Sylvia Mansilla

Marc Bataller

Dr José Portugal

A nuclear budding mechanism in transiently arrested cells generates drugsensitive and drug-resistant cells

Keywords: Aneuploidy, HCT116 cells, p21 WAF, mitotic catastrophe, neosis, senescence

HCT116 (p53 +/+ ) human colon carcinoma cells treated with nanomolar concentrations of doxorubicin underwent transient senescence, synthesized DNA, showed endopolyploidization, increased their size and became multinucleated without a significant increase in mitosis. Nuclei underwent a budding process that involved the release of buds outside the nuclear membrane, and some of the buds seemed to escape from the polyploid cells. A clonogenic assay showed that some cells proliferated following the initial treatment. In general, cells ensuing after budding were not resistant to a variety of drugs, although some of them turned out to be resistant, indicating a potential selective advantage. Nuclear budding was accompanied by changes in protein levels in the giant cells, including inhibition of p53 and enhanced expression of p21 WAF1 and the meiosis-related Mos. The buds might be a mechanism for the segregation and elimination of redundant DNA, or for generating viable aneuploid cells with a potentially extended life span.

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

Introduction

Cell cycle progression is regulated by checkpoint controls, which protect the genome integrity.

These checkpoints prevent cell cycle progression until the preceding events are finished, or DNA damage has been repaired [START_REF] Suijkerbuijk | Preventing aneuploidy: The contribution of mitotic checkpoint proteins[END_REF][START_REF] Wang | Mitotic checkpoint defects in human cancers and their implications to chemotherapy[END_REF]. Mitosis is a short phase of the cell cycle strictly controlled by temporally expressed proteins. These usually act together with other post-transcriptional modified proteins that are present throughout the cell cycle [START_REF] Suijkerbuijk | Preventing aneuploidy: The contribution of mitotic checkpoint proteins[END_REF][START_REF] Chang | p21 Waf1/Cip1/Sdi1 -induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells[END_REF][START_REF] Murray | Recycling the cell cycle: cyclins revisited[END_REF]. The presence of weakened checkpoints is prevalent in tumor cells [START_REF] Suijkerbuijk | Preventing aneuploidy: The contribution of mitotic checkpoint proteins[END_REF][START_REF] Weaver | Decoding the links between mitosis, cancer, and chemotherapy: The mitotic checkpoint, adaptation, and cell death[END_REF], which can promote aneuploidy.

Apoptosis protects cells from aneuploidy [START_REF] Suijkerbuijk | Preventing aneuploidy: The contribution of mitotic checkpoint proteins[END_REF][START_REF] Castedo | Mitotic catastrophe constitutes a special case of apoptosis whose suppression entails aneuploidy[END_REF], but tumor cells usually have defective apoptotic pathways, thus they may enter mitosis that is prolonged for extended periods of time, or the cells may be committed to dying by mitotic catastrophe, which can be induced by caspasedependent or caspase-independent mechanisms [START_REF] Mansilla | Mitotic catastrophe results in cell death by caspasedependent and caspase-independent mechanisms[END_REF]. Moreover, some cells can overcome such a defective mitosis but they are halted in the ensuring G1 phase; an arrest that is considered to be p53-dependent [START_REF] Andreassen | Neither p21 WAF1 nor 14-3-3σ prevents G2 progression to mitotic catastrophe in human colon carcinoma cells after DNA damage, but p21 WAF1 induces stable G1 arrest in resulting tetraploid cells[END_REF][START_REF] Ferbeyre | Oncogenic ras and p53 cooperate to induce cellular senescence[END_REF]. Senescence is defined as a cell program of terminal growth arrest that can be activated after chemotherapy [START_REF] Roninson | Tumor cell senescence in cancer treatment[END_REF]. Senescence is considered largely p53-dependent, while the onset of mitotic catastrophe is not [START_REF] Chang | Role of p53 and p21 waf1/cip1 in senescence-like terminal proliferation arrest induced in human tumor cells by chemotherapeutic drugs[END_REF][START_REF] Gewirtz | Accelerated senescence: an emerging role in tumor cell response to chemotherapy and radiation[END_REF]. Several DNA-binding drugs have been reported to produce mitotic catastrophe in both cells containing wild-type p53 and those bearing mutated or deleted p53 genes [START_REF] Roninson | If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells[END_REF][START_REF] Mansilla | Mitotic catastrophe as a consequence of chemotherapy[END_REF][START_REF] Eom | Two distinct modes of cell death induced by doxorubicin: apoptosis and cell death through mitotic catastrophe accompanied by senescence-like phenotype[END_REF][START_REF] Hyzy | Antitumour imidazoacridone C-1311 induces cell death by mitotic catastrophe in human colon carcinoma cells[END_REF][START_REF] Vakifahmetoglu | DNA damage induces two distinct modes of cell death in ovarian carcinomas[END_REF]. Mitotic catastrophe occurs in tumor cells treated with various antitumour drugs or radiation [START_REF] Mansilla | Mitotic catastrophe results in cell death by caspasedependent and caspase-independent mechanisms[END_REF][START_REF] Roninson | If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells[END_REF][START_REF] Mansilla | Mitotic catastrophe as a consequence of chemotherapy[END_REF][START_REF] Hyzy | Antitumour imidazoacridone C-1311 induces cell death by mitotic catastrophe in human colon carcinoma cells[END_REF][START_REF] Vakifahmetoglu | DNA damage induces two distinct modes of cell death in ovarian carcinomas[END_REF][START_REF] Lock | Dual modes of death induced by etoposide in human epithelial tumor cells allow Bcl-2 to inhibit apoptosis without affecting clonogenic survival[END_REF][START_REF] Niida | Specific role of Chk1 phosphorylations in cell survival and checkpoint activation[END_REF][START_REF] Ianzini | Mitotic Catastrophe[END_REF][START_REF] Bataller | Mithramycin SK modulates polyploidy and cell death in colon carcinoma cells[END_REF]. These antitumour agents can also limit tumor growth through accelerated senescence arrest [START_REF] Roninson | Tumor cell senescence in cancer treatment[END_REF][START_REF] Gewirtz | Accelerated senescence: an emerging role in tumor cell response to chemotherapy and radiation[END_REF][START_REF] Roninson | If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells[END_REF][START_REF] Chang | Molecular determinants of terminal growth arrest induced in tumor cells by a chemotherapeutic agent[END_REF]. Mitotic catastrophe occurs as a A c c e p t e d M a n u s c r i p t 3 consequence of the uncoupling of the onset of mitosis from DNA replication, but how the resulting lethality is regulated is not completely known. Because it is sometimes followed by caspase-dependent cell death, it may be also considered a special case of apoptosis [START_REF] Mansilla | Mitotic catastrophe as a consequence of chemotherapy[END_REF][START_REF] Castedo | Cell death by mitotic catastrophe: a molecular definition[END_REF].

Senescence-like growth arrest may be followed by mitotic catastrophe once the cells re-enter the cell cycle after drug treatment [START_REF] Roninson | If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells[END_REF][START_REF] Mansilla | Mitotic catastrophe as a consequence of chemotherapy[END_REF]. Senescence may also follow mitotic catastrophe if polyploid cells are arrested in G1 or G2 after mitotic catastrophe [START_REF] Roninson | If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells[END_REF][START_REF] Demidenko | Mechanism of G1-like arrest by low concentrations of paclitaxel: next cell cycle p53dependent arrest with sub G1 DNA content mediated by prolonged mitosis[END_REF], but the arrest is not permanent when, for example, cells lack p53 function, or the p53 and p21 WAF1 protein levels decrease after treatment with certain drugs [START_REF] Mansilla | Mitotic catastrophe results in cell death by caspasedependent and caspase-independent mechanisms[END_REF][START_REF] Chang | Role of p53 and p21 waf1/cip1 in senescence-like terminal proliferation arrest induced in human tumor cells by chemotherapeutic drugs[END_REF][START_REF] Roninson | If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells[END_REF][START_REF] Bataller | Mithramycin SK modulates polyploidy and cell death in colon carcinoma cells[END_REF][START_REF] Chang | Molecular determinants of terminal growth arrest induced in tumor cells by a chemotherapeutic agent[END_REF][START_REF] Blagosklonny | Mitotic arrest and cell fate: why and how mitotic inhibition of transcription drives mutually exclusive events[END_REF], or as a result of alterations in the phosphorilation of Chk1 [START_REF] Niida | Specific role of Chk1 phosphorylations in cell survival and checkpoint activation[END_REF]. Therefore, cells can re-enter the cell cycle, increasing their polyploidy before they eventually die. Some of those cells may escape cell death and undergo further polyploidization [START_REF] Rajaraman | Stem cells, senescence, neosis and self-renewal in cancer[END_REF]. DNA damage may provide the molecular background to trigger entry into an endocycle instead of mitosis [START_REF] Erenpreisa | Endopolyploidy in irradiated p53-deficient tumour cell lines: Persistence of cell division activity in giant cells expressing Aurora-B kinase[END_REF], which also explains the presence of large multinucleated cells. These cells may facilitate cell survival [START_REF] Ivanov | Endopolyploid cells produced after severe genotoxic damage have the potential to repair DNA double strand breaks[END_REF], and their presence has thus been linked to a poor response to chemotherapy. G2 arrest caused by DNA damage may provide the molecular conditions to trigger the onset of endoreduplication, while p21 WAF1 may concomitantly suppress the DNA damage response [START_REF] Ullah | Differentiation of trophoblast stem cells into giant cells is triggered by p57/Kip2 inhibition of CDK1 activity[END_REF]. It has been suggested that giant senescent cells can undergo a type of cell division known as neosis [START_REF] Rajaraman | Stem cells, senescence, neosis and self-renewal in cancer[END_REF][START_REF] Sundaram | Neosis: a novel type of cell division in cancer[END_REF], which is characterized by a peculiar karyokinesis that occurs via nuclear budding, followed by asymmetric intracellular cytokinesis that would produce small cells, termed Raju cells, with stem-cell-like characteristics and extended life span [START_REF] Rajaraman | Stem cells, senescence, neosis and self-renewal in cancer[END_REF]. It has been hypothesized that neosis is a mechanism of recurrent growth of resistant tumor cells after chemotherapy [START_REF] Rajaraman | Stem cells, senescence, neosis and self-renewal in cancer[END_REF].

During neosis, the spindle checkpoint would not be activated, which may give rise to aneuploidy [START_REF] Rajaraman | Stem cells, senescence, neosis and self-renewal in cancer[END_REF]. The prospect that endopolyploid tumor cells may have survival potential [START_REF] Erenpreisa | Segregation of genomes in polyploid tumour cells following mitotic catastrophe[END_REF] remains controversial [START_REF] Castedo | Cell death by mitotic catastrophe: a molecular definition[END_REF]. It has not been established whether neosis is a mechanism that evades cell death via mitotic catastrophe and whether Raju cells could facilitate tumor self-renewal, and thus allow recurrent growth of resistant tumor cells after chemotherapy. Aberrant chromosomal structures in aneuploid cells can generate nuclear projections referred to as 'buds', while the frequency of nuclear budding and micronucleation is increased by inactivating p53, suggesting that p53 would minimize the probability of forming broken chromosomes [START_REF] Shimizu | Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase[END_REF]. Some of the formed buds may lead to the appearance of micronuclei enriched in extrachromosomically amplified DNA. In addition, their formation may be followed by the elimination of redundant DNA, which will be consistent with the hypothesis that a non-selective DNA damage can lead to the elimination of extrachromosomal material from human cancer cells [START_REF] Shimizu | Nonselective DNA damage induced by a replication inhibitor results in the selective elimination of extrachromosomal double minutes from human cancer cells[END_REF]. We sought to gain new insights into the relationship between mitotic catastrophe, senescence and the presence of multinucleated cells, sometimes also containing micronuclei. We have used HCT116 human colon carcinoma cells that contain wild-type 53, treated with low nanomolar concentrations of doxorubicin, which do not trigger the apoptotic response. We examined whether a nuclear budding can generate aneuploid Raju cells. Moreover, we also explored whether the HCT116 (p53 +/+ ) cells that recover, after budding, from a treatment with moderate concentrations of doxorubicin were relatively resistant to treatment with a variety of antitumor agents and become invasive cells or whether they were committed to cell death. Our results suggest that after a nuclear budding procedure some cells recover their growing capacity.

It seems that excess DNA was extruded from the nuclei. Cells that eliminated the buds were proliferating and increased their time span. However, these buds should not be considered Raju cells (i.e. small aneuploid cells with stem cell characteristics).

Materials and methods

Drugs

Doxorubicin, etoposide (VP-16), camptothecin, cisplatin and paclitaxel were were purchased from Sigma (St. Louis, MO). WP631 was a generous gift of Dr. Waldemar Priebe (MD Anderson Cancer Center, Texas). Drug stocks were prepared as 1 mM solutions in sterile 150 mM NaCl or 100% DMSO, maintained at -20°C, and brought to the final concentration using cell culture media just before use.

Cell culture and drug treatments

HCT116 (p53 +/+ ) human colon carcinoma cells were maintained in 50% Dulbecco's MEM (Gibco, Invitrogen, Prat de Llobregat, Spain)/50% Ham's F12 (Lonza, Barcelona, Spain) medium, supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, 100 g/ml streptomycin, at 37 °C in a humidified atmosphere with 5% CO 2 . Exponentially growing cells subcultured at a density of 2.5 x 10 4 cells/ml were incubated with low nanomolar doses of doxorubicin at 37 °C for times indicated in the legend to figures.

Cytotoxicity assays

The effect of the different drugs on HCT116 cell growth was determined by the MTT method using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (Sigma), following 72-h treatment, as described elsewhere [START_REF] Villamarín | A comparative analysis of the time-dependent antiproliferative effects of daunorubicin and WP631[END_REF].

Cell cycle analysis and flow cytometry

A c c e p t e d M a n u s c r i p t 5 After treatment with doxorubicin for various periods of time, the cells were harvested, fixed with 70% ethanol, stained with PI (Propidium iodide, Sigma) and nuclei analyzed with a Coulter Epics-XL flow cytometer (Coulter Corporation, Hialeah, FL) as described elsewhere. [START_REF] Villamarín | A comparative analysis of the time-dependent antiproliferative effects of daunorubicin and WP631[END_REF] To analyze the mitotic fraction, fixed cells were incubated with the anti-phospho-histone H3 (ser 10) antibody (Upstate, Millipore, Madrid, Spain) followed by Cy2-conjugated secondary antibody (Jackson ImmunoResearch, Newmarket, UK). Stained cells were then counterstained with PI and analyzed for Cy2 and PI fluorescence by flow cytometry.

Determination of DNA synthesis

DNA synthesis was assayed by the incorporation of BrdU (5'-Bromo-2'-deoxy-uridine, Roche Diagnostics, Barcelona, Spain) using a fluorescence-conjugated antibody against BrdU (BD Biosciences, Madrid, Spain), and co-stained with PI.

Confocal microscopy, time-lapse imaging and immunofluorescence

HCT116 cells in exponential growth phase were stained, after drug treatment, with either DAPI (Sigma) or Draq5 (Molecular Probes, Invitrogen, Prat de Llobregat, Spain) and rinsed with drug free medium before analysis. Confocal laser microscopy was performed with a Leica confocal TCS-Sp2 AOBS microscope system (Leica Microsystems; Heidelberg, Germany) using the 488 nm line of an argon laser. Cell were contained in a temperature-controlled box (PeCon, Erbach, Germany) stabilized at 37 °C. Time-lapse sequences were collected using Nomarski differential interference microscopy, every 3 min for 72 h, using Leica confocal software (v. 2.61).

Assignment of senescence-like growth arrest

Senescence-like growth arrest was determined using two complementary criteria. Cell cycle analysis of cells by cytometry was used to detect elevated forward and site scatters, which reflect increased cell size and granularity, [START_REF] Biroccio | Inhibition of c-Myc oncoprotein limits the growth of human melanoma cells by inducing cellular crisis[END_REF] and the presence of senescence-associated -Gal staining at pH 6.0 (SA--Gal+ cells) [START_REF] Dimri | A biomarker that identifies senescent human cells in culture and in aging skin in vivo[END_REF] was observed by phase-contrast microscopy.

Western analysis of protein levels

Protein was extracted from doxorubicin-treated and control cells as described elsewhere [START_REF] Bataller | Mithramycin SK modulates polyploidy and cell death in colon carcinoma cells[END_REF].

Total protein was quantified by the Bradford assay (Bio-Rad, Hercules, CA). About 30 g of denatured proteins was subjected to electrophoresis on SDS-polyacrylamide gels, blotted onto Optitran BA-S85 membranes (Schleicher & Schuell, Dassel, Germany), probed with the specific antibodies -p53 (Santa Cruz, Quimigen, Spain), p21 WAF1 (Calbiochem, Merck, Madrid, Spain), cyclin B1 (Santa Cruz), cyclin D1 (Santa Cruz), Mos (Santa Cruz) and -actin (Sigma) used as a normalization control-, incubated with secondary antibodies (Jackson ImmunoResearch), and detected by chemiluminescence using Luminol (Sigma). The final fate of doxorubicin-treated HCT116 (p53 +/+ ) cells was analyzed at different times by using a clonogenic assay, according to a protocol described elsewhere [START_REF] Plumb | Cell sensitivity assays: clonogenic assay[END_REF], with minor modifications that included using 5% Giemsa (Sigma) for cell staining.

Clonogenic assay

Results

Transient senescence in HCT116 colon carcinoma cells treated with doxorubicin

We have analyzed the effects of nanomolar concentrations of doxorubicin on HCT116 cells bearing wild type p53. Treatment with 125 nM doxorubicin for 72 h (equivalent to its IC 50 ) followed by either incubation in free-drug medium after withdrawal of the drug, or a continuous treatment with the drug for more than 13 days produced similar effects on cell distribution in the various phases of the cell cycle (Fig. 1A). Between 24 and 72 h treatments about half of the cell population was halted in G2/M and underwent senescence (see below). One day after withdrawal of the drug, some HCT116 cells were polyploid (N > 4). A similar effect was observed after 4-days continuous treatment (cf. left and right panels in Fig. 1A). Timedependent monitoring of the fate of these cells showed they were distributed like in Fig. 1A (72h + 10 days), which may be ascribed to the recovery of cell cycle distribution after treatment.

According to cell staining with Trypan blue, about 20% cells, either in the floating or adhered population, were dying throughout the experimental analysis. The presence of polyploid cells was more evident during continuous treatments (Fig. 1A, right panel) and there was a shift of the peaks corresponding theoretically to G1 and G2 distribution, which may indicate that cells arrested in these phases were not the canonical 2N and 4N cells, but cells with some degree of polyploidization.

Figure 1B shows cytofluorimetric dot plots of the forward versus side scatter parameter.

The relatively high values revealed the presence of senescence among the cells treated with doxorubicin for the first 72 h, but not afterwards. The presence of senescent cells was confirmed by the specific lysosomal -gal staining of senescent cells, at pH 6, upon treatment with doxorubicin (Fig. 1C). Senescence was not observed after 72-h treatment irrespective of whether cells were maintained under continuous treatment with doxorubicin, or they were allowed to grow in fresh drug-free medium after withdrawal of the drug. This was understood to indicate they were re-entering the cell cycle (see Fig. 1A).

Endoreduplication rather than mitosis generated giant multinucleate cells

Having established the presence of a transient senescence-like arrest in doxorubicin-treated cells, we next sought to discern whether cells transiently arrested in G1 were entering the Sphase, in which DNA synthesis took place (measured by BrdU incorporation), and whether A c c e p t e d M a n u s c r i p t 7 those in G2 phase were entering mitosis. To this end, cells treated with doxorubicin were tested for the presence of H3pser10, a histone variant only expressed during mitosis, by using a specific antibody, thus to substantiate that the cells that were synthesizing DNA did not enter several rounds of mitosis, but they were underwent endoreduplication (Fig. 2). Up to about 48-h treatments, DNA synthesis was active from both G1 and G2-like aneuploid cells, and also in cells with a > 4N content (Fig. 2), regardless of whether they were allowed to grow in fresh medium after withdrawal of the drug, or they were under continuous drug treatment. However, after longer treatments with doxorubicin, DNA synthesis was inhibited. The presence of changes in the levels of several proteins that might participate in cell arrest in G1 and G2 will be considered in a following paragraph. Furthermore, at no time did mitosis increase compared to control cells (Fig. 2). Nevertheless, it is noteworthy that after 7 days under continuous treatment with doxorubicin a few endopolyploid cells had entered mitosis (bottom-right panel in Fig. 2).

Nuclear budding in drug-treated cells is related to de-polyploidization and cell recovery

Under the microscope, doxorubicin-treated HCT116 (p53 +/+ ) cells were large and contained more than one nucleus (Fig. 3B). Moreover, there was a time-dependent increase in the presence of peri-nuclear buds in all the cells -some of which are indicated by arrows in several panels of Fig. 3-. Sometimes, they covered almost the entire cytoplasm (Fig. 3F). These "bubble-like" cytoplasmic structures were not lysosomes since they stained negatively with Lysotracker (Molecular Probes) (not shown) but nuclear buds. After 8-days continuous treatment, the morphology of some surviving cells was similar to that of untreated HCT116 cells in respect to cell size, although they tended to grow in clusters (Fig. 3G). In addition, some living cells were unattached (floating), as judged by the absence of Trypan blue staining in this population (cf. panels A and H in Fig. 3). This observation indicated that some of the cells recovered after treatment or, alternatively, that budding nuclei may result in the generation of daughter cells that were released to the media. Such cells may hypothetically be Raju cells, so they may possess stem cell properties [START_REF] Rajaraman | Stem cells, senescence, neosis and self-renewal in cancer[END_REF].

Therefore, we examined whether the "bubble-like" structures surrounding the cell nuclei, which sometimes covered almost the whole cell cytoplasm (Fig. 3F), were due to budding, and whether these structures contained genetic material. A time-lapse video analysis of drug-treated cells that documents that nuclear budding was a dynamic process. This is presented as supplementary information (Supplementary movie 1). In this video, budding progression can be observed as "bubbles" moving away from the nucleus.

We next examined whether budding removed the excess of DNA in cells overcoming senescence or mitotic catastrophe, or it was a step towards the formation of aneuploid cells with Figure 4A shows the presence of giant doxorubicin-treated cells containing four macronuclei, which stained with DAPI, while Fig. 4B shows the staining of multinucleated cells (upper part of the panel), which can contain a macronucleus surrounded by some small micronuclei. As mentioned above, the micronuclei and some of the buds that might be produced via budding in the vicinity of the larger nuclei contained DNA, as indicated by their staining with DAPI (indicated by arrows in Figs. 4B and4C), while, in other cases, they did not appear to contain any nucleic acid (Fig. 4D). Both the presence/absence of DNA, and the peri-nuclear location of the emerging buds were confirmed by time-lapse video microscopy using the fluorescent Draq5 dye, which can detect smaller quantities of DNA (Fig. 4F). Figures 4E and4F show photograms taken from the time-lapse video, which allowed us to compare the bright field image of a doxorubicin-treated cell (panel E) alongside the same cell stained with Draq5 (panel F). DNA was labeled in both the nucleus and most of the peri-nuclear buds. A complete time-lapse video is shown as Supplementary movie 2.

Several changes in protein levels concur with endoreduplication and nuclear budding

Time-dependent changes in p53, p21 WAF , cyclin D1, cyclin B1 and Mos protein levels after treatment with doxorubicin are shown in Fig. 5. While p21 WAF1 levels increased upon treatment and remained high and they did not change in 3 days treatment, the level of p53 increased slightly in control (untreated cell) after 48 h treatment, while they remained low in drug-treated cells.

Cyclin B1 levels decreased upon treatment, and only about 19% remained after removal of the drug followed by cell growing in drug-free medium, while about 21% remained after 7-days continuous treatment. This decrease in cyclin B1 levels may be related to the absence of mitosis and the observed increase in polyploidization [START_REF] Ianzini | Lack of p53 function promotes radiation-induced mitotic catastrophe in mouse embryonic fibroblast cells[END_REF]. Deregulation of cyclin B1 may be mediated by changes in p53 protein levels, which is a post-transcriptional regulator of cyclin B1 [START_REF] Ianzini | Lack of p53 function promotes radiation-induced mitotic catastrophe in mouse embryonic fibroblast cells[END_REF]. In the absence of regulation by p53, p21 WAF1 remained high (Fig. 5) after treatment and throughout the budding process. Levels of cyclin D, which is mostly associated to G1 phase, increased (Fig. 5), which may indicate that cells recovering after treatment may be entering a G1-like phase before re-cycling. The levels of Mos increased after treatment, which indicates that cells "leaving" a senescence state were sensed for they high DNA content. These results suggest that nuclear budding is to some extent controlled genetically. In line with the changes observed in protein levels, polyploid cells could arise as a consequence of DNA synthesis in the absence of

After nuclear budding some cells recover, proliferate and they might grow to be multidrugresistant cells

As described above, most of the doxorubicin-treated cells remained alive after the budding procedure (Fig. 3 G-H). A clonogenic assay was performed by seeding cells at different time intervals during the treatments (Fig. 6A). After 72 h continuous treatments colonies (≥50 cells)

were not observed in concordance with these cells were senescent (Fig. 1 B-C). After budding, the generated cells proliferated, although with different efficiencies that depended on whether they grew in the absence or presence of the drug (Fig. 6A).

Both adherent and floating cells were harvested at the times in which proliferation was observed during the clonogenic assays, plated in fresh drug-free medium, and allowed to grow for additional 24 h. Afterwards, six antitumor drugs with different mechanisms of action (doxorubicin, WP631, etoposide (VP-16), camptothecin, paclitaxel and cisplatin) were incubated with such cells for 72 h, and their survival was measured by the MTT method (Fig. 6B). Only continuous treatment with several of these drugs disclosed a significant increase of drug-resistant cells (Student's t-test, Fig. 6B). This outcome was not seen when cells were allowed to grow in fresh drug-free medium after withdrawal of the drugs (Fig. 6B). The anthracyclines (doxorubicin and WP631), of which the former is considered a topoisomerase II poison, like etoposide (VP16), are among the drugs to which the HCT116 became less sensitive.

After budding, cells were also less sensitive to paclitaxel, a mitotic inhibitor. On the other hand, cells growing after budding were neither more resistant to the topoisomerase I inhibitor camptothecin nor to the DNA-alkylating agent cisplatin (Fig. 6B).

These results indicate that surviving cells were susceptible to a variety of drugs, and resistant cells only arose following the previous treatment with nanomolar concentrations of doxorubicin when the contact with the new drugs was continuous. This cell behavior may be independent of whether the cells arose from the micronuclei shown in Figs. 3 and4, or as the results of nuclear budding, which tentatively would generate daughter cells more resistant to antitumor drugs.

Discussion

The relevance of senescence and non-apoptotic routes to cell death has been overshadowed by the high interest in apoptosis. Evidence is accumulating that senescence may play a key role in chemotherapy outcome [START_REF] Chang | p21 Waf1/Cip1/Sdi1 -induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells[END_REF][START_REF] Gewirtz | Accelerated senescence: an emerging role in tumor cell response to chemotherapy and radiation[END_REF][START_REF] Chang | Molecular determinants of terminal growth arrest induced in tumor cells by a chemotherapeutic agent[END_REF]. Low doses of doxorubicin are a well-known inductor of A c c e p t e d M a n u s c r i p t 10 senescence in HCT116 cells containing wild-type 53 [START_REF] Chang | p21 Waf1/Cip1/Sdi1 -induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells[END_REF][START_REF] Gewirtz | Accelerated senescence: an emerging role in tumor cell response to chemotherapy and radiation[END_REF]. Both senescence and mitotic catastrophe may result in the presence of giant multinuclate cells. Genetic destabilization can be produced when they re-enter the cell cycle after a prolonged p21 WAF1 induction [START_REF] Chang | p21 Waf1/Cip1/Sdi1 -induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells[END_REF]. Here, we have shown that endopolyploidization occurs in the presence of DNA synthesis (Fig. 2) without cells entering mitosis, so giant cells can emerge from G1-like or G2-like aneuploid cells. These results are in agreement with a recent report that indicates that induction of senescence by doxorubicin leads to increased genomic instability in HCT116 cells [START_REF] Sliwinska | Induction of senescence with doxorubicin leads to increased genomic instability of HCT116 cells[END_REF]. After treatments with low nanomolar concentrations of doxorubicin, p21 WAF1 levels were higher than in control cells, an enhancement that was observable from 24 h onwards (Fig. 5). In fact, there was overexpression of p21 WAF1 throughout all the budding process, which is consistent with the role of this protein in the development of polyploidy [START_REF] Chang | p21 Waf1/Cip1/Sdi1 -induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells[END_REF][START_REF] Niculescu | Effects of p21 (Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication[END_REF] and its role of this protein in protecting cells against doxorubicin-induced mitotic catastrophe [START_REF] Lin | Mechanisms underlying the pro-survival pathway of p53 in suppressing mitotic death induced by adriamycin[END_REF]. Accordingly, p21 WAF1 -mediated depletion of some checkpoint control proteins may result in endoreduplication (Fig. 5 and ref. [START_REF] Chang | p21 Waf1/Cip1/Sdi1 -induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells[END_REF]), while p21 WAF1 also maintained the halt in G2 in absence of cells entering mitosis, even though p53 levels decreased during long treatments. The levels of Mos, a protein involved in the control of meiosis [START_REF] Kalejs | Upregulation of meiosis-specific genes in lymphoma cell lines following genotoxic insult and induction of mitotic catastrophe[END_REF], were enhanced, which would indicate the presence of some type of control of the budding process, in agreement with the synergy that has described between the Mos/mitogen-activated protein kinase pathway and the loss of p53 function in the occurrence of chromosome instability [START_REF] Fukasawa | Synergy between the Mos/mitogen-activated protein kinase pathway and loss of p53 function in transformation and chromosome instability[END_REF]; in our case a time-dependent decrease in p53 levels (Fig. 5). The increase in Mos levels occurred in parallel to the decrease in p53 (Fig. 5), which is coherent with the previously observed Mos increase observed in p53 -/-cells [START_REF] Fukasawa | Synergy between the Mos/mitogen-activated protein kinase pathway and loss of p53 function in transformation and chromosome instability[END_REF].

At first glance, the observation of multinucleated giant cells in absence of mitosis and in the presence of DNA synthesis (Fig. 2) is at variance with previous observations of the behavior of cells treated with low concentrations of doxorubicin, which cells entered mitosis and showed abnormal mitotic figures. We did not observe a release from p21 WAF1 -mediated growth arrest upon drug-treatment, which may explain why cells did not enter mitosis and mitotic catastrophe [START_REF] Bunz | Requirement for p53 and p21 to sustain G2 arrest after DNA damage[END_REF]. Induction of p21 WAF1 in cells containing wild-type p53 may result in genetic destabilization [START_REF] Chang | p21 Waf1/Cip1/Sdi1 -induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells[END_REF][START_REF] Broude | p21 (CDKN1A) is a negative regulator of p53 stability[END_REF]. However, the p21 WAF1 levels did not maintain growth arrest and senescence; tentatively because of the changes in other protein levels (Fig. 5). The p21 WAF1 induction was, in this case, observed while p53 was down-regulated, which indicates that the formation of endopolyploid cells might be not an exclusive characteristic of cells lacking wild-type p53, but it can be induced by low doses of some antitumor agents in p53 +/+ cells.

The production of buds was sometimes so intense that cells appear to be filled by "bubblelike" structures (Fig. 3F). A mode of cell division that may be related to budding has been A c c e p t e d M a n u s c r i p t 11 described and referred to as neosis, which is defined as a parasexual reduction division resulting in polyploid-multinucleate cells [START_REF] Sundaram | Neosis: a novel type of cell division in cancer[END_REF]. According to this novel mode of cell division, budding nuclei would distribute genetic material to small daughter cells. Such small aneuploid cells (Raju cells) may have an extended life span and be multidrug-resistant tumor cells [START_REF] Rajaraman | Stem cells, senescence, neosis and self-renewal in cancer[END_REF].

Although our results show that at the end of a prolonged treatment some cells can recover (Fig. 3G andH), the budding process did not provide evidence as to whether the small buds leaving the nuclei were Raju-like cells. An additional problem in considering the possibility that the buds may generate actual Raju daughter cells is our lack of understanding of how the buds could acquire the minimum cell organelles needed for them to be considered as potential stem cells. Time-lapse videos, shown as supplementary material, indicated that cytoplasm was not included during the budding process. We favor an alternative explanation based on the idea that a portion of the cell population might escape death through de-polyploidization. This could mean that some "extra DNA" was extruded from the nuclei via budding. Cells would thus bear more acceptable level of aneuploidy/polyploidy. This would be concordant with reports that some inhibitors of replication increase micronucleation and formation of buds containing autonomous replicating extrachromosomal structures that are lost spontaneously in absence of selection [START_REF] Shimizu | Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase[END_REF]. Some buds were stained with the DNA-binding dyes DAPI or Draq5 (Fig. 4). They could contain chromosome fragments, which are difficult to envisage as potential independent cells. We conclude that cells recovering after budding were some of the former large aneuploid cells rather than 'new' cells produced by neosis. An enhancement in the levels of the meiosis-related protein Mos was observed, which agrees with the view that cells somehow detect the presence of extra DNA (as it occurs during the first meiotic division [START_REF] Kalejs | Upregulation of meiosis-specific genes in lymphoma cell lines following genotoxic insult and induction of mitotic catastrophe[END_REF]).

As mentioned above, what is peculiar in the present study is that a nuclear budding procedure might reduce the polyploidy of giant cells bearing wild-type p53 protein, the levels of which were reduced by drug treatment. In contrast, a significant proportion of the buds leaving the nuclei in doxorubicin-treated cells did not contain DNA. This experimental observation would agree with a somewhat stochastic liberation of material, yet the presence of meiotic genes suggests that some sort of control could exist. These observations are reminiscent of a selective elimination of extrachromosomal 'double minutes' by replication inhibitors [START_REF] Shimizu | Nonselective DNA damage induced by a replication inhibitor results in the selective elimination of extrachromosomal double minutes from human cancer cells[END_REF].

A clonogenic assay (Fig. 6A) showed that after the budding process most of the surviving HCT116 cells proliferated for a long time, yet they displayed different degrees of susceptibility to a variety of drugs (Fig. 6B). Cells that recovered from the initial treatment were significantly more resistant to several antitumor drugs when they were incubated continuously for 33 days.

A c c e p t e d M a n u s c r i p t 12 Conversely, when those cells were allowed to recover in fresh drug-free medium after withdrawal of the drug they were drug sensitive (Fig. 6).

One known role of p53 is to avert aneuploidy by blocking endoreduplication. Low nanomolar concentrations of doxorubicin produced a transient increment in p53 levels, which was followed by a reduction of p53, compared to untreated cells (Fig. 5). This might occur through a direct effect of doxorubicin on gene transcription [START_REF] Chang | Molecular determinants of terminal growth arrest induced in tumor cells by a chemotherapeutic agent[END_REF][START_REF] Mansilla | Sp1 transcription factor as a target for anthracyclines: Effects on gene transcription[END_REF]. The enhancement of p21 WAF1 levels in tumor cells during chemotherapy has been considered to contribute to carcinogenesis and tumor progression, as a consequence of genetic destabilization [START_REF] Niculescu | Effects of p21 (Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication[END_REF], but also to the growth arrest during senescence [START_REF] Chang | p21 Waf1/Cip1/Sdi1 -induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells[END_REF][START_REF] Roninson | Tumor cell senescence in cancer treatment[END_REF]. The results presented here point out that increased ploidy, which is also associated with p21 WAF1 , does not necessarily lead to more invasive cells.

Although not yet directly proven, our observations suggest that nuclear budding does not generate Raju cells -which are deemed to be rather tumorogenic [START_REF] Rajaraman | Stem cells, senescence, neosis and self-renewal in cancer[END_REF]-, nor does it improve the life span of the cell. Additional elucidations of the mechanisms that control endoreduplication should provide a more complete understanding of cell death and resistance to antitumor agents. In this context, it is remarkable that cells that survived the primary treatment with moderate concentrations of doxorubicin only developed drug resistance when they were maintained under continuous contact with a selecting drug (Fig. 6B), an observation with potential clinical relevance that will merit further consideration. The clonogenic capacity was determined as the percentage of colonies containing ≥ 50 cells, after a posterior incubation for the times indicated at the left side. After 72 h treatments there was no proliferation in keeping with the presence of senescence (see also, Figs. 1B and1C). After 72-h treatments with doxorubicin followed by 30 days incubation in fresh drug-free medium: 21.0%, 26.0 and 48.7% colonies were quantified after 10 days, compared to the control (100%), while 33 days of continuos doxorubicin-treatment rendered 20.9, 27.0 and 56.6% colonies respectively.

(B) Analysis of the HCT116 cell proliferation using cells that survived to the treatment with moderate doses of doxorubicin. Cells recovering after treatment with doxorubicin for 72 h

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

 8 survival capacity. An additional problem with the second possibility is how these aneuploid nuclei can obtain enough cytoplasm organelles components to become Raju cells.

  could be the consequence of the inhibition of mitosis-associated proteins by p21 WAF1[START_REF] Chang | p21 Waf1/Cip1/Sdi1 -induced growth arrest is associated with depletion of mitosis-control proteins and leads to abnormal mitosis and endoreduplication in recovering cells[END_REF].

Fig. 1 .

 1 Fig. 1. (A) Flow cytometry analyses of HCT116 (p53 +/+ ) colon carcinoma cells treated with a 125 nM doxorubicin (equivalent to its IC 50 ). The experiments displayed correspond to either 72 h incubation with the drug followed by incubation in fresh drug-free medium after withdrawal of the drug (left panel) or continuous treatments for the times stated (right panel). Although in drugtreated cells the different peaks are market as and 2N or 4N for the convenience of uniform plotting, they may contain a higher, aneuploid, DNA content (see main text for further details). (B) Detection of senescence-like arrest in doxorubicin-treated HCT116 cells by cytofluorimetric dot plots of forward (x-axis) versus side (y-axis) scatter parameters.High values of both parameters are observed after 48 h and 72 h treatments. (C) Microscopic analysis of SA--gal expression at pH 6 (a subrogate marker of senescence) in doxorubicin-treated HCT116 cells for 48h shows the presence of senescence.

Fig. 2 .

 2 Fig. 2. (A) Bromodeoxyuridine (BrdU) incorporation in the absence (control), or during treatment with 125 nM doxorubicin, shows the presence of DNA synthesis (BrdU-positive cells) depending on the phase of the cell cycle (PI staining), which suggests cells were labeled in S phase and they underwent endoreduplication in the absence of mitosis (see panel (B)). (B) Assay for the simultaneous detection of diploid and polyploid cells by PI and cells in mitosis by an antibody against H3pS10 (specific marker Ser-10-phosphorylated histone H3). The percentage of mitotic cells was rather small, even though increased ploidy was evident, which indicates the presence of endoreduplication.

Fig. 3 .

 3 Fig. 3. Microscopic analysis of HCT116 (p53 +/+ ) cells treated with 125 nM doxorubicin show the presence of giant cells with macronuclei and peri-nuclear "bubble" structures (indicated by arrows) formed by a budding process. (A) Untreated cells. (B, C and F) Cells treated for 72 h, followed by incubation in fresh drug-free medium for additional 4 days, 7 days or 12 days respectively, or (D) Cells treated continuously during 6 days. Panels E and F show some examples of cells in which budding ended in "bubble-like" structures covering almost the entire cytoplasm. (G and H) Cells that recovered after treatment with doxorubicin were allowed to grow for 33 days in fresh drug-free medium after withdrawal of the drug. Panel G shows attached cells, while those in panel H are floating cells, which were alive according to their exclusion of Trypan blue (bars correspond to 50 m). See also Fig. 4 and the two time-lapse videos shown as Supplementary material on line.

Fig. 4 .

 4 Fig. 4. Morphological analysis of doxorubicin-treated HCT116 cells stained with the DNAbinding dyes DAPI or Draq5. (A) Cells treated with 125 nM doxorubicin show macronuclei, which stained with DAPI. (B) Cells treated with 125 nM doxorubicin for 72 h and allowed to grow in fresh drug-free medium for additional 4 days show the presence of peri-nuclear buds. (C and D) HCT116 cells treated continuously with doxorubicin for 72 days also show the presence of buds. The presence of buds in some cells is indicated by arrows, (bars correspond to 50 m). (E) Bright-field photogram of a time-lapse microscopy analysis of HCT116 cells treated with 125 nM doxorubicin for 72 h, allowed to grow in fresh drug-free medium during 3 days, and recorded afterwards. (F) Image of the cell shown in panel E stained with Draq5. Fluorescence is observed in the nucleus and inside most of the peri-nuclear budding structures. The complete time-lapse video is shown as Supplementary material on line.

Fig. 5 .

 5 Fig. 5. Western blot analysis of changes in protein levels in HCT116 (p53 +/+ ) treated with a low (125 nM) doxorubicin concentration for the times indicated at the top of the figure. For the sake of comparison, the distribution of the cells in the different phases of the cell cycle is also displayed at the top. Lane a: control, untreated, cells, lanes b-d: protein levels after continuous treatment with 125 nM doxorubicin, lanes e and f: cells treated with 125 nM doxorubicin for 72 h were allowed to grow for additional 1 or 4 days in drug-free medium after withdrawal of the drug, lanes g and h: cells treated continuously with 125 nM doxorubicin for 4 or 7 days respectively.

Fig. 6 .

 6 Fig. 6. Analysis of the fate of HCT116 (p53 +/+ ) cells recovering after treatment with doxorubicin. (A) Clonogenic assays showing the proliferation of HCT116 cells treated with 125 nM doxorubicin for different periods of time. The times indicated at the top correspond to the moment at which doxorubicin-treated cells were re-seeded (1x10 3 cells in 60 mm Petri dishes).

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Acknowledgements

We thank Dr. Lidia Bardia for confocal microscopy support and advice. Supported by grants from the former Spanish Ministry of Education and Science: BFU2007-60998, and the FEDER program of the European Community. M.B. is recipient of a fellowship from the 'Parc Cientific de Barcelona-CSIC'.


Ana Gomes

PhD, REQUIMTE Eduarda Fernandes

Artur M S Silva

Clementina M M Santos

Diana C G A Pinto

José A S Cavaleiro

José L F C Lima

Anti-inflammatory potential of 2-styrylchromones regarding their interference with arachidonic acid metabolic pathways

Keywords: 2-Styrylchromones, Cyclooxygenase, 5-Lipoxigenase, Leukotriene B 4, Dual inhibitors, Inflammation

A c c e p t e d M a n u s c r i p t 2 Abstract Cyclooxygenases (COXs) are the key enzymes in the biosynthesis of prostanoids. COX-1 is a constitutive enzyme while the expression of COX-2 is highly stimulated in the event of inflammatory processes, leading to the production of large amounts of prostaglandins (PGs), in particular PGE 2 and PGI 2 , which are pro-inflammatory mediators.

Lipoxygenases (LOXs) are enzymes that produce hydroxy acids and leukotrienes (LTs). 5-LOX metabolizes arachidonic acid to yield, among other products, LTB 4 , a potent chemoattractant mediator of inflammation.

The aim of the present work was to evaluate the anti-inflammatory potential of 2styrylchromones (2-SC), a chemical family of oxygen heterocyclic compounds, vinylogues of flavones (2-phenylchromones), by studying their COX-1 and COX-2 inhibitory capacity as well as their effects on the LTB 4 production by stimulated human polymorphonuclear leukocytes (PMNL). Some of the tested 2-SC were able to inhibit both COX-1 activity and LTB 4 production which makes them dual inhibitors of the COX and 5-LOX pathways. The most effective compounds in this study were those having structural moieties with proved antioxidant activity (3',4'-catechol and 4'-phenol substituted B-rings). This type of compounds may exhibit anti-inflammatory activity with a wider spectrum than that of classical non-steroidal anti-inflammatory drugs (NSAIDs) by inhibiting 5-LOX productmediated inflammatory reactions, towards which NSAIDs are ineffective.

Introduction

Arachidonic acid is the most abundant polyunsaturated fatty acid in the phospholipid bilayer of cell membranes. This second-messenger molecule is released by phospholipase A 2 in activated cells and further metabolized in different eicosanoids by the cyclooxygenase and lipoxygenase pathways (Fig. 1). Cyclooxygenases (COXs) are the key enzymes in the biosynthesis of prostanoids. In mammalian cells, COXs exist in at least two isoforms (COX-1 and COX-2).

COX-1 is a constitutive enzyme, expressed in almost every cell type, responsible for the physiological production of prostaglandins (PGs) i.e., PGI 2 , PGE 2 , PGF 2α , and PGD 2 , in diverse organs and thromboxane A 2 in platelets. COX-2, on the other hand, is usually absent in most tissues (with the exception of kidney, parts of the brain, and gravid uterus where COX-2 is constitutive) but its expression can be readily induced by numerous stimuli like such as growth factors, tumor promoters, or cytokines [START_REF] Kam | Cyclo-oxygenase isoenzymes: physiological and pharmacological role[END_REF][START_REF] Cipollone | Cyclooxygenase and prostaglandin synthases in atherosclerosis: Recent insights and future perspectives[END_REF]. The earliest expression of COX-2 is detected in stromal cells, but it also can be found in multiple cells, like epithelial, endothelial, and stromal cells in several types of tumors [START_REF] Wang | Cyclooxygenases, prostanoids, and tumor progression[END_REF]. In addition, COX-2 is highly expressed by cells that are involved in inflammation (e.g., neutrophils, macrophages, monocytes, mast cells, synoviocytes), emerging as the isoform primarily responsible for the synthesis of the prostanoids involved in pathological processes, such as acute and chronic inflammatory states. These inflammationrelated cell types can be stimulated by lipopolysaccharide, phorbol esters, cytokines, or growth factors, producing large amounts of PGs, in particular PGE 2 and PGI 2 , which are pro-A c c e p t e d M a n u s c r i p t 4 inflammatory mediators that increase vascular permeability and promote edema at the sites of inflammation. Furthermore, these PGs are involved in the nociception of the inflammatory pain [START_REF] Davies | The Role of Arachidonic-Acid Oxygenation Products in Pain and Inflammation[END_REF]. Thus, COX-2 isoform has been associated with acute and chronic inflammatory disorders [START_REF] Vane | Anti-inflammatory drugs and their mechanism of action[END_REF] as well as in the carcinogenesis and tumor progression [START_REF] Wang | Cyclooxygenases, prostanoids, and tumor progression[END_REF].

Lipoxygenases (LOXs) are enzymes that produce hydroxy acids and leukotrienes (LTs). From the LOXs existent in the mammalian tissues, 5-LOX, which is mainly found in cells of myeloid origin, i.e., polymorphonuclear leukocytes (PMNL), mast cells, macrophages, is the most implicated in inflammatory and allergical disorders [START_REF] Jampilek | 5-lipoxygenase, leukotrienes biosynthesis and potential antileukotrienic agents[END_REF]. 5-LOX metabolizes arachidonic acid to yield 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), which is further metabolized to the bioactive 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) by PMNL 5-hydroxyeicosanoid dehydrogenase [START_REF] Powell | Metabolism of 5(S)-Hydroxy-6,8,11,14-Eicosatetraenoic Acid and Other 5(S)-Hydroxyeicosanoids by a Specific Dehydrogenase in Human Polymorphonuclear Leukocytes[END_REF], and various LTs (LTA 4 -LTE 4 ) [START_REF] Samuelsson | Leukotrienes and Lipoxins -Structures, Biosynthesis, and Biological Effects[END_REF]. 5-oxo-ETE and LTB 4 , produced by PMNL, are potent chemoattractant mediators of inflammation. LTB 4 stimulates neutrophil chemotaxis, enhances neutrophil-endothelial interactions, and stimulates neutrophil activation, leading to degranulation and the release of mediators, enzymes, and superoxide radicals [START_REF] Busse | Leukotrienes and inflammation[END_REF], while 5-oxo-ETE is much more active than LTB 4 as an eosinophil chemoattractant [START_REF] Stamatiou | 5-oxo-ETE induces pulmonary eosinophilia in an integrin-dependent manner in brown Norway rats[END_REF]. LTC 4 , LTD [START_REF] Davies | The Role of Arachidonic-Acid Oxygenation Products in Pain and Inflammation[END_REF] and LTE 4 , also known as Cys-leukotrienes, have shown to be essential mediators in asthma pathophysiology [START_REF] Sharma | The role of leukotrienes in the pathophysiology of inflammatory disorders: is there a case for revisiting leukotrienes as therapeutic targets?[END_REF].

Although the major pathophysiological implication of LTs was considered to be the bronchial asthma, these eicosanoids contribute to the pathogenesis of other human inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, Crohn's disease, and psoriasis (see [START_REF] Sharma | The role of leukotrienes in the pathophysiology of inflammatory disorders: is there a case for revisiting leukotrienes as therapeutic targets?[END_REF][START_REF] Werz | Therapeutic options for 5-lipoxygenase inhibitors[END_REF][START_REF] Khanapure | Eicosanoids in inflammation: Biosynthesis, pharmacology, and therapeutic frontiers[END_REF] for reviews). Recently, the 5-LOX pathway has also been associated with atherosclerosis, osteoporosis and certain types of cancer like prostate cancer [START_REF] Werz | Therapeutic options for 5-lipoxygenase inhibitors[END_REF].

A c c e p t e d M a n u s c r i p t 5 Non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used remedy in inflammatory disorders. However, they cause several adverse effects, the most important being gastric injury up to gastric ulceration, renal failure and asthma [START_REF] Bertolini | Selective COX-2 inhibitors and dual acting anti-inflammatory drugs: critical remarks[END_REF]. On the other hand, the COX-2 selective drugs, generically known as COXIBs, have recently been a cause of controversy due to the enhanced cardiovascular risk they carry [START_REF] Khanapure | Eicosanoids in inflammation: Biosynthesis, pharmacology, and therapeutic frontiers[END_REF]. Thus, alternative therapeutic solutions, with similar anti-inflammatory potency but with fewer side effects, are needed, especially for the control of chronic inflammatory diseases, which implicate longer therapies.

2-Styrylchromones (2-SC) are a chemical family of oxygen heterocyclic compounds, vinylogues of flavones (2-phenylchromones), whose occurrence in nature has been reported [START_REF] Gerwick | Hormothamnione, a novel cytotoxic styrylchromone from the marine cyanophyte Hormothamnion enteromorphoides grunow[END_REF][START_REF] Gerwick | 6-Desmethoxyhormothamnione, a new cytotoxic styrylchromone from the marine cryptophyte Chrysophaeum taylori[END_REF]. Natural and synthetic 2-styrylchromones have been tested in different chemical and biological systems, showing activities with potential therapeutic applications [START_REF] Doria | Antiallergic agents. III. Substituted trans-2-ethenyl-4-oxo-4H-1-benzopyran-6-carboxylic acids[END_REF][START_REF] Desideri | Synthesis and anti-rhinovirus activity of 2styrylchromones[END_REF][START_REF] Brion | Preparation of 2-styryl-4chromanones as anticancer agents[END_REF][START_REF] Karton | Synthesis and biological activities of flavonoid derivatives as A3 adenosine receptor antagonists[END_REF][START_REF] Fernandes | 2-Styrylchromones as novel inhibitors of xanthine oxidase. A structure-activity study[END_REF][START_REF] Fernandes | Hepatoprotective activity of polyhydroxylated 2-styrylchromones against tert-butylhydroperoxide induced toxicity in freshly isolated rat hepatocytes[END_REF][START_REF] Filipe | Polyhydroxylated 2styrylchromones as potent antioxidants[END_REF][START_REF] Gomes | 2-Styrylchromones: novel strong scavengers of reactive oxygen and nitrogen species[END_REF]. The antiinflammatory potential of 2-styrylchromones, concerning their interference with the arachidonic acid metabolic pathways, has not been explored so far. However, this may represent a promising field of research considering the potent inhibition of eicosanoids production by certain flavonoids containing structural similarities with 2-SC (see [START_REF] Werz | Inhibition of 5-lipoxygenase product synthesis by natural compounds of plant origin[END_REF] for review). Therefore, the purpose of this work was to evaluate the inhibition of COX-1 and COX-2 activities, in a cell-free system, as well as the inhibition of LTB 4 production, in human PMNL, by a group of 2-SC (Fig. 2).

-Please place fig. 1 2-Styrylchromones 1A-1D, 2A-2D and 3A-3D were synthesised by the three step Baker-Venkataraman method, starting from the O-protected 2'-hydroxyacetophenones and cinnamoyl chlorides [START_REF] Santos | Synthesis of new hydroxy-2-styrylchromones[END_REF]. The first step involves the O-acylation of appropriate 2'-hydroxyacetophenones with cinnamoyl chloride derivatives to give 2'-cinnamoyloxyacetophenones. These intermediates were converted into 5-aryl-3-hydroxy-1-(2-hydroxyaryl)-2,4-pentadien-1-ones by the basecatalysed Baker-Venkataraman rearrangement [START_REF] Baker | Molecular rearrangement of some o-acyloxyacetophenones and the mechanism of the production of 3-acylchromones[END_REF][START_REF] Mahal | Synthetical experiments in the chromone group. Part XIV. The action of sodamide on 1-acyloxy-2-acetonaphthones[END_REF][START_REF] Price | 2-Styrylchromones -Biological Action, Synthesis and Reactivity[END_REF]. The cyclodehydration of these βdiketones with a mixture of DMSO and a catalytic amount of iodine or p-toluenesulfonic acid gives polybenzyloxy-2-SC, which were debenzylated by treatment with hydrogen bromide in acetic acid at reflux, to give the expected hydroxy-2-SC 1A-1D, 2A-2D and 3A-3D.

Equipment

A c c e p t e d M a n u s c r i p t 7 A microplate reader (Synergy HT, BIO-TEK), was used to perform the spectrophotometric readings in all the assays.

Determination of LTB 4 production by human PMNL

Human PMNL were isolated from peripheral blood of healthy volunteers as previously described [START_REF] Costa | Inhibition of human neutrophil oxidative burst by pyrazolone derivatives[END_REF]. Neutrophil suspensions (5 x 10 6 cells/mL) in HBSS were pre-incubated at 37ºC for 10 min with the 2-SC (25 and 10 µM) or with the lipoxygenase inhibitor, NDGA (1 µM). The cells were subsequently incubated with A23187 (5 µM) and arachidonic acid (10 µg/mL) for 8 min. The reactions were stopped by the addition of cold methanol. Samples were subsequently centrifuged at 13,000 x g for 1 min, and the supernatants were stored at -70ºC until analysis. The amount of LTB 4 in the samples was measured using the above mentioned commercial EIA kit, according to the manufacturer's instructions. The results were expressed as the percent inhibition of control LTB 4 production. At least four determinations were done for each experiment.

COX-1 and COX-2 inhibition assays

The inhibition of COX-1 (ovine) and COX-2 (human recombinant) by 2-SC was determined in a cell-free system by quantifying the levels of PGF 2α , produced by catalysis of arachidonic acid, using the above mentioned specific EIA kit according to the manufacturer's instructions.

The COX inhibitors indomethacin and celecoxib were used as positive controls. The results were expressed as the percent inhibition of control COX-1 or COX-2 activity. At least four determinations were done for each experiment. The reductive potential of 2-SC was determined according to a previously described method [START_REF] Mathew | In vitro antioxidant activity and scavenging effects of Cinnamomum verum leaf extract assayed by different methodologies[END_REF]. Test compounds, in 1 mL of a ultrapure water/DMSO 1:4 solution, were mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and potassium ferricyanide [K 3 Fe(CN) 6 ] (2.5 mL, 1% w/v). The mixture was incubated at 50ºC for 20 min. A portion (2.5 mL) of trichloroacetic acid (10% w/v) was added to the mixture, which was then centrifuged for 10 min at 3000g. The upper layer of the solution (2.5 mL) was mixed with ultrapure water (2.5 mL) and FeCl 3 (0.5 mL, 0.1% w/v), and the absorbance was measured at 700 nm. Higher absorbance of the reaction mixture indicated greater reducing power. Ascorbic acid was used as standard compound.

Determination of metal chelating activity

The metal chelating activity was assessed by monitoring the changes in UV/Vis spectrum of 2-SC after addition of Fe(II) [START_REF] Guo | Iron-binding properties of plant phenolics and cranberry's bio-effects[END_REF]. Titration experiments were performed by sequential additions of 2 µL of FeCl 2 solution (300 µM stock solution) to 300 µL of a 10 µM (final concentration) 2-SC solution. The titrations were performed in phosphate buffer 20 mM, pH 7.2.

Cytotoxicity

A c c e p t e d M a n u s c r i p t 9

The effect of the 2-SC on the cell viability was assessed the trypan blue exclusion method and by the release of LDH (as a measure of cell membrane integrity). The LDH activity was determined by following the rate of oxidation of NADH at 340 nm.

Statistical analysis

Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Dunnett's multicomparison test using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com". When comparing with control group, values of P less than 0.05 were considered significant.

Pearson correlation tests were performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com".

Results

Inhibition of LTB 4 production by human PMNL

All the tested 2-SC showed to have inhibitory effects on the LTB 4 production by human PMNL. 2-SC from group 1 were more effective inhibitors of LTB 4 production than the correspondent compounds from the other groups. Compounds 1A, 1C, and 1D, at the concentration of 25 μM, were able to inhibit the LTB 4 production with a ≈ 90% effect, while the compound 1B showed a 66.0±12.4% effect at the same concentration (Fig. 3). These compounds were still very effective at the lowest tested concentration (10 μM), 1C being the strongest A c c e p t e d M a n u s c r i p t 10 inhibitor (80.9±10.1% effect, P<0.001) and 1B the weakest inhibitor (38.5±11.8% effect, P<0.01). 1A showed a 63.9±9.2% effect (P<0.001) and 1D showed a 62.3±7.9% effect (P<0.001). The compounds from group 2 were more effective inhibitors than the correspondent compounds from group 3. 2C was the most effective compound from group 2, reaching a 78.0±9.0% effect at the concentration of 25 μM, while 3D was the most effective compound from group 3, showing a 48.9±10.3% effect at the same concentration (Fig. 3). These compounds were still significantly effective at the concentration of 10 μM, where 2C showed an effect of 50.6±11.9% (P<0.001) and 3D showed an effect of 42.2±9.6% (P<0.01). The compounds 1B, 2B, and 3B were the weakest inhibitors in the respective groups. The 5-LOX inhibitor NDGA (1 μM) reached a 76.1±11.0% inhibitory effect (Fig. 3).

-Please place fig. 3 here-3.2 Inhibition of COX-1 and COX-2 in a cell-free system All 2-SC from group 1 where able to inhibit COX-1. 1A significantly inhibited the enzyme's activity at the concentrations of 100 μM (46.0±5.6%) and 250 μM (74.4±12.8%). 1B inhibition was only significant at 250 μM (54.2±12.6%). Due to solubility issues, compounds 1C and 1D could only be tested at a maximum concentration of 100 μM, with 1D showing a significant inhibition (24.6±6.4%). From group 2, only 2A and 2B where shown to inhibit COX-1, when tested at the concentration of 250 μM, 2A being a significant inhibitor (66.5±11.4%) (Fig. 4). No -Please place fig. 4 here-

Reducing power

From the tested 2-SC, compounds 1A-1D and 2B were able to reduce, significantly, ferric ion.

Compounds from group 1 were shown to be more efficient reducers than ascorbic acid (Fig. 5).

-Please place fig. 5 here-

Metal chelating activity

The UV-Vis absorption spectrum of 2-SC 1A-1D shown a common absorption peak at 380 nm which presented bathochromic shift upon addition of growing amounts of Fe(II), increasing gradually until the 400 nm (Fig. 6).

No relevant changes were observed in the UV-Vis spectrum of 2-SC from groups 2 and 3 upon addition of Fe(II).

-Please place fig. 6

Correlations between the inhibition of LTB4 production and scavenging activity

Significant correlations were found between the inhibition of LTB 4 production, described in 3.1, and the scavenging activity for singlet oxygen ( 1 O 2 ), and peroxynitrite anion (ONOO -) (these assays were performed in a previous work [START_REF] Gomes | 2-Styrylchromones: novel strong scavengers of reactive oxygen and nitrogen species[END_REF]).

Please place Table 1 here 4 Discussion

In this work, 2-SC were tested for their anti-inflammatory potential, through their capacity to interfere with the arachidonic acid metabolic pathways, in particular, the COX-1 and COX-2 pathways, studied in a cell-free system, and 5-LOX pathway, studied in a cellular system using human PMNL. All the studied compounds where shown to inhibit the LTB 4 production by human leukocytes, especially those with a catechol substituted B-ring. This biological effect of 2-SC is shown for the first time, in the present study. As previously mentioned, 2-SC are vinylogues of flavones. Accordingly, Yoshimoto et al. [START_REF] Yoshimoto | Flavonoids: potent inhibitors of arachidonate 5-lipoxygenase[END_REF] had previously found that the flavone structure represents a pharmacophore for 5-LOX inhibitory activity. In a recent theoretical study A c c e p t e d M a n u s c r i p t 13 [START_REF] Redrejo-Rodriguez | Lipoxygenase inhibition by flavonoids: semiempirical study of the structure-activity relation[END_REF], a correlation was found between the lipoxygenase inhibitory activity of flavonoid molecules and their planar character, which probably facilitates the access, through a hydrophobic cavity, to the catalytic site of the enzyme. Considering this rationale, the planar character of 2-SC [START_REF] Gerwick | 6-Desmethoxyhormothamnione, a new cytotoxic styrylchromone from the marine cryptophyte Chrysophaeum taylori[END_REF][START_REF] Price | 2-Styrylchromones -Biological Action, Synthesis and Reactivity[END_REF][START_REF] Silva | Novel (E)-and (Z)-2styrylchromones from (E,E)-2 '-hydroxycinnamylideneacetophenones -Xanthones from daylight photooxidative cyclization of (E)-2-styrylchromones[END_REF], conferred by the styryl link between the B-ring and the C-ring, may contribute to their effectiveness as 5-LOX inhibitors.

Several mechanisms, other than a direct inhibition of 5-LOX, can explain the observed inhibition of LTB 4 production by 2-SC. These include the inhibition of phospholipase A 2 , and thus of the arachidonic acid release, the inhibition of LTA 4 hydrolase, or the blockade of 5lipoxygenase activating protein. Nevertheless, it is conceivable that 2-SC, in conformity with flavonoids and other phenolic compounds, act as redox 5-LOX inhibitors. Catalytically active 5-LOX requires the conversion of Fe(II) to Fe(III), conferred by certain lipid hydroperoxides (LOOH). Most 5-LOX inhibitors act at the catalytic domain by reducing or chelating the activesite iron or by scavenging radical intermediates in the redox cycle of the iron [START_REF] Jampilek | 5-lipoxygenase, leukotrienes biosynthesis and potential antileukotrienic agents[END_REF][START_REF] Werz | Inhibition of 5-lipoxygenase product synthesis by natural compounds of plant origin[END_REF]. Accordingly, the reduction of ferric ion is a possible mechanism of inhibition of LTB 4 production by 2-SC, since the most effective compounds (1A-1D) also behaved as strong reducers (Fig. 3 vs. Fig. 5). Indeed, significant correlation was found between the inhibition of LTB 4 production and the reducing power of the tested compounds (Table 1). On the other hand, the spectral changes of those compounds upon addition of Fe(II) indicate the formation of iron complexes. The similar changes in all the compounds indicate that the Fe(II)-binding site is between the ortho-hydroxyl groups in the B-ring, considered to be one "iron binding motif" in flavonoid molecules [START_REF] Guo | Iron-binding properties of plant phenolics and cranberry's bio-effects[END_REF][START_REF] Mira | Interactions of flavonoids with iron and copper ions: a mechanism for their antioxidant activity[END_REF]. Furthermore, the Fe(II) titration curves reveal the formation of 1:2 Fe (II):compound complexes (Fig. 6). Besides indicating a possible mechanism of inhibition of LTB 4 production, the metal chelating capacity of the 2-SC from group 1, shown here for the first time, is also a very A c c e p t e d M a n u s c r i p t 14 important indicator of their antioxidant activity. Finally, the capacity of some 2-SC to scavenge reactive oxygen species (ROS) and reactive nitrogen species (RNS), demonstrated in a previous study [START_REF] Gomes | 2-Styrylchromones: novel strong scavengers of reactive oxygen and nitrogen species[END_REF], is also likely to contribute to their LTB 4 production inhibitory effect. Noteworthy, significant correlations were found between the inhibition of LTB 4 production observed in the present study and the scavenging activity for singlet oxygen ( 1 O 2 ), and peroxynitrite anion (ONOO -) reported before (Table 1).

2-SC 1A, 1B, 1D and 2A where shown to significantly inhibit COX-1. This is also the first time that such biological activity of 2-SC is disclosed. On the other hand, none of the tested compounds was able to inhibit COX-2. Still, it is conceivable that this kind of effect might be seen at higher concentrations in accordance to what has been previously observed with a structural-similar compound (7,3',4'-trihydroxyflavone) [START_REF] Selvam | Cyclooxygenase inhibitory flavonoids from the stem bark of Semecarpus anacardium Linn[END_REF]. However, due to solubility issues, the studied 2-SC could not be tested at higher concentrations. On the other hand, several flavonoids have previously shown to inhibit COX-2 expression (see [START_REF] Gomes | Molecular mechanisms of antiinflammatory activity mediated by flavonoids[END_REF] for review). The flavonoid's structural features considered by others [START_REF] Rosenkranz | SAR: flavonoids and COX-2 inhibition[END_REF] as relevant for this kind of activity are shared by some of the tested 2-SC, specifically compounds 1A and 2A. Thus, 2-SC, particularly the two referred compounds, may still be COX-2 inhibitors in vivo despite the negative results obtained in the present study.

The mechanism through which 2-SC inhibit COX-1 is likely to consist in the scavenging of the radical intermediates involved in COX enzyme catalysis, especially the phenoxy radical formed on a tyrosine residue, according to what has been previously suggested for other compounds with antioxidant moieties [START_REF] Dannhardt | Structural approaches to explain the selectivity of COX-2 inhibitors: Is there a common pharmacophore?[END_REF]. This mechanism is consistent with the fact that the only effective 2-SC where those which have shown higher ROS and RNS scavenging effects in our previous study [START_REF] Gomes | 2-Styrylchromones: novel strong scavengers of reactive oxygen and nitrogen species[END_REF]. The capacity of some of the tested 2-SC to inhibit both COX-1 and LOX pathways plays in favour of their potential use as effective and secure anti-inflammatory drugs, when compared to the commonly used NSAIDs and COXIBs. It is well known that NSAIDs may cause several adverse effects such as gastric ulceration, renal failure and asthma. On the other hand, the safety of selective COXIBs has been recently questioned due to the apparent association of these drugs with an increased risk of cardiovascular events. The cardiovascular adverse effects are probably due to a reduction on the levels of PGI 2 , an important anti-thrombotic eicosanoid, in concomitance to an excess of thromboxane A 2 , which has the opposite effect [START_REF] Antman | Cyclooxygenase inhibition and cardiovascular risk[END_REF][START_REF] Solomon | Selective cyclooxygenase 2 inhibitors and cardiovascular events[END_REF][START_REF] Grosser | Biological basis for the cardiovascular consequences of COX-2 inhibition: therapeutic challenges and opportunities[END_REF].

Considering gastric effects, COX-2 selective inhibitors can delay the repair of existing gastrointestinal damage caused by ulcers, most likely by reducing the synthesis of PGs that are thought to play an important role in the healing process [START_REF] Fiorucci | Dual inhibitors of cyclooxygenase and 5-lipoxygenase. A new avenue in anti-inflammatory therapy?[END_REF]. In fact, the inhibition of COX pathway has to be interpreted with careful because of the diversity of COX-derived mediators with different properties. Indeed, the PGE 2 , which has been mostly considered a proinflammatory mediator, has, on the other hand, given proves of involvement in anti-inflammatory and pro-resolving responses [START_REF] Vancheri | The lung as a privileged site for the beneficial actions of PGE(2)[END_REF][START_REF] Harizi | Cyclooxygenase-2-issued prostaglandin E-2 enhances the production of endogenous IL-10, which down-regulates dendritic cell functions[END_REF][START_REF] Serhan | Resolution phase of inflammation: Novel endogenous anti-inflammatory and proresolving lipid mediators and pathways[END_REF]. Furthermore, the COX inhibition shunts the arachidonic acid metabolism toward the 5-LOX pathway enhancing the gastric mucosal damages due to the augmented production of LTB 4 [START_REF] Fiorucci | Dual inhibitors of cyclooxygenase and 5-lipoxygenase. A new avenue in anti-inflammatory therapy?[END_REF][START_REF] Hudson | Enhanced Gastric-Mucosal Leukotriene-B(4) Synthesis in Patients Taking Nonsteroidal Antiinflammatory Drugs[END_REF] and inducing adverse reactions in patients with asthma as a result of the cys-LTs overgeneration [START_REF] Fiorucci | Dual inhibitors of cyclooxygenase and 5-lipoxygenase. A new avenue in anti-inflammatory therapy?[END_REF].

According to Bertolini et al. [START_REF] Bertolini | Selective COX-2 inhibitors and dual acting anti-inflammatory drugs: critical remarks[END_REF], dual acting anti-inflammatory drugs may represent a breakthrough in the treatment of rheumatic diseases, in view of several important arguments: (i) the same molecule (i.e., one drug alone) inhibits both COXs and 5-LOX; (ii) the inhibition of both COX isoforms ensures a high anti-inflammatory efficacy and the concurrent preservation of the cardiovascular protective effects; (iii) the simultaneous inhibition of 5-LOX prevents pro-A c c e p t e d M a n u s c r i p t 16 inflammatory and gastrointestinal damaging effects of leukotrienes. From our point of view, the use of dual inhibitors of the COX/5-LOX pathways seems advantageous to the treatment of other inflammatory disorders beyond rheumatic diseases. In fact, this type of compounds may exhibit anti-inflammatory activity with a wider spectrum than that of classical NSAIDs by inhibiting 5-LOX product-mediated inflammatory reactions towards which NSAIDs are ineffective [START_REF] Fiorucci | Dual inhibitors of cyclooxygenase and 5-lipoxygenase. A new avenue in anti-inflammatory therapy?[END_REF].

Importantly, it has been demonstrated that the antioxidant effect of many NSAIDs may contribute to their therapeutic effectiveness [START_REF] Costa | Inhibition of human neutrophil oxidative burst by pyrazolone derivatives[END_REF][START_REF] Fernandes | The metabolism of sulindac enhances its scavenging activity against reactive oxygen and nitrogen species[END_REF][START_REF] Fernandes | In vitro scavenging activity for reactive oxygen and nitrogen species by nonsteroidal anti-inflammatory indole, pyrrole, and oxazole derivative drugs[END_REF][START_REF] Costa | Dipyrone and aminopyrine are effective scavengers of reactive nitrogen species[END_REF]. Thus, the strong antioxidant effects of 2-SC will certainly contribute to their anti-inflammatory efficacy in vivo.

In conclusion, in the present study, several of the tested 2-SC were shown, for the first time, to prevent LTB 4 production by human leukocytes and to inhibit COX-1 activity. These effects, together with the well known antioxidant properties of these compounds may become a new therapeutic option in the treatment of inflammatory processes.

1C 1D

  All the chemicals and reagents were of analytical grade. Hanks' balanced salt solution (HBSS), Dulbecco's phosphate buffered saline, DMSO, nordihydroguaiaretic acid (NDGA), calcium ionophore (A23187), indomethacin, iron (II) chloride, iron (III) chloride, ascorbic acid, and arachidonic acid were obtained from Sigma-Aldrich (Steinheim, Germany). The "Leucotriene B 4 Enzyme Immunoassay (EIA) Kit" and "COX Inhibitor Screening Assay" were obtained from Cayman Chemical Co, Ann Arbor, MI, USA. Celecoxib was an offer from Pfizer.Potassium ferricyanide [K 3 Fe(CN) 6 ] and trichloroacetic acid were obtained from Merck (Darmstadt, Germany).

8 2. 5

 85 A c c e p t e d M a n u s c r i p t Determination of the reducing power

other 2 -

 2 SC was able to inhibit COX-1. None of the studied 2-SC inhibited COX-2 at the tested concentrations (10 and 25 μM). Indomethacin (1 μM) inhibited COX-1 (26.6±6.6%) and COX-2 A c c e p t e d M a n u s c r i p t 11 (92.6±2.1%) and the selective COX-2 inhibitor celecoxib (10 μM) could only inhibit this isoenzyme (68.2±3.0%).

  None of the 2-SC caused an increased in the release of LDH compared to the control. The cell viability, assessed by the trypan blue exclusion method, was above 95% for all the compounds.

Fig. 1

 1 Figure captions

Fig. 3

 3 Fig. 3 Inhibition of human PMNL production of LTB 4 by 2-SC (25 μM) and NDGA (1 μM) determined by EIA. Each value represents mean±SEM of at least 4 experiments performed in

Fig. 4

 4 Fig. 4 Inhibition of COX-1 activity by 2-SC, determined by EIA. Each value represents

Fig. 5

 5 Fig.5Reducing activity of 2-SC and ascorbic acid (AA). All the compounds were tested at the

Fig. 6

 6 Fig. 6 UV-Vis absorption spectrum of 10 µM of compounds 1A, 1B, 1C, and 1D in the

Table 1 -

 1 Pearson correlations between the inhibition of LTB 4 production and the reducing activity and between the LTB 4 production and the scavenging activity against 1 O 2 (IC 30 ) and ONOO -(with and without bicarbonate) (IC 50 )[START_REF] Gomes | 2-Styrylchromones: novel strong scavengers of reactive oxygen and nitrogen species[END_REF] of the tested 2-SC. *significant at p < 0.05; **significant at p < 0.01. a) without bicarbonate; b) with bicarbonate.

Figure 1 A

 1 Figure 1

Figure 2 A

 2 Figure 2

Acknowledgements: The authors acknowledge FCT and FEDER financial support for the project POCI/QUI/59284/2004 and the Organic Chemistry Research Unit (nº 62; Univ. Aveiro).

Ana Gomes acknowledges FCT and FSE her PhD grant (SFRH/BD/23299/2005).