Tom Juncker

Marc Schumacher

Mario Dicato

Dr Marc Diederich
email: marc.diederich@lbmcc.lu

UNBS1450 from Calotropis procera as a regulator of signaling pathways involved in proliferation and cell death

Keywords: Calotropis procera, cardenolide, Na + /K + ATPase, UNBS1450, nuclear factor B (NF-B), apoptosis, autophagy

Despite significant progress in oncology therapeutics in the last decades, the urge to discover and to develop new, alternative or synergistic anti-cancer agents still remains. For centuries it has been known that the coarse shrub Calotropis procera is a very promising source of ascaricidal, schizonticidal, anti-bacterial, anthelmintic, insecticidal, anti-inflammatory, antidiarrhoeal, larvicidal and cytotoxic chemicals. Different compounds like norditerpenic esters, organic carbonates, the cysteine protease procerain, alkaloids, flavonoids, sterols as well as numerous types of cardenolides have provided this plant for centuries with scientists' interest.

The chemical class of cardenolides and their related bioactivity evaluation and structure activity relationship (SAR) studies pointed out their therapeutic utility and led to the discovery of promising drug candidates. Recently the cardiotonic steroid UNBS1450 01 (derived from 2-oxovoruscharin 02) from Calotropis procera, was shown to additionally exert an anti-cancer activity. UNBS1450 01 has been proven to be a potent sodium pump inhibitor, showing anti-proliferative and cell death-inducing activities. This anti-cancer potential of UNBS1450 01 is achieved by disorganisation of the actin cytoskeleton after binding to the sodium pump at the cellular membrane, by inducing autophagy-related cell death, by repressing NF-B activation as well as by down-regulating c-Myc in cancer cells. We aim to review pharmacologically important chemical extracts from Calotropis procera and focus more specifically on the anti-cancer activities of UNBS1450 01.

A c c e p t e d M a n u s c r i p t -1 -

1.) Calotropis procera as a source of natural drugs

1.1.) Calotropis procera

According to literature, the origin of synthetic drugs development lies within the upcoming of The Industrial Revolution. The potentially more active synthetic products and the increasing economic power of pharmaceutical companies were the main reasons for a preference for these new products compared to the natural compounds from mineral, plant and animal sources used for ages.

However, a significant number of these synthetic drugs are obtained from natural precursors ; it is even estimated that 11 % of the drugs considered as basic and essential by the World Health Organisation (WHO) are strictly of plant origin. Digoxin, quinine, quinidine, vincristine, vinblastine, atropine, morphine and codeine might be the most prominent.

Furthermore, according to Yue-Zhong Shu, 60 % of current or future anti-tumor and antiinfectious drugs are of natural origin [START_REF] Shu | Recent natural products based drug development: a pharmaceutical industry perspective[END_REF].

Calotropis procera, a member of the Asclepiadaceae, is a woody, broadleaf evergreen coarse shrub, 3 to 5 m tall, widely growing in the tropics. It is distributed in arid to semiarid regions of the Caribbean, Central America, South America, Africa, India and Israel, mainly appearing on plains and in the uplands. For decades, C. procera, also referred to as "The Apple of Sodom" has been used especially in traditional folk medicine because of its pharmacologically active compounds found in its roots, bark, leaves and mainly in its latex which exudates from damaged leaves. Meanwhile, chemical extracts from C. procera have been shown to have ascaricidal, schizonticidal, anti-bacterial, anthelmintic, insecticidal, antiinflammatory, anti-diarrhoeal, larvicidal, cytotoxic and analgesic effects, thus explaining a growing demand in today's medical research for the different parts of the plant [START_REF] Sharma | Evaluation of in vitro schizontocidal activity of plant parts of Calotropis procera--an ethnobotanical approach[END_REF][START_REF] Dewan | Preliminary studies on the analgesic activity of latex of Calotropris procera[END_REF][START_REF] Kumar | Anti-diarrhoeal activity of the latex of Calotropis procera[END_REF][START_REF] Kumar | In vivo and in vitro effect of latex of Calotropis procera on gastrointestinal smooth muscles[END_REF].

1.2.) The latex of Calotropis procera

More than 12000 plant species contain latex, a milky fluid in which occurs a wide range of proteins, and for some 1000 species also cis -polyisoprene that is the polymeric hydrocarbon of rubber [START_REF] Yeang | Allergenic proteins of natural rubber latex[END_REF]. As the latex is abundant in the green parts of the plant, it is thought to be produced and accumulated as a defense strategy against viruses, fungi and insects. Indeed, besides proteins involved in rubber biosynthesis, latex fluids have been shown to contain proteins implicated in plant defense and oxidative metabolism [START_REF] Azarkan | Carica papaya latex is a rich source of a class II chitinase[END_REF] [START_REF] Nallamsetty | Purification and biochemical characterization of a highly active cysteine protease ervatamin A from the latex of Ervatamia coronaria[END_REF]. Today, latex is A c c e p t e d M a n u s c r i p t -2 -considered as a promising source of pharmacologically active molecules that might be chemically modified to improve their effectiveness.

With regards to C. procera, although the pharmacological potential of its latex has well been proven, only few active latex molecules have been identified until today ; most often latex is either used as an entity or is only poorly processed, mainly in order to eliminate rubber compounds. Usually, the crude latex from C. procera is collected from non-cultivated plants by small incisions near the younger leafs and is mixted 1:1 (v/v) into distilled water. The material is then kept under constant, gentle, agitation in order to prevent it from precipitating.

It is maintained at room temperature until being centrifuged at 5000 x g, 10 min at 4 °C. The precipitated material is pooled apart while the supernatant is further dialysed using membranes of 8000 Da cut-off, thus keeping the protein fraction, before being lyophilized.

Generally, authors use both, the dialysis liquid containing low molecular weight compounds, called the dialysis latex (DL), and the non dialyzable latex (NDL) obtained after exhaustive dialysis and containing concentrated proteins.

Soares et al., were among the first to show benefic, antinociceptive effects of fractionated latex (NDL) [START_REF] Soares | Antinociceptive activity of Calotropis procera latex in mice[END_REF]. After acetic acid induced writhes in mice, the latex was capable of inhibiting up to 100 % the abdominal constrictions in a dose dependant manner (12,5 to 50 mg/kg).

Morphine was used as a positive control (54,5 % of inhibition). Similar results were obtained for formalin-induced paw licking for an hot plate test. In contrast to morphine, they found that the opioid antagonist nalaxone did not alter the latex-induced antinociceptive effect.

In 2006, Freitas et al ., went a bit further and discovered pro-and anti-inflammatory potentials of dialysed and non dialysable latex [START_REF] Freitas | Enzymatic activities and protein profile of latex from Calotropis procera[END_REF] ; dialised latex of C. procera was shown to increase induced leukocyte, especially neutrophil, migration whereas anti-inflammatory and antinociceptive activities were associated to non dialysable latex.

Since C. procera has been known to be effective against several human diseases, the aim of the work of Ramos et al ., was to elucidate larvicidal activities of different fractions of the plant's latex [START_REF] Ramos | Latex constituents from Calotropis procera (R. Br.) display toxicity upon egg hatching and larvae of Aedes aegypti (Linn.)[END_REF]. Their evaluation was based on egg hatching and larval development of Aedes aegypti. A 10 mg/mL concentration of NDL was able to completely block egg hatching ; the inhibiting potential of the DL fraction was significantly lower as a 5 times greater concentration (50 mg/mL) was needed in order to achieve 0 % of egg hatching. Both, NDL as well as DL, also triggered severe larvae mortality in a dose and exposure time dependant manner. Chromatography analysis revealed different protein and amino acids of NDL and DL latex, thus delivering promising outlooks for future preparations of C. procera latex as a larvicidal compound. After being triturated 1:1 with gum acacia, DL administrated orally to albino rats and guinea pigs inhibited carrageenan and Freund's adjuvant induced edema as well as granuloma formation. Further anti-inflammatory activities of DL resulted in fluid exudation inhibition and in reduced UV-induced erythema onset [START_REF] Sangraula | Evaluation of anti-inflammatory activity of latex of Calotropis procera in different models of inflammation[END_REF]. Similar results were obtained by Arya et al .

Shade dried DL, soxhlated to get methanolic extracts and triturated with gum acacia as seen before, showed anti-inflammatory activity against histamine, serotonin, compound 48/80, bradykinin and prostaglandin mediated inflammatory reactions in induced rat paw edema [START_REF] Arya | Antiinflammatory efficacy of extracts of latex of Calotropis procera against different mediators of inflammation[END_REF]. The same research team also demonstrated that DL, triturated this time in normal saline, is able to play an antioxidant and anti-hyperglycemic role in alloxan-induced diabetes in rats [START_REF] Roy | Antioxidant and protective effect of latex of Calotropis procera against alloxan-induced diabetes in rats[END_REF]. Contradictory to these findings, there is some evidence that DL is also capable of inducing pro-inflammatory effects. In 2003, Shivkar et al . proved DL to be able to produce dose dependent inflammatory responses resulting in rat paw edema similar to carrageenan.

Further studies suggested biogenic amines, especially histamine, to be involved in DL driven inflammation [START_REF] Shivkar | Histamine mediates the pro-inflammatory effect of latex of Calotropis procera in rats[END_REF].

Similarly, by extracting DL sequentially with petroleum ether and methanol followed by a silica gel chromatography fractionation, Choedon et al ., applied an DL treatment on X15-myc transgenic, hepatocellular carcinoma developing mice [START_REF] Choedon | Anticancer and cytotoxic properties of the latex of Calotropis procera in a transgenic mouse model of hepatocellular carcinoma[END_REF]. Not only did they discover an in vivo chemopreventive effect of DL, they also noticed that DL has cytotoxic effects on Huh-7 and COS-1 cancer cell lines. By chromatography separation, they isolated a polar fraction responsible for the observed cytotoxic potential. Apoptosis analysis revealed induced DNA fragmentation as a mechanism.

1.3.) Ethanolic, methanolic and aqueous extracts

Phytochemically, the plant has been investigated since the 1960's, especially for cardenolides [17] [18], triterpenoids [19] [20], anthocyanins [START_REF] Tiwari | Study of anthocyanins from the flowers of some medicinal plants[END_REF] and hydrocarbons [START_REF] Carruthers | Hydrocarbons from Calotropis procera in northern Australia[END_REF].

Besides the use of plant latex, aqueous, methanolic, ethanolic and other organic plant extracts from different parts of the plant, namely flowers, buds, roots, stems and leaves, were among the very first approaches to develop natural drugs for classical and alternative medicine. In 1979, Malik and Chughtai described antimicrobial activity against pathogenic bacteria [START_REF] Malik | Antimicrobial activity of Calotropis procera. A preliminary study[END_REF].

Moreover, organic extracts of C. procera were also shown to possess nematocidal [START_REF] Nandal | Preliminary screening of some weeds and shrubs for their nematocidal activity against Meloidogyne javanica[END_REF] [25],

A c c e p t e d M a n u s c r i p t -4 -larvicidal [START_REF] Girdhar | Mosquito control by Calotropis latex[END_REF], antifertility [START_REF] Prakash | Effect of oral administration of forty two indigenous plants extracts on early and late pregnancy in albino rats[END_REF] and even anti-cancer potentials [START_REF] Dhar | Screening of Indian plants for biological activity : Part I[END_REF] More specific spectral data analysis of organic extracts of the plant revealed the presence of a norditerpenyl ester, Calotropterpenyl, two pentacyclic triterpenoids, calotropursenyl acetate and calotropfriedelenyl acetate as well as an organic carbonate, namely 2-propenyl-2'hydroxyethyl carbonate [START_REF] Ansari | Norditerpenic ester and pentacyclic triterpenoids from root bark of Calotropis procera (Ait) R[END_REF] [38] (Figure 1).

1.4.) Procerain

By further method standardizations and working condition optimizations, the next step in characterizing the plant's pharmaceutical potentials consisted in the isolation of new, specific By default, plants are usually rich sources of various kinds of proteases, enabling protection, development of the plant, ripening of its fruits [START_REF] Brady | Fruit ripening[END_REF], nutritional reserve, degradation of seed storage proteins thus allowing germination [START_REF] Kembhavi | The two cysteine endopeptidases of legume seeds: purification and characterization by use of specific fluorometric assays[END_REF], activation of proenzymes as well as elimination of defective proteins [START_REF] Rudenskaya | Taraxalisin --a serine proteinase from dandelion Taraxacum officinale Webb s.l[END_REF]. Proteolytic enzymes from plant sources, are of interest for the pharmaceutical research as the have been proven to be active over wide ranges of temperature and pH [START_REF] Dubey | Procerain, a stable cysteine protease from the latex of Calotropis procera[END_REF]. Consequently, an important number of proteases have been isolated from lattices, fruits and seeds. Among these, most belong to the cysteine super family, one of five catalytic types of proteases [START_REF] Boller | Roles of proteolytic enzymes in interaction of plant and other organisms[END_REF]. Procerain was isolated by applying a 50 % ammonium fractionation of crude, gumless latex followed by CM-sepharose and SP-sepharose cation exchange chromatographies. Different physical and chemical properties were then analysed : an SDS-PAGE electrophoresis revealed an average Mr of 28,8 kDa for procerain, putting it in the common molecular mass range of 20-35 kDa for plant cysteine proteases. No carbohydrate could have been determined by the phenol sulfuric acid method [START_REF] Hounsell | Chemical methods of analysis of glycoproteins[END_REF] thus differing procerain from glycoproteins. An isoelectric point of 9,32, defined by isoelectric focusing on polyacrylamide gel, indicates a predominance of basic amino acid residues. Total amino acid analysis revealed the presence of seven cysteines, one free and six forming disulfides [START_REF] Ellman | Tissue sulfhydryl groups[END_REF], eight tryptophan and twenty tyrosine residues. In addition to proteolytic activity against different natural substrates such as casein, azocasein, azoalbumin and haemoglobin, procerain also showed hydrolytic activity against synthetic substrates. The enzyme's activity was best over a broad pH 7,0-9,0 range and together with its high stability with respect to temperature, denaturants and organic solvents, this makes it a very useful tool for food and pharmaceutical industry.

1.5.) Cardenolides

Since the 1960's it has been known that the principal toxic components in C. procera, capable of causing death in mammals when reaching high concentrations, are cardenolides [START_REF] Watt | Medicinal and Poisonous Plants of Southern and Eastern Africa[END_REF].

Chemically, cardenolides, cardiac glycosides named after their capacitiy to influence heart beat, are 5H,14-hydroxy -unsaturated -lactones widely distributed in plants. In 1983, Erdman was among the first to measure individual nutrient and cardenolide concentrations in unextracted as well as in extracted C. procera [START_REF] Erdman | Nutrient and cardenolide composition of unextracted and solventextracted Calotropis procera[END_REF]. Several cardenolides were positively identified : ascleposide, calactin, calotoxin, calotropin, calotropagenin, coroglaucigenin, proceroside, usharidin and uzarigenin. Recently, with the aim to use cardenolides as potential anti-cancer agents, the list of cardiotonic glycosides found in Calotropis procera has been enlarged by 2-oxovoruscharin 02, a derivative of voruscharin [START_REF] Van Quaquebeke | Identification of a novel cardenolide (2''-oxovoruscharin) from Calotropis procera and the hemisynthesis of novel derivatives displaying potent in vitro antitumor activities and high in vivo tolerance: structure-activity relationship analyses[END_REF]. However, due to its high toxicity observed in mice, chemical modifications were necessary in order to improve tolerance in vivo. The outcome, named UNBS1450 01, not only showed reduced toxicity, but also a severely increased therapeutic anti-cancer activity [START_REF] Mijatovic | UNBS1450 : A New Hemi-Synthetic Cardenolide With Promising Anti-Cancer Activity[END_REF].

Figure 2 gives a short overview of how Calotropis procera can be used in pharmaceutical science, either by applying rather simple methods, to extract for example latex, or by more sophisticated approaches seeking to isolate specific compounds like cardenolides. However, recent studies suggest that this Na + pump inhibition is not necessary to obtain an inotropic effect on myocytes [START_REF] Xie | Na+-K+--ATPase-mediated signal transduction: from protein interaction to cellular function[END_REF]. By interacting with cardiac glycosides, the Na + -K + pump complex has been shown to act as a signalosome. Various intracellular signaling pathways are responsive to cardiac glycoside binding ; Ca 2+ could play the role of a second messenger, the phosphatidylinositide 3'-kinase and protein kinase B can be activated as well as the SRC-EGFR-RAS-RAF-ERK cascade. In this way, cardiac glycosides may influence cell proliferation, cell differentiation and finally cell death by apoptosis (Figure 3).

2.) Bioactivity of cardenolides

2.2.) Cardenolide derivatives and their structure-activity-relationship

Recently, Toxicaroside D 03, isolated via bioassay-guided fractionation from a ethanolic stem extract of the rainforest tree Antiaris toxicaria, exhibited cell proliferation inhibition in different cancer cell lines together with 10 other cardenolides purified from the same source.

Concerning structure-activity relationship, a reduction of 19-CHO and mono-glycosidation of the 3-hydroxy group resulted in a higher cytotoxicity to different cancer cell lines by factor 2 and over 100 respectively. In contrast, a -hydroxylation of carbon 12 reduced the induction of apoptosis [START_REF] Jiang | Cardenolides from Antiaris toxicaria as potent selective Nur77 modulators[END_REF]. However, an additional acetoxy group did not modify the cytotoxicity [START_REF] Ueda | Antiproliferative activity of cardenolides isolated from Streptocaulon juventas[END_REF][START_REF] Zhao | Bioactive cardenolides from the stems and twigs of Nerium oleander[END_REF]. These observations had been confirmed in a previous publication [START_REF] Ueda | Antiproliferative activity of cardenolides isolated from Streptocaulon juventas[END_REF]. Structural modification of the core unit as an additional alcohol group or an epoxy group at C8 or a double bond between C16 and C17 led to a decrease of cytotoxicity [START_REF] Zhao | Bioactive cardenolides from the stems and twigs of Nerium oleander[END_REF]. Additionally, Ueda They proved that di-glycosidation did not alter or even decrease the activity. Hence, the hydroxyl groups at C1 and C5 were important for the anti-proliferative property. These authors concluded that bioactivity of the isolated cardenolides-set depends on the The research group of O'Doherty developed a novel stereoselective synthetic method to glycosylate the aglycone core units of cardenolides. The comparative study of the synthesized mono-, di-or tri-saccharide derivatives 05-07 of digitoxigenin 04 showed that the linkage of a monosaccharide increased the bioactivity [START_REF] Zhou | The de novo synthesis of oligosaccharides: application to the medicinal chemistry SAR-study of digitoxin[END_REF], which confirmed the previously described studies by Yao and Kadota [START_REF] Jiang | Cardenolides from Antiaris toxicaria as potent selective Nur77 modulators[END_REF][START_REF] Ueda | Antiproliferative activity of cardenolides isolated from Streptocaulon juventas[END_REF]. The higher anti-proliferative properties of the trisaccharide derivatives of digoxin and gitoxin underlined this result [START_REF] Lopez-Lazaro | Digitoxin inhibits the growth of cancer cell lines at concentrations commonly found in cardiac patients[END_REF]. In detail, the stereocenter at C2' of the sugar unit plays a crucial role in cytotoxicity, as the unnatural epimers of the cardenolides loose almost their complete apoptotic activity against cancer cells [START_REF] Langenhan | Enhancing the anticancer properties of cardiac glycosides by neoglycorandomization[END_REF].

The anti-inflammatory bioactivity of cardenolides proved to be sensible on chemical modifications of the core unit. Although, an additional acetoxy function at C16 doesn't alter the observed properties, an additional alcohol group at C8, modification of the C14 hydroxyl group or a double bond between C16 and C17 resulted in a lower anti-inflammatory activity [START_REF] Zhao | Bioactive cardenolides from the stems and twigs of Nerium oleander[END_REF].

A low anti-proliferative activity is not necessary for the reversal of multi-drug resistance (MDR) as one cardenolide, even exhibiting a cytotoxicity at a concentration range up to 77

mM, might serve as potential future lead MDR-cancer reversal agent due to the calcein accumulation in MDR human ovarian cancer 2780AD cells [START_REF] Zhao | Bioactive cardenolides from the stems and twigs of Nerium oleander[END_REF]. This compound had been isolated first by Neumann and its structure determination had been carried out by the research teams of Reichstein and Yamauchi [START_REF] Neumann | Über Glykoside des Oleanders[END_REF][START_REF] St Janiak | Alpha"-, "beta"-and "delta"anhydrodigitoxigenin[END_REF][START_REF] Abe | Triterpenoid glycosides from bark of Meliosma lanceolata[END_REF] (Figure 4 and Figure 5).

Interestingly, in the case of the hemi-synthetic derivative 01 of 2"-oxovoruscharin 02, the reduction of the carboxyl function into a primary alcohol did not result in a higher cytotoxicity but the in vivo toxicity was reduced by factor 10 [49] (Figure 4).

The design of novel anti-cancer drugs derived from natural products is still a matter in the development of potent cancer therapeutics. There is still a need and potential in further investigation of natural sodium pump inhibitors.

2.3.) Pharmaceutical activity of cardenolides

With chronic heart failure being a widespread health problem in modern society, there is an urge for new therapy drugs. A c c e p t e d M a n u s c r i p t -10 -

3.) UNBS1450 and its anti-cancer activity

3.1.) The impact of UNBS1450 on the Na + .K + -ATPase isozymes Several isozyme forms are known for Na + ,K + -ATPases, depending on tissue and cell type : 4

 and 3  isoforms have been identified so far. Until today, severely increased expression of the 1 subunit has been associated to the clinical patterns of non-small cell lung cancers (NSCLC) and glioblastoma [70] [71].

For both studies, sodium pump inhibition assays were performed using a heterologous protein expression system. Given the advantage of producing high amounts of recombinant Na + /K + -ATPases, baculovirus-transfected Sf-9 insect cells expressing 11, 21 and 31

isozymes were used to analyse the inhibitory capacities of several cardenolides, among which showed cytotoxic effects on rat glioma, mouse mammary carcinoma and melanoma cells but at a far less extent when compared to UNBS1450 01 anti-tumor effects in human cancer cells.

This feature could relate to the double mutation observed in the alpha-1 subunit of rodent sodium pump.

More recently, the observed findings were confirmed : UNBS1450 01 is also capable of repressing in vitro growth of A549 tumor cells as well as growth and migration of glioblastoma cell lines.

UNBS1450's in vivo anti-cancer activity has been investigated so far by tumor growth analysis in xenograft models. Generally, by administering UNBS1450 01 chronically to nude mice bearing orthotopic xenografts of NSCLC and glioblastoma cell lines, cytotoxicity of this hemi-synthetic cardenolide has been determined by intraperitoneal or per os injections. In both cases, the maxium tolerated dose (MTD) had been previously elucidated acutely in healthy mice. When compared to classical cardenolides such as ouabain and digoxin as well as to its precursor, 2-oxovoruscharin 02, UNBS1450 01 showed significantly increased MTD values : for ouabain and digoxin, MTD values were 5 mg/kg and 10 mg/kg respectively, whereas for UNBS1450 01 the MTD value raised up to 120 mg/kg. As a result, chronic administration of UNBS1450 01 was realized with different concentrations, from a 32 to an 8fold reduced MTD [START_REF] Mijatovic | The cardenolide UNBS1450 is able to deactivate nuclear factor kappaBmediated cytoprotective effects in human non-small cell lung cancer cells[END_REF]. Interestingly, UNBS1450 01 at concentrations from 5 to 20 mg/kg was proven to be able to increase grafted mice survival. In control mice, about 20 days were sufficient to bring the mouse population down to 45 %. In contrast, mice which had been chronically administrated (12 times) with 10 mg/kg of UNBS1450 01 showed best results, with a population down to 45 % only after 40 days. Higher concentrations (20 mg/kg) seemed to be less encouraging since the outcome was less promising than for 10 mg/kg, probably due to slight toxic effects.

Comparable results were obtained for per os administrations of UNBS1450 01, nevertheless requiring higher dosages. Oral uptake of UNBS1450 01 at 80 mg/kg significantly increased mouse survival. Whereas the 50 % survival barrier was broken shortly after 20 days posttumor graft in control mice, with 80 mg/kg of UNBS1450 01 this point was delayed to day 40.

A principal characteristic of many cancer cells is their high invasiveness ; glioblastoma cells for instance are active, "self-propelled" brain cancer cells, highly capable of invading brain tissue by shape and volume modifications [START_REF] Sontheimer | Malignant gliomas: perverting glutamate and ion homeostasis for selective advantage[END_REF]. Today it is believed that ion transport plays an important role in enabling cancer cell migration [START_REF] Ransom | Volume-activated chloride currents contribute to the resting conductance and invasive migration of human glioma cells[END_REF]. Until very recently, the impact of the Na + ,K + -ATPase in cancer cell migration remained unexplored. In early 2008, Lefranc et al .

A c c e p t e d

M a n u s c r i p t -13 -tried to establish a link between glioblastoma cell migratory invasion, sodium pump and cardenolide administration [START_REF] Lefranc | Targeting the alpha 1 subunit of the sodium pump to combat glioblastoma cells[END_REF]. At first, they demonstrated that in human glioblastoma cells the 1 subunit of the Na + ,K + -ATPase is overexpressed when compared to normal human brain tissue. Using immunofluorescent staining, they were able to show that 1 subunits colocalize with caveolin-1 in the cytoplasmic protrusions of U373-MG glioblastoma cells.

Additionally, UNBS1450 01 was able to exert a cytotoxic effect on human U373-MG cells ;

the same was true for rat C6 cells, but to a lesser extent. For U373-MG, concentrations of around 100 nM of both UNBS1450 01 as well as ouabain rendered cell survival nearly impossible. For rat C9 cells, ouabain was practically ineffective whereas high concentrations reaching 10 M were required to completely abolish cell viability. An impact of UNBS1450 01 on intracellular ATP concentrations in human glioblastoma cells had then been suggested.

Next, computer-assisted phase-contrast microscopy analysis permitted to gain further insights in cellular proliferation, morphology and motility as well as on ion homeostasis. They discovered that in the presence of 10 nM UNBS1450 01, U373 cells are well capable of undergoing mitosis, but fail to divide into daughter cells and to form lamellipodia.

Nevertheless, the observed, round-shaped U373 cells remained alive for several days (mitochondria staining). However, contrary to a possible hypothesis, the administration of 10 nM of UNBS1450 01 did not result in either an increased Ca 2+ nor an increased Na + concentration, which at the same time constitutes one of the major advantages of UNBS1450 when compared to other cardiac glycosides like digitalis. Increasing intracellular Ca 2+ favors arrhythmias in otherwise healthy people. The observed changings were probably due to irreversibly modified actin cytoskeleton, thus explaining reduced migratory capacities of these human glioblastoma cells (Figure 6).

3.4.) UNBS1450-induced cell death -by apoptosis

It has been reported in literature that cardiac steroid glycosides are a priori potentially able to induce cell death by apoptosis. For example it has been observed in vitro that different leukemia cell lines such as THP-1 [START_REF] Kurosawa | Distinct PKC isozymes regulate bufalin-induced differentiation and apoptosis in human monocytic cells[END_REF], K562 [START_REF] Numazawa | A cardiotonic steroid bufalin-like factor in human plasma induces leukemia cell differentiation[END_REF], HL-60 , U937 and ML1 [START_REF] Zhang | Distinct role of the Nterminal tail of the Na,K-ATPase catalytic subunit as a signal transducer[END_REF] undergo apoptosis when treated with different concentrations of Bufalin or Digoxin. However, when treated with rather low nanomolar doses of these cardiac glycosides, it might preferentially stimulate differentiation and proliferation. It is believed that nanomolar concentrations lead to -by autophagy

Recent studies on UNBS1450-induced cell death claim pro-autophagic effects to be responsible for the observed cytotoxic potential [START_REF] Lefranc | Targeting the alpha 1 subunit of the sodium pump to combat glioblastoma cells[END_REF]. It has been reported that during autophagy, besides apoptosis, one of the two self-destructive processes, intracellular organelles are enclosed in characteristic double-or multi-membraned vacuoles which finally fusion with lysosomes thus leading to degradation [START_REF] Maiuri | Self-eating and self-killing: crosstalk between autophagy and apoptosis[END_REF]. Together with apoptosis, cellular "self-eating" has the aim to eliminate superfluous, damaged or aged cells or organelles like mitochondria and endoplasmic reticulum. Moreover, being implicated in macromolecule catabolism, autophagy helps cells in overcoming stress situations, thus avoiding probable cell death by apoptosis for instance. Depending on the situation, autophagy sometimes constitutes an alternative pathway to apoptosis, sometimes a possibility to escape apoptosis.

In 2006, first studies indicating UNBS1450-induced autophagy in cancer cells appeared [START_REF] Mijatovic | Cardenolide-induced lysosomal membrane permeabilization demonstrates therapeutic benefits in experimental human non-small cell lung cancers[END_REF].

Working on NSCLC cell lines, cell death was indeed relevant after treatments with In the same context UNBS1450 01 has been assigned to induced cell death through autophagy in different human glioblastoma cell lines [START_REF] Lefranc | Targeting the alpha 1 subunit of the sodium pump to combat glioblastoma cells[END_REF]. In a first step, acridine orange staining was used in order to quantify acidic vesicular organelles whose abundance is tightly associated to autophagy. Indeed, the presence of nanomolar UNBS1450 01 significantly increased staining in U373-MG and U87-MG cells (from 3 to 42 and from 4 to 65 %). At the same time, the expressions of LC3 and beclin-1, two specific autophagy markers, were found to be stimulated by UNBS1450 01.

Besides lung cancer and glioblastoma cell lines, UNBS1450 01 has also been found to induce probably autophagy-related cell death in human prostate cancer cell lines by severe nucleolar targeting [START_REF] Mijatovic | Nucleolus and c-Myc: potential targets of cardenolide-mediated antitumor activity[END_REF]. In fact, UNBS1450 01 efficiently inhibited apoptosis-resistant PC-3 prostate cancer cell growth at 100 nM, without caspase-3 and caspase-9 activation and PARP cleavage. Intracellular Ca 2+ concentrations were not modified, in contrast to cellular ROS load. Not only did UNBS1450 01 (10 and 100 nM) severely increase ROS production, computer-assisted phase-contrast microscopy revealed that it again completely disorganized the actin cytoskeleton as seen before and disturbs nucleolar morphology. The nucleolus domain constitutes the site of ribosome biogenesis and is thus indispensable to cell survival. It has been reported that nucleolar regions are of great importance in cancer cells as it has been proven that tumor cells retain tumor suppressors, like p53 and MYC for instance, inside the nucleoli. The observed impairments of CDK and Myc protein expressions might thus be mechanisms of nucleolar disorganization and induced autophagy (Figure 7).

4.) Conclusion

Taken together, these results suggest that the novel hemi-synthetic cardenolide UNBS1450 01 from Calotropis procera is able to repress cancer cell proliferation and even induce cell death.

Despite the fact that the plant's benefical effects on many different pathologies have been admitted many centuries ago, the real breakthrough is still to come. Both, the entire latex as Figure 5 SAR studies on analogues of natural sodium pump inhibitors.

Summary of structure-activity relationship (SAR) results of natural sodium pump inhibitors and derivatives concerning the observed anti-cancer cytotoxicity and anti-oxidant properties.

The pharmacophores identified during the studies are highlighted in colour. [START_REF] Van Quaquebeke | Identification of a novel cardenolide (2''-oxovoruscharin) from Calotropis procera and the hemisynthesis of novel derivatives displaying potent in vitro antitumor activities and high in vivo tolerance: structure-activity relationship analyses[END_REF][START_REF] Jiang | Cardenolides from Antiaris toxicaria as potent selective Nur77 modulators[END_REF][START_REF] Ueda | Antiproliferative activity of cardenolides isolated from Streptocaulon juventas[END_REF][START_REF] Zhao | Bioactive cardenolides from the stems and twigs of Nerium oleander[END_REF][START_REF] Zhou | The de novo synthesis of oligosaccharides: application to the medicinal chemistry SAR-study of digitoxin[END_REF][START_REF] Lopez-Lazaro | Digitoxin inhibits the growth of cancer cell lines at concentrations commonly found in cardiac patients[END_REF][START_REF] Langenhan | Enhancing the anticancer properties of cardiac glycosides by neoglycorandomization[END_REF][START_REF] Neumann | Über Glykoside des Oleanders[END_REF][START_REF] St Janiak | Alpha"-, "beta"-and "delta"anhydrodigitoxigenin[END_REF][START_REF] Abe | Triterpenoid glycosides from bark of Meliosma lanceolata[END_REF] Table 1 Main endogenous and exogenous cardenolides and their pharmaceutical activity.

  latex fractionation, some authors used air dried latex ; in most examples, latex was extracted from the aerial parts of the plant before being dried under shade (DL, dried latex).

A c c e p t e d M a n u s c r i p t - 5 -

 5 and pharmacologically important proteins and compounds, especially with an enzymatic activity.

2. 1 .

 1 ) Effect of cardenolides on the sodium pump Frequently named sodium pump, the Na,K-ATPase is an integral-membrane protein with both an enzymatic and a transporter function : in the conformational state E1, the phosphorylated protein binds three intracellular Na + . These Na + ions are then released into the extracellular fluid, in exchange to 2 external K + ions and phosphate formation (state E2). With the Na + /K + gradient being implicated in the regulation of animal cell volume, in lyses prevention, in membrane potential control, in pH homeostasis and in energy delivery for ion transport, the sodium pump is essential for cell viability and is thus ubiquitously expressed throughout the different tissues[START_REF] Horisberger | Recent insights into the structure and mechanism of the sodium pump[END_REF] [START_REF] Faller | Mechanistic studies of sodium pump[END_REF].The Na,K-ATPase is a heterodimer formed of an  and  subunit. The  subunit, with a molecular mass of 106 kDa, contains a ten segment transmembrane domain (M) and three different cytosolic domains, namely a nucleotide binding domain (N) binding ATP, a phosphorylation domain (P) and an actuator domain (A). The abundantly glycosylated  subunit of 40 kDa has a single transmembrane segment, a short intracellular N-terminal and a larger extracellular site. In contrast to the catalyzing  subunit, the  subunit is required for the biogenesis and activity of the holoenzyme. A third subunit FXYD has been reported to be associated to the  and  subunits in some tissues.

A c c e p t e d M a n u s c r i p t - 7 -

 7 Endogenous and exogenous cardiac glycosides, for example cardenolides, act by binding to extracellular H 1 -H 2 , H 3 -H 4 and H 5 -H 6 loops of the catalytic  subunits of sodium pumps. It had been shown that these cardenolide compounds are tightly fixed to the E2 conformational enzyme state, leading to inactivation[START_REF] Erdmann | Ouabain-receptor interactions in (Na + +K + )-ATPase preparations from different tissues and species. Determination of kinetic constants and dissociation constants[END_REF]. E2-phosphoenzyme-cardiotonic compound complexes were found to be formed irreversibly. With varying Kd values, the inhibition complexes are probably internalized and degraded. Inhibitors of the Na + ,K + transporter lead to an increase of the cytosolic Na + concentration and are thus influencing the thermodynamics of the Na + ,Ca 2+ exchange pump finally resulting in an increased Ca 2+ concentration in sarcoplasmic reticulum[START_REF] Keller | F90927: a new member in the class of cardioactive steroids[END_REF] (Na + -Lag hypothesis).

  et al. isolated 15 cardenolides from the Vietnamese medicinal plant Streptocaulon juventas.

A c c e p t e d M a n u s c r i p t - 8 -

 8 hydrophilicity of the sugar unit linked to C3-OH because a free hydroxyl C2' and acetylating of C3'OH reduced the cytotoxicity[START_REF] Ueda | Antiproliferative activity of cardenolides isolated from Streptocaulon juventas[END_REF].

UNBS1450 01 .

 01 When compared to ouabain and digoxin, UNBS1450 01 showed a greater Na + /K + -ATPase isozyme inhibitory potential. The 31 dimer was mostly affected by an UNBS1450 inhibition, with a determined Ki of 3,2 nM. The 21 and 11 isozymes were also inhibited by UNBS1450 01, but to a lesser extent, with Ki values of 15 and 160 ± 70 nM respectively. Compared to classic cardenolides, the double-linked sugar moiety as well as the trans-conformational steroid structure seem to further potentiate the inhibitory activity of UNBS1450 01, rendering this cardenolide especially promising for additional anti-cancer studies based on modified cell proliferation, differentiation and cell death by inhibited sodium pump driven signaling. According to recent findings, sodium pump signaling through UNBS1450 01 binding leads to disorganization of the actin cytoskeleton, increased non-apoptotic cell death by autophagy, down-regulation of c-Myc in cancer cells, NF-B deactivation and general modifications of the nucleolar morphology.

3. 2 .

 2 ) The impact on the NF-B signaling pathway As many typical cancer cell lines, A549, non-small cell lung cancer cells, show constitutive NF-B pathway activation, and thus become able to escape cell death by apoptosis, necrosis, autophagy, senescence, mitotic catastrophe and paraptosis[START_REF] Okada | Pathways of apoptotic and non-apoptotic death in tumour cells[END_REF]. NF-B activation favores multidrug resistant cancer development.

A c c e p t e d M a n u s c r i p t - 11 -

 11 Mijatovic et al . were among the very first to investigate a possible repressive potential of cardiotonic steroids and especially cardenolides on constitutive NF-B activation[START_REF] Mijatovic | The cardenolide UNBS1450 is able to deactivate nuclear factor kappaBmediated cytoprotective effects in human non-small cell lung cancer cells[END_REF]. In their work they used a new hemi-synthetic derivative of 2-oxovoruscharin 02, a compound recently identified by themselves in Calotropis procera and isolated by liquid chromatography from methanolic extracts from the root barks of C. procera. In contrast to stereotyp cardenolides, the chemically modified 2-oxovoruscharin named UNBS1450 01 shows a double-linked sugar moiety and a trans-conformational steroid core.As a first result, they managed to show that UNBS1450 01 is able to influence NF-B activity in human A549 cells. In fact, a 24-hour treatment with UNBS1450 01 with a concentration of 10 nM was sufficient to point out induced accumulation of I-B, however not of I-B. For the latest, phosphorylation levels were decreased after 4 to 8 hours post-treatment. The expression of cdc34, an enzyme implicated in I-B ubiquitination, was also decreased. Further NF-B binding (Trans-AM) as well as NF-B reporter gene (luciferase reporter plasmid construct pNF-B-Luc) assays confirmed the in vitro inhibitory potential of UNBS1450 01 on A549 tumor cells. Secondly, preliminary in vivo studies revealed increased survival of A549 orthotopic xenograft-bearing nude mice when treated with different concentrations of the maximum tolerated dose of UNBS1450 01. 3.3.) The effect of UNBS1450 on cell proliferation and cancer Starting with in vitro anti-proliferation assays in 2005, Van Quaquebeke et al. analysed cytotoxicity levels of several cardenolides, among which UNBS1450 01, on 57 human cancer cell lines ; for each compound average IC 50 values were determined by MTT assays after 72 h of incubation. As reference drugs, a tubulin inhibitor (taxol) and a topoisomerase I inhibitor (SN-38) were chosen. Concerning the individual sensitivity of each human cancer cell line, significant differences were observed between UNBS1450 01, taxol and SN-38 suggesting a different antitumor mechanism for UNBS1450 01. In terms of average growth inhibition however, UNBS1450 01 was proven to be as potent as taxol, with a mean IC 50 of 2,7 nM for UNBS1450 01 and 2,5 nM for taxol. SN-38 was significantly less potent. Furthermore, when applicated on multidrug resistant tumor bladder cells, UNBS1450 01 showed rather astonishing capacities in inhibiting cell growth : vincristine and adriamycin resistant tumor cells forced taxol to reach concentrations of 10000 nM to become cytotoxic whereas UNBS1450 01 became effective at 45 nM. Complementary to these data, UNBS1450 01 alsoA c c e p t e d M a n u s c r i p t -12 -

A c c e p t e d M a n u s c r i p t - 14 -

 14 low-frequency oscillations of Ca 2+ concentrations favoring NF-B activation and protection against apoptosis whereas higher concentrations would lead to apoptosis due to sustained, high levels of Na + and Ca 2+[START_REF] Aizman | Ouabain, a steroid hormone that signals with slow calcium oscillations[END_REF].In contrast to many other cardiac steroids, UNBS1450 01 does not seem to induce cell death by apoptosis in vitro, at least when working with adherent cell lines (evidence provided by flow cytometry analyses after propidium iodide and Annexin V staining). To our knowledge, the entire studies on UNBS1450's anti-proliferative and anti-cancer activity has been carried out on solid tumor cell lines. UNBS1450 01 stimulation has been shown to induce nonapoptotic cell death[START_REF] Lefranc | Targeting the alpha 1 subunit of the sodium pump to combat glioblastoma cells[END_REF] [START_REF] Mijatovic | Cardenolide-induced lysosomal membrane permeabilization demonstrates therapeutic benefits in experimental human non-small cell lung cancers[END_REF].

UNBS1450 01 :

 01 a 24 h treatment with 100 nM of UNBS1450 reduced cell viability to about 70 %, when prolonged to 72 h, 10 nM were sufficient to obtain similar results. Consequently, PARP cleavage analysis permitted to exclude cell death through apoptosis, as PARP remained intact. Complementary performed Tunel assays ultimately confirmed the absence of apoptotic cell death. Cell cycle analysis revealed increased percentages of cells in S and G 2 phases whereas microscopy images clearly showed severe cytoplasmic vacuole formation. Further Mijatovic et al . made clear that UNBS1450 01 induces Hsp70 downregulation thus rendering possible lysosomal membrane permeabilization, typically inhibited in lung cancer cells by heightened levels of Hsp70 which constitute a prosurvival function. Lysosomal permeabilization is a known indicator of cell death through autophagy ; these findings of 2006 A c c e p t e d M a n u s c r i p t -15 -then strengthened the hypothesis of non-apoptotic cell death by "self-eating" in UNBS1450 treated cancer cells.

  well as organic extracts, might well have proven their usefulness in treating different clinical entities, only recent advances in research revealed the true potential lying within C. procera.By chemically modifying a novel cardenolide isolated from this tropical plant, researchers discovered with UNBS1450 01 a new and very promising weapon in cancer therapy.

A c c e p t e d M a n u s c r i p t - 16 -

 16 In contrast to many other anti-cancer agents, UNBS1450 01 does not induce apoptosis but autophagy, at least in solid tumor cells. Essentially by deactivating NF-B, downregulating Hsp70 and by modifying nucleolar structure, UNBS1450 01 leads to vacuole formation, actin cytoskeleton impairment and finally cell death.Further insights, particularly concerning its usefulness in leukemia treatment, still remain to be investigated.

  Figure 1 Illustrative representation of the different pharmacologically important potentials of Calotropis procera extracts.

Figure 2 Calotropis

 2 Figure 2Calotropis procera cartoon showing the three main approaches for using the plant's intrinsic pharmaceutical potential.

Figure 3 Schematic

 3 Figure 3Schematic representation of Na + /K + ATPase inhibition by cardiotonic steroids.Two conformationally different stades are known for the Na + /K + ATPase : in E1, enzyme phosphorylation on the phosphorylation domain P enables intracellular binding of 3 Na + in exchange to 2 K + , thus leading to stade E2. The 3 Na + are released in the extracellular fluid whereas 2 new K + are imported. Through hydrolysis, the enzymatic complex returns to stade E1 with P i release. Cardiotonic steroids (CS) like cardenolides become tightly bound to the different extracellular loops (H 1 -H 2 , H 3 -H 4 , H 5 -H 6 ) of the enzymatic  subunit. The holoenzyme is blocked in stade E2, no further ion exchange is possible.

Figure 4 Chemical

 4 Figure 4Chemical structure of a small selection of natural sodium pump inhibitors and its analogues.

A c c e p t e d M a n u s c r i p t

  In vitro and in vivo effects of UNBS1450.

Figure 7 Apoptosis

 7 Figure 7Apoptosis and autophagy related cell death induced by UNBS1450.

Figure A c c e p t e d M a n u s c r i p

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Figure

  Figure

Figure

  Figure

Figure A c c e p t e d M a n u s c r i p

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  [START_REF] Ayoub | Screening of plants used in Sudan folk medicine for anticancer activity[END_REF]. In 1987, Mascolo et al . further elucidated the biological properties of Calotropis procera and, using ruminant animal models, discovered anti-inflammatory, analgesic, antimicrobial and antipyretic activities in extracts from C. procera flowers. In the late 1980's,Fenado et al ., established a

	standard method to get crude aqueous extracts (CAE) from powdered C. procera flowers
	[30]. Similarly, in 1994 Asuzu and Onu managed to get crude methanolic extracts (CME)
	from C. procera using a Soxhlet apparatus [31]. Others used filtered ethanolic extracts to
	elucidate the plant's pharmacological potential [32] [33].
	Meanwhile, the ethanolic extracts of flowers, buds, roots, stems and leaves were discovered to
	have a schizontocidal potential in vitro. The different fractions of Calotropis procera showed
	dose-dependant inhibitory effects on chloroquine sensitive and resistant Plasmodium
	falciparum strains. Antimalarial screening had been performed according to the method of
	Ang et al . [34] ; parasitaemia had been adjusted to 1-1,5 % before different concentrations of
	C. procera fractions were added. After an incubation time of 36 h, each sample was analysed
	under the microscope.
	Furthermore, both, aqueous and methanolic extracts of powdered Calotropis procera flowers,
	were shown to possess time-dependent anthelmintic activities in vitro [35]. Based on
	nematode egg counting in sheep feces, the authors confirmed their observed results in vitro : a
	maximum of 88,4 % egg reduction (ECR, egg count percent reduction) had been recorded in
	sheep treated with CAE and 77,8 % in sheep treated with crude powder. CME had been
	proven to be less effective.
	Besides further insecticidal properties [36], organic C. procera extracts also possess
	interesting cytotoxic effects. Compared to the reference compound cisplatin (IC 50 0,9 g/mL),
	Smit et al . demonstrated that ethanolic flos extracts (IC 50 1,4 g/mL) have potent growth
	inhibition capacities when used on COLO 320 tumour cells.

  Concerning the different Calotropis species, since 1979 it has been known that the latex of Calotropis gigantea contains four cysteine proteases,

namely Calotropin FI, FII, DI and DII

[START_REF] Abraham | Studies on proteinases from Calotropis gigantea latex. I. Purification and some properties of two proteinases containing carbohydrate[END_REF]

. More recently, Dubey et al . purified a new cysteine protease from Calotropis procera, named procerain.

Acknowledgements

The authors thank C. Cerella and MH. Teiten for critical reading. TJ is a recipient of an Aide à la Formation-Recherche (AFR) fellowship and MS is a recipient of a Télévie fellowship. Dr Diederich's research at the Laboratoire de Biologie Moléculaire et Cellulaire du Cancer (LBMCC) is financially supported by « Recherche Cancer et Sang » foundation, by the « Recherches Scientifiques Luxembourg » association, by « Een Häerz fir kriibskrank Kanner » association, by the Action Lions « Vaincre le Cancer » association and by Télévie Luxembourg.