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.

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.

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.

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.

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.

Stabilization of potato tuber lipoxygenase on talc.

Potato tuber lipoxygenase, like many plant enzymes is a very labile protein and can lose activity during or after the purification procedure. In order to overcome this problem, we immobilized potato tuber lipoxygenase by adsorption on talc, a silicate support. Lipoxygenase adsorption greatly increased the long-term stability of the enzyme without modifying enzyme specificity. Moreover, adsorption on talc could be used to purify labile enzymes whose enzymatic activity decreases rapidly during purification stages. A multi-step reaction to produce large quantities (around 100 mg) of fatty acid hydroperoxides without enzyme inactivation was developed.

Linoleic acid peroxidation by Solanum tuberosum lipoxygenase was activated in the presence of human 5-lipoxygenase-activating protein.

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 100000xg 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.

Hydroperoxyeicosatetraenoic acids. Potent inhibitors of lymphocyte responses.

Arachidonic acid can be transformed into a series of HPETEs by the lipoxygenase enzyme activity of mouse peritoneal macrophages. These resulting HPETEs inhibit some mouse lymphocyte responses. When mice are injected with 15-L-HPETE, their splenocytes show a decreased [3H]thymidine uptake after lectin stimulation.

Expression of arachidonate platelet-type 12-lipoxygenase in human rheumatoid arthritis type B synoviocytes.

In the present study, we have demonstrated platelet-type 12-lipoxygenase (12-LOX) expression in human rheumatoid arthritis (RA) type B synoviocytes by reverse-transcription polymerase chain reaction (RT-PCR). The presence of 12-LOX mRNA in these cells was revealed by classical RT-PCR analysis using platelet-type 12-LOX cDNA primers and the PCR fragment (246 bp) was purified, amplified and sequenced. By sequence analysis, this fragment was determined to be 100% identical to that from platelet-type 12-LOX cDNA. Immunofluorescence data demonstrate that interleukin-1beta (IL-1beta) increases cellular 12-LOX protein. Other results associate specific inflammatory cytokines with the activity of 12-LOX in human RA type B synoviocytes. IL-1beta increased 12S-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) production (4-fold) and we also observed an increase in 12-HETE production (2.5-fold) after incubation of human RA type B synoviocytes with TNF alpha. In contrast to the action of IL-1beta on 12-HETE synthesis, IL-4 and IL-6 did not enhance 12-HETE production. This is the first demonstration of platelet-type 12-LOX cDNA derived from the mRNA of cultured human RA type B synoviocytes.

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.

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.

Cytotoxicity of arachidonic acid and of its lipoxygenase metabolite 15-hydroperoxyeicosatetraenoic acid on human breast cancer MCF-7 cells in culture.

Monolayer cultures of MCF-7 human breast cancer cell line were treated with arachidonic acid (AA) and 15-L-(s)-hydroperoxyeicosatetraenoic acid (15-L-(s)-HPETE) in a concentration range of 10(-5)-10(-11) M and their relative cytotoxic potential was determined. Both compounds had a time and dose-dependent cytotoxic effect on MCF-7 cells. 15-L-(S)-HPETE, the 15-lipoxygenase product of AA was the most effective.

Changes in lipoxygenase activities in human erythroleukemia (HEL) cells during diosgenin-induced differentiation.

A human erythroleukemia (HEL) cell line was used as a model to study dynamic changes in human 12-, 15-, 5-lipoxygenases, five lipoxygenase activating protein (FLAP), and leukotriene A4 (LTA4) hydrolase gene expression during megakaryocytic differentiation induced by diosgenin (Beneytout, J.L., Nappez, C., Leboutet, M.J. and Malinvaud, G., Biochem. Biophys. Res. Commun., 207 (1995) 398-404). The study was performed at the transcriptional level: 12- and 5-lipoxygenase mRNAs, FLAP mRNA and LTA4 hydrolase mRNA were detected before and after diosgenin treatment in HEL cells while 15-lipoxygenase mRNA was undetected. When HEL cells were incubated with arachidonic acid, 5-, 12-, 15-hydroxyeicosatetraenoic acids (HETE) and LTC4 were synthesized. In contrast, the diosgenin treatment induced the suppression of 12-lipoxygenase activity and only 5-, 15-HETEs and LTC4 were synthesized.

trans,trans-2,4-decadienal: cytotoxicity and effect on glutathione level in human erythroleukemia (HEL) cells.

The effects of trans,trans-2,4-decadienal (DDE), an isomer of a lipid peroxidation product were investigated on the human erythroleukemia cell line (HEL TIB 180). DDE strongly inhibits cell growth and affects cell viability without any differentiating effects. DDE treatment of HEL cells leads to a marked variation of the cellular glutathione level (GSH) and is involved in the beginning of DNA fragmentation.

Production of arachidonic acid metabolites by the colon adenocarcinoma cell line HT29 cl.19A and their effect on chloride secretion.

Eicosanoids were found in large amounts in the colonic mucosa of patients suffering from inflammatory bowel diseases and colonic adenocarcinoma. The aim of this study was to evaluate the role of the intestinal epithelial cells in the arachidonic acid metabolism and their functional response to certain eicosanoids. We used the human adenocarcinoma epithelial cell line HT29 cl.19A cell, which is an in vitro model of colon carcinoma and ion transport. These cells were found to express 5- and 15-lipoxygenase, leukotriene A4 hydrolase and cyclooxygenase-1 and -2 mRNAs. We observed an arachidonic acid metabolism via 5-lipoxygenase pathway despite the lack of FLAP mRNA expression and that certain eicosanoids such as hydroperoxy- and hydroxyeicosatetraenoic acids stimulate chloride secretion.

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.

Localization of 12-lipoxygenase mRNA in cultured oligodendrocytes and astrocytes by in situ reverse transcriptase and polymerase chain reaction.

We studied the distribution of 12-lipoxygenase mRNA in glial cells. First, mRNA was detected from cellular extracts by soluble-phase reverse transcriptase-polymerase chain reaction (RT-PCR). Taking into account that cell culture populations could not be 100% homogeneous, we then developed, for the first time, an in situ RT-PCR combined with immunocytochemistry with cell specific markers. Using this procedure we showed that 12-lipoxygenase mRNA was expressed both in mature oligodendrocytes and astrocytes.

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 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.