Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells

Accumulation of lipids in nonadipose tissues can lead to cell dysfunction and cell death, a phenomenon known as lipotoxicity. However, the signaling pathways and mechanisms linking lipid accumulation to cell death are poorly understood. The present study examined the hypothesis that saturated fatty acids disrupt endoplasmic reticulum (ER) homeostasis and promote apoptosis in liver cells via accumulation of ceramide. H4IIE liver cells were exposed to varying concentrations of saturated (palmitate or stearate) or unsaturated (oleate or linoleate) fatty acids. ER homeostasis was monitored using markers of the ER stress response pathway, including phosphorylation of IRE1alpha and eIF2alpha, splicing of XBP1 mRNA, and expression of molecular chaperone (e.g., GRP78) and proapoptotic (CCAAT/enhancer-binding protein homologous protein) genes. Apoptosis was monitored using caspase activity and DNA laddering. Palmitate and stearate induced ER stress, caspase activity, and DNA laddering. Inhibition of caspase activation prevented DNA laddering. Unsaturated fatty acids did not induce ER stress or apoptosis. Saturated fatty acids increased ceramide concentration; however, inhibition of de novo ceramide synthesis did not prevent saturated fatty acid-induced ER stress and apoptosis. Unsaturated fatty acids rescued palmitate-induced ER stress and apoptosis. These data demonstrate that saturated fatty acids disrupt ER homeostasis and induce apoptosis in liver cells via mechanisms that do not involve ceramide accumulation.     

Saturated free fatty acids,palmitic acid and stearic acid,induce apoptosis by stimulation of ceramide generation in rat testicular Leydig cell

In men, obesity has generally been associated with reduced plasma testosterone levels and with elevation of the plasma free fatty acids (FFAs). In this study, we investigated the effects of saturated FFAs including palmitic acid (PA) and stearic acid (SA), and polyunsaturated FFA arachidonic acid (AA) on the survival of rat testicular Leydig cell cultured in vitro. PA and SA markedly suppressed Leydig cell survival in a time- and dose-dependent manner. In contrast, AA stimulated the cell proliferation at 5-10 times of physiological concentration. The suppressive effect of PA and SA on cell survival was caused by apoptosis evidenced by DNA ladder formation and Annexin V-EGFP/propidium iodide staining of the cells. The apoptotic effect of PA was possibly mediated by ceramide generation because it could be completely blocked by ceramide synthase inhibitor fumonisin B1 and exogenous ceramide itself could directly induce apoptosis in vitro. Surprisingly, the apoptosis induced by PA could be partly prevented by AA. These results indicate that PA and SA induce apoptosis in testicular Leydig cells by ceramide production and these apoptotic effects may be a possible mechanism for reproductive abnormalities in obese men, and AA can partly prevent the apoptotic effect induced by saturated FFA.     

Protective effect of cyclo(his-pro) on streptozotocin-induced cytotoxicity and apoptosis in vitro

Cyclo(His-Pro) (CHP) is a naturally occurring, cyclic dipeptide structurally related to thyrotropin-releasing hormone (TRH). CHP was efficiently obtained from soybean meal by hydrolysis with flavourzyme and alcalase. In this study, the effects of CHP on streptozotocin (STZ)-induced beta-cell dysfunction and apoptosis were investigated in rat insulinoma cells (RINm5F) secreting insulin. When the RINm5F cells were treated with 2mM STZ, insulin secretion decreased to approximately 54% that of control cells. However, CHP treatment restored the insulin-secreting activity of RINm5F cells to approximately 71% that of the untreated control cells. Moreover, CHP significantly protected the cells from STZ-mediated cytotoxicity via reduction of nitric oxide (NO) production (2.3-fold) and lipid peroxidation (1.9-fold), which were induced by STZ. Moreover, CHP treatment also attenuated STZ-induced apoptotic events, such as activation of caspase-3, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation in RINm5F cells, indicating that CHP could protect the cells from apoptotic cell death induced by oxidative stress of STZ by increasing the expression of an anti-apoptotic protein, Bcl-2. These results suggest that CHP could be a candidate material for a protective and therapeutic agent against STZ-mediated cytotoxicity and apoptosis.     

Linoleic acid and antioxidants protect against DNA damage and apoptosis induced by palmitic acid

Polyunsaturated fats are the main target for lipid peroxidation and subsequent formation of mutagenic metabolites, but diets high in saturated fats are more strongly associated with adverse health effects. We show that the common saturated fatty acid, palmitic acid, is a potent inducer of DNA damage in an insulin-secreting cell line, and in primary human fibroblasts. Damage is not associated with upregulation of inducible nitric oxide synthase, but is prevented by two different antioxidants, alpha-lipoic acid and 3,3'-methoxysalenMn(III) (EUK134), which also partly prevent palmitic acid-induced apoptosis and growth inhibition. Since mutagenic metabolites can be formed from peroxidation of polyunsaturated fatty acids, co-administration of palmitic and a polyunsaturated fatty acid might be particularly harmful. Palmitic acid-induced DNA damage is instead prevented by linoleic acid, which is acting here as a protective agent against oxidative stress, rather than as a source of mutagenic metabolites. These results illustrate the complexity of the relationship of dietary fat intake to genotoxicity.     

雷诺嗪对高糖高脂诱导的NIT-1细胞凋亡的保护作用

目的 研究雷诺嗪对高糖高脂诱导的NIT-1胰岛β细胞凋亡的保护作用及Cleaved caspase-3表达的影响,探讨雷诺嗪保护胰岛β细胞的机制.方法 采用CCK-8法测定不同浓度的雷诺嗪对体外培养及高糖高脂诱导的NIT-1胰岛β细胞的增殖能力的影响,同时应用流式细胞术检测NIT-1细胞凋亡,Western blot检测凋亡因子Caspase-3活化片段Cleaved caspase-3蛋白的表达.结果 不同浓度的雷诺嗪对NIT-1胰岛β细胞保护作用呈剂量依赖性:低浓度雷诺嗪对细胞凋亡无明显保护作用,随浓度升高保护作用明显.在培养基中加入高脂高糖及高浓度的雷诺嗪(5μmol/L)共同培养24h,雷诺嗪组细胞凋亡率明显低于高脂高糖单独作用组(P＜0.01),同时相对于高糖高脂组,激活型Caspase3表达明显降低(P＜0.05).结论 雷诺嗪能抑制高糖高脂诱导的NIT-1胰岛β细胞凋亡,其分子机制可能是雷诺嗪对Caspase-3的激活作用.     

Sodium nitroprusside-induced mitochondrial apoptotic events in insulin-secreting RINm5F cells are associated with MAP kinases activation

Exposure of insulin-secreting RINm5F cells to the chemical nitric oxide donor sodium nitroprusside (SNP) resulted in apoptotic cell death, as detected by cytochrome c release from mitochondria and caspase 3 activation. SNP exposure also leads to phosphorylation and activation of enzymes involved in cellular response to stress such as signal-regulated kinase 2 (ERK2) and c-Jun NH(2)-terminal kinase 46 (JNK46). Both cytochrome c release and caspase 3 activation were abrogated in cells exposed to MEK and p38 inhibitors. Treatment of cells with the NO donors SNP, DETA-NO, GEA 5024, and SNAP resulted in phosphorylation of the antiapoptotic protein Bcl-2, which was resistant to blockade of MEK, p38, and JNK pathways and sensitive to phosphoinositide 3-kinase (PI3K) inhibition. In addition, transient transfection of cells with the wild-type PI3K gamma gene mimics the increased rate of Bcl-2 phosphorylation detected in NO-treated cells. The generation of phosphoinositides seems to participate in the process since Bcl-2 phosphorylation was not observed in cells overexpressing lipid-kinase-deficient PI3Kgamma. The potential of SNP toxicity directly from NO was supported by our finding that the NO scavenger carboxy-PTIO prevented cell death. We found no evidence to support the contention that oxygen radicals generated during cellular SNP metabolism mediate cell toxicity in RINm5F cells, since neither addition of catalase/superoxide dismutase nor transfection with superoxide dismutase prevented SNP-induced cell death. Thus, we propose that exposure to apoptotic concentrations of NO triggers ERK- and p38-dependent cytochrome c release, caspase 3 activation, and PI3K-dependent Bcl-2 phosphorylation.     

Uptake and metabolic conversion of saturated and unsaturated fatty acids in Hep2 human larynx tumor cells

Research on fatty acid metabolism in cultured human larynx tumor cells Hep2 was carried out.The cells were incubated with either a saturated (palmitic) or a polyunsaturated (linoleic, alpha-linolenic and eicosatrienoic (n-6)) radioactive fatty acid (0.66 pM, 24 h). The best incorporation capacity was observed in the linoleic acid followed by alpha-linolenic, palmitic and eicosatrienoic acids. All fatty acids tested were anabolized to higher derivatives within their own family. Palmitic acid was primarily monodesaturated rather than elongated, proving to have a very active A9 desaturase activity.With respect to polyunsaturated acid metabolism, the conversion of alpha-linolenic acid to higher homologs, although better than linoleic acid, occurred far less efficiently than that observed in other non-highly undifferentiated human tumor cells. This impairment in higher polyunsaturated fatty acid biosynthesis, reflected in the low levels of arachidonic acid in the fatty acid composition, would not reside in the A5 desaturation step since Hep2 cells can readily convert eicosatrienoic acid into arachidonic acid. Considering the potential regulatory role of specific polyunsaturated fatty acids in the cell proliferative control, the knowledge of the metabolism of fatty acids in this human tumor cell would be important for designing future experiments in order to clarify the mechanism involved in balance, proliferation and cell death.     

A role for aberrant protein palmitoylation in FFA-induced ER stress and β-cell death

Exposure of insulin-producing cells to elevated levels of the free fatty acid (FFA) palmitate results in the loss of β-cell function and induction of apoptosis. The induction of endoplasmic reticulum (ER) stress is one mechanism proposed to be responsible for the loss of β-cell viability in response to palmitate treatment; however, the pathways responsible for the induction of ER stress by palmitate have yet to be determined. Protein palmitoylation is a major posttranslational modification that regulates protein localization, stability, and activity. Defects in, or dysregulation of, protein palmitoylation could be one mechanism by which palmitate may induce ER stress in β-cells. The purpose of this study was to evaluate the hypothesis that palmitate-induced ER stress and β-cell toxicity are mediated by excess or aberrant protein palmitoylation. In a concentration-dependent fashion, palmitate treatment of RINm5F cells results in a loss of viability. Similar to palmitate, stearate also induces a concentration-related loss of RINm5F cell viability, while the monounsaturated fatty acids, such as palmoleate and oleate, are not toxic to RINm5F cells. 2-Bromopalmitate (2BrP), a classical inhibitor of protein palmitoylation that has been extensively used as an inhibitor of G protein-coupled receptor signaling, attenuates palmitate-induced RINm5F cell death in a concentration-dependent manner. The protective effects of 2BrP are associated with the inhibition of [(3)H]palmitate incorporation into RINm5F cell protein. Furthermore, 2BrP does not inhibit, but appears to enhance, the oxidation of palmitate. The induction of ER stress in response to palmitate treatment and the activation of caspase activity are attenuated by 2BrP. Consistent with protective effects on insulinoma cells, 2BrP also attenuates the inhibitory actions of prolonged palmitate treatment on insulin secretion by isolated rat islets. These studies support a role for aberrant protein palmitoylation as a mechanism by which palmitate enhances ER stress activation and causes the loss of insulinoma cell viability.     

Ataxia telangiectasia-mutated gene product inhibits DNA damage-induced apoptosis via ceramide synthase.

DNA double-stranded breaks (dsb) activate surveillance systems that identify DNA damage and either initiate repair or signal cell death. Failure of cells to undergo appropriate death in response to DNA damage leads to misrepair, mutations, and neoplastic transformation. Pathways linking DNA dsb to reproductive or apoptotic death are virtually unknown. Here we report that metabolic incorporation of 125I-labeled 5-iodo-2'deoxyuridine, which produces DNA dsb, signaled de novo ceramide synthesis by post-translational activation of ceramide synthase (CS) and apoptosis. CS activation was obligatory, since fumonisin B1, a fungal pathogen that acts as a specific CS inhibitor, abrogated DNA damage-induced death. X-irradiation yielded similar results. Furthermore, inhibition of apoptosis using the peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone did not affect CS activation, indicating this event is not a consequence of induction of apoptosis. ATM, the gene mutated in ataxia telangiectasia, is a member of the phosphatidylinositol 3-kinase family that constitutes the DNA damage surveillance/repair system. Epstein-Barr virus-immortalized B cell lines from six ataxia telangiectasia patients with different mutations exhibited radiation-induced CS activation, ceramide generation, and apoptosis, whereas three lines from normal patients failed to manifest these responses. Stable transfection of wild type ATM cDNA reversed these events, whereas antisense inactivation of ataxia telangiectasia-mutated gene product in normal B cells conferred the ataxia telangiectasia phenotype. We propose that one of the functions of ataxia telangiectasia-mutated gene product is to constrain activation of CS, thereby regulating DNA damage-induced apoptosis.     

Effects of ethanol on pancreatic beta-cell death: interaction with glucose and fatty acids

Western lifestyle plays an important role in the prevalence of type 2 diabetes by causing insulin resistance and pancreatic β-cell dysfunction, a prerequisite for the development of diabetes. High fat diet and alcohol are major components of the western diet. The aim of the present study was to investigate the effects of ethanol and fatty acids on β-cell survival and metabolism. We treated the rat β-cell line RINm5F with ethanol, a mixture of palmitic and oleic acids, or both. Reactive oxygen species (ROS) were determined by (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate) (CM-H2DCFDA) fluorescence assay, and mitochondrial activity was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction assay and by determining ATP production. Cell viability was assessed with a cell counter and trypan blue exclusion, and the mode of cell death by Hoechst33342 and propidium iodide staining. With both ethanol and fatty acid treatments, MTT reduction and ATP production decreased, whereas ROS production increased. Ethanol treatment had no effect on cell number, whereas fatty acid treatment reduced the cell number. Cell incubation with ethanol, fatty acids, or both increased the number of Hoechst 33342-positive nuclei. However, the majority of nuclei from fatty acid-treated cells were stained with propidium iodide, indicating a loss of plasma membrane integrity. We conclude that both ethanol and fatty acids generate cellular oxidative stress, and affect mitochondrial function in RINm5F β-cells. However, ethanol causes β-cell death by apoptosis, whereas fatty acids cause cell death predominantly by necrosis. It is not known whether these results are applicable to human β-cells.     

Effects of fatty acids on lysis of Streptococcus faecalis.

Palmitic, stearic, oleic, and linoleic acids at concentrations of 200 nmol/ml all inhibited autolysin activity 80% or more in whole cells or cell-free extracts. This concentration of the saturated fatty acids palmitic acid and stearic acid had little or no effect on the growth of whole cells or protoplasts. However, the unsaturated fatty acids oleic acid and linoleic acid induced lysis in both situations. This lytic effect is apparently not related to any uncoupling activity or inhibition of energy catabolism by unsaturated fatty acids. It is concluded that unsaturated fatty acids induce cell and protoplast lysis by acting as more potent membrane destabilizers than saturated fatty acids.     

Possible involvement of proteolytic degradation of tyrosinase in the regulatory effect of fatty acids on melanogenesis.

The purpose of this study was to investigate the mechanism of fatty acid-induced regulation of melanogenesis. An apparent regulatory effect on melanogenesis was observed when cultured B16F10 melanoma cells were incubated with fatty acids, i.e., linoleic acid (unsaturated, C18:2) decreased melanin synthesis while palmitic acid (saturated, C16:0) increased it. However, mRNA levels of the melanogenic enzymes, tyrosinase, tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2), were not altered. Regarding protein levels of these enzymes, the amount of tyrosinase was decreased by linoleic acid and increased by palmitic acid, whereas the amounts of TRP1 and TRP2 did not change after incubation with fatty acids. Pulse-chase assay by [35S]methionine metabolic labeling revealed that neither linoleic acid nor palmitic acid altered the synthesis of tyrosinase. Further, it was shown that linoleic acid accelerated, while palmitic acid decelerated, the proteolytic degradation of tyrosinase. These results suggest that modification of proteolytic degradation of tyrosinase is involved in regulatory effects of fatty acids on melanogenesis in cultured melanoma cells.     

Characterization of ceramide-induced apoptotic death in cerebellar granule cells in culture

Sphingomyelin signalling system has been involved in several examples of cell death through apoptosis. We have characterised the effect of exposure to the cell permeable ceramide analogue, C2-ceramide, on cultures of differentiated cerebellar granule cells. C(2)-ceramide was toxic to granule cells in a dose- and time-dependent way at concentrations higher than 10 microM. Ceramide exposure was accompanied by characteristic alterations of cell morphology, namely swollen cell bodies and punctuate appearance and arcuate direction of processes. The final outcome of ceramide exposure was a form of cell death largely apoptotic in nature. Hoechst stain, followed by counts of nuclei with normal appearance and size or with condensed chromatin and reduced size, revealed a large increase of the proportion of shrunken nuclei in treated cultures. In situ visualisation of fragmented DNA through the TUNEL technique, additionally marked cells undergoing apoptosis as a consequence of ceramide treatment. Accordingly, the DNA extracted from cultures exposed to C2-ceramide and subjected to agarose gel electrophoresis showed the peculiar ladder of fragmented low molecular weight DNA. Treatments with inhibitors of two caspases or of nitric oxide synthase were unable to rescue neurons exposed to ceramide, thus suggesting a neurotoxic action not primarily dependent on activation of death proteases or on nitric oxide production.     

Saturated free fatty acid release and intracellular ceramide generation during apoptosis induction are closely related processes

Apoptosis induced by cells from the immune system is frequently associated with an increase in the ceramide content of target cells, due to the activation of sphingomyelinases (SMase). Some studies have also reported the release of saturated and monounsaturated free fatty acids (FFA) from apoptotic cells. However, the possible relationship between these lipid biochemistry events has not been characterized. We have analysed for the first time the release of FFA triggered by tumor necrosis factor-alpha (TNF-alpha), Fas/CD95 or the perforin/granzyme system of cytotoxic T lymphocytes (CTL) and their relationship to intracellular ceramide generation. TNF-alpha- and Fas-induced apoptosis are associated with both intracellular ceramide generation from sphingomyelin (SM) and release of palmitic-derived FFA, with similar kinetics. Intracellular SMase activation and FFA release from target cells during Fas-induced apoptosis are much more rapid and efficient if Fas-based cytotoxicity is exerted by alloantigenic CTL. In the case of perforin/granzyme-based cytotoxicity exerted by CTL, intracellular ceramide generation and FFA release from target cells seem to depend on the type of lysis induction used. Importantly, the correlation between intracellular SMase activation and the release of palmitic acid-derived FFA from target cells has been observed in all types of cytotoxicity assayed. In addition, exogenous natural ceramide induces the rapid release of the same FFA, well before any apoptotic sign is detected, and FFA release during Fas-induced apoptosis is inhibited in SM-depleted cells by chronic fumonisin-B(1) treatment. These results demonstrate a novel connection between the release of palmitic acid-derived FFA and intracellular ceramide accumulation during apoptosis induction.     

Impact of mitochondrial beta-oxidation in fatty acid-mediated inhibition of glioma cell proliferation

Tetradecylthioacetic acid (TTA), which cannot be beta-oxidized, exerts growth-limiting properties in glioma cells. In order to investigate the importance of modulated lipid metabolism and alterations in mitochondrial properties in this cell death process, we incubated glioma cells both with TTA and the oxidizable fatty acid palmitic acid (PA), in the presence of L-carnitine and the carnitine palmitoyltransferase inhibitors etomoxir and aminocarnitine. L-carnitine partly abolished the PA-mediated growth reduction of glioma cells, whereas etomoxir and aminocarnitine enhanced the antiproliferative effect of PA. The production of acid-soluble products increased and the incorporation of PA into glycerolipids decreased after L-carnitine supplementation. L-carnitine was found to enhance the antiproliferative effect of TTA, but did not affect the incorporation of TTA into glycerolipids, or ceramide. PDMP, sphingosine 1-phosphate, desipramine, fumonisin B(1), and L-cycloserine were able not to rescue the glioma cells from PA and TTA-induced growth inhibition, suggesting that increased ceramide production is not important in the growth reduction. TTA-mediated growth inhibition was accompanied with an increased uptake of PA and increased incorporation of PA into triacylglycerol (TG). Our data suggest that mitochondrial functions are involved in fatty acid-mediated growth inhibition. Whether there is a causal relationship between TG accumulation and the apoptotic process remains to be determined.     

Anandamide induces apoptosis in human cells via vanilloid receptors. Evidence for a protective role of cannabinoid receptors

The endocannabinoid anandamide (AEA) is shown to induce apoptotic bodies formation and DNA fragmentation, hallmarks of programmed cell death, in human neuroblastoma CHP100 and lymphoma U937 cells. RNA and protein synthesis inhibitors like actinomycin D and cycloheximide reduced to one-fifth the number of apoptotic bodies induced by AEA, whereas the AEA transporter inhibitor AM404 or the AEA hydrolase inhibitor ATFMK significantly increased the number of dying cells. Furthermore, specific antagonists of cannabinoid or vanilloid receptors potentiated or inhibited cell death induced by AEA, respectively. Other endocannabinoids such as 2-arachidonoylglycerol, linoleoylethanolamide, oleoylethanolamide, and palmitoylethanolamide did not promote cell death under the same experimental conditions. The formation of apoptotic bodies induced by AEA was paralleled by increases in intracellular calcium (3-fold over the controls), mitochondrial uncoupling (6-fold), and cytochrome c release (3-fold). The intracellular calcium chelator EGTA-AM reduced the number of apoptotic bodies to 40% of the controls, and electrotransferred anti-cytochrome c monoclonal antibodies fully prevented apoptosis induced by AEA. Moreover, 5-lipoxygenase inhibitors 5,8,11,14-eicosatetraynoic acid and MK886, cyclooxygenase inhibitor indomethacin, caspase-3 and caspase-9 inhibitors Z-DEVD-FMK and Z-LEHD-FMK, but not nitric oxide synthase inhibitor Nomega-nitro-l-arginine methyl ester, significantly reduced the cell death-inducing effect of AEA. The data presented indicate a protective role of cannabinoid receptors against apoptosis induced by AEA via vanilloid receptors.     

Protective effects of docosahexaenoic acid in staurosporine-induced apoptosis: involvement of phosphatidylinositol-3 kinase pathway

Docosahexaenoic acid (22:6n-3, DHA) is highly enriched in neuronal membranes and is considered to be essential for proper brain function. We have previously demonstrated in Neuro 2A cells that DHA as a membrane component protects cells from apoptotic death induced by serum deprivation (Kim et al. 2000). In the present study we demonstrate that staurosporine (ST) induces apoptosis in Neuro 2A cells and DHA enrichment prior to the ST treatment significantly inhibits the apoptotic cell death, as evidenced by the reduction of caspase-3 activity, cleavage of pro-caspase-3 to active caspase-3, DNA strand-breaking and laddering. Enrichment of cells with other fatty acids such as oleic and arachidonic acids did not exert such an effect, indicating that the antiapoptotic effect was specific to DHA enrichment. Among the several protein kinase inhibitors, only phosphatidylinositol 3-kinase (PI3-K) inhibitors, wortmanin, and LY-294002 abolished the protective effect of DHA in ST-induced apoptosis. Concurrently, ST-treatment significantly decreased the phosphorylation status of Akt at Ser-473 and Thr-308 as well as Akt activity, and this reduction was partially prevented by DHA enrichment. The extent of the antiapoptotic effect of DHA correlated with a time-dependent increase in the phosphatidylserine (PS) content upon DHA enrichment. When cells were enriched with DHA in serine-free medium, the PS increase diminished and the DHA effect on caspase-3 activation as well as Akt phosphorylation in ST-induced apoptosis was no longer apparent, suggesting that DHA's role in accumulating membrane PS is an important component for the observed protection. In summary, DHA enrichment uniquely protects ST-induced apoptosis in a PS- and PI3-K-dependent manner. From these data, we suggest that the antiapoptotic effect of DHA is mediated at least in part through the PI3-K/Akt pathway, facilitated by DHA-induced PS accumulation.     

Caspases are the main executioners of Fas-mediated apoptosis, irrespective of the ceramide signalling pathway

Tumor necrosis factor alpha (TNF) or cytotoxic anti-Fas antibodies lead to the activation of apoptotic proteases (caspases) and to sphingomyelinase-mediated ceramide generation. Caspases and ceramide are both known to induce apoptosis on its own, but their relative contribution to Fas- and TNF-induced cell death is not well established. We report here that rapid apoptosis induced by TNF in U937 cells or anti-Fas in Jurkat cells, in the presence of cycloheximide, induced only a very low increase (<20%) in the cell ceramide content. Neither treatment with inhibitors of sphingomyelinases nor incubation of cells with fumonisin B1, which inhibits de novo ceramide synthesis, prevented TNF and Fas-mediated apoptosis. Increasing or depleting the cell ceramide content by prolonged culture in the presence of monensin or fumonisin B1, respectively, did not prevent TNF and Fas-mediated apoptosis. Treatment of cells with sphingomyelinase inhibitors did not affect to the activation of CPP32 (caspase-3) induced by TNF or anti-Fas antibodies. Chromatin condensation and fragmentation in cells treated with anti-Fas or TNF was abrogated by peptide inhibitors of caspases, which also inhibited Fas-, but not TNF-induced cell death. These results indicate that while ceramide does not seem to act as a critical mediator of TNF and Fas-induced apoptosis, it is generated as a consequence of CPP32 activation and could contribute to the spread of the intracellular death signal.     

Role of ceramide in mediating apoptosis of irradiated LNCaP prostate cancer cells

The sphingomyelin metabolites ceramide and sphingosine are mediators of cell death induced by gamma-irradiation. We studied the production of ceramide and the effects of exogenous ceramide on apoptosis in LNCaP prostate cancer cells that are highly resistant to gamma-irradiation-induced cell death. LNCaP cells can be sensitized to gamma-irradiation by tumor necrosis factor alpha (TNF-alpha) and, to a lesser degree, by the agonistic FAS antibody CH-11. TNF-alpha activated intrinsic and extrinsic apoptosis pathways and increased ceramide and sphingosine levels in irradiated LNCaP cells. CH-11 activated only the extrinsic apoptosis pathways and had a negligible effect on ceramide and sphingosine levels in irradiated LNCaP cells. Exogenous ceramide and bacterial sphingomyelinase sensitized LNCaP cells to radiation-induced apoptosis and had a synergistic effect on cell death after irradiation with TNF-alpha, but not with CH-11. Cell death effects after exposure to ceramide and irradiation were blocked by the serine protease inhibitor TLCK (Na-p-tosyl-L-lysine-chloromethylketone), but not by the caspase inhibitor z-VAD (2-val-Ala-Asp(oMe)-CH(2)F). During LNCaP cell apoptosis induced by exogenous ceramide, we observed activation of caspase-9, but not caspases-8, -3, or -7. The effect of ceramide occurred largely via the intrinsic mitochondrial apoptosis pathway and enhanced TNF-alpha, but not CH-11 effects on irradiated cells. The data show that ceramide enhanced activation of the intrinsic apoptotic pathway and enhanced cell death induced by TNF-alpha with or without gamma-irradiation. TNF-alpha and gamma-irradiation elevated levels of endogenous ceramide and activated the intrinsic cell death pathway.