Chantal Barthomeuf

Abstract: Since the improvement of chemotherapy with safe molecules is needed for a better efficacy without supplementary toxicity, we investigated the feasibility and tolerability of the combination of docetaxel and curcumin, a polyphenolic derivative extracted from Curcuma longa root.

Abstract: Meroterpenes are compounds of mixed biogenesis, isolated from plants, microorganisms and marine invertebrates. We have previously isolated and determined the structure for a series of meroterpenes extracted from the ascidian Aplidium aff. densum. Here, we demonstrate the chemical synthesis of three of them and their derivatives, and evaluate their biological activity on two bacterial strains, on sea urchin eggs, and on cancerous and healthy human cells.

Abstract: Marine plants and animals are sources of a huge number of pharmacologically active compounds, some of which exhibit antineoplastic activity of clinical relevance. However the mechanism of action of marine natural products (MNPs) is poorly understood. In this study, proton NMR spectroscopy-based metabolomics was applied to unravel biochemical disorders induced in human MCF7 breast cancer cells by 3 lead candidate anticancer MNPs: ascididemin (Asc), lamellarin-D (Lam-D), and kahalalide F (KF). Asc, Lam-D, and KF provoked a severe decrease in DNA content in MCF7 cells after 24-h treatment. Asc and Lam-D provoked apoptosis, whereas KF induced non-apoptotic cell death. Metabolite profiling revealed major biochemical disorders following treatment. The response of MCF7 tumor cells to Asc involved the accumulation of citrate (x17 the control level, P<0.001), testifying enzyme blockade in citrate metabolism, and the accumulation of gluconate (x9.8, P<0.005), a metabolite never reported at such concentration in tumor cells, probably testifying glycolysis shutdown. The response to Lam-D involved the accumulation of aspartate (x7.2, P<0.05), glutamate (x14.7, P<0.05), and lactate (x2.3, P<0.05), probably in relation with the targeting of the malate-aspartate shuttle, as discussed. The response to KF involved increased lipid accumulation (polyunsaturated fatty acids x9.8, P<0.05), and phospholipid and acetate derivative alterations. Altogether, this study demonstrates the potential of proton NMR spectroscopy-based metabolomics to help uncover metabolic targets and elucidate the mechanism of cytotoxicity of candidate antineoplastic MNPs.

Abstract: Complementary and alternative therapies for neoplastic diseases treatment and prevention receive increasing attention from the medical community. Prostate cancer (PC) is the most frequently diagnosed malignancy and the second major cause of male death in industrialized countries. The chemopreventive properties and clinical safety of curcumin, a polyphenolic derivative, have already been established. However, curcumin regimen value in addition to conventional hormone refractory (HR) PC treatment remains largely unknown. This review article summarizes mechanisms by which curcumin may decrease HRPC aggressive proliferation and potentiate activity of taxane therapy. Our analysis suggests that curcumin alone has a therapeutic value in HRPC. In combination with a taxane agent, this compound may enhance cytotoxicity and retard PC cell resistance to taxane. As a consequence, a rationale is provided for considering the possible benefits of curcumin regimen in combination with taxane therapy in HRPC patients.

Abstract: There is growing evidence that docetaxel, a microtubule-targeting agent like the other taxane paclitaxel, induces dual cytotoxicity mechanism according to dose level. Postgenomics screening technologies are now more and more applied to the elucidation of drug response mechanisms. Proton nuclear magnetic resonance spectroscopy-based pharmacometabolomics was here applied to get further insight into the response of human MCF7 breast carcinoma cells to docetaxel at high (clinical, 5 microM) and low (1 nM) doses. The global response to both doses was evaluated by nuclear morphology and DNA content, the latter as an index of cell proliferation and DNA ploidy. High dose provoked long-lasting cell cycle arrest in mitosis during the first 48 h of exposure to treatment and severe decrease in DNA content followed by significant amount of cell death. In contrast, at low dose, no long-lasting cell cycle arrest was observed on micrographies, and DNA content was decreased but less than at high dose (P < 0.05), without significant cell death. This response was compared to biochemical alteration assessed by pharmacometabolomics. Thirty metabolites were identified and quantified. Metabolite profiling at clinical dose revealed time-dependent disorders in derivatives of glycolysis, lipid metabolism and glutathione metabolism. Comparison between high and low doses was performed at 72 h and showed common traits including the accumulation of cytidinediphosphocholine (x 5.0 and x 6.9, respectively, P < 0.03), the decrease in phosphatidylcholine (x 0.3 and x 0.2, respectively, P < 0.03), and gluthathione (x 0.6 and x 0.6, respectively, P < 0.03). Despite that, significant dose-dependent differences were found in 12 of 30 measured metabolites. Among them, the most discriminant metabolites were polyunsaturated fatty acids (ratio of high-to-low dose of 14.8, P < 0.05), glutamate, myoinositol, and homocysteine (ratio < 0.4, P < 0.05). In addition, the mechanism for glutathione decrease was different. At high dose, it resulted from extensive consumption with precursor starvation (glutamate: -89%, P < 0.05) and increased glutathione S-transferase activity (x 5, P < 0.01), whereas at low dose, it resulted from glutathione biosynthesis blockade with homocysteine accumulation (+144%, P < 0.03) and decreased glutathione S-transferase activity (-70%, P < 0.01). Altogether, this pharmacometabolomics analysis provides further evidence of the varying cellular responses at high and low doses of docetaxel in MCF7 breast cancer cells.

Abstract: NMR spectroscopy-based metabolomics still needs development in quantification procedures. A method was designed for quantitative two-dimensional high resolution magic angle spinning (HRMAS) proton-NMR spectroscopy-based metabolite profiling of intact cells. It uses referencing of metabolite-related NMR signals to protein-related NMR signals and yields straightforward and automatable metabolite profiling. The method enables exploitation of only two-dimensionally visible metabolites and combination of one- and two-dimensional spectra, thus providing an appreciable number of screened metabolites. With this procedure, 32 intracellular metabolites were attributed and quantified in human normal fibroblasts and tumor cells. The phenotype of several tumor cell lines (MCF7, PC3, 143B, and HepG2) was characterized by high levels of glutathione in cell lines with the higher proliferation rate, high levels of creatine, low levels of free amino acids, increased levels of phospholipid derivatives (mostly phosphocholine), and lower lactate content in cell lines with the higher proliferation rate. Other metabolites such as fatty acids differed widely among tumor cell lines. The response of tumor cell lines to chemotherapy also was evaluated by differential metabolite profiling, bringing insights into drug cytotoxicity and tumor cell adaptive mechanisms. The method may prove widely applicable to tumor cell phenotyping.

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Abstract: Platelet-derived growth factor (PDGF)-dependent recruitment of mural cells such as pericytes and smooth muscle cells plays a central role in the maturation and stabilization of newly formed vasculature during angiogenesis. In this work, we show that the dietary flavones apigenin and luteolin may interfere with this event through their inhibitory effect on PDGF-dependent phosphorylation of PDGF receptor beta (PDGFR-beta) in smooth muscle cells. Inhibition of PDGFR-beta activity by apigenin and luteolin occurred at low concentrations of the molecules and resulted in the inhibition of extracellular signal-regulated kinase and Akt phosphorylation triggered by PDGF, as well as in a marked reduction of the migratory and invasive properties of these cells. Apigenin and luteolin also strongly inhibit the PDGF-dependent increase in vascular endothelial growth factor (VEGF) mRNA levels and the secretion of VEGF by smooth muscle cells as well as vessel formation in the mouse Matrigel plug assay, suggesting that the inhibitory effects of both molecules on smooth muscle cell function result in impaired angiogenesis. Overall, these results identify apigenin and luteolin as dietary-derived inhibitors of PDGFR-beta activity and suggest that this inhibitory effect may contribute to the chemopreventive properties of these molecules.

Abstract: The treatment of chemoresistant tumors represents an important challenge in the field of oncology. Primary or acquired overexpression of ATP-dependent transporters, in particular P-glycoprotein (Pgp, MDR1 protein), is a major cause of multidrug resistance and reduced patient survival. Sustained efforts have thereby been undertaken to find agents overcoming this resistance. This review provides a chemical and biological overview on bioactive metabolites from the marine field (natural molecules and analogues) that can overcome or circumvent resistance to ATP-dependent efflux pumps, their mechanisms of action and their structure-activity relationships. Their clinical relevance and status are presented. Active compounds (often microtubule-interacting agents) have been isolated from sponges and ascidians and, in lesser extent from cnidarians, and molluscs. The toxicity and the reversal activity can be uncoupled but, marine metabolites usually maintain high toxicity in multiresistant cancer cells. Certain display synergistic effects with clinically important anticancer drugs. The marine drug recently approved for cancer therapy [Trabectedin (Yondelis)] and those entered into clinical trials act on multiple targets and, circumvent or overcome chemoresistance through very unusual mechanisms of action. Pharmacological and clinical data suggest that metabolites from the marine field could provide new therapeutic options for patients with tumors resistant to conventional therapy.

Abstract: In animal models, methionine (MET) restriction in association with chloroethylnitrosoureas led to a substantial improvement. On this basis, we initiated a Phase I clinical trial of dietary MET restriction in association with chloroethylnitrosourea (cystemustine) treatment for patients with recurrent glioma or metastatic melanoma. Our purpose was 1) to determine the optimal MET-free diet duration for a maximum depletion of plasma MET and 2) to evaluate the feasibility of this association. A total of 10 patients received 4 cycles of 2 wk of an association of a MET-free diet of 1, 2, 3, or 4 consecutive days and cystemustine (60 mg/m(2)). For each cycle, plasma MET concentrations, nutritional status (weight, albumin, prealbumin) and toxicity were measured. Conversely, fed-state concentrations of plasma MET (12 AM) were reduced by dietary MET restriction, with an optimal depletion of 41% at the 1st day of MET-free diet without effect of the extending MET-free diet period. Indeed, we demonstrated the feasibility, that is, good diet acceptability and good tolerance (nutritional status and toxicity), of the association of a MET-free diet and cystemustine treatment. Based on these results, a Phase II clinical trial has been initiated to test the activity of the association of a 1-day MET-free diet with cystemustine treatment.

Abstract: Metastatic malignant melanoma have a bad prognosis (median survival: 6-8 months) mainly due to the development of lung, hepatic and brain metastases. In this study we have used the resazurin reduction test and FACS analysis to assess the cytostatic and cytotoxic effect of umbelliprenin from Ferula szowitsiana (Apiaceae) on human solid cancer cells and human primary fibroblasts. We have observed that the cell susceptibility to umbelliprenin decreases in the order M4Beu (metastatic pigmented malignant melanoma)>A549 (nonsmall cell lung carcinoma) approximately PC3 (androgen-resistant prostate carcinoma)>PA1 (ovary teratocarcinoma)>human primary fibroblasts approximately MCF7 (breast adenocarcinoma)>DLD1 (colon adenocarcinoma). M4Beu cell-proliferation is inhibited through cell-cycle arrest in G1 and induction of caspase-dependent apoptosis. The finding that the cytotoxic effect of umbelliprenin is markedly more pronounced in M4Beu cells than in primary fibroblasts, suggests a therapeutic margin. As M4Beu cell proliferation is more potently inhibited by umbelliprenin (IC50 12.3 microM) than by the citrus coumarin auraptene (7-geranyloxycoumarin, IC50 17.1 microM) previously reported capable of inhibiting the prevalence of lung metastasis in mice bearing B16BL6 murine melanoma, our data suggest that umbelliprenin orally administered and foods and folk medicines containing this coumarin, may afford protection against the development and early recurrence of malignant melanoma. In vivo investigations are needed to test these hypotheses.

Abstract: From a mixed assemblage of Lyngbya majuscula rich marine cyanobacteria, we isolated a series of cell growth inhibitory cyclic peptides. The structures of the two major components, laxaphycins A (1) and B (2), and of two minor peptides, laxaphycins B2 (3) and B3 (4), were determined by spectroscopic methods and degradative analysis. Absolute configurations of natural and nonproteinogenic amino acids were determined by a combination of hydrolysis, synthesis of noncommercial residues, chemical derivatization, and HPLC analysis. The organism producing the laxaphycins was identified as the cyanobacterium Anabaena torulosa. The antiproliferative activity of laxaphycins was investigated on a panel of solid and lymphoblastic cancer cells. Our results demonstrate that in contrast to laxaphycin A, laxaphycin B inhibits the proliferation of sensitive and resistant human cancer cell lines and that this activity is strongly increased in the presence of laxaphycin A. This effect appears to be due to an unusual biological synergism.

Abstract: The present study focuses on eudesmin (bicyclic lignan, 0.15 % of dry leaves) and diphyllin (arylnaphthalene lignan, 0.1 % of dry roots), both isolated from H. perforatum Kar. et Kir, a Rutaceae species endemic to Uzbekistan. We first compared their specificity for cancer cells with those of etoposide and podophyllotoxin by screening their cytotoxicity on 3 healthy cell-lines and 7 sensitive or resistant human solid cancer lines. We then tested their capacity to reverse P-glycoprotein-mediated multidrug resistance (MDR) by assaying dye and drug uptake in MDR1-transfected Madin-Darby canine kidney (MDCK-MDR1) and doxorubicine-resistant human breast carcinoma cells (MCF7/Dox). Eudesmin displays IC (50) values > 100 microM on all tested lines. Our data provide the first demonstration that this non-toxic lignan reverses Pgp-mediated drug efflux and supports the hypothesis that it may inhibit resistance mediated by MDR1 and MRP proteins. Even if its reversal activity is insufficient for clinical application, its capacity to accumulate [(3)H]-vinblastine in MDCK/MDR1 and MCF7/Dox cells suggests that eudesmin may positively affect the bioavailability and, thereby, the therapeutic potency of anticancer drugs in Pgp-overexpressing cells. Diphyllin exhibits IC (50) values ranging from 10 (- 6) to 10 (- 4) M. It is markedly less toxic than podophyllotoxin (IC (50) : 13 - 61 nM), but exhibits tumoricidal effects close to those of etoposide. Unfortunatly, it is 65-fold more toxic than etoposide on human primary fibroblasts. Consequently, it has no value as an anticancer drug. Its value as raw material for the hemisynthesis of anticancer drugs is discussed.

Abstract: Fagaronine derivatives syntheses were optimized and their effect on PC3 androgen-independent prostate cell line was evaluated. An assessment of the lipophilicity of the benzo[c]phenanthridine derivatives was achieved at pH 7.4 and et 6.7 by determining log D.

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Abstract: Although many data are available concerning anticarcinogenic effects of industrial conjugated linoleic acid (CLA), few studies have reported the antitumour properties of CLA mixtures originating from ruminant products. The aim of the present study was to investigate the in vitro antiproliferative effects of beef CLA mixtures on breast, lung, colon, melanoma and ovarian human cancer cell lines. For this purpose, four fatty acid (FA) extracts prepared from beef lipid and varying in their CLA composition, their corresponding purified CLA-enriched fractions, and mixtures of pure synthetic CLA, the composition of which reproduced that of the four selected beef samples, were tested on cancer cell lines. Cancer cells were exposed for 48 h to medium containing 100 microm-FA and their proliferation was determined by quantifying cellular DNA content (Hoechst 33342 dye). Compared with cells incubated without FA, the number of cancer cells was reduced from 25 to 67 % (P<0.0001) following FA treatment. Antiproliferative effects of CLA mixtures varied in magnitude according to the source of FA, the CLA composition and the cell lines. CLA mixtures naturally present in beef inhibited the proliferation of human cancer cell lines, a high content in cis-trans isomers allowing the most important antiproliferative effect. Beef total FA exhibited a greater growth-inhibitory activity than their corresponding CLA-enriched fractions. These results suggested that either beef FA other than beef CLA could possess antiproliferative properties and/or the existence of complementary effects of non-conjugated FA and CLA, which could favour the antiproliferative properties of beef total FA.

Abstract: Overexpression of the protein transporter P-glycoprotein (Pgp, MDR1) at the cell surface is a major cause of multidrug resistance (MDR) and poor response to treatment in cancer chemotherapy and therapy for leishmaniasis. The present study shows that conferone, a sesquiterpene coumarin ether isolated for the first time from Ferula schtschurowskiana, endemic in Uzbekistan, enhances the cell toxicity of vinblastine (VBL) in MDR1-transfected Madin-Darby canine kidney (MDCK-MDR1) cells. Conferone presents the advantage to mediate this effect at safe concentrations. At 10 microM, it efficiently competes with the photoactivatable cyclosporin A analogue (SDZ 212 - 122) for the binding to Pgp and accumulates [3H]-VBL to a higher extent than cyclosporin A or cnidiadin. [3H]-VBL accumulation is dose-dependent and correlates with the inhibition of Pgp photolabeling affinity, supporting the hypothesis that conferone sensitizes MDCK-MDR1 cells to VBL by competitively inhibiting drug efflux. In MDCK-MDR1 cells, [3H]-VBL accumulation appears to be almost completely dependent on inhibition of Pgp transport. However, the strict specificity of conferone to this efflux pump has to be demonstrated in cell lines expressing other protein transporters. Collectively, our findings identify conferone as a powerful modulator of Pgp transport and a promising molecule for the treatment of MDR malignancies and leishmaniasis. Complementary in vitro and in vivo studies are, however, needed to assess the value of conferone as a reversal drug in human therapy. Considering its high affinity for Pgp, conferone may have an additional usefulness as a tool for the design or the (hemi)synthesis of agents probing Pgp. To our knowledge, this is the first report identifying sesquiterpene coumarins from Ferula as possible drug candidates for the reversion of MDR encoded by the MDR1 gene or the synthesis of agents probing Pgp.

Abstract: Vascular endothelial growth factor (VEGF) and platelet-derived lipid sphingosine-1-phosphate (S1P) are two proinflammatory mediators which contribute to angiogenesis, in part through the synthesis of platelet-activating factor (PAF). The red grape skin polyphenolic extract (SGE) both prevents and inhibits angiogenesis in the Matrigel model, decreases the basal motility of endothelial and cancer cells, and reverses the chemotactic effect of S1P and VEGF on bovine aortic endothelial cells (BAECs) as well as the chemotactic effect of conditioned medium on human HT-1080 fibrosarcoma, human U-87 glioblastoma, and human DAOY medulloblastoma cells. Inhibition of VEGF- and S1P-mediated chemotaxis by SGE is associated with a down-regulation of ERK and p38/MAPK phosphorylation and a decreased in acute PAF synthesis. Notably, as do extracellular inhibitors of PAF receptor, SGE prevents S1P-induced PAF synthesis and the resulting activation of the S1P/endothelial differentiation gene-1 cascade. Given the key role of VEGF and S1P in inflammation, angiogenesis, and tumor invasion, SGE may therefore contribute to prevent (or to delay) the development of diseases associated with angiogenesis dysregulation, including cancer. The dual inhibition of S1P- and VEGF-mediated migration of endothelial cell and of serum-stimulated migration of U-87 cells suggests a usefulness of SGE against highly invasive human glioblastoma.

Abstract: OBJECTIVES: Mitoxantrone/prednisone was the 2002 palliative reference treatment for hormone-refractory prostate cancer (HRPC). Paclitaxel and carboplatin has demonstrated antitumor activity in HRPC. The therapeutic benefit of such treatment was compared with that of mitoxantrone. METHODS: A randomized Phase II study was conducted that included 40 patients with HRPC who had not undergone chemotherapy. Patients in arm A received paclitaxel (175 mg/m2 every 3-week cycle) and carboplatin (area under the curve of 5 every 3-week cycle). Patients in arm B received mitoxantrone (12 mg/m2 every 3-week cycle). All the patients treated were receiving low-dose prednisone. The primary endpoint was the prostate-specific antigen response. RESULTS: The prostate-specific antigen response to paclitaxel and carboplatin was significantly greater (40% [95% confidence interval 18.5% to 61.5%] versus 10% [95% confidence interval 1% to 32%], P = 0.031) and more durable (8.6 versus 2 months, P = 0.015) than the response to mitoxantrone. A tendency was noted for patients with measurable disease who were receiving paclitaxel and carboplatin to have a somewhat greater objective response rate than those who received mitoxantrone (23% [95% confidence interval 5.3% to 55%] versus no objective response, P = 0.060). The median overall survival was 14.5 months for the paclitaxel and carboplatin arm compared with 11.1 months for the mitoxantrone arm. The group given paclitaxel and carboplatin had significantly greater rates of sensitive neuropathy (50% versus 0%, P = 0.00026). CONCLUSIONS: The 3-week regimen of paclitaxel and carboplatin induced a greater and more durable prostate-specific antigen response than did mitoxantrone for HRPC treatment. The major additive toxicity induced was peripheral neuropathy due to paclitaxel. Investigations with paclitaxel and carboplatin regimens merit large Phase III studies.

Abstract: In the course of structure-activity relationship studies, diversely substituted 1-(beta-D-glucopyranosyl)-isoindigo derivatives were prepared from commercially available indolines. Their antiproliferative activities were evaluated toward a panel of human solid cancer cell lines (PC 3, DLD-1, MCF-7, M4Beu, A549, PA 1), a murine cell line (L929) and a human fibroblast primary culture to get an insight into the substitution pattern required for the best biological potencies.

Abstract: PURPOSE: Overexpression of P-glycoprotein (Pgp) encoded by the MDR1 gene is one of the major obstacles to successful cancer chemotherapy. The goal of this study was to evaluate if, among other natural coumarins, cnidiadin, a furanocoumarin present in traditional Chinese medications and in a spice commonly used in Greek food, inhibits Pgp transport activity and has the potential to reverse MDR1 multidrug resistance. METHODS: Using MDR1-transfected Madin-Darby canine kidney (MDCK-MDR1) cells as a model of cells expressing the human MDR1 phenotype, and verapamil or CsA or both as positive control, we tested the capacity of six natural coumarins (umbelliferone, esculin, esculetin, cnidiadin, angelicin and psoralen) to induce the accumulation of rhodamine-123 (R-123) and [3H]-vinblastine ([3H]-VBL) and to modulate the photolabeling of Pgp by SDZ 212-122, a diazirin cyclosporin A. The growth-inhibitory effect of cnidiadin and its capacity to enhance the cell toxicity of vinblastine (VBL) or vincristine (VCR) was then evaluated by the WST-1 assay in two cell lines overexpressing Pgp (MDCK-MDR1 and vincristine-resistant KB/VCR). RESULTS: Cnidiadin was the only tested coumarin capable of significantly accumulating R-123 and [3H]-VBL and inhibiting Pgp photolabeling in MDCK-MDR1 cells. The dose-dependent increase in [3H]-VBL uptake (IC50 26.5 microM) induced by cnidiadin in the dose range 1-100 microM correlated with inhibition of Pgp photolabeling. At 10 microM cnidiadin inhibited photolabeling by 59% and sensitized both MDCK-MDR1 and KB/VCR cells to vinca alkaloids. CONCLUSION: Cnidiadin is a cytotoxic agent capable in vitro of competitively inhibiting the binding and efflux of drug by Pgp and of enhancing the cell toxicity of vinca alkaloids in two cell lines (MDCK-MDR1 and mutant human carcinoma KB/VCR) overexpressing Pgp. This suggests that diet or traditional preparation containing cnidiadin may contribute to the reversal of MDR1 multidrug resistance and may affect the bioavailability of Pgp substrates orally administered. However, due to its cell toxicity, clinical interest in cnidiadin as a chemosensitizer appears to be limited.

Abstract: We have investigated new drug combinations of potential clinical value for treatment of hormone-refractory prostate cancer. Combinations of paclitaxel, carboplatin and mitoxantrone, and combinations of these three drugs with compounds targeting important pathways for cancer progression, 13-cis-retinoic acid and chelerythrine, were assessed. The drugs combinations were incubated for 72 h in steroid-free conditions with two androgen-independent cell lines, DU145 and PC3. Cytotoxicity assay was performed using resazurin and Hoescht 33342. Synergism and antagonism were measured by the combination index, and calculated for each combination by the median-effect method. All six compounds exhibited cytotoxic effects when tested alone. Paclitaxel exhibited the highest and 13-cis-retinoic acid the lowest effect on both cell lines. Paclitaxel demonstrated synergism or additivity with 13-cis-retinoic acid in both cell lines, whereas antagonistic effects were observed when it was tested in combination with carboplatin. Chelerythrine showed additive effects with mitoxantrone in both cell lines and with paclitaxel in PC3 cells. Our results suggest that combination of paclitaxel and 13-cis-retinoic acid, and of chelerythrine with mitoxantrone and paclitaxel, may have clinical value for the treatment of hormone-refractory prostate cancer.

Abstract: Conjugated linoleic acid (CLA), mainly c9,t11- and t10,c12-isomers, and polyunsaturated n-3 fatty acids (n-3 PUFA) have been shown to reduce tumor growth. This study compared, on a set of human tumor cells (breast, lung, colon, prostate and melanoma), the antiproliferative effects of: i) trans monounsaturated fatty acids (MUFA) vs. cis MUFA and MUFA vs. PUFA, ii) individual isomers of CLA vs. linoleic acid, iii) CLA-conjugated derivatives vs. their non-conjugated homologues and vs. CLA isomers. Tumor cells were exposed to medium containing individual FA (100 microM) for 48 h and their proliferation was determined by measuring the cellular DNA content (fluorescent Hoechst 33342 dye). The antiproliferative effects of FA varied with the type of cells and were mainly dependent on the degree of unsaturation and on the position and configuration of their double bonds. One isomer of CLA (t9,t11-18:2) and CLA-conjugated derivatives exhibited the strongest growth-inhibitory effect against cancer cells. These results suggest that ruminant products contain active compounds against human tumor cell proliferation.

Abstract: PURPOSE: Neoangiogenesis is critical to cancer proliferation and metastasis and constitutes an attractive target for cancer therapy. It has previously been demonstrated that hederacolchiside-A1 (HCol-A1), a triterpenoid saponin from Hedera colchica Koch, has antimelanoma potential. The goal of this study was to evaluate, in vitro, if in addition to its tumoricidal effect on melanoma cells, HCol-A1 might affect endothelial cell network formation. METHODS: We investigated whether HCol-A1 affects matrigel-induced tubulogenesis and inhibits the viability (WST-1 assay) of human umbilical vein endothelial cells (HUVECs). To provide structure-activity relationships (SAR), studies were conducted on HCol-A1, oleanolic acid and hederacolchiside A (HCol-A), a triterpenoid saponin which possess the same sugar sequence as Hcol-A1. Plasma membrane cholesterol sequestration was studied by labelling with [3H]cholesterol and assayed with HCol-A1-cholesterol complexes. HCol-A1 signalling was investigated using immunoassays. RESULTS: In contrast to HCol-A and oleanolic acid, HCol-A1 inhibited matrigel-induced angiogenesis at micromolar concentration. Plasma membrane cholesterol sequestration was found to be critical for this activity. Activation of the Ras/MEK/ERK cascade appears to be one of the mechanisms by which Hcol-A1 affects HUVEC network formation. The predominant activation of the Ha-Ras isoform, which decreases HUVEC-tolerance to apoptosis, might contribute to the high susceptibility of this cell line to HCol-A1. CONCLUSION: Since cholesterol sequestration affects cell confluence-dependent remodelling of endothelial membranes and vascular endothelial growth factor receptor-2 activity, these results raise the possibility that Hcol-A1 might slow-down cancer proliferation and metastasis in vivo by inhibiting critical aspects of neoangiogenesis. Further in vivo studies are needed to verify this hypothesis.

Abstract: We previously reported that hederacolchiside A1 (Hcol A1), a new oleanene saponin isolated from Hedera colchica Koch (Araliaceae) exhibits a preferential cytotoxicity on a pigmented melanoma cell line. The present study confirms the high susceptibility of melanoma cell lines to this drug and shows concentrations producing a 50% decrease in cell content (IC50 values) inversely proportional to the melanin content of each cell line. At cytotoxic concentrations, Hcol A1 induces membrane-damaging effects within 6 h, cytoplasmic vacuolization within 24 h, and non-apoptotic cell death within 48 h. Using a new high-resolution magic-angle spinning nuclear magnetic resonance method, we have shown for the first time that this hederasaponin specifically interacts with melanin. The dissociation constant (2.7 mM) is comparable to those observed with drugs known to interact with melanin. Taking into consideration that the IC50 values were inversely proportional to the melanin in each cell line, our data suggest that, in addition to the delayed membrane injury induced by this drug, the ability of Hcol A1 to bind melanin could contribute to the higher toxicity of Hcol A1 in pigmented melanoma cells.

Abstract: The plasminogen activator/plasmin system represents a key component of the proteolytic machinery underlying angiogenesis. In this work, we investigated the effect of Neovastat (AE-941), a naturally occurring multifunctional antiangiogenic agent that is currently in Phase III clinical trials, on tissue and urokinase plasminogen activator activities. We found that in vitro, Neovastat at 100 microg/ml markedly stimulates t-PA-mediated plasmin generation, while it slightly inhibits the generation of plasmin mediated by uPA. The stimulatory effect of Neovastat on t-PA activity was markedly increased by a heat treatment, resulting in a 15-fold increase in the rate of activation of plasminogen. Neovastat did not directly stimulate the activity of t-PA or plasmin towards exogenous substrates, suggesting that its effect requires the presence of plasminogen. Accordingly, kinetic analysis showed that Neovastat increases both the k(cat) of t-PA as well as its affinity for plasminogen by 10-fold. The stimulation of t-PA activity by Neovastat was also correlated with a direct interaction of Neovastat with plasminogen as monitored by the surface plasmon resonance technology. Overall, these results identify Neovastat as a potent stimulator of t-PA-dependent activation of plasminogen, further emphasizing its pleiotropic mechanism of action on several molecular events involved in angiogenesis.

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Abstract: The beneficial health effects of consumption of marine foods, in general, and of marine oils, in particular, are well recognized. Fish by-products constitute valuable sources of components, such as lipids for human consumption, and liver and roe from large cod (Gadus morhua)are currently exploited. This paper presents a new approach for extracting lipids from several cod by-products by using a pre-hydrolysis step with large spectrum proteases in order to disrupt tissues and cell membranes. Yields extraction for total lipids, phospholipids, EPA and DHA are compared to those obtained by organic extraction.

Abstract: PURPOSE: The quaternary benzophenanthridine alkaloid sanguinarine exhibits a broad range of activity, including cytotoxicity against various human tumour and normal cell lines. Here, we examined its potency as an anticancer drug. METHODS: The differential cytotoxicity against cancer versus normal cells was assessed in vitro by two fluorimetric assays (RRT and Hoechst 33342 dye DNA assays, respectively) in a panel of human solid cancer cell lines and a human fibroblast primary culture. The ability to induce apoptosis was demonstrated in PC3 human prostatic adenocarcinoma cells by analysis of morphological changes, internucleosomal DNA fragmentation, cellular poly(ADP-ribose) polymerase cleavage and caspase 3/7 activation. Production of reactive oxygen species was evaluated by the 2',7'-dichlorofluorescin diacetate assay. Depletion of cellular glutathione content was assessed with the monochlorobimane assay. RESULTS: Sanguinarine markedly inhibited the growth of all tested cells (IC(50) 0.9-3.3 microM) without differential cytotoxicity against normal versus cancer cells. In PC3 cells, continuous treatment with 5 microM sanguinarine induced an early (within 10 min) cellular reduced glutathione depletion insensitive to dithiothreitol or N-acetylcysteine treatment, followed by a caspase 3/7-dependent apoptotic response within 2 h. Complementary assays suggested that the glutathione depletion was initiated by direct reactivity of sanguinarine with reduced glutathione. CONCLUSIONS: Taken together, these results show that (1) sanguinarine exhibits no specificity for cancer cells, and (2) its strong cytotoxicity is probably due to a rapid apoptotic response induced by an early and severe glutathione-depleting effect. They also suggest that the clinical usefulness of this alkaloid as an anticancer drug is limited.

Abstract: Neovastat (AE-941) is an antiangiogenic drug isolated from marine cartilage. It interferes with several steps associated with the development of angiogenesis through its ability to induce endothelial cell apoptosis, and to inhibit matrix metalloproteinase activities and vascular endothelial growth factor-mediated signaling pathways, suggesting that Neovastat behaves as a multifunctional antiangiogenic drug. Neovastat is orally bioavailable, and shows significant antitumor and antimetastatic properties in animal models. An excellent safety profile with few side effects has been monitored in more than 800 patients who have been exposed to Neovastat, some of whom for more than 4 years. This indicates that Neovastat is suitable for long-term use, either alone or in combination with other anticancer therapies. Accordingly, Neovastat is currently under evaluation in three pivotal clinical studies with two phase III clinical trials in patients with lung and renal carcinoma, and a phase II clinical trial in patients with multiple myeloma is ongoing.

Abstract: Hederacolchisid A1, a new oleanolic acid monodesmoside, isolated from Hedera colchica K. Koch, an ivy species endemic in Georgia, was evaluated in vitro for antiproliferative activity on cancer cells versus normal cells in comparison to cisplatin. Investigations were made on six human cell lines (colon adenocarcinoma DLD-1, ovarian teratocarcinoma PA 1, lung carcinoma A 549, breast adenocarcinoma MCF7, prostatic adenocarcinoma PC 3 and malignant melanoma M4 Beu) versus normal human fibroblasts, by assaying both cellular metabolic activity (RTT test) and DNA content in living cells (test with Hoechst 33,342) after 48 h continuous contact. Results demonstrated the strong cytotoxicity of hederacolchisid A 1 on all cancer cells (IC50 from 4.5 to 12 microM). The antiproliferative effects on malignant melanoma M4 Beu (IC50 ca 4.5 microM) versus normal cells (IC50 ca 7.5 microM) suggests that, despite a lack of specificity for cancer cells, hederacolchisid A1 has potential anti-tumor applications. Comparison of the cytotoxicity of hederacolchisid A 1 with that of five other saponins from H. colchica, offers some new information about structure-activity relationships. It was observed that i) for a same sugar sequence, monodesmosides with oleanolic acid as aglycone exhibit higher cytotoxicity than those containing hederagenin, ii) the sugar sequence O-alpha-L-rhamnopyranosyl (1 --> 2)-alpha-L-arabinopyranoside at C3 induces strong cytotoxicity and might be identified as a basic sequence for anti-tumor activity of oleanolic acid monodesmosides. iii) a complementary glucopyranosyl moiety branched at C1 of arabinose increases the cytotoxicity against malignant melanoma M4 Beu, prostatic adenocarcinoma PC 3 and normal fibroblasts in a different manner for each type of monodesmoside. A slight increase whose amplitude was quite similar on cancers and normal cells, was observed with oleanolic acid monodesmoside. This increase was much higher with hederagenin monodesmoside and markedly elevated in normal cells than in cancer cells.

Abstract: A sulfoglycolipidic fraction (SF) isolated from the red microalga Porphyridium cruentum was analyzed for fatty acid composition and assayed for ability to inhibit, in vitro, the generation of superoxide anion in primed leucocytes and the proliferation of a panel of human cancer cell-lines. Results demonstrated that SF contained large amounts of palmitic acid (26.1%), arachidonic acid (C20: 4 omega-6, 36.8%), and eicopentaenoic (C20:5 omega-3, 16.6%) acids, and noticeable amounts of 16:1n-9 fatty acid (10.5%). It strongly inhibited both the production of superoxide anion generated by peritoneal leukocytes primed with phorbol myristate acetate (IC(50): 29.5 microg/mL), and the growth of human colon adenocarcinoma DLD-1 and to a lesser extent of human breast adenocarcinoma MCF-7, human prostate adenocarcinoma PC-3, and human malignant melanoma M4 Beu cell-lines, and therefore might have a chemopreventive or chemotherapeutic potential, or both. It was found markedly more cytotoxic than sulfoquinovosyldiacylglycerols from plant used as a standard (STD), due to a stronger ability to inhibit DNA alpha-polymerase (IC(50): 378 microg/mL, vs 1784 microg/mL for STD). After a 48-h continuous treatment, IC(50) values for growth inhibition were in the range of 20-46 microg/mL instead of 94 to >250 microg/mL for STD, and those for inhibition of metabolic activity were in the range of 34-87 microg/mL instead of >250 microg/mL for STD. The higher anti-proliferative effect was observed on colon adenocarcinoma DLD-1 cells, and the weaker effect was observed on breast adenocarcinoma MCF-7.

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Abstract: A water-soluble hydroxycinnamate-derived polymer (>1000 kDa) from Symphytum asperum Lepech. (Boraginaceae) strongly reduced the diphenylpicrylhydrazyl radical (IC(50) approximately 0.7 microg/mL) and inhibited the nonenzymatic lipid peroxidation of bovine brain extracts (IC(50) approximately 10 ng). This polymer exhibited only a low hydroxyl radical scavenging effect in the Fe(3+)-EDTA-H(2)O(2) deoxyribose system (IC(50) > 100 microg/mL) but strongly decreased superoxide anion generation in either the reaction of phenazine methosulfate with NADH and molecular oxygen (IC(50) approximately 13.4 microg/mL) or in rat PMA-activated leukocytes (IC(50) approximately 5 microg/mL). The ability to inhibit both degranulation of azurophil granules and superoxide generation in primed leukocytes indicates that the NADPH oxidase responsible for this later effect is inhibited, pointing to the Symphytum asperum polymer as a potent antiinflammatory and vasoprotective agent. At all concentrations tested (0-200 microg/mL), we observed no cytotoxicity on normal human fibroblasts and neither antiproliferative effects nor proliferation activation on neoplastic cells.

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Abstract: Pyrethrins, the most economically important natural insecticide, comprise a group of six closely related monoterpene esters. The industrial production is based on their extraction from Chrysanthemum cinerariaefolium (Pyrethrum) capitula. The world production of natural pyrethrins still falls short of global market demand stimulating the research in in vitro production as an alternative to conventional cultivation methods. The different biotechnological alternatives such as callus cultures, shoot and root cultures, plant cell suspension cultures, and bioconversion of precursors by means of enzymatic synthesis or genetically engineered microorganisms, as well as the progress achieved in methods for the identification and quantitation of insecticidal compounds have been reviewed. Although technology for plant cell culture exists, industrial applications have, to date, been limited due to both the low economical viability and technological feasibility at large scale. Bioconversion of readily available precursors looks more attractive, but more research is needed before this technology is used for the industrial production of pyrethrins.

Abstract: Prebiotic agents are food ingredients that are potentially beneficial to the health of consumers. The main commercial prebiotic agents consist of oligosaccharides and dietary fibres (mainly inulin). They are essentially obtained by one of three processes: 1) the direct extraction of natural polysaccharides from plants; 2) the controlled hydrolysis of such natural polysaccharides; 3) enzymatic synthesis, using hydrolases and/or glycosyl transferases. Both of these enzyme types catalyse transglycosylation reactions, allowing synthesis of small molecular weight synthetic oligosaccharides from mono- and disaccharides. Presently, in Europe, inulin-type fructans, characterised by the presence of fructosyl units bound to the beta-2,1 position of sucrose, are considered as one of the carbohydrate prebiotic references. Prebiotics escape enzymatic digestion in the upper gastrointestinal tract and enter the caecum without change to their structure. None are excreted in the stools, indicating that they are fermented by colonic flora so as to give a mixture of short-chain fatty acids (acetate, propionate and butyrate), L-lactate, carbon dioxide and hydrogen. By stimulating bifidobacteria, they may have the following implications for health: 1) potential protective effects against colorectal cancer and infectious bowel diseases by inhibiting putrefactive bacteria (Clostridium perfringens ) and pathogen bacteria (Escherichia coli, Salmonella, Listeria and Shigella ), respectively; 2) improvement of glucid and lipid metabolisms; 3) fibre-like properties by decreasing the renal nitrogen excretion; 4) improvement in the bioavailability of essential minerals; and 5) low cariogenic factor. These potential beneficial effects have been largely studied in animals but have not really been proven in humans. The development of a second generation of oligosaccharides and the putative implication of a complex bacterial trophic chain in the intestinal prebiotic fermentation process are also discussed.

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Abstract: Fructo-oligosaccharides (FOS) are new food ingredients that are able to beneficially affect the host by selectively stimulating the growth and/or activity of colonic bifidobacteria (concept of prebiotics). A commercial enzyme preparation was found to possess a high fructosyltransferase activity and could be used as a biocatalyst for the industrial production of FOS from sucrose. Under optimum conditions (pH: 5.5, temperature: 55 degrees C and 7 units of fructosyltransferase activity per gram sucrose), in presence of glucose (competitive inhibitor) the actual yield reached the theoretical value (up to 50%). Actually, FOS that are commercially available for their prebiotic properties belong to inulin type with low degree of polymerisation (DP:3 to 10). Our FOS were identified by both HPLC and 13C-NMR spectrometry as neo-FOS type (neo-kestose, neo-nystose and neo-fructofuranosylnystose), a new structure which is very close to inulin type (same linkage between fructosyl units). The neo-FOS may act as a prebiotic factor due to their structural similarity with inulin type.

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Abstract: Octastatin (vapreotide INN) is a somatostatin analogue being developed in gastro-enterologic, neuroendocrine and oncologic applications. The pharmaceutical form is a freeze-dried preparation for parental injection use. This study was intended to evaluate the stability of the freeze-dried products and to determine the optimally stable formulation. Two types of stabilizing agents (lactose and glutamic acid/sodium glutamate buffer) and three dosage forms (0.5, 1.5, 15 mg of vapreotide base) were investigated. The peptide content and chemical stability of cakes stored at 50 degrees C and 70% relative humidity were determined by HPLC and the regression analysis parameters calculated. Results indicate that the formulation with glutamate buffer is appropriate for long term storage.

Abstract: Octastatin (RC-160, vapreotide INN) is a somatostatin analogue being developed for use in oncological, enterologic and neuroendocrine applications. The pharmaceutical form is a freeze-dried preparation for parenteral injection use. Three dosage forms containing 0.5, 1.5 and 15 mg of vapreotide base have been investigated. Various freeze-drying conditions, stabilizing agents, membranes for sterile filtration and heating procedures have been examined. The formulation with glutamic acid-sodium glutamate buffer as a stabilizing agent, the type of membrane for filtration and the freeze-drying procedure have been found appropriate for subsequent industrial production. No evident degradation was observed either after manufacturing, or after a three-week accelerated stability study at 50 degrees C and 70% relative humidity.

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Abstract: A strain of Aspergillus ochraceus, a microorganism classically used for bioconversions of the steroid nucleus, was tested on solasodine glucosides. It induced specific modifications to the oside chain of the solamargine molecule, without modifying the genin nucleus. These modifications depended on the composition of the culture medium and the aeration, and led under certain conditions to a single isomer of the secondary glucoside beta 2-solamargine or Khasianine: o-alpha-L-rhamnopyranosyl-(1-->4 glu)-O-(3)-beta-D-glucopyranosyl solasodine.

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Abstract: Densitometric analysis has been performed on various standard protein and peptide solutions and on luteinizing hormone releasing hormone hydrolysate. After thin-layer chromatographic separation using two-dimensional separation on cellulose plates, the plates were mapped and each amino acid was assayed individually in order to obtain a specific integration profile of the hydrolysate. Densitometry proved to be a useful method for the screening of protein hydrolysates and is accurate enough for the assay of peptide hydrolysates. The hormone was hydrolysed with hydrochloric acid-trifluoroacetic acid (1:1, v/v) in the presence of thioglycolic acid. This limits the loss of tryptophan and does not interfere with the analysis. Results were sufficiently reliable, accurate and reproducible for routine analysis.

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Abstract: Amylase inhibitory activities have been sought in the powders of Phaseolus vulgaris pods and of Vicia faba bean hulls. Unlike to the bean, Phaseolus vulgaris pod has no amylase inhibitory activity and the presence of tannin could not be determined. Contrary, Vicia faba bean hull shows an obvious amylase inhibitory activity. It contains an appreciable content of tannins which may be responsible of this activity. The quantities of amylase whose are inhibited increase in some measure with the quantities of enzyme added. They are not in direct ratio to the quantities of extract, then, inhibition may be of no competitive type.

Abstract: Trypsin inhibitory activities of Phaseolus vulgaris L. pods and Vicia faba L. bean hulls have been studied according to the extraction conditions of the powder: solvent pattern, temperature and pH. The two vegetal powders have a trypsin inhibitory activity quite similar, weak but appreciable. The trypsin inhibitor of Phaseolus vulgaris L. pods is thermosensitive but stable at low pH. Inversely the Vicia faba L. bean hulls one is thermostable but it feels low pH.

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Abstract: A. niger LCF 9 synthesizes a new aspergillopeptidase of potential interest in therapeutics. The properties and operating range of the enzyme were determined. It is a semi-alkaline aspergillopeptidase (EC 3.4.23.4) with one endopeptidase activity. Its pI is 4.10, its molecular weight is 21000 Da and its A1%(1 cm) at 280 nm is 9.75. It rapidly hydrolyzes casein and hemoglobin. Its optimal pH is 7.8 and optimal temperature is 45 degrees C. It is thermally labile above 40 degrees C but can be stabilized by adding calcium ions. It is inhibited by phenylmethylsulfonyl fluoride (PMSF), by ethylenediaminetetraacetic acid (EDTA) and by certain metals ions, e.g. copper, manganese and cobalt ions. It has no dipeptidase or tripeptidase activity and its esterase activity is weak. It has a high collagenase activity and is to our knowledge the only aspergillopeptidase that is active towards benzoyl-arginine p-nitroanilide (BAPNA).

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Abstract: PURPOSE Previous reports have demonstrated a value of HRMAS-proton magnetic resonance spectroscopy (HRMAS-1H-MRS) for metabolomic analysis of cancer cells and identification of drug response pathways and biomarkers (1,2). In this study, HRMAS-1H-MRS was applied to human MCF7 breast carcinoma cells treated by the marine-derived depsipeptide Kahalalide F (KF). KF promotes oncosis cell death and, antitumor activity independent of multidrug resistance (MDR) and of p53 status. The hepatocellular carcinoma, NSCLC and melanoma phase II clinical trials finished in 2006. Because it modulate lysosomal function, we suspected KF to strongly affect membranar phospholipid remodeling pathways. METHODS To test this hypothesis, exponentially growing human breast MCF7 carcinoma cells were treated by KF (5 µM) in kinetic mode. The cell survival was determined with the resazurin reduction test that determines the amount of fluorescent resorufin produced by living cells. Treated and control cells were submitted to proton NMR spectroscopy with a "Brucker DX500" equipped with a high resolution magic angle spinning (HRMAS) probe. Sequences were used after water signal suppression. Spectra were recorded in 1D mode for qualitative response profiles and in 2D mode ([H,H]-TOCSY] for quantification of cell metabolites. RESULTS. 31 metabolites were identified and quantified. As expected, strong alterations of phospholipid metabolism leading to rapid blockade of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PdtEth) biosynthesis and degradation pathways, were detected as early as 6h after MCF7 treatment. These alterations were time-dependent and correlated with Krebs cycle and glycolysis disturbance. Significant changes in the content of osmolytes devoted to protection of cell homeostasis, most notably the sodium transporter myoinositol, and irreversible accumulation of phosphoethanolamine were further observed. CONCLUSION The present data demonstrate that in vitro, human breast carcinoma response to KF involves early and profound alterations of phospholipid metabolism leading to progressive krebs-cycle and glycolysis blockade and to myoinositol accumulation. In MCF7 cultures, myoinositol is a biomarker of KF response. Further studies are needed to determine its value as biomarker of clinical response.

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Abstract: Docetaxel (Taxotère®) is a microtubule-stabilizing agent with antitumour activity in various tumor types, including advanced and metastatic breast cancer. Docetaxel induces apoptosis through decreased protein kinase C (PKC) activity and cell cycle blockade with decreased structural and antioxidative gene expression. Metabolic changes in response to taxanes were previously shown to involve lipid accumulation. However, the cytotoxic mechanisms of docetaxel remain partially unknown. The identification of metabolic pathways and biomarkers of the response is an important challenge for cancer treatment. We thus used high resolution magic angle spinning (HRMAS) 1H-NMR spectroscopy, a well known metabolomics tool, in an attempt to obtain such information in human MCF7 breast tumor cells treated by docetaxel. MCF7 cells were exposed to docetaxel at clinical dose (5 μM) and followed at various intervals of time (12h, 36h and 72h). Cytotoxicity was assayed using Hoescht 33342. Proton 1D saturationrecovery and 2D TOCSY spectra were acquired on untreated and treated intact cell pellets. A new quantitative procedure, applied to 2D NMR data, permitted, in combination with 1D data, to quantify variations of thirty metabolites. A 72h-exposition to docetaxel decreased MCF7 cell survival to 8.3% (P < 0.01). Among the quantified metabolites, the most significant variations were: a decrease of total glutathione pool by 39% (P < 0.05) and of lactate by 53% (P < 0.05). We observed strong variations ofincrease of polyunsaturated fatty acids by 1600 % (P < 0.01), phosphoethanolamine by 119 % (P < 0.01) and cytidinediphosphocholine by 470 % (P < 0.01), and a decrease of glycerophosphoethanolamine by 44 % (P < 0.01), glycerophosphocholine by 54 % (P < 0.01) and phosphatidylcholine (PtdCho) by 74 % (P < 0.05). Taken together our data support a downregulation of PtdCho biosynthesis, at the cholinephosphotransferase (CPT) level, and glycolysis disturbances thus reducing diacylglycerol (DAG) biosynthesis. This suggests that docetaxel induces apoptosis through a reduction of DAG level and a down-regulation of PKC activity, as was previously reported for farnesol-induced apoptosis. Moreover, since PtdCho is involved in the removal of ceramides, its strong decrease may promote an accumulation of ceramides that may contribute to induce apoptosis.

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