+172.5 and +180 % vs. control mice respectively; P < 0.001, Figure 5C). However, no significant change in protein carbonyl and 4-HNE content was detected between C26 and C26-Antiox mice. We next determined total antioxidant capacities in plasma, using the ORAC test. Total antioxidant capacities of C26-mice did not significantly differ from those of control mice (1315 ± 343 vs. 1479 ± 196 µmol eq Trolox/L). Unexpectedly, this parameter decreased drastically (P < 0.001) in C26-Antiox mice compared to control and C26-PBS mice (1479 ± 196 vs. 823 ± 174 µmol eq Trolox/L, P < 0.001

Figure 5A

). These results suggest that the antioxidant cocktail decreased the total antioxidant capacities without impacting systemic OS in C26-mice.

Antioxidants accelerated tumor growth and reduced tumor lipid peroxidation

To understand if the deleterious effects observed previously in supplemented mice were tumordependent, we analyzed the effects of antioxidant supplementation on tumor growth and OS. We observed that tumor weight did not differ between both C26-PBS and C26-Antiox mice (Figure 6A). Furthermore, Ki67 staining, a marker of proliferation rate, was significantly higher in C26-Antiox mice compared to C26-PBS (+ 14.3%, P < 0.01; Figure 6B andC). However, we highlight that tumor weights were early reached in C26-Antiox mice since these animals presented a median survival of 18 days, whereas this median was not reached in C26-PBS mice within 22 days when all C26-Antiox mice were died (see Figure 1B). These results suggest that antioxidant supplementation enhanced C26 tumor growth in our experimental model. In line with this hypothesis, we focused on tumor proliferation and cell cycle regulation. We found that the expression of p21 but not p27, two inhibitors of cell cycle, was downregulated in tumor of C26-Antiox mice when compared to C26-PBS mice (-45%, P < 0.05, Figure 6D). To determine how antioxidant can modulate oxidative damage in tumor, we quantified 4-HNE and carbonylated proteins. No significant difference in carbonyled proteins content was denoted between C26-PBS and C26-Antiox mice (Figure 6E). We found that 4-HNE content was markedly lower in tumors of supplemented animals compared to C26-PBS mice (-66%, P < 0.001; Figure 6F). Ultimately, local inflammation within tumor seemed to be unaffected in response to antioxidant supplementation, as evidenced by the unchanged expression of TNF-α and IL-6 at the mRNA level (Figure S1). All these data would finally support the paradigm, that antioxidants accelerate tumor proliferation and modulate OS profiles within tumor.

DISCUSSION

Cancer cachexia is a devastating disorder that developed in 50-80% of patients with advanced stages of cancer. Common symptoms are: anemia, hypogonadism, systemic inflammation, respiratory failure, heart atrophy, altered liver metabolism, adipose tissue depletion and excessive loss of skeletal muscle [START_REF] Dodson | Muscle wasting in cancer cachexia: clinical implications, diagnosis, and emerging treatment strategies[END_REF][START_REF] Argiles | Molecular mechanisms involved in muscle wasting in cancer and ageing: cachexia versus sarcopenia[END_REF][START_REF] Johns | Muscle wasting in cancer[END_REF]. Although muscle wasting, the key feature of cachexia, is mainly responsible for the death of many cancer patients, it remains a poorly understood mechanism.

Several pieces of evidence suggest that OS is involved in the establishment and evolution of this catabolic process [START_REF] Mantovani | Cachexia and oxidative stress in cancer: an innovative therapeutic management[END_REF]. In the present study we demonstrated that supplementation with antioxidants reduced mice survival, failed to prevent muscle atrophy and promoted tumor growth in C26 tumor-bearing mice.

We found that antioxidants accelerated cachexia development and reduced life span in C26 mice.

To our knowledge no studies have assessed the effects of antioxidant supplementation on the survival rate of cachectic animals. Nevertheless, recently, a meta-analysis concluded that the results of intervention studies with antioxidants, performed from 1966 till 2009, have no effects on colon cancer incidence and recurrence [START_REF] Pais | Do antioxidants prevent colorectal cancer? A meta-analysis[END_REF]. Even worse, they found that combination of β-carotene with other antioxidants was associated with an increase in mortality. Elevated systemic levels of TNF-α, Interleukine-6 and Interferon-γ have been reported in patients experiencing cachexia and in several rodent models of cancer cachexia [START_REF] Balkwill | Evidence for tumour necrosis factor/cachectin production in cancer[END_REF][START_REF] Matthys | Severe cachexia in mice inoculated with interferon-gamma-producing tumor cells. International journal of cancer[END_REF][START_REF] Strassmann | Suramin interferes with interleukin-6 receptor binding in vitro and inhibits colon-26-mediated experimental cancer cachexia in vivo[END_REF][START_REF] Zhou | Reversal of cancer cachexia and muscle wasting by ActRIIB antagonism leads to prolonged survival[END_REF]. There is considerable evidence that TNF-α plays a pivotal role in muscle loss during cancer cachexia. Thus transplantation of Chinese Hamster Ovary cells, transfected with human TNF-α gene, in nude mice produced a syndrome resembling to cachexia [START_REF] Oliff | Tumors secreting human TNF/cachectin induce cachexia in mice[END_REF]. In our model the circulating levels of TNF-α were significantly higher in C26 mice supplemented or not with antioxidants. The unchanged expression of TNF-α within Gas muscles is not surprising, as usually skeletal muscle is not a major source of cytokines production during cachexia but rather undergo catabolism in response to tumorderived cytokines. TNF-α induces the activation of NF-κB, leading to the induction of the ubiquitin-proteasome system [START_REF] Li | NF-kappaB mediates the protein loss induced by TNF-alpha in differentiated skeletal muscle myotubes[END_REF]. We found that the p65 subunit of NF-κB was phosphorylated on the Ser536 residue, notably, in muscles of no-supplemented mice. The decrease of p65 phosphorylation levels observed in supplemented animals did not result in the reduction of MAFbx and MuRF-1 mRNA relative expression and protein ubiquitination, indicating that the ubiquitinproteasome system was activated regardless the phosphorylation status of p65. Additionally the expression of IKB-α, the specific inhibitor of NF-κB, remained unchanged in skeletal muscle of the three experimental groups (data not shown). This may suggest the existence of other compensatory mechanisms, than NF-κB signaling, which promote proteasome system induction.

Our interpretation is consistent with the findings of a recent report, showing that p65 binding to DNA is not required for the regulation of genes expression involved in the development of muscle atrophy in cachectic mice bearing C26 tumor [START_REF] Cornwell | C26 cancerinduced muscle wasting is IKKbeta-dependent and NF-kappaB-independent[END_REF]. Moreover, antioxidants have not only failed to slow the catabolic process but they, most likely, aggravated muscle wasting by suppressing Phospho-Akt expression, which is directly involved in protein synthesis. One striking finding of this study, was the flagrant decrease of plasma Mstn concentrations in cachectic C26 mice. Since Mstn is a negative regulator of muscle growth [START_REF] Grobet | A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle[END_REF][START_REF] Grobet | Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle[END_REF], the loss of skeletal muscle in response to catabolic stimuli during cachexia could lead to a, protective, down-regulation of systemic Mstn levels. Moreover, as Mstn is mainly produced and secreted by skeletal muscle [START_REF] Lee | Regulation of muscle mass by myostatin[END_REF], it is expected that Mstn levels will decline in response to muscle mass decrease. This interpretation is coherent with our data showing a positive correlation between plasma Mstn concentrations and skeletal muscle mass. Such results are in line with a recent study reporting a decrease in plasma Mstn levels in cachectic patients with lung or colorectal cancer [START_REF] Loumaye | Role of activin a and myostatin in human cancer cachexia[END_REF]. Redox imbalance and chronic OS were found in cancer patients, notably in those with colon cancer [START_REF] Obtulowicz | Oxidative stress and 8-oxoguanine repair are enhanced in colon adenoma and carcinoma patients[END_REF]. Others have found that protein degradation in skeletal muscle was mediated through the upregulation of proteasome subunits and this was an OS-dependent mechanism [15]. Muscle oxidative damages were increased only in supplemented mice, without any change in the expression of MnSOD and CuZnSOD. On the other hand, catalase expression was up-regulated but seemed ineffective and insufficient to counteract muscle OS. We speculate that antioxidants provoked an unbalance in the redox state within muscle in favor of OS, thus creating a favorable microenvironment for the development and enhancement of muscle wasting. Furthermore, both C26 experimental groups exhibited systemic OS but the decrease in total antioxidants capacities was observed in supplemented mice only. This decrease, could be attributed to either, a pro-oxidant effect produced synergistically by the antioxidants cocktail or the consumption of antioxidants by another tissue, like tumor.

Although, tumor weights were similar in both C26 groups, this do not rule out the possibility that tumor of supplemented mice has developed faster, since they died earlier. Also it is plausible that the premature death of supplemented mice was tumor-dependent. Interestingly, we found that the percentage of tumor proliferating cells was greater in supplemented group and this was associated with a depression of p21 expression, suggesting that antioxidants have abolished potent stimuli for p21 induction and cell cycle arrest. All these elements plead in favor of an enhancement of tumor growth in supplemented mice. Interestingly, we found that 4-HNE deceased exclusively within tumor in response to antioxidant supplementation. Clinically, variable amounts of lipid peroxidation products were found in colorectal cancer tissues of patients [START_REF] Skrzydlewska | Lipid peroxidation and antioxidant status in colorectal cancer[END_REF] and the decrease in HNE levels was positively associated with tumor proliferation [START_REF] Barrera | Oxidative stress and lipid peroxidation products in cancer progression and therapy[END_REF]. In order to provide explanations about the selective decrease of oxidative damage within tumor, we attempted to propose antioxidant compounds that could be potentially more problematical in our cachexia model. Our mixture contained mainly polyphenols, vitamin E and C. Recently, Sayin et al have elegantly demonstrated that supplementation with vitamin E accelerated lung cancer progression in mice, reduced ROS amounts within tumor and, the subsequent, oxidative damage level [START_REF] Sayin | Antioxidants accelerate lung cancer progression in mice[END_REF].

Accordingly, Yam and coworkers demonstrated, in a model of mice bearing Lewis Lung Carcinoma (LLC), that vitamin E and C reduced the anticancer activity of omega-3 enriched diet by decreasing in situ oxidation of fatty acids [START_REF] Yam | Suppression of tumor growth and metastasis by dietary fish oil combined with vitamins E and C and cisplatin[END_REF]. Thus, we speculate that vitamin E and C, wellknown to inhibit lipid peroxidation, could be potential candidates responsible for the decrease of 4-HNE content in C26 tumor of supplemented mice. Other major components of our antioxidant mixture were polyphenols, namely, catechins, resveratrol and quercetin. On the one hand supplementation with resveratrol does not affect tumor growth and muscle wasting in distinct models of cancer cachexia [START_REF] Busquets | Resveratrol does not ameliorate muscle wasting in different types of cancer cachexia models[END_REF]. On the other hand, administration of either catechins or quercetin, into cachectic rodent bearing tumor, reduced the magnitude of muscle atrophy and tumor weights [START_REF] Wang | Green tea polyphenol EGCG suppresses lung cancer cell growth through upregulating miR-210 expression caused by stabilizing HIF-1alpha[END_REF][START_REF] Camargo | Inhibition of tumor growth by quercetin with increase of survival and prevention of cachexia in Walker 256 tumor-bearing rats[END_REF]. It should be highlighted that these studies focused mostly on muscle wasting but lacked direct analyzes on tumor proliferation and modulation of systemic and local OS. As ROS may exert a double-faced role in cancer [START_REF] Schieber | ROS function in redox signaling and oxidative stress[END_REF], antioxidants could be beneficial in other experimental models, despite the deleterious effects observed in this study. For example, we have previously shown that dietary antioxidants decreased tumor oxidative damage and reduced proliferation in prostate cancer [START_REF] Gueritat | Exercise training combined with antioxidant supplementation prevents the antiproliferative activity of their single treatment in prostate cancer through inhibition of redox adaptation[END_REF]. Thus, inhibition of OS could either enhance or slow tumor growth depending on tumor type/localization.

One of the limitations of our study was the identification of the antioxidant compound responsible for the acceleration of cachexia development. At the same time, we cannot deny the fact that using these antioxidants in combination, but not in an isolated manner, is perhaps behind the harmful effects observed in mice. We believe that the experimental model employed in the present study, could be more reflective to what happening clinically with cancer patients who privilege the use of an antioxidant cocktail, as they believe that these compounds are natural and safe. The usefulness of antioxidants supplementation in cancer and cancer cachexia remains questionable and data emerging from interventions studies are controversial and incoherent [START_REF] Van Zandwijk | a randomized trial of vitamin A and N-acetylcysteine in patients with head and neck cancer or lung cancer. For the EUropean Organization for Research and Treatment of Cancer Head and Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 Neck and Lung Cancer Cooperative Groups[END_REF][START_REF] Lesperance | Mega-dose vitamins and minerals in the treatment of non-metastatic breast cancer: an historical cohort study[END_REF][START_REF] Watson | Oxidants, antioxidants and the current incurability of metastatic cancers[END_REF][START_REF] Mantovani | Cyclooxygenase-2 inhibitors and antioxidants in the treatment of cachexia[END_REF][START_REF] Mantovani | Randomized phase III clinical trial of five different arms of treatment for patients with cancer cachexia: interim results[END_REF]. The self-prescribe antioxidants in cancer patients is recurrent and starts to be an alarming phenomenon, especially when antioxidants are used randomly and inappropriately. More than 50% of patients with cancer, increase their consumption of antioxidants complements, without medical prescription, after diagnosis of cancer [START_REF] Velicer | Vitamin and mineral supplement use among US adults after cancer diagnosis: a systematic review[END_REF]. In the present study, we used an antioxidant cocktail available at the European market as "Over The Counter" medicament and doses given to mice were adjusted to be adequate with supplier's recommendations. Another important detail is that individual doses of each antioxidant compound were not ultra-physiologic comparing to the most of studies conducted on animals. The present study clearly indicates that the daily use of antioxidant complements in colon cancer cachexia reduced survival and enhanced cachexia-associated complications in C26 mice. Patients with cancer, especially those with cachexia, may be more cautious and careful

The impact of PA on tumor growth

PA constitutes a primary component of the standard care treatment in the primary and secondary prevention of numerous chronic illnesses like, cardiovascular diseases [START_REF] Giannuzzi | Physical activity for primary and secondary prevention. Position paper of the Working Group on Cardiac Rehabilitation and Exercise Physiology of the European Society of Cardiology[END_REF]. The last ten years, studies investigating the impact of PA on cancer have increased exponentially giving birth to a new field, today, known as "exercise-oncology". Mainly motivated by the evidence that PA could decrease cancer incidence, with a linear dose-response relationship in breast and colon cancer, researchers aimed to investigate the impact of exercise in patients with diagnosed cancer [START_REF] Ballard-Barbash | Physical activity, biomarkers, and disease outcomes in cancer survivors: a systematic review[END_REF]. Emerging data from breast, colon and prostate cancer indicate that physically active patients exhibited less recurrence and mortality, highlighting a possible role for PA in the suppression of tumor growth (Friedenreich et al., 2016;Meyerhardt et al., 2006b). addressed the impact of exercise on tumor incidence, growth and metastasis. Most these studies were performed on breast, intestinal, liver, lung and prostate cancer (Ashcraft et al., 2016). We might underscore that since the systematic review of Ashcraft and collaborators (April 2016), at least, five studies have been published and, therefore, were not included into the analysis. After adjusting to integrate all studies, we found that exercise inhibited tumor growth in 64% of studies, accelerated growth in 11% (i.e. subcutaneous mammary and melanoma) and did not induce significant changes in 25% of cases. Nevertheless, there is a high heterogeneity in the type, the moment of intervention, duration and frequency of exercise as well as the location/type of tumor.

Type and location of tumor:

Only few studies have investigated the impact of exercise on intramuscular tumor, as most of them were performed subcutaneously to monitor days [START_REF] Gershbein | Influence of stress of lesion growth and on survival of animals bearing parenteral and intracerebral leukemia L1210 and Walker tumors[END_REF]. Recently, Hiroux et al reported that 4 days of voluntary wheel running was sufficient to reduce the growth of colon C26 tumor injected intramuscularly in Balb/c mice [START_REF] Hiroux | Physical Activity Counteracts Tumor Cell Growth in Colon Carcinoma C26-Injected Muscles: An Interim Report[END_REF]. This was probably due to decreased tumor proliferation, as evidenced by the reduction in Ki-67 staining on tumor sections [START_REF] Hiroux | Physical Activity Counteracts Tumor Cell Growth in Colon Carcinoma C26-Injected Muscles: An Interim Report[END_REF]. Authors wanted to assess the effect of direct muscle contraction on tumor growth. This could be interesting from a mechanistic point of view, nevertheless, muscle cannot mimic the natural evolution of colon or breast cancer as it does not accurately represent the orthotopic microenvironment of the tumor. For example, voluntary wheels running tended to increase tumor growth, by 21%, in mice bearing orthotopic mammary cancer [START_REF] Jones | Effect of aerobic exercise on tumor physiology in an animal model of human breast cancer[END_REF], whereas the same modality of PA reduced tumor mass in mice bearing intra-peritoneal mammary cancer cells [START_REF] Zhu | Energetics and mammary carcinogenesis: effects of moderate-intensity running and energy intake on cellular processes and molecular mechanisms in rats[END_REF] No studies have investigated the impact of exercise on orthotopic intramuscular sarcoma, but two studies have addressed the effect of forced swimming exercise on subcutaneous sarcoma-180 tumor growth [START_REF] Assi | The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Tumor-Skeletal Muscle Crosstalk in Cancer-Ph[END_REF][START_REF] Sasvari | The effect of regular exercise on development of sarcoma tumor and oxidative damage in mice liver[END_REF]. Both studies demonstrated that an eight to ten weeks continuous swim training (1h/day; before and after tumor implantation), reduced the size of sarcoma compared to non-trained animals. Contrary to these findings, we have shown that 14 weeks of continuous voluntary running on wheels, promoted intramuscular LS tumor growth, compared to inactive counterparts that stopped running the day of injection. Tumor growth was mainly due to an increase in cellular mitosis and proliferation. Accordingly, the bulging tumor mass in active mice exacerbated skeletal muscle dysfunction in terms of strength and endurance. Unfortunately, it cannot be possible to compare between our study and those performed on sarcomas or even with those showing deleterious effects in mammary and melanoma cancer, since there is a difference in several essential parameters including: (1) type of tumor (e.g.

LS vs. ascites sarcoma/melanoma), (2) tumor cell line strain (human vs. mouse), ( 3) location of tumor (intramuscular vs. subcutaneous or intra-peritoneal), ( 4) modality of exercise (forced vs. voluntary), ( 5) duration of exercise (14 weeks vs. 8-to-10 weeks) and ( 6) frequency of exercise (spontaneous access/ 7 days vs. 30min-1 hour/day/5 days).

Modality, intensity and the moment of intervention with exercise: One of the important factors to take into account when designing a preclinical study is whether to perform a forced or voluntary exercise training. Although a current opinion associates voluntary running on wheels with addictive or stereotypic behavior that negatively affects animal welfare, this remains highly speculative [START_REF] Richter | Resting Is Rusting: A Critical View on Rodent Wheel-Running Behavior[END_REF]. Running on wheels is considered as a low-intensity exercise but it is a powerful tool to study biological mechanisms underlying PA, especially in cancer, where the ability of patients to perform high-intensity exercise could be compromised [START_REF] Stene | Effect of physical exercise on muscle mass and strength in cancer patients during treatment--a systematic review[END_REF]. Clinically, the intensities of exercise vary between 50 to 100% according to the physical ability of a cancer patient [START_REF] Sasso | A framework for prescription in exercise-oncology research[END_REF], meaning that in some cases the intervention with forced exercise could be safe and associated with health-related benefits. Thus, the choice between forced or voluntary PA in preclinical models must be done according to the primary endpoints of the study. Different intensities of exercise may produce different outcomes on tumor biology. For example, Almeida and coworkers found that swimming with 50% workload reduced tumor growth, whereas swimming with 80% workload was not effective [START_REF] Almeida | Swim training suppresses tumor growth in mice[END_REF]. Additionally, the moment of intervention with PA is an important predictor of tumor growth. For example, Zhu et al. reported that wheels running after tumor inoculation reduced mammary cancer growth, regardless the levels of PA before injection [START_REF] Zhu | Energetics and mammary carcinogenesis: effects of moderate-intensity running and energy intake on cellular processes and molecular mechanisms in rats[END_REF].

In contrast, Pedersen et al. demonstrated that wheel running before tumor implantation was important to regress melanoma growth [START_REF] Pedersen | Voluntary Running Suppresses Tumor Growth through Epinephrine-and IL-6-Dependent NK Cell Mobilization and Redistribution[END_REF]. However, the common factor between both studies is that maintaining PA reduces tumor mass. Our findings are in contrast with these studies, showing that mice who practiced voluntary running on wheels before and after tumor implantation developed bigger intramuscular tumors compared to those who ceased running the day of injection.

The outcomes of physical exercise on tumor growth are conditioned by the type and localization of tumor. Our study is the first to show that maintaining a regular PA was deleterious in mice bearing orthotopic intramuscular tumor (SW872) and, thus, propose new mechanistic insights on the effects of PA on tumors that could occur naturally in muscles. Perhaps, the implantation of the same human LS cells (SW872) subcutaneously, will produce different effects in response to PA. In this light, orthotopic in vivo models should be preferentially used to predict the impact of exercise on a given tumor type. Also the modality, intensity and the moment of intervention with exercise, are all factors that affect tumor growth; thus, should be scheduled and designed to answer a clinical scenario. At the moment, the heterogeneity of the employed animal models precludes a significant conclusion, about a modality or intensity of exercise that could be valuable to all cancer types. Our study fulfill must recommendations for preclinical exercise oncology research defined recently by the team of Lee Jones (Ashcraft et al., 2016). We have used an orthotopic model with human tumor; we have included appropriate control groups; the modality of exercise is adapted to the clinical condition, where lower-extremities sarcoma patients are less likely to perform high-intensity exercise; we have assessed both systemic and intra-tumor molecular mechanisms to explain how PA could modulate tumor biology.

Respecting these parameters in the design of preclinical studies will allow the categorization of studies and facilitate interpretation.

Type of exercise: Aerobic and resistance are the two types of exercise that could be prescribed for cancer patients. The American college of sports medicine recommends for healthy individuals: 20-to 60 min of aerobic exercise 3 to 5 times per week at 55% Vo2R and 20 repetitions of resistance exercise with an intensity corresponding to 12-to-16 rate of exertion (20 is the maximum exertion rate) 2 to 3 times per week. Cancer patients may at least practice 150 min of moderate-intensity aerobic exercise or aerobic combined to resistance exercise [START_REF] Schmitz | American College of Sports Medicine roundtable on exercise guidelines for cancer survivors[END_REF]. Combination of aerobic and resistance exercise, mainly tested in breast and prostate cancer patients, seemed to improve quality of life, social physique anxiety, fatigue, physical fitness, muscle strength and lean body mass and decrease systemic inflammation [START_REF] Galvao | Combined resistance and aerobic exercise program reverses muscle loss in men undergoing androgen suppression therapy for prostate cancer without bone metastases: a randomized controlled trial[END_REF][START_REF] Milne | Effects of a combined aerobic and resistance exercise program in breast cancer survivors: a randomized controlled trial[END_REF]. However, aerobic exercise could have a wider applicability in cancer, as patients showed a trend to engage in significantly more physical activity in aerobic training comparing to resistance exercise [START_REF] Mina | A randomized trial of aerobic versus resistance exercise in prostate cancer survivors[END_REF]. This could be due to cancer-related fatigue and muscle weakness, making aerobic exercise easier to practice and suitable to patient's situation. Dennett et al. have recently reviewed 42 randomized controlled trials and found a doseresponse relationship between moderate-intensity aerobic exercise and improved quality of life/reduced fatigue [START_REF] Dennett | Moderate-intensity exercise reduces fatigue and improves mobility in cancer survivors: a systematic review and metaregression[END_REF]. Therefore, convincing evidences exist about the beneficial effects of aerobic exercise alone or in combination with resistance exercise in cancer survivors. Nonetheless, there is no convincing evidence about the impact of resistance exercise alone on patient's quality of life. Even some preclinical studies reported limitedness and deleterious effects of resistance training in animals bearing aggressive cancers [START_REF] Das Neves | Loss of strength capacity is associated with mortality, but resistance exercise training promotes only modest effects during cachexia progression[END_REF][START_REF] Khamoui | Aerobic and resistance training dependent skeletal muscle plasticity in the colon-26 murine model of cancer cachexia[END_REF]. In our study, mice performed low-intensity aerobic exercise by running ~ 5.5 km/day, but such type/modality of exercise enhanced intramuscular LS growth. Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 Seemingly, in particular types of intramuscular cancers, aerobic exercise could produce negative effects, however, this suggestion needs to be confirmed in the future. At the moment, aerobic exercise appears to be a convenient type of PA for most cancer patients.

Focus on potential molecular mechanisms modulated by PA in cancer

Systemic and intra-tumor redox profiles: The ability of exercise to mimic an antioxidant response has been suggested as a mechanism to counteract the role of ROS in both tumor growth and muscle wasting (de Sousa et al., 2016). Rats trained on treadmill during 4 weeks exhibited reduced prostate tumor size and increased tumor doubling-time (Gueritat et al., 2014). Although intra-tumor protein carbonyls were unaffected, the levels of oxidative DNA damage (i.e. 8-oxoG) and lipid peroxidation (i.e. 4-HNE) were significantly reduced. These events were probably due to a decrease in intra-tumor ROS, as the activation of the redox-sensitive kinase ERK1/2 was also attenuated. Accordingly, since ERK1/2 plays an important role in promoting tumor growth, its inhibition by exercise was positively reverberated on the rate of tumor cell proliferation (Gueritat et al., 2014). Similarly, Sasvari et al. and Deminice et al. reported that forced exercise was able to decrease tumor growth and systemic oxidative damage, as evidenced by the low levels of protein carbonyls and lipid peroxidation in liver extracts of animals bearing sarcoma or walker 256 carcinoma, respectively [START_REF] Deminice | Resistance exercise prevents impaired homocysteine metabolism and hepatic redox capacity in Walker-256 tumor-bearing male Wistar rats[END_REF][START_REF] Sasvari | The effect of regular exercise on development of sarcoma tumor and oxidative damage in mice liver[END_REF]. Interestingly, the combination of forced exercise with chemotherapy enhanced the anticancer efficacy of the treatment by increasing intra-tumor oxidative damages [START_REF] Lemke | Slowing down glioblastoma progression in mice by running or the anti-malarial drug dihydroartemisinin? Induction of oxidative stress in murine glioblastoma therapy[END_REF]. Indeed, it seems that moderate-intensity exercise alone may rather induce a slight increase in intra-tumor ROS levels, leading to hormetic-like up-regulation of antioxidant genes expression and, therefore, reducing tumor OS. Whereas when combining moderate exercise with anticancer therapies, the amounts of ROS generated by both strategies could reach toxicity threshold; thus, causing further oxidative damages and favoring pro-apoptotic over anti-apoptotic pathways. Such mechanism is under study in our laboratory, in a model combining both treadmill exercise and radiotherapy. In our study, we did not found any change in the expression of antioxidant enzymes GPx-2, catalase, MnSOD and CuZnSOD at both mRNA and/or protein level. Accordingly, the levels of 4-HNE adducts were similar in tumor tissues of both active and inactive mice. This suggests that oxidative profiles are unlikely to be modulated by low-intensity exercise. Seemingly, more intensive boosts of exercise may be needed to induce enzymatic antioxidant changes in skeletal muscle [START_REF] Macdougall | Muscle performance and enzymatic adaptations to sprint interval training[END_REF] and, therefore, within intramuscular tumors. A low-to-moderate intensity of PA is likely to promote cardiopulmonary adaptations, but a high-intensity exercise induces enzymatic adaptations in skeletal muscle [START_REF] Sasso | A framework for prescription in exercise-oncology research[END_REF]. For example, a repetitive endurance training on treadmill with incrementing intensities (1 week; 30 min/day; 20 m/min and, then, 4 weeks; 45 min/day; 25m/min) could allow the production of moderate amounts of ROS intramuscularly and permit hormetic-like changes in the antioxidant response.

Insulin metabolism: One of the essential findings in this thesis was that maintaining PA for 14 weeks was sufficient to decrease basal insulin levels, which coincided with enhanced intramuscular LS growth. Previous studies have demonstrated that voluntary running on wheels decreased circulating insulin levels in tumor-bearing rats and mice [START_REF] Yan | Effects of non-motorized voluntary running on experimental and spontaneous metastasis in mice[END_REF][START_REF] Zhu | Energetics and mammary carcinogenesis: effects of moderate-intensity running and energy intake on cellular processes and molecular mechanisms in rats[END_REF]. Yan and Demars found that 11 weeks of continuous voluntary running on wheels (9 weeks before and 2 weeks after tumor challenge) reduced insulin levels but have no effects on lung LLC tumor growth and spontaneous metastasis in mice [START_REF] Yan | Effects of non-motorized voluntary running on experimental and spontaneous metastasis in mice[END_REF]. LS is considered as an adipocytic tumor [START_REF] Stock | Adipocytic tumors[END_REF] that preferentially rises in skeletal muscle (Peterson et al., 2003). Intramuscular LS could be particularly sensitive to insulin, since the latter regulates several aspects of cell proliferation/differentiation in both adipocytes and skeletal muscle cells [START_REF] Engelman | Constitutively active mitogen-activated protein kinase kinase 6 (MKK6) or salicylate induces spontaneous 3T3-L1 adipogenesis[END_REF][START_REF] Zetser | p38 mitogen-activated protein kinase pathway promotes skeletal muscle differentiation. Participation of the Mef2c transcription factor[END_REF]. Font de Mora and colleagues have demonstrated that alteration of insulin signaling in 3T3-L1 adipocytes induced dedifferentiation via the activation of ERK1/2 MAPK, well-known for its role in inducing cell proliferation and, therefore, blocking differentiation (Font de [START_REF] Font De Mora | Mitogen-activated protein kinase activation is not necessary for, but antagonizes, 3T3-L1 adipocytic differentiation[END_REF]. Interestingly, 3T3-L1 cells were able to form liposarcoma-like tumors in nude mice, which become more aggressive by blocking differentiation [START_REF] Mariani | JUN oncogene amplification and overexpression block adipocytic differentiation in highly aggressive sarcomas[END_REF]. Insulin treatment promoted differentiation of the LS cell lines Lisa-2, MLS-402 and MLS-1765 [START_REF] Codenotti | Caveolin-1, Caveolin-2 and Cavin-1 are strong predictors of adipogenic differentiation in human tumors and cell lines of liposarcoma[END_REF]Wabitsch et al., 2000). Importantly, Codenotti et al. found that the expression of cavin family proteins, that stabilize the insulin receptor and enhance insulin signaling, were most likely expressed in the least aggressive welldifferentiated LS tumors. Accordingly, the authors proposed that the decrease in insulin signaling may promote LS tumor growth [START_REF] Codenotti | Caveolin-1, Caveolin-2 and Cavin-1 are strong predictors of adipogenic differentiation in human tumors and cell lines of liposarcoma[END_REF]. Data in this field are scarce, nevertheless, these studies may shed light about an eventual role for insulin in decreasing LS proliferation and aggressiveness. Mistakenly, the role of insulin could be confused with that of IGF-1. Although both factors control several aspects of cell survival and metabolism, IGF-1 and insulin may regulate to a different extent cellular proliferation and differentiation in vivo (Dupont and LeRoith, 2001). Notably, IGF-1 most likely activates genes related to cell division, chromosome partitioning and protein translation, all of which essential for cellular growth, whereas insulin was likely to be less involved in mitogenesis (Dupont et al., 2001). This specific biological response could be due to a significant difference in their mitogenic signaling response, as the IGF-1 receptor is ten times more efficient in promoting DNA synthesis than the insulin receptor [START_REF] Lammers | Differential signalling potential of insulin-and IGF-1-receptor cytoplasmic domains[END_REF]. Therefore, studies showing potential role for IGF-1 in promoting oncogenesis must be interpreted carefully [START_REF] Grille | The protein kinase Akt induces epithelial mesenchymal transition and promotes enhanced motility and invasiveness of squamous cell carcinoma lines[END_REF][START_REF] Olmos | Targeting insulin-like growth factor signaling: rational combination strategies[END_REF], as it do not necessarily reflects the same mechanisms for insulin in cancer.

We have supposed that insulin may delay intramuscular LS tumor growth through the activation of P38 MAPK-P21 route. P38 and P21 were activated in the small tumors of inactive mice and coincided with significantly higher levels of circulating insulin. Our cell culture experiments demonstrated evidence that insulin is capable to activate p-P38 Thr180/Tyr182 and P21 expression, in the LS cell line SW872, and that blocking P38 activity abrogates P21 expression.

An excellent example, is given by Conejo et al. showing that insulin blocked cell cycle and restored differentiation through reactivating the expression of P21 in a P38-dependent fashion, within highly proliferative Ras-transformed myoblast cells [START_REF] Conejo | Insulin restores differentiation of Ras-transformed C2C12 myoblasts by inducing NF-kappaB through an AKT/P70S6K/p38-MAPK pathway[END_REF]. Therefore, insulin may block proliferation through its ability to induce differentiation. Our data suggest that insulin, at moderate levels, could be a possible activator of the P38 MAPK-P21 pathway in LS tumors and perhaps in other adipocytic malignancy. Finally, we must recall that the potential effects of insulin on LS tumors were observed after eight weeks of inactivity and, therefore, could be transitory. The impact of a longer period of inactivity on insulin and tumor growth stills unknown and deserves to be studied in the future. Although its inferior mitogenicity comparing to IGF-1, some studies have shown a pro-oncogenic role for high insulin levels in cancer [START_REF] Mauro | Estradiol increases IRS-1 gene expression and insulin signaling in breast cancer cells[END_REF][START_REF] Panno | Effect of oestradiol and insulin on the proliferative pattern and on oestrogen and progesterone receptor contents in MCF-7 cells[END_REF]; thus, our suggestion that moderate insulin levels could act as an anti-oncogene could be specific for adipocytic/muscular tumors but not generalized to other cancer types.

The impact of antioxidants on tumor growth

Likely to exercise training that could induce muscular antioxidant adaptations [START_REF] Macdougall | Muscle performance and enzymatic adaptations to sprint interval training[END_REF] 2014), exogenous antioxidants may induce similar effects and, therefore, could substitute exercise in advanced stages of cancer cachexia, where it is application is intolerable. Nutritional support including antioxidant supplementation, is used to reduce the adverse side-effects of anticancer therapies, like muscle wasting, mucositis and fibrosis (Borek, 2004a). Also, it is thought that antioxidants could be depleted during the evolution of cancer and/or in response to radiation and chemotherapeutic drugs (Santiago-Arteche et al., 2012). Thus, antioxidants could be proposed to limit toxicity in normal tissues and avoid severe malnutrition of patients. Mechanistic preclinical studies have shown that ROS play a central role in promoting tumorigenesis and other cancerrelated complications like muscle wasting (Assi and Rebillard, 2016). Seemingly, the evolution of cancer toward more advanced stages and the development of cancer cachexia could be associated to higher ROS levels [START_REF] Fortunati | Pro-inflammatory cytokines and oxidative stress/antioxidant parameters characterize the bio-humoral profile of early cachexia in lung cancer patients[END_REF]. These observations appear to go in the same direction and suggest that antioxidants are beneficial in cancer and related cachexia.

However, preclinical and clinical findings have obtained contradictory results rising doubts about the clinical usefulness of antioxidants in cancer. To be beneficial, antioxidant intervention must reduce toxicity and potentiate the anticancer activity of the treatment (Sagar, 2004), nevertheless, such evidence still lacking. The point of debate was the ability of antioxidants to protect tumor cells by quenching ROS and, therefore, reducing the anticancer activity of treatments (Borek, 2004a, b). Latterly, several publications have alarmed against the use of antioxidants, as there is increasing evidence about their negative impact on radiotherapy efficiency; this becomes unsurprising when we know that X-ray and γ-radiation mediate two third of their tumor-killing effects by increasing ROS generation [START_REF] Coia | Patterns of care study decision tree and management guidelines for esophageal cancer[END_REF]. In the middle of this scientific storm with or against antioxidants, clinicians have decided to prohibit the use of such compounds in cancer patients (D'Andrea, 2005). Such measure was insufficient to restrict the use of antioxidants, as survey studies clearly indicate that between 25 to 60% of cancer patients use antioxidant supplements without informing their treating physician [START_REF] Sandler | Use of vitamins, minerals, and nutritional supplements by participants in a chemoprevention trial[END_REF]Velicer and Ulrich, 2008). Unfortunately, this clinical reality imposes the implementation of new preclinical studies to better understand the mechanisms borrowed by antioxidants in cancer and provide valuable information about the type of antioxidant that could be used in cancer therapy, the time of intervention with antioxidants and the type of cancer in which antioxidants could be beneficial more than others. The association of preclinical with clinical/epidemiological data, may help clinicians to provide useful advices for cancer patients about their antioxidant supplementation and, therefore, better control the random use of such compounds.

Type/stage of tumor and the moment of intervention with antioxidants:

The past three years, high-quality studies gave multiple strikes against antioxidants use in cancer.

The Swedish team of Martin Bergo demonstrated that vitamin E or NAC were both able to reduce survival and accelerate lung cancer progression in mice (Sayin et al., 2014). They also showed that NAC enhanced malignant melanoma metastasis to lymph nodes and lungs in mice with Braf and Pten mutation (Le Gal et al., 2015). Additionally, NAC increased the migration and invasive capacities of seven malignant melanoma cell lines (Le Gal et al., 2015). Similarly, Piskounova et al. have elegantly showed that NAC increased the number of melanoma cells in blood and metastatic burden in NSG mice bearing aggressive melanoma xenografts from patients [START_REF] Piskounova | Oxidative stress inhibits distant metastasis by human melanoma cells[END_REF]. Consistent with these findings, we have found that supplementation with an antioxidant mixture containing vitamins, oligoelements and polyphenols accelerated total body and skeletal muscle weight loss, reduced survival and enhanced C26 colon tumor growth in cachectic mice (Assi et al., 2016). Indeed, malignant colon cancer cells may produce high levels of H2O2 near the toxicity threshold, and giving antioxidants may reduce H2O2 rates and promote proliferation [START_REF] Laurent | Controlling tumor growth by modulating endogenous production of reactive oxygen species[END_REF]. Despite the heterogeneity in the employed animal models, these studies may share two characteristics: (1) the use of an aggressive cancer model and ( 2) the deleterious effects of antioxidant supplementation in term of tumor or metastatic burden.

On the other hand, we have previously observed that antioxidants reduced the volume and delayed the growth of low-grade AT-1 prostate adenocarcinoma in rats (Gueritat et al., 2014).

Similar findings were obtained in mice bearing differentiated breast MCF-7 tumor [START_REF] Liao | Growth inhibition and regression of human prostate and breast tumors in athymic mice by tea epigallocatechin gallate[END_REF] or Dalton's lymphoma ascites [START_REF] Sharma | alpha-Tocopherol prevents lymphoma by improving antioxidant defence system of mice[END_REF]. Clinically, histological studies found a decrease in the expression/activity of antioxidant enzymes in precancerous stages or inflamed tissues propitious for cancer development [START_REF] Bostwick | Antioxidant enzyme expression and reactive oxygen species damage in prostatic intraepithelial neoplasia and cancer[END_REF][START_REF] Cullen | Expression of antioxidant enzymes in diseases of the human pancreas: another link between chronic pancreatitis and pancreatic cancer[END_REF][START_REF] Sander | Oxidative stress in malignant melanoma and nonmelanoma skin cancer[END_REF], in contrast, advanced grades of cancer were most likely associated with increased expression/activity of antioxidant enzymes in tumor tissues [START_REF] Durak | Adenosine deaminase, 5' nucleotidase, xanthine oxidase, superoxide dismutase, and catalase activities in cancerous and noncancerous human laryngeal tissues[END_REF][START_REF] Portakal | Coenzyme Q10 concentrations and antioxidant status in tissues of breast cancer patients[END_REF][START_REF] Sander | Oxidative stress in malignant melanoma and nonmelanoma skin cancer[END_REF][START_REF] Strzelczyk | The activity of antioxidant enzymes in colorectal adenocarcinoma and corresponding normal mucosa[END_REF]. The combination of preclinical and clinical data, let us suppose that intervention with antioxidants could be plausible at early stages of cancer but harmful in advanced stages. Early stages of cancer are associated with a decrease in Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 antioxidant response, which may allow ROS to induce damage and malignant transformation; thus a precocious intervention with antioxidants could impede the pro-cancerous ROS actions.

During the progression of cancer and the subsequent development of cachexia, malignant cells enhance their metabolic activity to meet their increasing energetic needs, resulting in a higher generation of ROS [START_REF] Fortunati | Pro-inflammatory cytokines and oxidative stress/antioxidant parameters characterize the bio-humoral profile of early cachexia in lung cancer patients[END_REF][START_REF] Panieri | ROS homeostasis and metabolism: a dangerous liason in cancer cells[END_REF]. To subsist in such condition, cancerous cells will implement pathways to increase the intracellular pool of antioxidants and protect them self against excessive oxidative damage; thus, a supplementation with antioxidants in late stages of cancer will fortify tumor protection and help it to evade apoptosis.

Type of antioxidants:

The progression of cancer is associated with chronic inflammation, a condition that promotes high intramuscular ROS formation and the subsequent skeletal muscle wasting [START_REF] Arthur | Oxidative stress as a therapeutic target during muscle wasting: considering the complex interactions[END_REF]. Targeting this organ with antioxidants could reduce damage/atrophy and improve its function. Accordingly, the use of vitamins and cysteine-based compounds was efficient to rescue muscle wasting in response to immobilization, sarcopenia and ageing (Servais et al., 2007) (Derbre et al., 2012;Sinha-Hikim et al., 2013). However, the presence of tumor during cancer may change the cumulative outcome, as tumor also needs antioxidants to buffer ROS damage [START_REF] Weinberg | Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity[END_REF]. This ambivalence of ROS in both tumor and skeletal muscle, makes intervention with antioxidants unsure. Previous preclinical studies have taught us that the use of vitamins is complicated in such situation, oral administration of vitamin C and/or E could promote tumor growth and cachexia by reducing the capacity of chemotherapeutic agents, like cisplatin, to induce lipid peroxidation within tumor (Hardman et al., 2002;[START_REF] Yam | Suppression of tumor growth and metastasis by dietary fish oil combined with vitamins E and C and cisplatin[END_REF]. We and others have also shown that vitamins most likely enhanced tumor and/or metastasis burden in animals (Assi et al., 2016;Le Gal et al., 2015;Sayin et al., 2014), by modulating OS profiles.

Polyphenols seem to be more promising agents in cancer, as their structural and chemical properties allow them to affect several tumor weaknesses. Such compounds are able to control the expression of genes involved in inflammation and cell cycle progression/arrest, through modulating epigenetic profiles [START_REF] Choi | Epigallocatechin-3-gallate, a histone acetyltransferase inhibitor, inhibits EBV-induced B lymphocyte transformation via suppression of RelA acetylation[END_REF]. Paradoxically, polyphenols could be spontaneously transformed into pro-oxidant radicals and consequently induce cellular senescence (Mileo and Miccadei, 2016a). Structural analysis have also demonstrated the ability of some polyphenols, like EGCG, to recognize specific receptors expressed on tumors, making the delivery of anticancer drugs with EGCG-pre-coated capsules a plausible strategy to selectively target tumor tissue and spare normal ones [START_REF] Lu | Augmented cellular uptake of nanoparticles using tea catechins: effect of surface modification on nanoparticle-cell interaction[END_REF]. Preclinical studies of cancer cachexia indicated that the use of polyphenols including, catechins, resveratrol and quercetin, was safe and, even, in some cases rescued muscle wasting and cancer cachexia evolution, probably by decreasing tumor growth (Busquets et al., 2007;Camargo et al., 2011;[START_REF] Shadfar | Oral resveratrol therapy inhibits cancer-induced skeletal muscle and cardiac atrophy in vivo[END_REF][START_REF] Velazquez | Quercetin supplementation attenuates the progression of cancer cachexia in ApcMin/+ mice[END_REF].

Duration of intervention and systemic antioxidant status: Lines of evidence suggest that supplementation must target patients with systemic antioxidant deficiency, as this may reflect depletion of these compounds from the body and need for supplementation to avoid malnutrition [START_REF] Hercberg | The SU.VI.MAX Study: a randomized, placebo-controlled trial of the health effects of antioxidant vitamins and minerals[END_REF]Mantovani et al., 2003b;Mantovani et al., 2012). For safety reasons, supplementation should be preferentially done after completion of anticancer therapies to avoid interactions with tumor or with treatments (D'Andrea, 2005). Supplementation that lasted 10 days to 6 months produced beneficial effects in deficient patients even when high doses of antioxidants were administrated [START_REF] Maccio | A randomized phase III clinical trial of a combined treatment for cachexia in patients with gynecological cancers: evaluating the impact on metabolic and inflammatory profiles and quality of life[END_REF][START_REF] Madeddu | Randomized phase III clinical trial of a combined treatment with carnitine + celecoxib +/-megestrol acetate for patients with cancer-related anorexia/cachexia syndrome[END_REF][START_REF] Mantovani | A phase II study with antioxidants, both in the diet and supplemented, pharmaconutritional support, progestagen, and anti-cyclooxygenase-2 showing efficacy and safety in patients with cancer-related anorexia/cachexia and oxidative stress. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF][START_REF] Mantovani | Randomized phase III clinical trial of five different arms of treatment in 332 patients with cancer cachexia[END_REF], whereas intervention with longer periods remains unsure and could produce deleterious effects specifically in individuals with adequate antioxidant status [START_REF] Federico | Effects of selenium and zinc supplementation on nutritional status in patients with cancer of digestive tract[END_REF][START_REF] Hercberg | The SU.VI.MAX Study: a randomized, placebo-controlled trial of the health effects of antioxidant vitamins and minerals[END_REF].

Importantly, the presence of systemic oxidative damage do not necessarily reflects the redox state in other organs (e.g. skeletal muscle) and should not be mistakenly interpreted as a need for antioxidant supplementation. In our study, C26 tumor-bearing mice exhibited high systemic oxidative damage content but skeletal muscle was protected probably because of an increase in muscular catalase expression (Assi et al., 2016). Intriguingly, administration of antioxidants did not affect systemic oxidative damage but, even worsened muscular protein oxidation. One limitation of our study, was the no-assessment of circulating vitamins before and during supplementation and, therefore, there is a lack of information about their bioavailability. The chow provided for animals contained vitamin A and E, meaning that mice were probably not deficient and received an overload of antioxidants, which was apparently responsible for the deleterious effects observed here. Therefore, systemic oxidative damage could not be a synonym for antioxidant deficiency. However, to date, measuring blood antioxidants levels seems to be the most adapted to the clinical context and could be used as a diagnostic tool to identify patients eligible to (Hardman et al., 2002;[START_REF] Salganik | Dietary antioxidant depletion: enhancement of tumor apoptosis and inhibition of brain tumor growth in transgenic mice[END_REF]Sayin et al., 2014;[START_REF] Yam | Suppression of tumor growth and metastasis by dietary fish oil combined with vitamins E and C and cisplatin[END_REF]. The reduction in oxidative damage may lead to apoptosis inhibition. In TgT121 mice that develop spontaneous brain tumor, antioxidant-poor-diet (depleted from vitamin

A and E) increased oxidative DNA damage and the rate of apoptosis (i.e. DNA fragmentation), while antioxidant-rich-diet produced the opposite effect and enhanced tumor growth (Hardman et al., 2002). Indeed, antioxidants may suppress apoptosis by removing DNA damage signatures like 8-oxoG and γ-H2AX that activate P53 (Sayin et al., 2014). Lipid peroxidation is also an important event that could lead to apoptosis, as oxidation of phospholipids on the mitochondrial membrane, like cardiolipins, disturbs mitochondrial transmembrane potential and promotes apoptosis [START_REF] Kagan | Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors[END_REF]. Also oxidation of poly-unsaturated fatty acids at the plasma membrane, induces a change in the structural conformation of lipid bilayer, increases fluidity, disturbs osmotic balance and may cause death [START_REF] Wong-Ekkabut | Effect of lipid peroxidation on the properties of lipid bilayers: a molecular dynamics study[END_REF]. In the present thesis, we found that lipid peroxidation adducts 4-HNE were decreased in C26 tumor of supplemented mice and coincided with higher proliferation rate and inhibition of P21 expression (Assi et al., 2016). We were not able to identify which compound was responsible, as we have used a mixture of antioxidants; nevertheless, we suppose that vitamin C and E could play a key role in such mechanism, given the kinetic capacity of vitamin E to scavenge ROS (k ~ 10 5 -10 6 M -1 s -1 ) and the role of vitamin C in regenerating it [START_REF] Forman | How do nutritional antioxidants really work: nucleophilic tone and para-hormesis versus free radical scavenging in vivo[END_REF]. Also, lipid peroxidation is usually associated with reduced GSH content [START_REF] Jain | Vitamin E supplementation restores glutathione and malondialdehyde to normal concentrations in erythrocytes of type 1 diabetic children[END_REF]. We did not assess the levels of GSH in C26 tumors, but lines of evidence indicate that vitamin E may also increase the intra-tumor pool of GSH by sparing its utilization for the scavenging of lipid peroxidation radicals, supporting the possible involvement of vitamin E in the enhanced tumor growth observed in our model [START_REF] Jain | Vitamin E supplementation restores glutathione and malondialdehyde to normal concentrations in erythrocytes of type 1 diabetic children[END_REF]Le Gal et al., 2015;[START_REF] Piskounova | Oxidative stress inhibits distant metastasis by human melanoma cells[END_REF]. In addition to the modulation of intra-tumor redox profiles, protein carbonyls and 4-HNE content were increased in the plasma of C26 mice comparing to non-tumor mice, but the administration of antioxidants did not induce further changes in such parameters (Assi et al., 2016). It is interesting to note that the use of the term "ROS" in most studies or performing techniques like DCFDA, to measure global quantity of ROS could hide many important information. ROS is a large term encompassing many species that differ in their chemical characteristic, reactivity and biological actions. Since the available techniques to measure intracellular O2 .-and H2O2 are limited [START_REF] Nauseef | Detection of superoxide anion and hydrogen peroxide production by cellular NADPH oxidases[END_REF], future methodological studies may focus on the development of new assays allowing a reliable quantification and identification of "which ROS" are implicated to a larger extent in mediating tumor proliferation and/or apoptosis.

Inflammation and skeletal muscle wasting: Previous studies have reported that the progression of cancer is associated with systemic inflammation and the subsequent development of muscle wasting (Khal et al., 2005;[START_REF] Wyke | NF-kappaB mediates proteolysis-inducing factor induced protein degradation and expression of the ubiquitin-proteasome system in skeletal muscle[END_REF]Zhou et al., 2010).

For example, Zhou et al. reported that C26-mice exhibited high circulating TNF-α, and IL-1β levels and developed muscle atrophy through the mobilization of the UPS (Zhou et al., 2010).

Accordingly, we found that the implantation of C26 tumors induced high circulating TNF-α levels and activated the UPS in the skeletal muscle, as evidenced by the expression of MuRF-1 and

MAFbx transcripts and poly-ubiquitinated protein content (Assi et al., 2016). Nevertheless, all these parameters remained unchanged in response to antioxidant supplementation. ROS are important signal transducers in mediating muscle atrophy and inducing muscular oxidative damage [START_REF] Arthur | Oxidative stress as a therapeutic target during muscle wasting: considering the complex interactions[END_REF]. Previous studies have reported an increase in intramuscular lipid peroxidation and protein carbonyls of cachectic animals bearing tumors [START_REF] Barreiro | Both oxidative and nitrosative stress are associated with muscle wasting in tumour-bearing rats[END_REF][START_REF] Mastrocola | Muscle wasting in diabetic and in tumor-bearing rats: role of oxidative stress[END_REF][START_REF] Springer | Inhibition of xanthine oxidase reduces wasting and improves outcome in a rat model of cancer cachexia. International journal of cancer[END_REF]. Contrary to these studies, C26 tumor did not induce changes in oxidative damage profiles within skeletal muscle. It was interesting to note that antioxidants supplementation increased intramuscular protein carbonyls content, probably reflecting higher ROS production (Assi et al., 2016). We suppose that such redox change, was behind the suppression of p-Akt ser473 in muscles. Our interpretation is consistent with the data of Tan et al.

showing that chronic OS could decrease p-Akt ser473 levels by directly oxidizing the protein's improve their clinical application to better control tumor growth and spare normal tissue from adverse side-effects is of real importance and needs preclinical studies to understand the underpinning molecular mechanisms (Ashcraft et al., 2016;Sagar, 2004). In this work we shed the light on new and/or neglected mechanisms modulated by both PA and antioxidants in cancer.

Despite the heterogeneity in the employed animal models, preclinical and clinical data are most likely to support a beneficial effect of PA in cancer progression and growth (Ashcraft et al., 2016;Friedenreich et al., 2016). We believe that the interpretation of some preclinical data in the field could be nuanced by the design of the study, for example, we cannot conclude that PA is beneficial in colon cancer if the tumor was not implanted orthotopically, as outcomes could be much different. In the first study, we determined the impact of voluntary wheel running on intramuscular LS growth in mice. Our main motivations were (1) the particularity of LS as a tumor naturally occurring in skeletal muscle that affects the musculoskeletal apparatus and reduce the capacity of patients to practice PA and (2) the molecular interest of investigating the effect of direct muscle contraction on intramuscular tumor growth. We have reported for the first time that PA enhanced intramuscular LS growth and accelerated skeletal muscle dysfunction. Our results did astonish surgeons operating intramuscular sarcoma, but even comforted their speculations that active patients may tend to develop more aggressive forms of the disease. These preclinical data and clinical observations must be shored by tangible clinical data; thus, a retrospective study will be launched in the aim to correlate the levels of PA, performed by each patient, with the corresponding histological phenotype of intramuscular sarcoma. If future clinical results are in accordance with our findings, it means that patients with intramuscular LS may reduce their levels of PA before undergoing surgery. We have found that eight weeks of inactivity increased circulating insulin levels. Also, we linked such change to the intra-tumor activation of P38 MAPK-P21 route and the subsequent delay of tumor growth. Nonetheless, we ignore the modifications that insulin metabolism could undergo during longer periods of inactivity and its reverberation on tumor growth. Therefore, the reduction of PA in patients should be temporary and restricted to the preoperative period. This thesis is the first to assess the impact of PA on intramuscular LS and several issues remain to be elucidated in the future. Patients usually receive conventional anticancer therapies and preclinical studies aiming to study the impact of PA on cancer must take such condition into account. As LS patients could receive neoadjuvant chemotherapy before chirurgical intervention, the combination of PA with chemotherapeutic agents should be tested in mice bearing intramuscular LS. In this regard, previous studies demonstrated that exercise combined to chemotherapy may produce different outcomes comparing to exercise alone and, probably, enhance the anticancer response [START_REF] Betof | Modulation of murine breast tumor vascularity, hypoxia and chemotherapeutic response by exercise[END_REF][START_REF] Lemke | Slowing down glioblastoma progression in mice by running or the anti-malarial drug dihydroartemisinin? Induction of oxidative stress in murine glioblastoma therapy[END_REF].

Form a molecular point of view, we have studied the impact of PA on tumor growth using an aggressive cell line of LS (SW872). Tumors of both active and inactive mice developed a LS of grade 3, which did not allow us to evaluate the progression of tumor. Future preclinical studies may use less aggressive cell lines, like Lisa-2, to determine whether PA could affect tumor progression. A striking finding of this thesis was to propose an anti-oncogene role for insulin in intramuscular LS. Other recent observations may also support a role for insulin in inhibiting LS proliferation [START_REF] Codenotti | Caveolin-1, Caveolin-2 and Cavin-1 are strong predictors of adipogenic differentiation in human tumors and cell lines of liposarcoma[END_REF]. Insulin has been looked as a pro-oncogene (Boyd, 2003) and its role is usually regrouped or confounded with that of IGF-1, which is not correct in term of signaling and biological response (Dupont et al., 2001). Our findings that P38 but not Akt was activated in the regressed tumors, may suggest that insulin activates non-classical pathways in a tissue and context-dependent manner. These tissue-specific effects of insulin have been discussed previously and are thought to be responsible for the divergence in insulin/IGF-1 actions in vivo (Dupont and LeRoith, 2001). However, the role of insulin in cancer deserves further indepth studies and must be taken into account by exercise-oncology studies.

In addition to PA, the high prevalence of antioxidants use among cancer patients and the limitedness of applying PA programs in some patients with advanced stages of cancer (increase the dependence on nutritional support), incited us to study the usefulness of antioxidant supplementation in a model of malignant cancer. individual antioxidants or the combination of two antioxidants on cancer growth, progression and metastasis (Hardman et al., 2002;[START_REF] Laurent | Controlling tumor growth by modulating endogenous production of reactive oxygen species[END_REF]Le Gal et al., 2015;[START_REF] Piskounova | Oxidative stress inhibits distant metastasis by human melanoma cells[END_REF]Sayin et al., 2014;[START_REF] Yam | Suppression of tumor growth and metastasis by dietary fish oil combined with vitamins E and C and cisplatin[END_REF]. In this thesis, we reported the first clear preclinical evidence that commercial antioxidant supplements are deleterious in an advanced stage of cancer. These findings must dissuade patients against the random use of such compounds.

We suppose that antioxidants are most likely deleterious in the late stages of cancers. If such hypothesis is true, this means that patients with early stages could benefit from antioxidants to delay the progression of the disease or the appearance of secondary symptoms like, inflammation and muscle atrophy. In this light, polyphenols could be the next generation of antioxidants that should retain the attention of future studies, given their multifunction character suitable with the multifactorial nature of cancer. For example, a preclinical study investigating the impact of polyphenols in two models of cachectic and non-cachectic colon cancer could be of particular interest.

At the moment, based on our study and the studies that we have reviewed recently (Assi and Rebillard, 2016), antioxidant supplementation could be useful for patient with systemic antioxidant deficiencies to alleviate symptoms like mucositis, fibrosis, nephrotoxicity and ototoxicity, but should be preferentially done after complete eradication of the tumor (Borek, 2004a, b;[START_REF] Maccio | A randomized phase III clinical trial of a combined treatment for cachexia in patients with gynecological cancers: evaluating the impact on metabolic and inflammatory profiles and quality of life[END_REF][START_REF] Madeddu | Randomized phase III clinical trial of a combined treatment with carnitine + celecoxib +/-megestrol acetate for patients with cancer-related anorexia/cachexia syndrome[END_REF][START_REF] Mantovani | A phase II study with antioxidants, both in the diet and supplemented, pharmaconutritional support, progestagen, and anti-cyclooxygenase-2 showing efficacy and safety in patients with cancer-related anorexia/cachexia and oxidative stress. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research[END_REF]. While, patients with adequate antioxidant status or undergoing anticancer therapies must avoid supplementation (French Speaking Society of [START_REF] Clinical | Clinical nutrition guidelines of the French Speaking Society of Clinical Nutrition and Metabolism (SFNEP): Summary of recommendations for adults undergoing non-surgical anticancer treatment[END_REF]Harvie, 2014). Another parameter that could be neglected by researchers and clinicians, is to provide an effective psychological support for patients to openly discuss about their fears and self-prescribe antioxidants.

Clinicians must be aware of the advances in research to inform their patients about the dangerous repercussions of inappropriate antioxidant use and, eventually, give useful advices concerning the type of antioxidant that could be suitable to patient's clinical case. Mutually, researchers must also design future studies in harmony with the clinical context nowadays.

Based on our results, the decrease in intra-tumor OS profiles could accelerate its growth especially in advanced stages of cancer cachexia. Perhaps, practicing a low-intensity PA, like wheel running, will not induce the same decrease in tumor oxidative damage as seen with antioxidant supplementation. Contrary to resistance exercise, recent studies demonstrated that the intervention Assi, 2016 with such modality of PA was unable to effectively reverse the time course of cachexia but was at least safe and partially preserved muscle function in C26 tumor-bearing mice [START_REF] Coletti | Spontaneous physical activity downregulates pax7 in cancer cachexia[END_REF][START_REF] Khamoui | Aerobic and resistance training dependent skeletal muscle plasticity in the colon-26 murine model of cancer cachexia[END_REF]. However, applying forced exercise in cancer cachexia is useless and even deleterious (Argiles et al., 2012). At the moment, given the complexity to manage cancer cachexia symptoms and the non-possibility to intervene with high-intensity PA or antioxidants (when patients are with adequate antioxidant status), future studies are still warranted to determine the feasibility and safety of low-intensity exercise in both animals and, then, in patients with advanced stages of cancer that develop anemia, cardiovascular dysfunction and respiratory troubles [START_REF] Grande | Exercise for cancer cachexia in adults. The Cochrane database of systematic reviews[END_REF]. Such strategy may be not sufficient to significantly impact tumor growth but could be at least an acceptable solution to alleviate fatigue, improve functional capacities and maybe slow muscle wastage. Reactive oxygen species (ROS) control several aspects of carcinogenesis as they can either promote tumor growth and progression or senescence and apoptosis. In advanced stages of cancer, ROS can also drive the development of other cancerrelated complications like, muscle wasting. Physical activity (PA) and antioxidant supplementation have been proposed as two adjuvant strategies to better control tumor growth, ameliorate performance and alleviate secondary symptoms related to cancer itself or to the heavy anticancer therapies. However, several issues remain to be elucidated. First of all, although PA could reduce colon, breast and prostate cancer growth and progression, its impact remains unknown on orthotopic intramuscular tumors like liposarcoma, which directly affect the musculoskeletal apparatus and reduce physical function. Secondly, given the limitedness of PA application in some advanced stages of cancer, patients may increase their dependency on nutritional and antioxidant complements as an alternative strategy, but such practice has spark a lot of polemic and inconsistent results. In this thesis, we have addressed the effectiveness of PA and antioxidants in two distinct animal models of cancer. Using in vivo and in vitro approaches, we found that voluntary PA accelerated the growth of intramuscular liposarcoma tumors and exacerbated skeletal muscle dysfunction, mainly, by decreasing circulating insulin levels and the subsequent activation of the tumor suppressor pathway "P38 MAPK-P21". We also demonstrated that nutritional doses of commercial antioxidants enhanced colon tumor growth, total body/skeletal muscle weight loss and caused premature death of mice. Such mechanism was due to selective changes in oxidative damage profiles, which decreased in tumor but increased in skeletal muscle, in a way driving tumor growth and skeletal muscle wasting/dysfunction. Clinically, it seems that (1) patients with intramuscular liposarcoma may, at least, not increase their levels of PA or undergo hospital-supervised exercise program, during the preoperative period; until the confirmation of our findings with clinical data and ( 2) patients with advanced stages of cancer must be very careful against the use of antioxidants as it could lifethreatening. Accordingly, health agencies in France, Europe and USA prohibit the use of synthetic antioxidant supplements without dietary counseling by a cancer patient's physician and/or nutritionist.

The present

Keywords-Reactive oxygen species; Physical activity; Antioxidant supplements; Tumor growth; Skeletal muscle wasting Les espèces réactives de l'oxygène (ERO) contrôlent plusieurs aspects de la carcinogenèse, étant donné leur capacité à promouvoir la prolifération mais aussi à induire la sénescence et l'apoptose. Dans les stades avancés de cancer, les ERO peuvent également participer au développement de l'atrophie musculaire, engendrant une détérioration de la qualité de vie des patients. L'activité physique (AP), connue pour augmenter les défenses antioxydants de l'organisme et la supplémentation en antioxydants sont considérées comme deux stratégies susceptibles de moduler la croissance tumorale, d'améliorer la performance physique et de réduire les effets secondaires liés à la maladie et aux traitements anticancéreux. Néanmoins, plusieurs questions restent aujourd'hui sans réponse. En effet, bien que l'AP puisse réduire la progression du cancer du sein, du colon et de la prostate, son impact sur les cancers se développant au sein du muscle (ex : le liposarcome), donc soumis aux contractions musculaires répétées, reste méconnu. En utilisant des approches in vivo et in vitro, nous avons démontré que maintenir une AP régulière accélère la croissance du liposarcome intramusculaire, probablement, via la réduction des taux circulants d'insuline et l'inhibition de la voie « P38 MAPK-P21 ». Pour les patients ne pouvant pas pratiquer d'AP, la consommation d'antioxydants pourrait être un moyen de réduire les ERO tumorales et musculaires et ainsi être prometteuse. Toutefois, les études actuelles restent très controversées. Nos travaux ont montré dans un modèle murin de cachexie cancéreuse que des doses nutritionnelles d'un produit antioxydant commercialisé en France, étaient suffisantes pour accélérer la croissance de la tumeur colique, favoriser la perte de masse corporelle totale et musculaire et engendrer la mort prématurée des souris. L'ensemble de nos résultats suggère que la pratique d'AP et la consommation en antioxydants peuvent induire des effets différents selon le type de tumeur et leur implantation, nécessitant une prise en charge individualisée des patients. En effet, (1) les patients atteints d'un LS intramusculaire devraient éviter de pratiquer une AP durant la période préopératoire mais ces résultats doivent être confirmés par une étude clinique, (2) les patients avec un stade avancé de cancer devraient être vigilants quant à l'utilisation de suppléments antioxydants ; une telle pratique pouvant avoir des répercussions dangereuses sur leur santé. inversement le muscle peut produire des facteurs qui agissent sur la tumeur et moduler son comportement 4-6 . Un dialogue direct tumeur-muscle pourrait avoir lieu dans des cas particuliers lorsque la tumeur se développe dans le muscle squelettique lui-même. La preuve que la PA peut réduire la croissance, la progression, la récidive et la mortalité associée au cancer s'accumule chez les patients atteints de cancer du sein, de la prostate, du côlon et de l'oesophage 7-9 . Les patients physiquement actifs ont également montré une amélioration de la QV globale et de la performance physique. Ces résultats confirment la conviction que PA doit constituer une partie du traitement anticancéreux primaire. On sait peu de choses sur les mécanismes moléculaires exacts par lesquels l'exercice pourrait servir de médiateur à ses effets protecteurs. Cependant, les changements dans l'inflammation systémique et la capacité de PA à imiter une réponse antioxydante peuvent jouer un rôle clé 10,11 36-38 . Fait intéressant, la PA semble également retarder la récurrence de la maladie, soulignant un rôle possible dans la suppression de la croissance tumorale 7,8,39

Mots clé-Espèces

Western Blot

Les tissus ont été broyés dans de l'azote liquide et la poudre obtenue a été utilisée pour effectuer des expériences. Les échantillons ont été lysés et des expériences d'immunoblot ont été réalisées comme décrit précédemment 47 . Les anticorps primaires suivants ont été utilisés: anti-Hsc70 (sc-7298, Santa-Cruz), anti-Phospho ERK1 / 2 Thr202 / Tyr204 (4370S, Cell Signaling Technologies), total anti-ERK (sc-292838, Santa-Cruz) Anti-phospho JNK Thr183 / Tyr185 (sc-6254, Santa-Cruz), anti-JNK total (sc-7354, Santa-Cruz), anti-phospho p38 Thr180 / Tyr182 (9211S, Cell Signaling Technologies), total anti-p38 (9212S , Cell Signaling Technologies), anti-p21 Waf1 / Cip1 (2947S, Cell Signaling Technologies), anti-p27 (2552S, Cell Signaling Technologies), anti-Phospho p53 ser15 (9284S, Cell Signaling Technologies), anti-p53 total (9282S, Cellulaire Signaling Technologies), anti-caspase-3 clivée (9664S, Cell Signaling Technologies), anti-Bax (2772, Cell Signaling Technologies) et anti-Bcl2 (2870, Cell Signaling Technologies). L'analyse de la densitométrie a été effectuée à l'aide du logiciel Image Gauge v 4.0.

Traitement de l'insuline et inhibition de la p38 MAPK in vitro

Les cellules SW872 ont été ensemencées à une densité de 300 x 10

Résultats principaux et discussion

Les souris actives ont montré une augmentation de la croissance de la tumeur intramusculaire

Étant donné que la fonction musculo-squelettique était sérieusement altérée chez les souris tumorales actives, nous nous sommes demandé qu'un changement dans la masse tumorale intramusculaire pourrait être en partie responsable; Ainsi, nous avons concentré la prochaine partie pour étudier l'impact de l'AP sur la croissance tumorale. De manière inattendue, les tumeurs chez les souris LSA étaient, visiblement, beaucoup plus grandes que chez les souris LSI (Figure 1a). Nous avons constaté que le poids tumoral moyen des souris LSA était supérieur au groupe inactif (3,55 ± 0,89 vs. 2,24 ± 0,99 g, P = 0,03; Figure 1b). L'analyse de l'histopathologie sur les sections tumorales a révélé que les deux groupes ont développé un LS différencié de grade 3 (Figure 1c). Cependant, le nombre de cellules tumoral en cours de division mitotiques a été élevé dans la tumeur des animaux actifs (38,44 ± 9,97 vs. 25,36 ± 7,26 1B). En outre, ni les souris C26-PBS ni les souris C26-Antiox n'ont développé une anorexie car aucun changement dans l'apport alimentaire n'a été observé dans les deux groupes par rapport au groupe témoin (Figure 1C). Sur la base de divers critères de développement de la cachexie du cancer, nous avons constaté que toutes les souris supplémentées ont développé une cachexie sévère, tandis que les souris C26-PBS ont été réparties entre la cachexie légère, modérée et sévère (Figure 1D).

Ces résultats ont démontré que les antioxydants naturels ont accélérés le développement de la cachexie du cancer du côlon et ont provoqué la mort prématuré des souris portant la tumeur C26.

Les antioxydants ont exercé un effet pro-oxydant dans le muscle squelettique des souris C26

Comme le montre la Figure 3A, nous avons observé que les souris C26-Antiox présentaient des niveaux supérieurs de protéines carbonylées par rapport aux animaux témoins (+ 63%, P <0,001). Fait intéressant, aucun changement n'a été observé chez les souris C26-PBS par rapport au témoin.

Contrairement aux protéines carbonylées, aucun changement dans la teneur de 4-HNE, marqueur de peroxydation lipidique, n'a été détecté entre les trois groupes expérimentaux (Figure 3B). L'expression des enzymes antioxydantes, la superoxyde dismutase de magnésium (MnSOD) et la superoxyde dismutase de cuivre-zinc (CuZnSOD) est restée inchangée entre tous les groupes de souris (Figure 3C). Alors que

  My best friends, Ali Farhat, Mohamad Chamas, Stanlislas Zafihita and Yvan Dietrich, I met you at different moments in my life; Ali and Mohamad we shared the same bench for several years at the school and the university in Lebanon; Since my arrival to France, I first met Stanlislas during my Master's studies at the Réunion Island and, then, Yvan at the Veterinary School of Nantes. Because of you I knew that true friendship exits and I simply want to thank each one of you.

FIGURE I :

 I FIGURE I: Introductive scheme showing the different interactions between tumor and skeletal muscle, which could be modulated by factors like, PA and antioxidants ......................................

FIGURE 1 :

 1 FIGURE 1: Disproportionation of O2.-by the "Ping-Pong" reaction; O2.-reduces the oxidized metal ion and then oxidizes the reduced metal ion. .......................................................................

FIGURE 2 :

 2 FIGURE 2: Main enzymatic reactions involved in ROS neutralization ........................................

FIGURE 3 :

 3 FIGURE 3: Reduction of free radicals by the GPx-GSH system ...................................................

FIGURE 4 :

 4 FIGURE 4: Reduction of disulfide bonds by TRX ..........................................................................

FIGURE 5 :

 5 FIGURE 5: Interrelationship between GSH, vitamin C and vitamin E. ........................................

FIGURE 6 :

 6 FIGURE 6: OS profiles in biological system .................................................................................

FIGURE 7 :

 7 FIGURE 7: Three main steps of lipid peroxidation. ......................................................................

FIGURE 8 :

 8 FIGURE 8: Example of oxidative modifications that proteins can undergo in response to high ROS production. .............................................................................................................................

FIGURE 9 :

 9 FIGURE 9: The biological cellular response in relation to intracellular ROS levels and redox potential ..........................................................................................................................................

FIGURE 10 :

 10 FIGURE 10: Oxidative stress as a hallmark of cancer .................................................................

FIGURE 11 :

 11 FIGURE 11: Cancer cells engage different biological responses according to the doses of nitric oxide produced by iNOS. Low-to-moderate doses enhance tumor growth but higher doses produce the opposite effects ...........................................................................................................

FIGURE 12 :

 12 FIGURE 12: Alteration of mitochondrial bioenergetics profiles maintains redox homeostasis in tumor cell and promotes its growth. ...............................................................................................

FIGURE 13 :

 13 FIGURE 13: Main ROS-dependent mechanisms driving tumor growth and progression ............

FIGURE 14 :

 14 FIGURE 14: Main ROS-dependent mechanisms driving tumor regression and death .................

FIGURE 15 : 52 FIGURE 16 :

 155216 FIGURE 15: The impact of tumor derived-factors on multiple organs during cancer cachexia...52 FIGURE 16: The main axis by which tumor-derived chemicals induce ROS production, oxidative damage and muscle degradation ....................................................................................................

FIGURE 17 :

 17 FIGURE 17: The identified pathways for iNOS/NO. in muscle atrophy and dedifferentiation .....

FIGURE 18 :

 18 FIGURE 18: Mitochondrial dysfunction in wasted muscles ..........................................................

FIGURE 19 :

 19 FIGURE 19: Regeneration of GSH molecules through the pentose phosphate pathway ..............

FIGURE 20 :

 20 FIGURE 20: Main protein synthesis pathways inhibited in skeletal muscle during cancer cachexia ..........................................................................................................................................

FIGURE 21 :

 21 FIGURE 21: ROS as second messenger in the activation of catabolic pathways within skeletal muscle in response to inflammatory and atrophic stimuli. ............................................................

FIGURE 22 :

 22 FIGURE 22: Main ROS-dependent molecular events leading to calcium accumulation and calpain activation in skeletal muscle cells, in response to inflammatory or catabolic stimuli ......

FIGURE 23 :

 23 FIGURE 23: Scheme recapitulating the central role of ROS in both carcinogenesis and related skeletal muscle wasting. .................................................................................................................

FIGURE 7 :

 7 FIGURE 7: Three main steps of lipid peroxidation.From (Neda Mimica-Dukić, 2012)

  NO . is a free radical produced enzymatically by NO synthase (NOS) from L- arginine. NOS exists in three different isoforms: Type I NOS and Type III NOS (eNOS) expressed constitutively, and Type II NOS also called inducible NOS (iNOS) expressed exclusively in the

FIGURE 11 :

 11 FIGURE 11: Cancer cells engage different biological responses according to the doses of nitric oxide produced by iNOS. Low-to-moderate doses enhance tumor growth but higher doses produce the opposite effects.Adapted from[START_REF] Vannini | The dual role of iNOS in cancer[END_REF]

FIGURE 13 :

 13 FIGURE 13: Main ROS-dependent mechanisms driving tumor growth and progression (recapitulates main findings of studies cited in the text)

FIGURE 14 :

 14 FIGURE 14: Main ROS-dependent mechanisms driving tumor regression and death (recapitulates main findings of studies cited in the text)

FIGURE 16 :

 16 FIGURE 16: The main axis by which tumor-derived chemicals induce ROS production, oxidative damage and muscle degradation

FIGURE 18 :

 18 FIGURE 18: Mitochondrial dysfunction in wasted muscles. From(Assi and Rebillard, 2016)

FIGURE 19 :

 19 FIGURE 19: Regeneration of GSH molecules through the pentose phosphate pathway[START_REF] Kohen | Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reactions, and methods for their quantification[END_REF]

FIGURE 20 :

 20 FIGURE 20: Main protein synthesis pathways inhibited in skeletal muscle during cancer cachexia. Adapted from(Tisdale, 2009)

FIGURE 21 :

 21 FIGURE 21: ROS as second messenger in the activation of catabolic pathways within skeletal muscle in response to inflammatory and atrophic stimuli. From(Assi and Rebillard, 2016)

FIGURE 22 :

 22 FIGURE 22: Main ROS-dependent molecular events leading to calcium accumulation and calpain activation in skeletal muscle cells, in response to inflammatory or catabolic stimuli (recapitulates main findings of studies cited in the text)

  , ROS are essential for cell survival but in response to pathological stimuli, their high production can promote tumorigenesis. The development and evolution of tumor is associated with the release of inflammatory mediators, which bind skeletal muscle cells to promote local ROS formation and the subsequent muscle degradation. Paradoxically, even if ROS cause cancer development, they may also provide the cure. Indeed, when excessive doses of ROS are generated, the extent of oxidative damages within tumor rises, favoring death over survival response. Tumor apoptosis is a good way to lower the level of circulating catabolic mediators and recover muscle mass. Given the central role of ROS in the pathogenesis of cancer and related muscle atrophy, several clinical and Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016

FIGURE 25 :

 25 FIGURE 25: Hypothetical scheme representing the potential beneficial and harmful effects of ROS in a dose-dependent manner within skeletal muscle

FIGURE 26 :

 26 FIGURE 26: Exercise-induced antioxidant, metabolic, hormonal and angiogenic adaptations as well as myokines production (recapitulates findings of studies cited in the text)

FIGURE 27 :

 27 FIGURE 27: Main factors modulating by physical activity, leading to reduced cancer progression and muscle wasting

  at home. Recently, Muller et al. performed a 4-week rehabilitation program consisting of a land and aquatic-based exercises as well as group exercise training and sport. They found that sarcoma

FIGURE 28 :

 28 FIGURE 28: PubMed search with "Cancer" & "Antioxidants" showing the production of scientific papers in the field over more than two decades

FIGURE 29 :

 29 FIGURE 29: The ambivalence and dual role of ROS between tumor and skeletal muscle as well as the poor selectivity of antioxidant vitamins for tumor tissues, complicates their use in cancer cachexia

FIGURE 30 :

 30 FIGURE 30: Polyphenols as promising compounds in the treatment of tumor growth and muscle wasting

FIGURE 31 :

 31 FIGURE 31: Redistribution of selenium and zinc between tumor and skeletal muscle, promotes the progression of cancer cachexia. Adapted from(Siren & Siren, 2010)

FIGURE 32 :

 32 FIGURE 32: Simplified model showing (•) the plasmatic AO values corresponding to each patient and (■)the suitable measure that could be applied to avoid undesirable side-effects

  . The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016

FlashGel

  system (Lonza, France). 1µg of total extracted RNA was used to perform the Reverse Transcription (RT) reaction (IScript reverse transcription, 170-8840). Sybergreen real-time PCR was carried out in a final volume of 10 µl per reaction containing: 5µl of neo-synthetized cDNA (diluted at 1/5), 0.2 µl of primer pair at 10 µM each (Sigma Aldrich and Integrated DNA Technologies) and 4.8 µl of GOTaq® qPCR Master Mix (A6001, Promega, Madison, USA). All experiments were monitored in a CFX Real-Time system (Bio-Rad, Marnes-La-Coquelle, France).

  for 30 min followed by 3 x 10 s of sonication. The homogenates were transferred to microcentrifuge tubes and centrifugated at 12000 g for 12 min at 4°C. The protein concentration of the supernatant was determined by a Lowry assay using a serial dilution of bovine serum albumin (BSA) to draw the standard curve. Samples were then diluted in SDS-PAGE sample buffer [50 mM Tris•HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.1% bromophenol blue], and heated 5 min at 95°C. Samples containing 40-100 µg of proteins were resolved on 10-15% SDS-PAGE. The proteins were transferred at 240 mA for 90 min onto a 0.2-µm nitrocellulose membrane.Membranes were blocked with 5% BSA or nonfat dry milk in TBST (Tris-buffered saline/0.1% for one hour at room temperature (RT). Membranes were incubated overnight at 4°C with the appropriate primary antibodies: anti-phospho-AKT (9271S), anti-AKT (9272S), antiphospho-NFκB (p65) subunit (3033S), anti-NF-κB (p65) subunit (8242S), anti-ubiquitin (3933S), anti-p21 (2947S) and anti-p27 (2552S) (1:1000; Cell Signaling, Ozyme, Saint Quentin-Yvelines, France); anti-catalase (sc-50508) and anti-HSC70 (sc-7298) (1:2000; Santa Cruz technologies, Santa Cruz, CA, USA); anti-MnSOD (ADI-SOD-110-F; 1:500; Enzo Life Sciences, Lyon, France) and anti-CuZnSOD (6F5; 1:2000; Novus Biologicals, Bio-techne, Lille, France); anti-α-sarcomeric actin (A2172; 1:5000; Sigma Aldrich) . Blots were washed with TBST and incubated for one hour at RT with infrared dye-conjugated secondary antibodies (LI-COR, Lincoln, NE, USA). After washing, blots were scanned using the Odyssey Imaging System (LI-COR, Lincoln, NE, USA).

Figure 2C ,FIGURE 1 :

 2C1 Figure 2C, both C26-PBS and C26-Antiox mice exhibited lower muscles weight compared to control mice (except for soleus). Remarkably, EDL and Gas muscles weight of C26-Antiox mice were significantly lower than those of C26-PBS mice(6.6 ± 1.3 and 79.4 ± 10.8 mg, respectively,

  µm, respectively vs. 41.2 ± 3.1 µm, P < 0.05, Figure 2B). A hanging wire grip test was also performed to determine how C26 tumor and antioxidants affect animals' muscle strength. Interestingly, the impulse score decreased dramatically in C26-PBS and C26-Antiox mice (6.32 ± 1.81 and 3.85 ± 2.14 N.sec, respectively) compared to control (18.47 ± 7.11 N.sec; Figure 2D) at 22 days or when mice reached 20% of body weight loss. No difference in impulse score was observed between the three groups at 10 th days post-injection. Contrary to our speculations, antioxidant complements were unsuccessful to slow muscle wasting.

FIGURE 2 :

 2 FIGURE 2: C26 inoculation induced muscle wasting in both C26-PBS and C26-Antiox groups. A) EDL sections stained with the Gomori method, showing structure of individualized muscle fibers; view ×400. B) Fibers diameter measurements. C) Skeletal muscle weights. D) Muscle strength. Three trials per animal were performed for each evaluation and the best time of latency was retained to calculate the holding impulse. Data are mean ± SEM (n=8-10/group) a: statistical difference between control and C26-PBS; b: statistical difference between control and C26-Antiox; c: statistical difference between C26-PBS and C26-Antiox.

FIGURE 3 :

 3 FIGURE 3: Antioxidants suppressed Akt activation in skeletal muscle without impacting the ubiquitin-proteasome system induction. A) Plasma levels of TNF-α. B) Plasma concentrations of Mstn. C) Immunoblot analysis of NF-κB (p65) phosphorylation on Ser 536 and total NF-κB (p65) content. D) Immunoblot analysis of the phosphorylated form of Akt on ser 473 and total Akt. E) RT-qPCR analysis of the transcript levels of MAFbx and MuRF-1, normalized to the reference gene 18S. F) Immunoblot analysis of protein polyubiquitination content. (C-F) All these experiments were performed on gastrocnemius muscles. Data are mean ± SEM (n=8-10/group) a: statistical difference between control and C26-PBS; b: statistical difference between control and C26-Antiox; c: statistical difference between C26-PBS and C26-Antiox

  Figure 4C) compared to control. These data indicate that antioxidant supplementation could up-regulate, in part, oxidative damage in skeletal muscle ongoing atrophy.

FIGURE 4 :

 4 FIGURE 4: Antioxidant supplementation induced muscle oxidative stress. Immunoblot analysis of A) protein carbonyls content, B) 4-HNE content and C) the expression of antioxidant defense enzymes (MnSOD, CuZnSOD and Catalase). Expression of all proteins was normalized to actin, which was used as a load control. (A-C) Data are mean ± SEM (n=8-10/group) a: statistical difference between control and C26-PBS; b: statistical difference between control and C26-Antiox; c: statistical difference between C26-PBS and C26-Antiox.

FIGURE 5 :

 5 FIGURE 5: Supplemented mice exhibited a reduced systemic antioxidant capacities. A) Plasma total antioxidant capacities measured using the ORAC test. Western blotting analysis of B) protein carbonyls and C) 4-HNE content in plasma. Data are mean ± SEM (n=8-10/group) a: statistical difference between control and C26-PBS; b: statistical difference between control and C26-Antiox; c: statistical difference between C26-PBS and C26-Antiox.

FIGURE 6 :

 6 FIGURE 6: Antioxidants accelerated tumor growth in C26-Antiox mice. A) Tumor weights. B) Ki-67 staining on tumor sections; view ×200. C) Percentage of Ki-67 positive nuclei. D) Immunoblot analysis of p21 and p27 expression. Immunoblot analysis of E) protein carbonyls and F) 4-HNE content. All these experiments were performed on C26-tumors. Hsc-70 was used as a load control. Data are mean ± SEM (n=8-10/group) a: statistical difference between control and C26-PBS; b: statistical difference between control and C26-Antiox; c: statistical difference between C26-PBS and C26-Antiox.

  Preclinical studies are crucial to evaluate the safety and biological feasibility of a given mode of exercise (time/frequency/type) in a specific type of cancer. As clinical analysis are usually limited to the systemic level, animal studies will allow us to in-depth investigate the impact of PA on tumor and broaden our understanding about the intra-tumor molecular mechanisms modulated by PA. Such approach is complementary to clinical studies and facilitates the prediction of the impact of a given PA program on cancer patients clinically. Data from preclinical studies in combination with epidemiological observations, will allow the design of more oriented clinical trials with well-defined objectives. Recently, Ashcraft et al. identified 53 preclinical studies that

5 .

 5 PA and antioxidants in cancer: contribution of the present thesis and future perspectives PA and antioxidant supplementation are two adjuvant strategies used in cancer care;

  Ph.D. thesis indicates that the impact of PA and antioxidant supplementation on cancer should not be underestimated. PA and antioxidant supplements, were deleterious in two distinct animal models of cancer. Two main mechanisms were identified: (1) A possible role for insulin in the activation of the tumor suppressor pathway P38 MAPK-P21 within intramuscular LS and (2) the selective modulation of oxidative damage profiles by antioxidants where they increased in skeletal muscle and decreased in colon tumor, in a way that promotes cancer cachexia development. Clinically, these findings suggest that (1) LS patients may, at least, not increase their PA or undergo hospitalsupervised exercise during the preoperative period, until the consolidation of our results with clinical data and (2) cancer patients, especially, those with late stages of the disease must be very careful against the use of commercial antioxidant supplements, as it could be lifethreatening. In spite of the deleterious effects observed in our models, we must recall that PA and antioxidants are still necessary to improve the quality of life of cancer patients and allow hard treatment completion; at present, the safest way to reach this goal seems to be by adapting and individualizing the modality of PA and type of antioxidant according to the clinical case of each patient. One of the most important challenge in the redox biology field resides in the measurement and identification of reactive species, as such approach will significantly improve our understanding on the pathophysiology of oxidative stress in cancer. Finally, in this thesis we addressed the impact of tumor on skeletal muscle (tumor-mediated muscle wasting) and vice-versa (muscle contraction on tumor growth); we must emphasize

  mitose / mm2, P = 0,04; Figure1d) et coïncidait avec une augmentation du taux de prolifération, évaluée par la coloration02 ± 4,1 vs. 68,49 ± 3,3%, P = 0,018; Figure1e et f). La nécrose est un facteur qui pourrait favoriser la croissance tumorale dans un certain nombre de cancers48 . Cependant, le pourcentage de zones nécrotiques dans la tumeur a tendance à augmenter dans le groupe actif, mais n'a atteint aucune différence statistique (Figure1get h). En outre, les deux groupes ont développé un faible niveau de métastase pulmonaire (Figure 1j et k). Les nodules tumoraux dans les poumons étaient principalement localisés autour des vaisseaux sanguins (flèche, Figure 1i). Conformément à la croissance accrue de la tumeur chez les souris actives, ces animaux semblaient développer un stade avancé de la maladie par rapport à leurs homologues inactifs, car seules les souris LSA présentaient une perte significative de la masse de tissu adipeux viscéral, corrélée négativement avec le poids de la tumeur (données non présentées). Ces résultats indiquent que les souris qui conservent une course volontaire présentent de Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 plus grandes tumeurs intramusculaires et manifestent des événements de cachexie précoce, sans modification des métastases pulmonaires.

Figure 1 .

 1 Figure 1. La croissance de la tumeur intramusculaire est modulée par l'AP. (A) Les images montrent une différence dans la taille de la tumeur intramusculaire entre les souris actives et inactives. (B) Poids moyen de la tumeur à huit semaines PI. (C) Sections tumorales colorées avec H & E (voir 200 ×) montrant une architecture de cellules tumorales indifférenciées et des cellules en cours de mitose. (D) Décompte mitotique obtenu à partir des sections tumorales présentées en (C). Dix champs distincts ont été comptés par section de tumeur à l'aide d'un microscope à deux têtes et les résultats ont été exprimés en (nombre mitotique / mm2). (E) Sections de tumeurs colorées avec DAPI (bleu) et Ki-67 (marron) (voir 200 ×). Une section tumorale par souris a été réalisée dans la partie la plus large du tissu. (F) Pour chaque section, le nombre de cellules positives Ki-67 a été compté dans cinq champs distincts et normalisé au nombre total de noyaux, afin de déterminer le pourcentage de cellules en cours de prolifération. (G) Sections de tumeurs colorées avec H & E (voir 50 ×). Les zones nécrotiques étaient délimitées par une ligne rouge. (H) Pourcentage de zones nécrotiques tumorales par zone tumorale totale. (I) Sections pulmonaires colorées avec H & E (voir 50 ×). Les nodules de tumeur métastatique ont été principalement retrouvés autour des vaisseaux sanguins (flèche noire) et indiqués par la lettre "T". (J) Le nombre total de nodules tumoraux métastatiques. (K) Le pourcentage de zone métastatique par zone pulmonaire totale. Les résultats sont exprimés en moyenne ± SEM (LSA: n = 9; LSI: n = 8); A: LSA vs LSI. La différence statistique entre LSA et LSI a été testée avec un test t étudiant. Les résultats ont été considérés comme significatifs à P <0,05.

3. 2 .

 2 Les tumeurs de souris actives présentaient une faible phosphorylation de p38 MAPK et une baisse d'expression de p21 Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Sur la base de nos résultats, il semble que la prolifération soit le principal mécanisme par lequel PA a modulé la croissance des tumeurs LS. Nous avons analysé l'expression de l'ARNm de nombreux gènes bien connus pour être liés au cycle cellulaire. Seule l'expression de l'inhibiteur du cycle cellulaire p21, mais pas celle de p27, p16 INK4 et p14 ARF , était 2 fois plus élevée dans les tumeurs de souris LSI par rapport à la LSA (1 ± 0,35 vs. 2,04 ± 0,5, P = 0,02; Figure 2a). En conséquence, le contenu protéique de p21, mais pas celui de p27, p53 et Phospho-p53 ser15 , était significativement élevée dans les tumeurs LSI (+ 46,77%, P = 0,015; Figure 2b et c). Les protéines MAPK activées régulent l'expression des gènes du cycle cellulaire et ont été décrites précédemment pour jouer un rôle important dans la progression de la tumeur et la suppression 44 . Ainsi, nous avons évalué l'état de phosphorylation (activation) des trois MAPK principaux, ERK1 / 2, JNK et p38. La phosphorylation de ERK1 / 2 Thr202 / Tyr204 et JNK Thr183 / Tyr185 était fluctuante inter / intragroupe et ne différait pas significativement entre LSA et LSI (Figure 2d et e). Fait intéressant, les niveaux de Phospho-p38 Thr180 / Tyr182 étaient nettement plus élevés dans les tumeurs de souris LSI par rapport à LSA (+ 86,05%, P = 0,02; Figure 2d et e). Ensemble, ces données indiquent que p38 MAPK a été activé dans les tumeurs des souris inactives et a coïncidé avec une plus grande expression de p21 à la fois au niveau transcriptionel et traductionnel.Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016

Figure 2 .

 2 Figure 2. L'AP affecte les voies de signalisation régulant la prolifération. (A) Expression relative des gènes du cycle cellulaire dans les tumeurs de souris LSA et LSI, déterminées par RTqPCR. (B) Analyse d'immunoblot pour p21, p27, p53 et Phospsho-p53 ser15 . (C) Analyse de la densitométrie pour les transferts présentés en (B). (D) Analyse d'immunoblot pour Phospho-p38 Thr180 / Tyr182 , Phospho-ERK1 / 2 Thr202 / Tyr204 et Phospho-JNK Thr183 / Tyr185 et leurs formes de protéine totale respectives. (E) Analyse de la densitométrie montrant le rapport "Phosphorylé: forme totale" pour p38, ERK1 / 2 et JNK. (F) Analyse d'immunoblot pour la teneur en protéines caspase-3, Bax et Bcl-2 clivées. (G) L'analyse de la densitométrie correspondant aux transferts montrés en (F). Toutes ces expériences ont été réalisées sur des extraits de tumeurs. Hsc70 a été utilisé comme contrôle de charge. Les résultats sont exprimés en moyenne ± SEM (LSA: n = 9; LSI: n = 8); A: LSA vs LSI. La différence statistique entre LSA et LSI a été testée avec un test t étudiant. Les résultats ont été considérés comme significatifs à P <0,05.

Figure 3 .

 3 Figure 3. Modulation des taux d'insuline circulants chez les souris portant une tumeur en fonction de leurs niveau de PA. (A) Expression relative de gènes antioxydants dans la tumeur, mesurée par RTqPCR. (B) Immunoblot montrant la teneur en protéines de 4-HNE, un marqueur de peroxydation lipidique. (C) Analyse de la densitométrie pour le 4-HNE. (D) Analyse d'immunoblot pour le marqueur de dégâts à l'ADN, γH2AX, dans des extraits de tumeurs. (E) Quantification de la densitométrie correspondant à γH2AX. Les niveaux de transcription des gènes inflammatoires dans (F) la tumeur et (G) l'environnement péri-tumoral évalué par RTqPCR. (H) Quantité de protéines TNF-a (pg / mL) dans le tissu péritumoral, mesurées à l'aide d'un kit ELISA spécifique. Niveaux circulants de (I) IL-6 (pg / mL) et (J) d'insuline (ng / mL) dans des échantillons de plasma de souris en état de non-jeûne, déterminé par des kits ELISA spécifiques. Hsc70 a été utilisé comme contrôle de charge. Les résultats sont exprimés en moyenne ± SEM (LSA: n = 9; LSI: n =

Figure 4 . 4 .Etude 2 : 1 . 2 .

 44212 Figure 4. Régulation de l'expression de p21 par p38 MAPK en réponse au traitement par l'insuline in vitro. (A) Modifications de la phosphorylation p38 Thr180 / Tyr182 et de l'expression de p21 avec le temps dans les cellules SW872 exposées à l'insuline. (B) Analyse de la densitométrie correspondant aux blots présentés en (A) (n = 2). (C) Analyse de western blot pour la phosphorylation de p38 Thr180 / Tyr182 et l'expression de p21 à 10 min et 4 h de traitement à l'insuline. Quantification de la densitométrie montrant (D) le rapport de "Phosphorylation: forme totale" de p38 (n = 4) et (E) l'expression de p21 (n = 4). (F) Analyse RTqPCR pour l'expression de l'ARNm p21 à 1, 2 et 4 h de traitement avec l'insuline, en présence ou en l'absence de SB203580 (n = 3) (G) Analyse de western blot pour la phosphorylation p38 Thr180 / Tyr182 et l'expression de p21 à 4 h de traitement d'insuline, en présence ou non de SB203580. (H) Quantification de la densitométrie pour l'expression de p21 (n = 3). Hsc70 a été utilisé comme contrôle de charge. (A-B) Les expériences ont été effectuées deux fois à partir de deux cultures cellulaires indépendantes. (C-H) Toutes les expériences ont été effectuées au moins trois fois à partir de trois cultures cellulaires indépendantes. Les résultats sont exprimés en moyenne ± SEM; E: Mock vs insulin, f: Insuline sans SB contre insuline-traité avec SB. (C-E) La

Figure 1 .Figure 2 .

 12 Figure 1. Les antioxydants ont provoqué la mort prématurée de souris C26. A) Changements de poids corporel au cours de la cachexie. B) La médiane de la survie animale déterminée par la méthode de Kaplan-Meier, les résultats sont exprimés en pourcentage C) Mesures de l'apport alimentaire pendant le protocole. D) Classification de la gravité de la cachexie en «légère, modérée ou sévère» en fonction de la perte totale de poids corporel et de l'atrophie musculaire. Les données sont moyennes ± SEM (n = 8-10 /

Assi,

  Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016chez les souris C26-Antiox, car ces animaux présentaient une survie médiane de 18 jours, alors que cette médiane n'était pas atteinte chez les souris C26-PBS dans les 22 jours où toutes les souris C26-Antiox étaient mortes (voir Figure1B). Ces résultats suggèrent que la supplémentation en antioxydant a accélérée la croissance de la tumeur C26 dans notre modèle expérimental. Conformément à cette hypothèse, nous nous sommes concentrés sur la prolifération tumorale et la régulation du cycle cellulaire. Nous avons constaté que l'expression de p21, mais pas de p27, deux inhibiteurs du cycle cellulaire, était diminuée dans la tumeur des souris C26-Antiox par rapport aux souris C26-PBS (-45%, P <0,05, Figure6D). Pour déterminer comment les antioxydants modulent l'état redox de la tumeur, nous avons quantifié 4-HNE et les protéines carbonylées. Aucune différence significative dans la teneur en protéines carbonylées n'a été notée entre les souris C26-PBS et C26-Antiox (Figure4E). Nous avons constaté que la teneur en 4-HNE était nettement plus faible dans les tumeurs d'animaux supplémentés par rapport aux souris C26-PBS (-66%, P <0,001; Figure4F). En fin de compte, l'inflammation locale dans la tumeur n'a pas été affectée en réponse à la supplémentation en antioxydant, comme en témoigne l'expression inchangée du TNF-α et de l'IL-6 au niveau de l'ARNm (Données supplémentaires). Toutes ces données soutiendraient finalement le paradigme, que les antioxydants accélèrent la prolifération tumorale et modulent les profils du SO dans la tumeur. Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016

Figure 4 . 4 .

 44 Figure 4. Les antioxydants ont accéléré la croissance tumorale chez les souris C26-Antiox. A) Poids des tumeurs. B) coloration Ki-67 sur les sections tumorales; Voir × 200. C) Pourcentage de noyaux Ki-67 positifs. D) Analyse immunologique des expressions p21 et p27. Analyse par immunotransfert de E) protéines carbonyl et F) 4-HNE contenu. Toutes ces expériences ont été réalisées sur des tumeurs C26. Hsc-70 a été utilisé comme contrôle de charge. Les données sont moyennes ± SEM (n = 8-10 / groupe) a: différence statistique entre le témoin et C26-PBS; B: différence statistique entre contrôle et C26-Antiox; C: différence statistique entre C26-PBS et C26-Antiox.4. Conclusion et perspectives cliniques

TABLE 4 :

 4 FIGURE 24: The vicious circle between physical inactivity, tumor development and muscle wasting ........................................................................................................................................... 73 FIGURE 25: Hypothetical scheme representing the potential beneficial and harmful effects of ROS in a dose-dependent manner within skeletal muscle .............................................................. 75 FIGURE 26: Exercise-induced antioxidant, metabolic, hormonal and angiogenic adaptations as well as myokines production .......................................................................................................... 78 FIGURE 27: Main factors modulating by physical activity, leading to reduced cancer progression and muscle wasting .................................................................................................... 85 FIGURE 28: PubMed search with "Cancer" & "Antioxidants" showing the production of scientific papers in the field over more than two decades ............................................................. 94 FIGURE 29: The ambivalence and dual role of ROS between tumor and skeletal muscle as well as the poor selectivity of antioxidant vitamins for tumor tissues, complicates their use in cancer cachexia .

XVI | P a g e ......................................................................................................................................... 97 FIGURE 30: Polyphenols as promising compounds in the treatment of tumor growth and muscle wasting ......................................................................................................................................... 100 FIGURE 31: Redistribution of selenium and zinc between tumor and skeletal muscle, promotes the progression of cancer cachexia .............................................................................................. 102 FIGURE 32: Simplified model showing (•) the plasmatic AO values corresponding to each patient and (■) the suitable measure that could be applied to avoid undesirable side-effects .... 105 TABLE 1: List of main oxygen, nitrogen and chlorine derivatives in the biological system ......... 10 TABLE 2: Main characteristics of chosen antioxidant enzymes .................................................... 11 TABLE 3: Studies showing the double-faced role of ROS in sarcoma-bearing mice .................... 47 Clinical studies emphasizing the impact of sarcoma on physical function and the potential effects of PA in sarcoma patients .................................................................................... 88 TABLE 5: List of main clinical studies with antioxidants alone or in combination with pharmacological agents on cancer patients with muscle atrophy (cachectic patients). .............. 107 Box 1: Role of creatine kinase in skeletal muscle .......................................................................... 56 Introduction 3 | P a g e Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad

1. Oxidative stress and cancer: an introduction

  Altered redox balance is an important hallmark of cancer. In 2009, Luo et al. added on the list of common cancer characteristics a new set of hallmarks that they called "Stress phenotype of cancer". Among these factors we cite, DNA damage stress, mitotic stress, metabolic stress and OS,

2016 Chapter III. ROS, Cancer and Related Muscle Wasting

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Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -

  exercise could mediate its anticancer effects through modulating oxidative profiles within tumor itself. Rats bearing AT-1 prostate tumor exhibited a reduction in tumor growth after undergoing a 4-week endurance exercise training on treadmill. This was mainly due to the decrease

	in lipid peroxidation (i.e. 4-HNE, TBARS) and oxidative DNA damage (i.e. 8-oxoG) within tumor
	tissues

Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 that

  , do not necessarily contradict the findings of Betof et al. and Faustino-Rocha et al., since a decrease in tumor angiogenesis is expected after the repair of tumor vasculature and breaking the vicious circle of HIF-1α activation. Feasibility of endurance exercise intervention in breast cancer patients undergoing neoadjuvant chemotherapy was demonstrated but clinical molecular pathways should be in-depth investigated in future studies

TABLE 4 :

 4 Clinical studies emphasizing the impact of sarcoma on physical function and the potential effects of PA in sarcoma patients

Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016

Chapter IV.

Physical activity, Cancer and Related Muscle Wasting 90

| P a g e Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad

Assi, 2016

  Physical activity reduces cancer progression, recurrence and related mortality, with convincing evidence in breast, colon and prostate cancer. These beneficial effects are observed when moderate-intensity of PA is practiced briskly and regularly. The underlying molecular mechanisms modulated by PA are not fully understood, however, accumulating evidence suggest that the antioxidant response of exercise may counteract the development of cancer by either reducing or increasing ROS levels within tumor. Also, in hormone-dependent cancers like breast, endometrial, ovarian and prostate, PA may induce a decrease in hormones levels and the subsequent pro-oncogenic signaling. Metabolic alterations observed in cancer patients such as hyperinsulineamia, dyslipidemia and obesity could be all improved by the practice of PA. Other pieces of evidence point the importance of PA in repairing tumor blood vessels/reducing hypoxia, potentiating the effects of chemotherapy and enhancing the anti-tumor immune response. All these events contribute to the regression of tumor growth and the magnitude of related muscle wasting; thus, resulting in a better QOL, muscle fitness and allowing

Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 ►Key Messages from Chapter IVthe completion of anticancer treatment. Nonetheless, the impact of PA on intramuscular tumors has not retained much attention and deserves more studies, especially that the particular location of such tumors may produce different and unexpected outcomes in response to PA. Unfortunately, cachectic patients with advanced stages of cancer, presenting anemia and respiratory difficulties,

Antioxidants, Cancer and Related Muscle wasting 97

Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Chapter V. | P a g e Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016

TABLE 5 :

 5 List of main clinical studies with antioxidants alone or in combination with pharmacological agents on cancer

	Data are presented in the table as mean values that reached statistical significant difference (P<0.05). No statistical different values	are replaced with NSD. a: α-lipoic acid (ALA: 600 mg/ day) and Carbocysteine (CS: 2.7 g/day); b: N-acetylcysteine (NAC: 1800	mg/day), ALA (200 mg/day), CS (2.7 g/day), Vit A (30000 IU/day), Vit C (500 mg/day) and Vit E (70 mg/day); c: Polyphenols	(PLP: 300-400 mg/day), ALA (300 mg/day), CS (2.7 g/day), Vit A (30000 IU/day), Vit C (500 mg/day) and Vit E (400 mg/day). *:	Cachectic patients with high ROS levels and low GPx activity exhibited poor performance status. H&N: Head and Neck cancer.

Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 patients with muscle atrophy (cachectic patients). From (Assi and Rebillard, 2016) Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016

Table 1 .

 1 All treatments were administrated by oral gavage. The composition of the antioxidant cocktail is presented in Twenty two days after inoculation of C26 cells or when mice have lost more than 20% of body weight, animals were anesthetized with a ketaminexylazine-butorphanol cocktail. Soleus, Extensor digitorum longus (EDL), rectus femoris, gastrocnemius (Gas) muscles and tumor were weighed and immediately frozen in liquid nitrogen

	laboratory "Movement, Sport and health Sciences" accredited for live animal experimentation
	(N°A35-047-34, delivered by the Department of Veterinary Services, French Ministry of
	Agriculture). Mice were maintained on a 12:12-h dark-light cycle. Water was available ad libitum
	and standard laboratory chow was provided, changed and monitored daily. The animals were
	divided into three experimental groups: control group that received a daily supplement of 250 µl
	of PBS (n=10); C26 tumor-bearing group that received 250 μl of PBS (n=10); and C26 tumor-

Brittany (N°R-2012-AR-01) and conducted in accordance with the current ethical standards of the European Community (Directive 2010/63/EU). Seven-week-old male Balb/c mice were obtained from Janvier Labs (Le Genest Saint Isle, France) and housed in the Animal Care Facility of the bearing group that was daily supplemented with an antioxidant mixture rich in catechins, curcumin, quercetin and vitamin C (81.5 mg/kg of body weight) dissolved in 350-500 μl of PBS (n=10). The volume of the daily supplement received by each mouse in this group was daily adjusted with body Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 weight change.

TABLE 1 : Composition of antioxidant mixture.

 1

Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016

TABLE 2 : List of primers used for RTqPCR analysis.

 2 Total proteins were extracted from Gas muscles and tumor tissues in a lysis buffer containing 50 mM Tris-HCl, 10 mM EDTA, 1% SDS, 1% NP-40, 0.25% sodium deoxycholate, 50 mM NaF, 100

Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 2.7. Western blot analysis

  [START_REF] Gershbein | Influence of stress of lesion growth and on survival of animals bearing parenteral and intracerebral leukemia L1210 and Walker tumors[END_REF] observed that rats bearing intramuscular walker 256 carcinoma exhibited reduced tumor size after forced swimming for 15 min daily, during 10

Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 tumor growth kinetics.

Main mechanisms modulated by antioxidants in cancer and related cachexia Systemic and intra-tumor oxidative damage profiles:

  Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 undergo supplementation, since those with systemic antioxidant deficiency appear to benefit the most from supplementation without adverse side-effects. Reducing oxidative damage and/or inducing antioxidant defense, are the two main mechanisms through which antioxidant compounds accelerate tumor growth in advanced stages of cancer. A body of evidence indicates that vitamin A,

4.

C and/or E could increase tumor growth by decreasing oxidative DNA damage and/or lipid peroxidation

antioxidants could induce selective redox change in an organ-dependent fashion, during

  Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016structure, in muscle cells(Tan et al., 2015). In this thesis, we demonstrated for the first time that cancer cachexia. As mentioned above, the capacity of antioxidants to promote tumor proliferation through attenuating oxidative damage has been validated in several models of animals with malignancy. Herein, we add to such mechanism the ability of antioxidants to enhance oxidative damage in muscle, which could negatively affect proteins involved in muscle anabolism like Akt.

	This

discriminatory modification of oxidative damage between tumor and skeletal muscle was probably the main cause behind faster tumor growth and skeletal muscle loss and

  , therefore, earlier cachexia development and premature death. Such data, support our idea that antioxidants are most likely harmful in advanced stages of cancer. Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-MohamadAssi, 2016

	Discussion and Perspectives
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  Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016

used an antioxidant mixture commercially available at the French market, for

  To better represent the clinical context, we have whom composition is similar to that used by cancer patients.

We found that antioxidants enhanced colon cancer growth, promoted weight and skeletal muscle loss and caused premature death of cachectic mice.

  Numerous high-quality preclinical studies demonstrated detrimental effects of

Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016

  Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad

	Discussion and Perspectives
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  réactives d'oxygène ; Activité physique ; Antioxydants ; Croissance tumorale ; Atrophie Le fardeau des maladies non transmissibles, y compris: le dysfonctionnement cardiovasculaire, le diabète de type 2 et certains types de cancer augmente dans les pays développés et représentent plus de 60% des 56 millions de décès qui surviennent chaque année(OMS, 2015). Le cancer est la première cause de mortalité en France et le deuxième après, les maladies cardiaques, dans le monde entier(INCa, 2015). Les soins contre le cancer sont un problème majeur au plan socioéconomique et de santé. Chaque année, le gouvernement français verse plus de 5 milliards d'euros sur le traitement du cancer et les soins cliniques. En 2014, 155 millions d'euros ont été alloués à la recherche sur le cancer par des organisations institutionnelles et des fondations nationales de bienfaisance. Selon le récent rapport publié en 2015 par l'Institut national français pour le cancer (INCa, 2015), 385 000 nouveaux cas ont été diagnostiqués avec un cancer à un âge médian de 67 à 68 ans. Les types les plus fréquents sont les cancers pulmonaires, prostatiques et colorectaux chez les hommes. Le cancer du sein reste le plus récurrent chez les femmes, suivi du cancer colorectal et du poumon. En 2015, 149 500 personnes (56% d'hommes) ont succombé au cancer en France. Parmi ceux-ci, le cancer du poumon et du sein était la cause principale qui a mené à la mort chez les hommes et les femmes respectivement (INCa, 2015).

	Une augmentation de mortalité pourrait être dû à un certain nombre de complications de santé
	associées au cancer qui réduisent la tolérance aux thérapies anticancéreuses 1 . Les deux principaux
	facteurs limitants sont l'altération du statut nutritionnel et de l'activité des patients (OMS, 2004). En fait,
	les stades avancés du cancer sont souvent associés à une malnutrition et à une perte excessive de masse
	musculaire squelettique entraînant une déficience fonctionnelle et un risque élevé d'inactivité 1 . En effet,
	le «Plan de cancer 2014-2019» lancé par le gouvernement Français a souligné l'importance d'améliorer la
	qualité de vie des patients (QV) et la performance physique dans l'achèvement des traitements
	anticancéreux. Dans cette perspective, les thérapies adjuvantes comme l'activité physique (AP) et le
	soutien nutritionnel pourraient être utiles lorsqu'ils sont utilisés en combinaison avec des traitements
	conventionnels.
	Dans cette optique, le dialogue entre la tumeur et le muscle squelettique est un événement
	majeur survenu pendant le cancer 2 . Nous supposons que deux voies principales pourraient mener la
	diaphonie entre la tumeur et le muscle squelettique; Dans ce manuscrit, nous avons choisi de leurs

musculaire Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Introduction nommer le dialogue directe ou indirecte. Une interaction tumeur-muscle indirecte se produit par la libération mutuelle de facteurs tumoraux ou spécifiques du muscle dans la circulation systémique. Dans un tel état, la tumeur pourrait sécréter des médiateurs qui induisent une atrophie musculaire 3 ou Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016

activité physique et la croissance du liposarcome: comment la contraction musculaire régulière peut avoir un impact direct sur la tumeur intramusculaire? 1. Contexte de l'étude L

  . D'une manière intrigante, bien que le muscle squelettique soit le premier organe sollicité lors de l'exercice physique, l'impact de la PA sur les tumeurs qui se développent à l'intérieur du muscle lui-même et, par conséquent, affecte directement sa fonction n'a pas reçu beaucoup d'attention. En conséquence, les patients atteints de cancers des membres inférieurs comme le liposarcome ne bénéficient pas de conseils concernant le niveau d'AP qui pourrait être pratiqué pendant la période de traitement. Nous pourrions souligner que les ERO sont également produites au sein de la tumeur et contrôlent sa réponse biologique15,16 . Des antioxydants ont été proposés pour contrer ces mécanismes ERO-dépendants et réduire la croissance tumorale ainsi que l'ampleur du Dans cette thèse de doctorat, nous avons testé l'impact de l'AP et des antioxydants dans deux modèles distincts de cancer, conçus pour être le plus proche possible du contexte clinique de nos jours. Dans la première étude, nous avons abordé l'impact de l'AP régulière sur le développement du liposarcome intramusculaire et, dans le deuxième modèle, nous avons évalué l'efficacité de la supplémentation en antioxydants chez les souris cachectiques portant un cancer du côlon sous-cutané. Ces modèles nous ont permis de comprendre l'impact de deux stratégies d'adjuvant sur: la croissance tumorale, la masse / fonction du muscle squelettique et la diaphonie directe / indirecte entre la tumeur et le muscle squelettique.La tumeur induit principalement une atrophie musculaire via la libération de médiateurs inflammatoires, ce qui augmente la production de ROS dans le muscle et son catabolisme subséquent22 .Compte tenu de l'importance du muscle squelettique dans la production de mouvement et le maintien de la posture ainsi que de la stabilité des articulations, son dépérissement altère sérieusement la fonction physique et diminue ainsi la probabilité d'achèvement du traitement anticancéreux23 . Il a été suggéré que l'AP pourrait constituer une partie des soins primaires contre le cancer pour mieux contrôler l'évolution de la tumeur et le dépérissement musculaire / faiblesse / fatigue ainsi que permettre aux thérapies rigides d'aller de l'avant24 . Un ensemble de preuves indique que la PA est bénéfique pour réduire la progression du cancer, la récidive et la mortalité associée, notamment dans le cancer du sein, du colon et de la prostate AP régulière sur la croissance tumorale et le dysfonction du muscle squelettique dans un modèle orthotopique de souris portant des tumeurs intramusculaires.Dans des cas particuliers, la capacité des patients atteints de cachexie cancéreuse à se déplacer et à pratiquer l'exercice pourrait être limitée en raison de l'anémie et de la dysfonction cardiovasculaire30 ; 'individualiser le traitement selon les besoins du patient33 . Même si les médecins interdisent la supplémentation avec des antioxydants pendant le traitement34 , ce n'est pas un secret que les patients spécialement ceux atteints de cancer du côlon et du poumon prennent des antioxydants au hasard21 . 'activité physique (AP) est une stratégie adjuvante qui pourrait être prescrite pour les patients atteints de cancer avant, pendant et après les traitements anticancéreux conventionnels pour alléger les symptômes secondaires, préserver la force musculaire, permettre aux patients de supporter les traitements lourds et optimiser la récupération36 . Un ensemble de preuves indique que l'AP peut réduire environ 25% le risque de cancer du côlon et du sein, avec des preuves possibles dans le cancer de la prostate, des poumons et des ovaires

	Cette pratique peut nuire à la santé des patients 35 et pourrait être la motivation principale de
	l'augmentation exponentielle de la recherche sur ce sujet, ces quelques dernières années. L'utilisation
	d'antioxydants dans le cancer est un problème contemporain qui nécessite d'autres recherches. À cet
	égard, le deuxième objectif était de déterminer l'effet des antioxydants commercialisés sur la croissance
	tumorale et un dysfonctionnement musculaire grave dans un modèle de souris cachectiques portant une
	tumeur sous-cutanée.
	Sur la base de ce qui a été dit ici, nous avons décidé de tester l'efficacité de deux stratégies
	d'adjuvants dans deux modèles animaux distincts de cancer ayant une pertinence translationnelle
	significative.
	Ce travail nous a permis de:
	gaspillage musculaire. Malheureusement, les résultats obtenus à partir d'études précliniques et cliniques
	de cancer étaient controversés 17-20 . L'hétérogénéité de ces résultats dépendait en grande partie (1) du Par conséquent, des stratégies alternatives sont nécessaires. Des antioxydants ont été proposés pour
	type / localisation de la tumeur, (2) du type / dose d'antioxydants, (3) du statut antioxydant basal du contrer le rôle des ROS dans la tumorigénèse et le gaspillage musculaire et pour fournir aux patients
	patient et (4) d'autres facteurs susceptibles de fausser nos conclusions, telles que l'auto-prescription des déficients un soutien nutritionnel adéquat 31 . Cependant, les résultats de la littérature ne sont pas
	suppléments antioxydants, une pratique hautement récurrente chez les patients atteints de cancer du concluants, probablement parce que des études antérieures ont essayé de standardiser le même
	poumon et du colon 21 .

Dans certains cas, la capacité des patients atteints d'un stade avancé du cancer (patients cachectiques) à poursuivre un programme d'AP hospitalisé pourrait être limitée en raison de l'anémie, de la dysfonction cardiovasculaire et de l'état pro-catabolique avancé

12

. Par conséquent, une stratégie alternative à l'AP pourrait être utile pour ralentir la détérioration de la santé globale des patients. D'un point de vue moléculaire, une telle condition est habituellement associée à une inflammation chronique due à la libération prolongée de produits chimiques dérivés de tumeurs, qui dérègle l'équilibre redox dans le corps

13

. Cela favorise la formation excessive d'espèces réactifs d'oxygène (ERO) dans le muscle squelettique et sa dégradation subséquente

14

. Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Objectifs de la thèse 7 . Les principaux mécanismes par lesquels la PA peut ralentir la progression du cancer sont principalement: la modulation des profils de dégâts oxydatifs de la tumeur et la réduction de l'inflammation systémique, des niveaux d'insuline en circulation ainsi que l'hypoxie / angiogenèse tumorale 11,25-27 . Même certains scientifiques pensent que les muscles exercés peuvent libérer des myokines capables d'induire des effets anticancéreux 4,28 . Lorsque nous savons que le muscle est d'abord sollicité pendant l'exercice et que l'AP est susceptible d'être bénéfique pour le cancer, il est intriguant qu'aucune étude antérieure ait évalué l'impact de l'AP sur les tumeurs intramusculaires. Surtout que ces formes particulières de malignité sont très agressives et affectent directement l'appareil musculo-squelettique. Nous pouvons supposer que l'architecture, l'organisation et la contractilité cellulaires uniques, ainsi que le haut niveau de production d'énergie / ROS et les changements dans l'acidité du pH 29 forment un sanctuaire dans lequel la tumeur peut se comporter différemment en réponse à exercice. Sur la base de ces interrogations, le premier but de cette thèse était d'étudier l'impact de l'traitement à une population hétérogène de patients présentant un statut clinique différent et des besoins antioxydants 32 . À ce jour, il devient clair que la meilleure façon d'utiliser de tels produits est Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 d► Étudier l'interaction réciproque entre la tumeur et le muscle squelettique dans différents cancers et comprendre comment cela pourrait être modulé en réponse à l'AP ou aux antioxydants ► Identifier les mécanismes redox-indépendants et les mécanismes sensibles à l'AP ou à la supplémentation d'antioxydant Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 Etude 1 : L'

  situés dans des tissus mous profonds41 . Il est le plus souvent trouvé dans les cuisses des patients42 . Cette localisation particulière à l'intérieur du muscle squelettique constitue une masse gênante qui peut entraver la capacité du muscle à produire une force suffisante et à assurer des besoins physiques de base. En effet, les survivants du sarcome de l'extrémité inférieure présentent une détérioration de la qualité physique43 . Cependant, l'impact de l'AP sur les tumeurs intramusculaires et la dysfonction de signalisation impliquées dans la croissance / progression de la tumeur11 . Dans cette optique, les membres de la famille des mitogen activated protein kinase (MAPK) constituent une cible majeure. En effet, il a été suggéré que l'AP pourrait moduler la croissance tumorale par la régulation des voies MAPK 2 , qui à son tour contrôle l'expression et la stabilisation d'un grand nombre d'activateurs / inhibiteurs du cycle cellulaire44 . Néanmoins, comme chaque type de tumeur possède des caractéristiques spécifiques, les voies moléculaires mobilisées en réponse à l'AP peuvent également différer45,46 . Par exemple, en réponse à la même modalité d'AP, les tumeurs «dans le muscle » pourraient réagir différemment par rapport à celles situées «à l'extérieur du muscle». Dans le présent travail, nous étions intéressés à étudier l'utilité de l'AP dans le contexte d'une interaction directe entre le muscle squelettique et le squelette. Nous avons abordé pour la première fois l'impact de l'AP normale sur la croissance intramusculaire du LS d'un point de vue moléculaire. immédiatement utilisé pour effectuer des mesures d'hématocrite et d'hémoglobine, soit placé dans des tubes revêtus d'EDTA pour la collecte du plasma. Les poumons ont été examinés pour les nodules tumoraux métastatiques en effectuant des sections en série. Trois sections de 4 μm séparées par une distance de 40 μm ont été réalisées par poumon et colorées avec H & E. Le nombre de nodules tumoraux métastatiques dans les poumons a été compté en observant des lames sur un microscope optique à deux têtes. Les sections ont été ensuite analysées par le logiciel d'image NDP.

	2.2. Lignée cellulaire SW872 de liposarcome et injection intramusculaire
	Les cellules SW872 humaines (passage 27) ont été obtenues auprès de Cell Line Services (CLS,
	Allemagne). L'authenticité a été vérifiée par CLS en utilisant la méthode de profilage ADN STR. Les cellules
	ont été maintenues dans une atmosphère humidifiée à 37 ° C; 5% de CO2 et cultivé dans du DMEM
	contenant de L-glutamine 2 mM, 10% de sérum bovin foetal (FBS) inactivé par la chaleur, ainsi que la
	pénicilline 100 μg / ml et la streptomycine 100 U / ml. Le jour de l'injection, les cellules SW872 ont été
	culotés après centrifugation (500 g pendant 6 min à 25 ° C) puis reprises avec du DMEM. Les souris ont
	reçu une inoculation intramusculaire de cellules SW872 (2 × 10 6 cellules dans 100 μl, passage 32) ou du
	souris ont été réparties de manière homogène en quatre groupes (n = 9 / groupe) en fonction des niveaux
	d'AP, au cours des six dernières semaines. Ensuite, les animaux ont reçu une injection intramusculaire de
	musculaire apparentée est encore mal compris; Par conséquent, il n'y a aucune recommandation cellules SW872 de liposarcome ou de DMEM seul (véhicule). Les roues d'activité ont été bloquées le jour
	concernant les niveaux d'AP à conseiller les patients atteints de sarcomes des membres inférieurs avant de l'injection pour les souris des groupes contrôle inactif (CTLI) et liposarcome inactif (LSI), tandis que les 2.4. Extraction d'ARN et RT-qPCR
	ou pendant tout traitement (par exemple, chirurgie, chimiothérapie et radiothérapie). souris des groupes contrôle actif (CTLA) et liposarcome actif (LSA) ont maintenu une AP jusqu'à la fin du Les tissus ont été broyés dans de l'azote liquide et la poudre obtenue a été utilisée pour effectuer
	L'exercice-oncologie reste un domaine avec plusieurs zones inexplorées et on sait peu de choses sur les protocole (Figure 1a). Au bout de huit semaines post-injection (PI), tous les animaux ont été anesthésiés des expériences. L'ARN total a été extrait en utilisant le Trizol® (Sigma Aldrich). Les quantités d'ARN ont

. À ce jour, 64% des études précliniques d'exercice-oncologie montrent un effet positif sur l'incidence, la croissance ou la progression des tumeurs, quel que soit le type / la localisation de la tumeur et la modalité / intensité de l'AP

40

. Curieusement, la majorité de ces études ont été réalisées sur des animaux porteurs de tumeurs en sous-cutanée, afin de surveiller la cinétique de la croissance tumorale; Alors que seulement quelques rapports ont utilisé des modèles orthotopiques et aucun n'a accordé d'attention à l'impact de PA sur les tumeurs intramusculaires 40 .

Le liposarcome (LS) est le type le plus fréquent de sarcome de tissu mou (20%) qui provient d'adipocytes et de fibroblastes mécanismes moléculaires exacts par lesquels l'exercice pourrait induire ses effets protecteurs. Cependant, on pense que les changements médiés par l'exercice sur l'inflammation systémique, les hormones, l'angiogenèse, l'état redox, le système immunitaire et la sensibilité à l'insuline pourraient influer sur les Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 voies 2. Conception de l'étude et techniques utilisées 2.1. Ethique et expérimentation animale Toutes les procédures décrites ci-dessous ont été effectuées conformément aux recommandations de la communauté européenne (directive 2010/63 / UE) et ont été approuvées par le ministère Français de l'enseignement supérieur et de la recherche, conformément au comité local d'éthique de la recherche de Rennes (autorisation no. APAFS n ° 581-2015050411405743). Des souris mâles « nude » de quatre semaines ont été obtenues auprès de Harlan (Rossdorf, Allemagne) et logées dans l'animalerie du laboratoire "Mouvement, Sport et Santé" (contrat A35-047-34). Au cours des six premières semaines, toutes les souris (n = 36) ont effectué une AP volontaire sur des roues d'activité (Intellibio, France) placées individuellement dans leurs cages. Les roues ont été connectées à des compteurs qui ont enregistré la distance journalière effectuée par chaque souris. Avant l'inoculation, les avec un cocktail de Ketamine-Xylazine et ont été sacrifiés. Les tumeurs, les tissus péri-tumorales, les poumons et les muscles squelettiques ont été récupérés et immédiatement congelés dans de l'azote liquide ou fixés dans du paraformaldéhyde à 4% (PFA). Le sang a été obtenu par ponction intracardiaque, Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 soit véhicule, sur le côté intérieur du membre postérieur droit pour s'assurer que le volume a été injecté dans le muscle squelettique mais pas par voie sous-cutanée ou dans le fascia. Le poids corporel et l'apport alimentaire ont été surveillés chaque semaine au début du protocole et, puis, deux fois par semaine lorsque les tumeurs ont commencé à devenir visibles. 2.3. Analyse histologique Les tissus tumoraux et pulmonaires ont été fixés par du PFA pendant 24 h et ensuite incorporés dans la paraffine. Des sections de tissu de 4 μm d'épaisseur ont été obtenues en utilisant le microtome LEICA et montées sur des lames de verre. Les tumeurs ont été colorées avec: Hematoxyline et Eosine (H & E) pour l'analyse de l'histopathologie, CD31 pour évaluer la densité des vaisseaux sanguins et Ki-67 pour déterminer le taux de prolifération des cellules tumorales. été déterminées par spectrophotométrie en utilisant le NanoDrop et la qualité de l'ARN a été contrôlée sur un gel d'agarose 1,2% en utilisant le système d'électrophorèse FlashGel (Lonza). La réaction de transcription inverse (RT) a été effectuée sur 1 ug d'ARN total (transcription inverse IScript, 170-8840).

  Ensuite, les expériences de PCR en temps réel par Sybergreen ont été réalisées sur les produits RT dans un volume final de 10 μl contenant: 5 μl d'ADNc (dilué à 1/5), 0,2 μl d'amorces (10 μM) et 4,8 μl de SYBR® Green Supermix (1725271, Biorad). Les expériences ont été effectuées dans la machine CFX Real-Time (Biorad). L'expression des gènes cibles a été normalisée par rapport aux gènes de références et l'expression relative a été calculée en utilisant la méthode ΔΔCt sur le logiciel CFX-Manager.

Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016

  test ANOVA à deux voies a été utilisé pour comparer les quatre groupes. D'autres interactions ont été analysées avec un test Fisher LSD. Lorsque les données n'ont pas suivi une distribution normale, les valeurs logarithmiques (log) ont été calculées puis une ANOVA à deux voies a été effectuée sur les valeurs log. La différence significative entre deux groupes a été examinée par un test-t student pour les tissus tumoraux, pulmonaires et péri-tumoraux. Un test non paramétrique Mann-Whitney a été utilisé au lieu du test-t, lorsque les données n'ont pas réussi à passer le test de normalité. Les résultats ont été considérés comme étant statistiquement différents à P <0,05.

	3 cellules / puits dans une
	plaque de six puits, incubées pendant la nuit, et ensuite exposées à 100 nM d'insuline (I6634-50MG, Sigma
	Aldrich) à différents moments (5, 10 et 30 Min; 1, 2 et 4 h). Les niveaux phosphorylés de p38 ainsi que
	l'expression de p21 au niveau de l'ARNm et protéique ont été évalués par RTqPCR et immunoblot,
	respectivement. Pour les expériences d'inhibition de p38, les cellules SW872 ont été prétraitées pendant
	2 h avec 10 μM de SB203580 (5633, Cell Signaling), un inhibiteur spécifique de l'activité p38. Ensuite, les
	cellules ont été traitées avec de l'insuline pendant 1, 2 et 4 h pour effectuer l'analyse RTqPCR et pendant
	4 h pour effectuer les expériences d'immunoblot.
	2.7. Analyses statistiques

La survie médiane a été déterminée par la méthode de Kaplan-Meier et analysée par le test logrank. Les données ont été présentées comme moyenne ± écart moyen. La normalité et la variance ont été testées avant l'analyse statistique. Pour le poids corporel, l'apport alimentaire, l'impulsion et la vitesse maximale, un

  dans les groupes de souris C26, les mesures du poids corporel n'ont plus été prises en compte dans l'interprétation des données, ce qui explique pourquoi les mesures du poids corporel ont été arrêtées aux 13ème et 20ème jours pour les souris C26-Antiox et C26-PBS, respectivement. Ici, nous avons montré que la supplémentation d'antioxydant a causé 50% et 100% de décès pour les souris C26 à 18 jours et 22 jours après l'injection, respectivement. En comparaison, seulement 20% des souris C26-PBS sont mortes après 22 jours (Figure

Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 produire

  chez les animaux et, ensuite, chez les patients présentant des stades avancés de cancer qui développent une anémie, un dysfonctionnement cardiovasculaire et des troubles respiratoires 81 . Une telle stratégie pourrait être au moins une solution acceptable pour atténuer la fatigue et améliorer les capacités fonctionnelles

Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016 32 la maladie Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016

Assi, Mohamad. The Impact of Physical Activity and Antioxidants on Tumor-Skeletal Muscle Crosstalk during Cancer : Deciphering Signaling Pathways Involved in Tumor Growth and Muscle Wasting -2016

Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-MohamadAssi, 2016

Acknowledgments

III | P a g e Acknowledgments IV | P a g e ACKNOWLEDGMENTS The authors thank Pascale Bellaud (H2P2 platform, University Rennes 1, Rennes), Dany Saligaut, Morgane Chevallier, Brice Martin and Erwan Salaun for technical help. The authors' responsibilities were as follows-AR, FD: designed the research; AR: had primary responsibility for obtaining ethical approval; MA, FD, LLO, AR: conducted the research; MA, FD, AR: interpreted the data; MA, FD: performed statistical analysis; MA drafted the manuscript; FD, AR: contributed to subsequent versions; AR: had responsibility for the final; and all authors: read and approved the final manuscript. FUNDING: Mohamad Assi is a recipient of a PhD research fellowship (n°2012/22) from the Brittany Region Council. This study was supported by CRITT santé Bretagne.

Tumor-Skeletal Muscle Crosstalk in Cancer-Ph.D. thesis-Mohamad Assi, 2016 regarding the use of antioxidants, as the prescription of such compounds should be done under the credible advice of a nutritionist or cancer patient's physician. Assi, 2016Talotta, F., Cimmino, A., Matarazzo, M.R., Casalino, L., De Vita, G., D'Esposito, M., Di Lauro, R., Verde, P., 2009. An autoregulatory loop mediated by miR-21 and PDCD4 controls the AP-1 activity in RAS transformation. Oncogene 28,[73][74][75][76][77][78][79][80][81][82][83][84] Tan, P.L., Shavlakadze, T., Grounds, M.D., Arthur, P.G., 2015. Differential Tang, H., Chen, Y., Liu, X., Wang, S., Lv, Y., Wu, D., Wang, Q., Luo, M., Deng, H., 2016. Downregulation of HSP60 disrupts mitochondrial proteostasis to promote tumorigenesis and progression in clear cell renal cell carcinoma. Oncotarget. Tardif, N., Klaude, M., Lundell, L., Thorell, A., Rooyackers, O., 2013. Autophagic-lysosomal pathway is the main proteolytic system modified in the skeletal muscle of esophageal cancer patients. The American journal of clinical nutrition 98, 1485-1492. Temel, J.S., Abernethy, A.P., Currow, D.C., Friend, J., Duus, E.M., Yan, Y., Fearon, K.C., 2016. Anamorelin in patients with non-small-cell lung cancer and cachexia (ROMANA 1 andROMANA 2

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