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Many natural components of plants extract are studied for their beneficial effects on health and particularly on carcinogenesis chemoprevention. In this study, we investigated the effect of citral (3,7-dimethyl-2,6-octadienal), a key component of essential oils extracted from several herbal plants, on the proliferation rate, cell cycle distribution, and apoptosis of the human breast cancer cell line MCF-7. The effects of this compound were also tested on cyclo-oxygenase activity. Citral treatment caused inhibition of MCF-7 cell growth (IC(50)-48 h: 18 x 10(-5)m), with a cycle arrest in G(2)/M phase and apoptosis induction. Moreover, we observed a decrease in prostaglandin E(2) synthesis 48 h after citral treatment. These findings suggest that citral has a potential chemopreventive effect.

International audience; Recently, sedimentation field-flow fractionation (SdFFF) was used to study the specific kinetics of diosgenin-induced apoptosis in K562 cells. Here, we propose a new SdFFF cell separation application in the field of cancer research concerning the correlation between induction of a biological event (i.e. apoptosis) and cell status (i.e. cell cycle position). SdFFF isolated subpopulations depending on the cell cycle position allowing the study of apoptosis kinetics and extent. Results showed that cells in G0/G1 phases (F3 cells) underwent significant and earlier apoptosis than cells in the active part of the cell cycle (S/G2/M phases). Results shed light on the correlation between differences in apoptosis kinetics and cell cycle stage when exposure to the inducer began. SdFFF monitoring and size measurement also led to the description of different subpopulations demonstrating complex variations in density between fractions associated with differences in biological processes.

International audience; Differentiation induction as a therapeutic strategy has, so far, the greatest impact in hematopoietic malignancies, most notably leukemia. Diosgenin is a very interesting natural product because, depending on the specific dose used, its biological effect is very different in HEL (human erythroleukemia) cells. For example, at 10 microM, diosgenin induced megakaryocytic differentiation, in contrast to 40 microM diosgenin, which induced apoptosis in HEL cells previously demonstrated using sedimentation field-flow fractionation (SdFFF). The goal of this work focused on the correlation between cyclooxygenase-2 (COX-2) and thromboxane synthase (TxS) and megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, the technique of SdFFF, having been validated in our models, was used in this new study as an analytical tool that provided us with more or less enriched differentiated cell fractions that could then be used for further analyses of enzyme protein expression and activity for the first time. In our study, we showed the implication of COX-2 and TxS in diosgenin-induced megakaryocytic differentiation in HEL cells. Furthermore, we showed that the analytical technique of SdFFF may be used as a tool to confirm our results as a function of the degree of cell differentiation.

Anti-cancer differentiation therapy could be one strategy to stop cancer cell proliferation. We propose a new sedimentation field flow fractionation (SdFFF) cell separation application in the field of cancer research. It concerns the study of megakaryocytic differentiation processes after a short exposure to an inducting agent (diosgenin). Washout process and early dual SdFFF separation--removing the influence of diosgenin and decreasing the influence of undifferentiated cells--resulted in the preparation of an enriched population to study the mechanism and kinetics of megakaryocytic differentiation. A short exposure to diosgenin was able to induce complete differentiation leading to maximal maturation which ended naturally after 192h incubation without the influence of a secondary effect of diosgenin. The study of isolated undifferentiated cells also showed that no resistance to diosgenin was observed. This result suggested different sensitivities to differentiation induction, and SdFFF cell separation would be of great interest to explore this phenomena.

Apoptosis is one of the most important phenomena in cell biology. Pre-apoptotic cells, defined as cells engaged in early stages of apoptosis, could be used as a cellular tool to study apoptosis pathways. The human 1547 osteosarcoma cell line and diosgenin (a plant steroid) association was selected as an in vitro cellular apoptosis model. In a previous study, using this model, we demonstrated that SdFFF monitored apoptosis induction as early as 6h after incubation. In this study, we investigated the capacity of Sedimentation Field-Flow Fractionation (SdFFF) to sort an enriched population of pre-apoptotic cells from 1547 cells incubated for 6 h with 40 microM diosgenin. In that way, two different separation devices which differed especially in channel thickness, 125 and 175 microm, were used and compared. Results showed, for the first time, that SdFFF is an effective method to obtain an enriched pre-apoptotic sub-population. These results suggest, as a new application, that SdFFF could be an included tool in the study of apoptotic mechanisms or the kinetic action of apoptotic drugs.

Inflammatory cytokines or soluble factors are essential in the pathogenesis of rheumatoid arthritis (RA). Leflunomide is an effective disease modifying antirheumatic drug (DMARD) in RA. The objective of the present study was to evaluate for the first time the effects of A77 1726 on cytokine (interleukin (IL)-8, IL-10, IL-11 secretion and tumor necrosis factor-alpha soluble receptor I (sTNFRI)) shedding in human RA fibroblast-like synoviocytes (FLS). At 100 microM, we observed an increase in IL-10 secretion, a decrease in IL-11 release and no effect on sTNFRI shedding and IL-8 secretion in IL-1beta-stimulated human RA FLS. Furthermore, at this dose, our results also confirmed that A77 1726 decreased IL-6 and prostaglandin E2 (PGE2) synthesis while it increased IL-1 receptor antagonist secretion (IL-1Ra). The mitogen-activated protein kinases (MAPKs) represent an attractive target for RA because they can regulate cytokine expression. At 100 microM, the effect of A77 1726 on IL-10 and IL-11 secretion seemed to be associated with the status of p38 MAPK activation. Our results confirmed the immunoregulatory action of leflunomide in the cytokine network involved in RA pathogenesis. It could shift the balance from cytokine mediated inflammation to cytokine directed inhibition of the inflammatory process.

International audience; Prostate cancer is the most common malignant cancer in men and the second leading cause of cancer deaths. Previously, we have shown that 2′-hydroxy-4-methylsulfonylchalcone (RG003) induced apoptosis in prostate cancer cell lines PC-3 and DU145. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent, some cancer cells are resistant to TRAIL treatment. PC-3 and LNCaP prostatic cancer cell lines have been reported to be resistant to TRAIL-induced apoptosis. Here, we show for the first time that RG003 overcomes TRAIL resistance in prostate cancer cells. RG003 can enhance TRAIL-induced apoptosis through DR5 upregulation and downregulation of Bcl-2, PI3K/Akt, NF-?B, and cyclooxygenase-2 (COX-2) survival pathways. When used in combined treatment, RG003 and TRAIL amplified TRAIL-induced activation of apoptosis effectors and particularly activation of caspase-8 and the executioner caspase-3, leading to increased poly-ADP-ribose polymerase cleavage and DNA fragmentation in prostate cancer cells. Furthermore, we showed that RG003 reduced COX-2 expression in cells. Previously, we showed that COX-2 was involved in resistance to an apoptosis mechanism; then, its inhibition by RG003 could render cells more sensitive to TRAIL treatment. We showed that nuclear factor-?B activation was inhibited after RG003 treatment. This inhibition was correlated with reduction in COX-2 expression and induction of apoptosis. Overall, we conclude, for the first time, that RG003 can enhance TRAIL-induced apoptosis in human prostate cancer cells. The significance of our in-vitro study with RG003 and TRAIL combined is very encouraging, suggesting the relevance of testing this combined treatment in xenograft animal models.

International audience; Differentiation therapy is a means to treat cancer and is induced by different agents with low toxicity and more specificity than traditional ones. Diosgenin, a plant steroid, is able to induce megakaryocytic differentiation or apoptosis in human HEL erythroleukemia cells in a dose-dependent manner. However, the exact mechanism by which diosgenin induces megakaryocytic differentiation has not been elucidated. In this study, we studied the involvement of Sonic Hedgehog in megakaryocytic differentiation induced by diosgenin in HEL cells. First, we showed that different elements of the Hedgehog pathway are expressed in our model by qRT-PCR. Then, we focused our interest on key elements in the Sonic Hedgehog pathway: Smoothened receptor, GLI transcription factor and the ligand Sonic Hedgehog. We showed that Smoothened and Sonic Hedgehog were overexpressed in disogenin-treated cells and that GLI transcription factors were activated. Then, we showed that SMO inhibition using siSMO or the GLI antagonist GANT-61, blocked megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, we demonstrated that Sonic Hedgehog pathway inhibition led to inhibition of ERK1/2 activation, a major physiological pathway involved in megakaryocytic differentiation. In conclusion, our study reports, for the first time, a crucial role for the Sonic Hedgehog pathway in diosgenin-induced megakaryocytic differentiation in HEL cells.

Peripheral aromatization of androgens exerts estrogenic actions in many tissues. In this study, osteoarthritis synoviocytes were examined to clarify the possible action of adrenal androgen on synovial cell. Synoviocytes from postmenopausal women are able to express aromatase mRNA. By sequence analysis, the PCR fragment (485 bp) was determined to be 100% identical to that of human placental aromatase cDNA. Moreover, this study demonstrates that adrenal androgen, androstenedione, is converted to estrone (E(1)) and estradiol (E(2)) in synoviocytes by aromatase which is positively regulated by glucocorticoids such as dexamethasone. E(2) production reduced significantly IL-6 secretion. These data provide preliminary evidence that in situ estrogen production by synoviocytes may have a role in OA susceptibility. However the role of E(2) in OA is not clear and remains to be determined.

The present investigation describes the ability of human 5-lipoxygenase-activating protein (FLAP) to activate a plant 5-lipoxygenase. The presence of an active recombinant human FLAP in the 100 000× g membrane fraction of infected Sf9 cells led to a specific increase in 9-hydroperoxyoctadecadienoic acid (9-HPOD) synthesis (+68%) or in 5-hydroperoxyeicosatetraenoic acid (5-HPETE) synthesis (+68%), after action of Solanum tuberosum tuber 5-lipoxygenase ( S.t. LOX) on linoleic acid (natural plant lipoxygenase substrate) or on arachidonic acid. On the contrary, the presence of non-transfected membranes obtained from non-infected Sf9 cells led to an inhibition of lipoxygenase activity. MK-886, a potent inhibitor of leukotriene biosynthesis, blocked the FLAP dependent S.t .LOX activation after preincubation with FLAP transfected membranes. In conclusion, this study demonstrates that a recombinant human FLAP can stimulate a lipoxygenase other than mammalian 5-lipoxygenase ( S.t. LOX) by using different polyunsaturated fatty acids as substrates.

International audience; Cyclooxygenase-2 (COX-2) has been found to be highly expressed in many types of cancers and to contribute to tumorigenesis via the inhibition of apoptosis, increased angiogenesis and invasiveness. In hematological malignancies, COX-2 expression was found to correlate with poor patient prognosis. However, the exact role of COX-2 expression in these malignancies, and particularly in erythroleukemias, remains unclear. The aim of this work was to describe and understand the relationships between COX-2 expression and apoptosis rate in erythroleukemia cells after apoptosis induction by several anticancer agents. We used three different erythroleukemia cell lines in which COX-2 expression was modulated by transfection with either COX-2 siRNA or COX-2 cDNA. These cellular models were then treated with apoptosis inducers and apoptosis onset and intensity was followed. Cell signalling was evaluated in unstimulated transfected cells or after apoptosis induction. We found that COX-2 inhibition rendered erythroleukemia cells more sensitive to apoptosis induction and that in cells overexpressing COX-2 apoptosis induction was reduced. We demonstrated that COX-2 inhibition decreased the pro-survival Akt signalling and activated the negative regulator of Akt signalling, phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Conversely, in COX-2 overexpressing cells, Akt signalling was activated and PTEN was inhibited. In these last cells, inhibition of casein kinase 2 or Akt signalling restored sensitivity to apoptotic agents. Our findings highlighted that COX-2 can positively regulate Akt signalling mostly through PTEN inhibition, partly via casein kinase 2 activation, and enhances survival of erythroleukemia cells exposed to anticancer agents.

International audience; Limited success has been achieved in extending the survival of patients with metastatic and hormone-refractory prostate cancer (HRPC). There is a strong need for novel agents in the treatment and prevention of HRPC. In the present study, the apoptotic mechanism of action of RG003 (2'-hydroxy-4- methylsulfonylchalcone) and RG005 (4'-chloro-2'-hydroxy-4- methylsulfonylchalcone) in association with intracellular signalling pathways was investigated in the hormone-independent prostate carcinoma cells PC-3 and DU145. We showed that these compounds induced apoptosis through the intrinsic pathway but not through the extrinsic one. We showed that synthetic chalcones induced an activation of caspase-9 but not caspase-8 in PC-3 cells. Even if both chalcones induced apoptosis in PC-3 cells, a dominant effect of RG003 treatment was observed resulting in a disruption of δm, caspase-9 and caspase-3 activation, PARP cleavage and DNA fragmentation. Furthermore, in regard to our results, it is clear that the simultaneous inhibition of Akt and NF-κB signalling can significantly contribute to the anticancer effects of RG003 and RG005 in PC-3 prostate cancer cells. NF-κB inhibition was correlated with the reduction of COX-2 expression and induction of apoptosis. Our results clearly indicate for the first time that RG003 and RG005 exert their potent anti proliferative and pro-apoptotic effects through the modulation of Akt/NF-κB/COX-2 signal transduction pathways in PC-3 prostate cancer cells with a dominant effect for RG003

International audience; Erythroleukemia is generally associated with a very poor response and survival to current available therapeutic agents. Cyclooxygenase-2 (COX-2) has been described to play a crucial role in the proliferation and differentiation of leukemia cells, this enzyme seems to play an important role in chemoresistance in different cancer types. Previously, we demonstrated that diosgenin, a plant steroid, induced apoptosis in HEL cells with concomitant COX-2 overexpression. In this study, we investigated the antiproliferative and apoptotic effects of cyclopamine and jervine, two steroidal alkaloids with similar structures, on HEL and TF1a human erythroleukemia cell lines and, for the first time, their effect on COX-2 expression. Cyclopamine, but not jervine, inhibited cell proliferation and induced apoptosis in these cells. Both compounds induced COX-2 overexpression which was responsible for apoptosis resistance. In jervine-treated cells, COX-2 overexpression was NF-κB dependent. Inhibition of NF-κB reduced COX-2 overexpression and induced apoptosis. In addition, cyclopamine induced apoptosis and COX-2 overexpression via PKC activation. Inhibition of the PKC pathway reduced both apoptosis and COX-2 overexpression in both cell lines. Furthermore, we demonstrated that the p38/COX-2 pathway was involved in resistance to cyclopamine-induced apoptosis since p38 inhibition reduced COX-2 overexpression and increased apoptosis in both cell lines.

International audience; One of the hallmarks of cancer is resistance to apoptosis. Elucidating the mechanisms of how cancer cells evade or delay apoptosis should lead to novel therapeutic strategies. Previously, we showed that HT-29 colorectal cancer cells undergoing apoptosis overexpressed cyclooxygenase-2 (COX-2), in a p38 dependent pathway, to delay ursolic acid-induced apoptosis. Here, we focused on elucidating the upstream signaling pathways regulating this resistance mechanism. The role of ATP as an extracellular signaling molecule took a long time to be accepted. In recent years, ATP and its analogs, via the activation of specific purinergic receptors, have been implicated in many biological processes including cell proliferation, differentiation and apoptosis. In the present report, we have demonstrated a novel role involving purinergic receptors and particularly the P2Y(2) receptor in resistance to ursolic acid-induced apoptosis in both colorectal HT-29 and prostate DU145 cancer cells. We found that ursolic acid induced an increase in intracellular ATP and P2Y(2) transcript levels. Upon activation, P2Y(2) activated Src which in turn phosphorylated p38 leading to COX-2 overexpression which induced resistance to apoptosis in both HT-29 and DU145 cells. Furthermore, Ca(2+)-independent PLA(2) (iPLA(2)) and Ca(2+)-dependent secretory PLA(2) (sPLA(2)) were responsible for arachidonic acid release, the substrate of COX-2. Our findings document that apoptosis triggering was dependent on protein kinase C (PKC) activation in both cell lines after ursolic acid treatment.

Previously, we demonstrated that diosgenin induced apoptosis in colorectal cancer cell lines HCT-116 and HT-29. HT-29 cells have been reported to be one of the most resistant colorectal cancer cell lines to TRAIL-induced apoptosis. In this study, we investigated the effect of diosgenin on TRAIL-induced apoptosis in HT-29 cells. We showed that diosgenin sensitizes HT-29 cells to TRAIL-induced apoptosis. Mechanisms underlying this sensitization mainly involved diosgenin-induced p38 MAPK pathway activation and subsequent DR5 overexpression. Furthermore, we showed that diosgenin alone, TRAIL alone or combination treatment increased COX-2 expression and that the use of a COX-2 inhibitor further increased apoptosis induction.

Colorectal cancer is one of the most common cancer types and the third leading cause of cancer-related death in the western world. Generally, colorectal cancers are resistant to anticancer drugs. Several lines of evidence support a critical role for cyclooxygenase-2 (COX-2) during colorectal tumorigenesis and its role in chemoresistance. In this study, we focused our interest on the role played by COX-2 in apoptosis induced in HT-29 human colorectal cancer cells by ursolic acid (UA), a triterpenoid found in a large variety of plants. We showed that UA-induced apoptosis and that COX-2 was overexpressed only in apoptotic cells. We demonstrated that this overexpression was mediated by the p38 MAP kinase pathway as inhibiting its activation using a p38-specific inhibitor, SB 203580, abrogated COX-2 expression. Inhibiting COX-2 expression either by using a p38-specific inhibitor or COX-2-specific siRNA increased apoptosis. These results demonstrated that COX-2 was involved in a resistance mechanism to UA-induced apoptosis in HT-29 cells. Cells undergoing apoptosis were able to trigger a resistance mechanism by overexpressing a protein such as COX-2 to delay their death. Furthermore, we demonstrated that this resistance mechanism was independent of PGE(2) production as the addition of the specific COX-2 activity inhibitor, NS-398, did not affect apoptosis in UA-treated cells.

Differentiation therapy could be one strategy for stopping cancer cell proliferation. A plant steroid, diosgenin, is known to induce megakaryocytic differentiation in human erythroleukemia (HEL) cells. In recent studies, the use of sedimentation field-flow fractionation (SdFFF) allowed the preparation of subpopulations that may differ in regard to sensitivity to differentiation induction. The specific goal of this study was to determine the relationship between cell cycle stage and sensitivity to megakaryocytic differentiation induction of HEL cells. After first confirming the capacity of diosgenin to specifically select targets, hyperlayer SdFFF cell sorting was used to prepare fractions according to cell cycle position from crude HEL cells. The sensitivities of these fractions to diosgenin-induced differentiation were then tested. The coupling of SdFFF cell separation to imaging flow cytometry showed that G1-phase cells were more sensitive to differentiation induction than S/G2M-phase cells, confirming the relationship between cell status at the start of induction, the extent of the biological event, and the potential of SdFFF in cancer research.

International audience; Classically described as a macroscale size-density based method, Sedimentation field flow fractionation (SdFFF) has been successfully used for cell sorting. The goal of this study was to develop a new SdFFF device for downscale applications, in particular for oncology research to rapidly monitor chemical biological event induction in a cell line. The development of a downscale SdFFF device required reduction of the separation channel volume. Taking advantage of a newly laboratory designed apparatus, channel volume was successfully decreased by reducing both length and breadth. To validate the apparatus and method, we used the well-known model of diosgenin dose-dependent induction of apoptosis or megakaryocytic differentiation in HEL cells. After a minute scale acquisition of a reference profile, the downscale device was able to perform fast, early, significant and reproducible monitoring of apoptosis and differentiation, two important biological mechanisms in the field of cancer research.

Summary An enzyme with at least dual activities, lipoxygenase and fatty acid lyase, has been isolated from Vicia sativa seeds. The enzyme utilizes directly linoleic acid as substrate. The enzyme had a pH optimum at 5.8 for the two activities and converted linoleic acid into two products : 9-hydroperoxylinoleic acid and trans-2, cis-4 decadienal. The enzyme does not act on 13- or 9- fatty acid hydroperoxide isomers. An enzymatic reaction for the biogenesis of trans-2, cis-4- decadienal is proposed. This involves the synthesis of an intermediate peroxyl radical due to oxygen insertion in carbon 9 of linoleic acid. This intermediate peroxyl radical may be converted into 9-HPOD and 2,4-decadienal.

International audience; Recently, the use of SdFFF in cancer research has been studied in order to better understand major phenomena implicated in cancer development and therapy: apoptosis and differentiation. In this report, we used SdFFF as a monitoring and cell separation tool to study the kinetics of apoptosis. Incubation of K562 cells with diosgenin, used as cellular model, led to a surprising apoptotic process occurring in two phases (after 24 and 48 h incubation), associated with specific p-ERK expression. Based on the capacity to sort apoptotic cells, results showed that SdFFF cell separation was an effective analytical tool to obtain different subpopulations regardless of the kinetics and extent of apoptosis. Results also showed that, after proper biological calibration of elution profiles, SdFFF can be used to monitor either the induction or the kinetics of a biological event.

Rheumatoid arthritis (RA) is characterized by persistent joint synovial tissue inflammation. Leflunomide is an immunomodulatory agent that has been approved for treatment of active RA. In the past few years, uses other than RA treatment have appeared. Leflunomide has been reported to show antitumor potential through inhibition of cancer cell proliferation. We thus tested the antiproliferative potential of leflunomide on HEL and K562 erythroleukemia cells. The findings summarized in this report demonstrate for the first time that low dose leflunomide prolonged survival and reduced apoptosis induced by several anticancer agents in erythroleukemia cells. We showed that in treated cells, leflunomide reduced the signalling pathways involved in promoting apoptosis by reducing p38 MAPK and JNK basal activity. On the other hand, leflunomide transiently activated the ERK signalling pathway and induced a sustained activation of Akt. We also showed that leflunomide reduced caspase-3 activity and DNA fragmentation induced by anticancer agents. By using an inhibitory strategy, we showed that inhibition of Akt activation but not ERK abolished the protective effect of leflunomide. Thus our findings suggested that leflunomide reduced apoptosis induced by anticancer agents through PI3K/Akt signalling activation.

Megakaryocytopoiesis is characterized by progressive polyploidization and acquisition of megakaryo-cytic markers. MAPK pathways play a key role during megakaryocytic differentiation of megakaryocyte precursors or leukemic cells. Apoptosis is the physiological fate of normal megakaryocyte after differentiation and maturation. The aim of this study was to investigate the signaling pathways involved in diosgenin-induced differentiation and the fate of diosgenin-differentiated HEL cells. The present report shows that diosgenin induced megakaryocytic differentiation of HEL cells through a combined activation of ERK and inhibition of the p38 MAPK pathways. Inhibition of ERK activation by a MEK inhibitor abrogated diosgenin-induced differentiation. Afterwards, differentiated cells showed a marked inhibition of expression of survival factors NF-kappaB, Akt and Bcl-xL and activation of caspase-3 together with PARP cleavage leading to apoptotic death of diosgenin-differentiated cells.

Megakaryocytopoiesis is characterized by progressive polyploidization and acquisition of megakaryo-cytic markers. MAPK pathways play a key role during megakaryocytic differentiation of megakaryocyte precursors or leukemic cells. Apoptosis is the physiological fate of normal megakaryocyte after differentiation and maturation. The aim of this study was to investigate the signaling pathways involved in diosgenin-induced differentiation and the fate of diosgenin-differentiated HEL cells. The present report shows that diosgenin induced megakaryocytic differentiation of HEL cells through a combined activation of ERK and inhibition of the p38 MAPK pathways. Inhibition of ERK activation by a MEK inhibitor abrogated diosgenin-induced differentiation. Afterwards, differentiated cells showed a marked inhibition of expression of survival factors NF-kappaB, Akt and Bcl-xL and activation of caspase-3 together with PARP cleavage leading to apoptotic death of diosgenin-differentiated cells.

Anticancer differentiation therapy could be one strategy to stop cancer cell proliferation. Human erythroleukemia (HEL) cell line, incubated with 10 microM diosgenin, underwent megakaryocytic differentiation. Thus, the association diosgenin/HEL could be used as a model of chemically induced cellular differentiation and anticancer treatment. The goal of this work was to determine the capacity of sedimentation field-flow fractionation (SdFFF) to sort megakaryocytic differentiated cells. SdFFF cell sorting was associated with cellular characterization methods to calibrate specific elution profiles. As demonstrated by cell size measurement methods, cellular morphology, ploidy, and phenotype, we obtained an enriched, sterile, viable, and functional fraction of megakaryocytic cells. Thus, SdFFF is proposed as a routine method to prepare differentiated cells that will be further used to better understand the megakaryocytic differentiation process.

Diosgenin is a steroidal sapogenin with antitumor properties. We previously showed that diosgenin induced apoptosis in human erythroleukemia (HEL) cells. In order to elucidate the mechanism of its apoptotic activity, we investigated the effect of diosgenin on nuclear factor-kappaB (NF-kappaB) binding and on three groups of human mitogen-activated protein kinases (MAPKs) in relation to diosgenin-induced apoptosis in different erythroleukemia cell lines (K562 and HEL). Our results showed that diosgenin decreased DNA binding of NF-kappaB in K562 and HEL cells after 48-h diosgenin treatment. This inhibition of NF-kappaB binding was correlated with strong apoptosis in both erythroleukemia cell lines. Diosgenin-induced apoptosis was associated with cyclo-oxygenase-2 (COX-2) up-regulation in HEL cells but not in K562 cells which are COX-2 deficient. Furthermore, diosgenin inhibited extracellular signal-regulated kinase (ERK) activation only in HEL cells. However, diosgenin activated p38 MAPK in both cell lines and activated c-jun NH2-terminal kinases (JNKs) only in HEL cells. Pre-treatment with a selective p38 inhibitor inhibited diosgenin-induced DNA fragmentation in K562 cells. For the first time, our results suggest that inhibition of NF-kappaB nuclear binding and p38 MAPK activation are involved in the diosgenin-mediated signal cascades in K562 cells for inducing/regulating DNA fragmentation.

Diosgenin is a steroidal sapogenin with antitumor properties. We previously showed that diosgenin induced apoptosis in human erythroleukemia (HEL) cells. In order to elucidate the mechanism of its apoptotic activity, we investigated the effect of diosgenin on nuclear factor-kappaB (NF-kappaB) binding and on three groups of human mitogen-activated protein kinases (MAPKs) in relation to diosgenin-induced apoptosis in different erythroleukemia cell lines (K562 and HEL). Our results showed that diosgenin decreased DNA binding of NF-kappaB in K562 and HEL cells after 48-h diosgenin treatment. This inhibition of NF-kappaB binding was correlated with strong apoptosis in both erythroleukemia cell lines. Diosgenin-induced apoptosis was associated with cyclo-oxygenase-2 (COX-2) up-regulation in HEL cells but not in K562 cells which are COX-2 deficient. Furthermore, diosgenin inhibited extracellular signal-regulated kinase (ERK) activation only in HEL cells. However, diosgenin activated p38 MAPK in both cell lines and activated c-jun NH2-terminal kinases (JNKs) only in HEL cells. Pre-treatment with a selective p38 inhibitor inhibited diosgenin-induced DNA fragmentation in K562 cells. For the first time, our results suggest that inhibition of NF-kappaB nuclear binding and p38 MAPK activation are involved in the diosgenin-mediated signal cascades in K562 cells for inducing/regulating DNA fragmentation.

International audience; In this paper, eight natural molecules structurally close to diosgenin (five saponins: diosgenin, hecogenin, tigogenin, sarsasapogenin, smilagenin; two steroidal alkaloids: solasodine, solanidine; one sterol: stigmasterol) have been tested for their biological activities on human 1547 osteosarcoma cells. Differences in activity were studied in term of proliferation rate, cell cycle distribution and apoptosis induction. By using molecular modelling, two structural characteristics were calculated: spatial conformation and electron transfer capacity. The second property has been investigated by the HOMO repartition and the corresponding energy. Correlation between the experimental and the theoretical data permit us to highlight the importance of the hetero-sugar moiety and the 5,6-double bond in the biological activity (apoptosis and cell cycle arrest) on the human 1547 cell line. The importance of conformation at C-5 and C-25 carbon atoms was also discussed.

International audience; Many natural components of plant extracts are studied for their beneficial effects for health and particularly on carcinogenesis chemoprevention. In the present study, we investigated the effects of diosgenin on erythroleukemia HEL cells. Our results demonstrated that diosgenin induced G2/M arrest of cell cycle progression through p21 up-regulation in a p53-independent pathway and strong induction of apoptosis in HEL cells. Apoptosis induction was accompanied by an increase in Bax/Bcl-2 ratio, PARP cleavage and DNA fragmentation. Moreover, we showed for the first time that diosgenin provoked a collapse of mitochondrial membrane potential with an increase in intracellular calcium levels. It is well known that [Ca2+]i increase is one of the major activators of cytosolic PLA2. In our study, we demonstrated that diosgenin treatment induced cPLA2 activation through translocation to the cellular membrane. Moreover, arachidonic acid metabolism activation led to cyclooxygenase-2 (COX-2) but not lipoxygenase overexpression. Surprisingly, we observed a COX-2 up-regulation associated with apoptosis induction by diosgenin. These findings suggest that diosgenin has a potential chemopreventive effect; future studies should evaluate the mechanism of COX-2 activation during diosgenin-induced apoptosis in cancer cell lines.

International audience; Apoptosis is one of the most important phenomena of cellular biology. Sedimentation field flow fractionation (SdFFF) has been described as an effective tool for cell separation, respecting integrity and viability. Because SdFFF takes advantage of intrinsic properties of eluted cells (size, density, shape or rigidity), we investigated the capacity of SdFFF in monitoring the early and specific biophysical modifications which occurred during cellular apoptosis induction. Then, we used, as an in vitro cellular apoptosis model, the association between human 1547 osteosarcoma cells and diosgenin, a plant steroid known to induce apoptosis. Four other molecules were studied: hecogenin, tigogenin, staurosporine and MG132. Our results demonstrated a correlation between SdFFF elution profile changes (peak shape modification and retention ratio evolution) and effective apoptosis induction. For the first time, we demonstrated that SdFFF could be used to monitor apoptosis induction as early as 6 h incubation, suggesting different applications such as screening series of molecules to evaluate their ability to induce apoptosis, or sorting apoptotic cells to study apoptosis pathway.

International audience; Previously, we demonstrated that a plant steroid, diosgenin, altered cell cycle distribution and induced apoptosis in the human osteosarcoma 1547 cell line. The objective of this study was to investigate if the antiproliferative effect of diosgenin was similar for different human cancer cell lines such as laryngocarcinoma HEp-2 and melanoma M4Beu cells. Moreover, this work essentially focused on the mitochondrial pathway. We found that diosgenin had an important and similar antiproliferative effect on different types of cancer cells. In addition, our new results show that diosgenin-induced apoptosis is caspase-3 dependent with a fall of mitochondrial membrane potential, nuclear localization of AIF and poly (ADP-ribose) polymerase cleavage. Diosgenin treatment also induces p53 activation and cell cycle arrest in the different cell lines studied.

Ursolic acid (UA) is a pentacyclic triterpene compound isolated from many kinds of medicinal plants and present in human diet. In this study, we investigated the pro-apoptotic effect of UA on HaCat derived keratinocyte cell line. Treatment with UA decreased the viability of HaCat cells in a concentration- and time-dependent manner. In addition, cell cycle analysis revealed that UA treated HaCat cells were blocked predominantly in G1 phase. Moreover, expression of p21WAF1, a cell cycle regulator, was increased by UA, indicating that UA-induced cell cycle arrest could be mediated through p21WAF1. During UA treatment, we also demonstrated that p53 was phosphorylated at serine 392 and translocated to the nucleus. It is well established that p53 achieves its tumor suppressor activity by inducing apoptosis on cells. To define the apoptotic process in our system, we examined effect of UA on caspase activities, and demonstrated caspase-3 activation. In conclusion, our results suggest that UA induces: i) cell cycle arrest concomitantly with the apparition of the apoptotic sub group G1 peak, and ii) cell death through apoptosis, which is mediated by caspase-3.

International audience; Non-steroidal anti-inflammatory drugs (NSAIDs) have been shown to produce an anti-proliferative and pro-apoptotic effect on different types of cancer cell lines. Previously, we demonstrated that high dose of NS-398 (100 microM), a selective cyclooxygenase-2 inhibitor, induced a cell cycle slowing or arrest and, in contrast to low dose (10 microM), a marked decrease in apoptosis in human 1547 osteosarcoma cells. In this study, we investigated particularly the effect of 100 microM NS-398 on p53 and p21 expression, caspase activities and nuclear factor-kappaB (NF-kappaB). We found a correlation between p53, p21 mRNA expression and NF-kappaB activation and, we observed an induction of heat shock protein 70 expression with a large decrease in caspase-3 activity after 100 microM NS-398 treatment. Moreover, the inhibition of apoptosis was correlated with an increase in bcl-2/bax ratio. Our new findings confirm the novel anti-apoptotic property of NS-398 at 100 microM, as we previously found, which contrasts to the described NS-398 pro-apoptotic effect on other cancer cell lines.

A selective cyclooxygenase-2 (COX-2) inhibitor, NS-398, was shown to produce an anti-proliferative and pro-apoptotic effect on different types of cell lines. We describe the presence of COX-1 and COX-2 pathways in the human osteosarcoma 1547 cell line, as well as the conflicting effects of NS-398 (10, 50 and 100 microM) on programmed cell death, PGE2 release and COX-2 expression in 1547 cells cultured under apoptotic conditions. We demonstrate a link between the effects of 10 and 100 microM NS-398 on cell apoptosis, PGE2 release, and expression of COX-2 in 1547 cells undergoing apoptosis. At 10 microM, NS-398 acted as a selective COX-2 inhibitor moderately increasing apoptosis without any effect on COX-2 expression. In contrast, at 100 microM, NS-398 induced a cell cycle slowing or arrest, strongly enhanced COX-2 expression which was associated with a high PGE2 release and a marked decrease in apoptosis. This latter property of NS-398 at 100 microM in 1547 human osteosarcoma cells is novel compared to the described NS-398 pro-apoptotic effect on other cell lines.