Diosgenin induces death receptor-5 through activation of p38 pathway and promotes TRAIL-induced apoptosis in colon cancer cells.

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.

Development of a downscale sedimentation field flow fractionation device for biological event monitoring.

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.

Roots of Daphne gnidium L. inhibit cell proliferation and induce apoptosis in the human breast cancer cell line MCF-7.

Daphne gnidium L. is a well-known Moroccan plant with cancer-related ethnobotanical use. In order to systematically evaluate its potential activity in breast cancer, four extracts from this plant of different polarity were tested for their antiproliferative effects on MCF-7 cells. The second aspect of this study related to understanding the nature and mechanism of the antiproliferative effect. Results from a viability assay showed the potent antiproliferative capacity of the hexane (IC(50)-48 h: 630 +/- 16 microg/ml), dichloromethane (IC(50)-48 h: 112 +/- 7 microg/ml) and ethyl acetate extracts (IC(50)-48 h: 263 +/- 9 microg/ml). On the other hand the methanol extract was inactive. LDH test revealed the cytotoxicity of the hexane extract as opposed to two others. The characterization of the ethyl acetate extract showed its dose-dependent pro-apoptotic effect. Surprisingly, we observed that activation of the inducible cyclooxygenase-2 followed the kinetics of apoptosis development. On the other hand, the dichloromethane extract showed a distinct effect on COX-2 activity as a function of the used dose. A low dose seemed to inhibit COX-2 activity whereas a high dose seemed to increase it. These findings suggest that Daphne gnidium L. might be of potential chemopreventive interest. Other studies are in hand to isolate the active agents responsible for the antiproliferative effect.

Analysis of relationship between cell cycle stage and apoptosis induction in K562 cells by sedimentation field-flow fractionation.

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.

Study of diosgenin-induced apoptosis kinetics in K562 cells by Sedimentation Field Flow Fractionation.

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.

Increased cyclooxygenase-2 and thromboxane synthase expression is implicated in diosgenin-induced megakaryocytic differentiation in human erythroleukemia cells.

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.

Sedimentation field flow fractionation to study human erythroleukemia cell megakaryocytic differentiation after short period diosgenin induction.

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.

Low dose leflunomide activates PI3K/Akt signalling in erythroleukemia cells and reduces apoptosis induced by anticancer agents.

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.

Pre-apoptotic sub-population cell sorting from diosgenin apoptosis induced 1547 cells by Sedimentation Field-Flow Fractionation. The effect of channel thickness on sorting performance.

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.

Megakaryocyte cell sorting from diosgenin-differentiated human erythroleukemia cells by sedimentation field-flow fractionation.

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.

SdFFF monitoring of cellular apoptosis induction by diosgenin and different inducers in the human 1547 osteosarcoma cell line.

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.

Effects of alumina and zirconium dioxide particles on arachidonic acid metabolism and proinflammatory interleukin production in osteoarthritis and rheumatoid synovial cells.

We describe a model which can be used for in vitro biocompatibility assays of biomaterials. We studied the in vitro response of human osteoarthritis or rheumatoid arthritis fibroblast-like synoviocytes to Al2O3 or ZrO2 particles by analysing the production of interleukin-1 (IL-1) and interleukin-6 (IL-6) and the metabolism of arachidonic acid via lipoxygenase and cyclo-oxygenase pathways. Our results show that, in these cells and under our experimental conditions, Al2O3 and ZrO2 did not significantly modify the synthesis of IL-1 and IL-6 or the metabolism of arachidonic acid.

A plant steroid, diosgenin, induces apoptosis, cell cycle arrest and COX activity in osteosarcoma cells.

Cyclooxygenases (COXs) are key enzymes in the conversion of arachidonic acid into prostanoids which are involved in apoptosis and inflammation. Two distinct COXs have been identified: COX-1 which is constitutively expressed and COX-2 which is induced by different products such as tumor promoters or growth factors. Previously, we demonstrated that a plant steroid, diosgenin, was a new megakaryocytic differentiation inducer of human erythroleukemia cells. In our study, we investigated the effect of diosgenin on the proliferation rate, cell cycle distribution and apoptosis in the human osteosarcoma 1547 cell line. The effects of this compound were also tested on COX expression and COX activities. Diosgenin treatment caused an inhibition of 1547 cell growth with a cycle arrest in G1 phase and apoptosis induction. Moreover, we found a correlation between p53, p21 mRNA expression and nuclear factor-kappaB activation and we observed a time-dependent increase in PGE2 synthesis after diosgenin treatment.

Aromatase in synovial cells from postmenopausal women.

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.

Dose-dependent modulation of apoptosis and cyclooxygenase-2 expression in human 1547 osteosarcoma cells by NS-398, a selective cyclooxygenase-2 inhibitor.

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.

Enhanced apoptosis in retrovirally transfected osteosarcoma cells after exposure to sodium butyrate.

We investigated the effects of sodium butyrate (NaBu) on two retrovirally transfected osteosarcoma cell lines (1547-TK and 1547-LacZ cells) compared to the corresponding untransfected cell line. The first finding was an inhibitory effect only on the proliferation of both transfected cell lines. This antiproliferative effect was associated with apoptosis induction, which was detected using techniques that monitor either characteristic biochemical or morphological processes. Our findings show that 1547-TK and 1547-LacZ cells were much more sensitive to NaBu treatment than untransfected 1547 cells as concerns both proliferation and apoptosis induction.

Arachidonate 15-lipoxygenase of reticulocyte-type in human rheumatoid arthritis type B synoviocytes and modulation of its activity by proinflammatory cytokines.

OBJECTIVE: Lipoxygenases (LOX) are lipid-peroxidating enzymes that are implicated in the pathogenesis of a variety of inflammatory disorders such as arthritis, psoriasis, and asthma. 15-LOX catalyzes the oxygenation of free arachidonic acid to 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), which is reduced to 15-hydroxyeicosatetraenoic acid (15-HETE). The biological role of 15-HETE is less clear. We sought to determine if cultured human rheumatoid synovial cells were able to express 15-LOX mRNA, leading to the synthesis of 15-HETE, and to examine the effect of different cytokines on 15-LOX activity. METHODS: Adherent synovial cells were obtained by enzymatic digestion of rheumatoid synovium, isolated from patients with rheumatoid arthritis (RA) undergoing hip synovectomy. Between passages 4 and 8, reticulocyte-type 15-LOX expression in these cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) in situ and confirmed by classical RT-PCR analysis followed by enzymatic digestion. The PCR fragment was purified, amplified, and sequenced. Cultured synovial cells were incubated with or without different cytokines and exogenous [1-(14)C] arachidonic acid metabolism of synoviocytes was analyzed by reverse phase high performance liquid chromatography (RP-HPLC). RESULTS: RT-PCR results showed that human RA type B synoviocytes expressed a reticulocyte-type 15-LOX. By sequence analysis, the PCR fragment (474 bp) was determined to be 100% identical to that of reticulocyte-type 15-LOX cDNA. Other results associated specific inflammatory cytokines with the activity of 15-LOX in these cells. RP-HPLC analysis showed that interleukin 4 (IL-4) increased 15-HETE production (2.4-fold); we also observed an increase in 15-HETE production (1.2-fold) after incubation of the cells with IL-1beta. CONCLUSION: Human RA type B synoviocytes are able to express 15-LOX mRNA leading to the synthesis of 15-HETE, which is modulated by various cytokines that play a major role in the pathophysiology of RA, especially IL-4 and IL-1.

Resistance to apoptosis and cyclooxygenase-2 expression in a human adenocarcinoma cell line HT29 CL.19A.

BACKGROUND: COX-2 expression increases the tumorigenic potential of enterocytes. Tumorigenic effect is partially linked to an inhibition of programmed cell death which is one of the most important components in maintaining intestinal epithelium integrity. MATERIALS AND METHODS: We analyzed apoptosis in HT29 cl.19A cells cultured over 3 weeks, in the presence (10%) or in the absence of fetal bovine serum (FBS), by analysis of genomic DNA fragmentation after agarose gel electrophoresis, morphological measurement of apoptosis using DAPI chromatin staining, and transmission electron microscopy (TEM) to identify apoptotic cellular morphological changes. RESULTS: Regardless of the methods used, no apoptotic signs were observed for each culture condition, even if cells were cultured 3 weeks in the absence of FBS. CONCLUSION: Using HT29 cl.19A cells (untransfected cells), we found that intrinsic or constitutive COX-2 expression in adenocarcinoma cell line was associated with spontaneous resistance to apoptosis.

Cyclooxygenase-2 up-regulation after FLAP transfection in human adenocarcinoma cell line HT29 cl.19A.

Five-lipoxygenase-activating protein (FLAP) is usually described as an essential protein to activate the leukotriene (LTs) synthesis via the 5-lipoxygenase pathway. In the enterocyte model HT29 cl.19A cell line, 5-lipoxygenase metabolism was found despite the lack of FLAP expression. Therefore HT29 cl.19A represents an original mammalian model to study FLAP-dependent leukotriene synthesis. In FLAP cDNA transfected HT29 cl.19A cells, FLAP expression led to an increase in cyclooxygenase pathway products (mainly PGE2) without an increase in 5-lipoxygenase metabolism. This increase in PGE2 synthesis was associated with a cyclooxygenase-2 upregulation in comparison to untransfected HT29 cl.19A cells. These results suggest a possible interaction between the two major pathways of arachidonic acid metabolism.

Cyclooxygenase-2 expression in human adenocarcinoma cell line HT29 cl.19A.

BACKGROUND: Overexpression of cyclooxygenase-2 (COX-2) has been implicated in carcinogenesis of human colorectal cancer which is one of the leading types of cancer in Western countries. MATERIALS AND METHODS: We analyzed the COX-2 expression and activity using RT-PCR, Western blot, immunocytochemistry, RP-HPLC and EIA analysis in 0% and 10% fetal bovine serum (FBS) cultured cells. RESULTS: HT 29 cl.19A cells exhibited a COX-2 expression called "constitutive" in the absence of FBS in culture media. This particular expression was not the result of a mutation of the HT29 cl.19A COX-2 gene promotor. CONCLUSION: In our study, the human colon adenocarcinoma cell line, HT29 cl.19A, expressed COX-2 abnormally. This expression appeared to be at the same time inducible by the action of classical exogenous inducers such as FBS or interleukin-1 beta and "constitutive" if none of these compounds were present.