SDZ PSC 833, the cyclosporine A analogue and multidrug resistance modulator, activates ceramide synthesis and increases vinblastine sensitivity in drug-sensitive and drug-resistant cancer cells.
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Resistance to chemotherapy is the major cause of cancer treatment failure. Insight into the mechanism of action of agents that modulate multidrug resistance (MDR) is instrumental for the design of more effective treatment modalities. Here we show, using KB-V-1 MDR human epidermoid carcinoma cells and [3H]palmitic acid as metabolic tracer, that the MDR modulator SDZ PSC 833 (PSC 833) activates ceramide synthesis. In a short time course experiment, ceramide was generated as early as 15 min (40% increase) after the addition of PSC 833 (5.0 microM), and by 3 h, [3H]ceramide was >3-fold that of control cells. A 24-h dose-response experiment showed that at 1.0 and 10 microM PSC 833, ceramide levels were 2.5- and 13.6-fold higher, respectively, than in untreated cells. Concomitant with the increase in cellular ceramide was a progressive decrease in cell survival, suggesting that ceramide elicited a cytotoxic response. Analysis of DNA in cells treated with PSC 833 showed oligonucleosomal DNA fragmentation, characteristic of apoptosis. The inclusion of fumonisin B1, a ceramide synthase inhibitor, blocked PSC 833-induced ceramide generation. Assessment of ceramide mass by TLC lipid charring confirmed that PSC 833 markedly enhanced ceramide synthesis, not only in KB-V-1 cells but also in wild-type KB-3-1 cells. The capacity of PSC 833 to reverse drug resistance was demonstrated with vinblastine. Whereas each agent at a concentration of 1.0 microM reduced cell survival by approximately 20%, when PSC 833 and vinblastine were coadministered, cell viability fell to zero. In parallel experiments measuring ceramide metabolism, it was shown that the PSC 833/vinblastine combination synergistically increased cellular ceramide levels. Vinblastine toxicity, also intensified by PSC 833 in wild-type KB-3-1 cells, was as well accompanied by enhanced ceramide formation. These data demonstrate that PSC 833 has mechanisms of action in addition to P-glycoprotein chemotherapy efflux pumping. (+info)
Mesalamine blocks tumor necrosis factor growth inhibition and nuclear factor kappaB activation in mouse colonocytes.
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BACKGROUND & AIMS: Derivatives of 5-aminosalicylic acid (mesalamine) represent a mainstay in inflammatory bowel disease therapy, yet the precise mechanism of their therapeutic action is unknown. Because tumor necrosis factor (TNF)-alpha is important in the pathogenesis of inflammatory bowel disease, we investigated the effect of mesalamine on TNF-alpha-regulated signal transduction and proliferation in intestinal epithelial cells. METHODS: Young adult mouse colon cells were studied with TNF-alpha, epidermal growth factor, or ceramide in the presence or absence of mesalamine. Proliferation was studied by hemocytometry. Mitogen-activated protein (MAP) kinase activation and IkappaBalpha expression were determined by Western blot analysis. Nuclear transcription factor kappaB (NF-kappaB) nuclear translocation was determined by confocal laser immunofluorescent microscopy. RESULTS: The antiproliferative effects of TNF-alpha were blocked by mesalamine. TNF-alpha and ceramide activation of MAP kinase were inhibited by mesalamine, whereas epidermal growth factor activation of MAP kinase was unaffected. TNF-alpha-stimulated NF-kappaB activation and nuclear translocation and the degradation of Ikappa-Balpha were blocked by mesalamine. CONCLUSIONS: Mesalamine inhibits TNF-alpha-mediated effects on intestinal epithelial cell proliferation and activation of MAP kinase and NF-kappaB. Therefore, it may function as a therapeutic agent based on its ability to disrupt critical signal transduction events in the intestinal cell necessary for perpetuation of the chronic inflammatory state. (+info)
Ceramide induces cytochrome c release from isolated mitochondria. Importance of mitochondrial redox state.
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In the present study we show that N-acetylsphingosine (C2-ceramide), N-hexanoylsphingosine (C6-ceramide), and, to a much lesser extent, C2-dihydroceramide induce cytochrome c (cyto c) release from isolated rat liver mitochondria. Ceramide-induced cyto c release is prevented by preincubation of mitochondria with a low concentration (40 nM) of Bcl-2. The release takes place when cyto c is oxidized but not when it is reduced. Upon cyto c loss, mitochondrial oxygen consumption, mitochondrial transmembrane potential (Delta Psi), and Ca2+ retention are diminished. Incubation with Bcl-2 prevents, and addition of cyto c reverses the alteration of these mitochondrial functions. In ATP-energized mitochondria, ceramides do not alter Delta Psi, neither when cyto c is oxidized nor when it is reduced, ruling out a nonspecific disturbance by ceramides of mitochondrial membrane integrity. Furthermore, ceramides decrease the reducibility of cyto c. We conclude that the apoptogenic properties of ceramides are in part mediated via their interaction with mitochondrial cyto c followed by its release and that the redox state of cyto c influences its detachment by ceramide from the inner mitochondrial membrane. (+info)
Genetic evidence for ATP-dependent endoplasmic reticulum-to-Golgi apparatus trafficking of ceramide for sphingomyelin synthesis in Chinese hamster ovary cells.
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LY-A strain is a Chinese hamster ovary cell mutant resistant to sphingomyelin (SM)-directed cytolysin and has a defect in de novo SM synthesis. Metabolic labeling experiments with radioactive serine, sphingosine, and choline showed that LY-A cells were defective in synthesis of SM from these precursors, but not syntheses of ceramide (Cer), glycosphingolipids, or phosphatidylcholine, indicating a specific defect in the conversion of Cer to SM in LY-A cells. In vitro experiments showed that the specific defect of SM formation in LY-A cells was not due to alterations in enzymatic activities responsible for SM synthesis or degradation. When cells were treated with brefeldin A, which causes fusion of the Golgi apparatus with the endoplasmic reticulum (ER), de novo SM synthesis in LY-A cells was restored to the wild-type level. Pulse-chase experiments with a fluorescent Cer analogue, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-D-erythro-sphingosine (C5-DMB-Cer), revealed that in wild-type cells C5-DMB-Cer was redistributed from intracellular membranes to the Golgi apparatus in an intracellular ATP-dependent manner, and that LY-A cells were defective in the energy-dependent redistribution of C5-DMB-Cer. Under ATP-depleted conditions, conversion of C5-DMB-Cer to C5-DMB-SM and of [3H]sphingosine to [3H]SM in wild-type cells decreased to the levels in LY-A cells, which were not affected by ATP depletion. ER-to-Golgi apparatus trafficking of glycosylphosphatidylinositol-anchored or membrane-spanning proteins in LY-A cells appeared to be normal. These results indicate that the predominant pathway of ER-to-Golgi apparatus trafficking of Cer for de novo SM synthesis is ATP dependent and that this pathway is almost completely impaired in LY-A cells. In addition, the specific defect of SM synthesis in LY-A cells suggests different pathways of Cer transport for glycosphingolipids versus SM synthesis. (+info)
Characterization of a novel mouse cDNA, ES18, involved in apoptotic cell death of T-cells.
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Using the modified screening approach in combination with expressed sequence tags, we have identified several novel cDNAs from mouse embryonic stem (ES) cells, whose expression is tissue-restricted and/or developmentally regulated. One of the cDNAs, ES18, is preferentially expressed in lymph node and thymus, and contains noteworthy features of transcriptional regulator. The expression of ES18 transcript was selectively regulated during the apoptosis of T-cell thymoma S49.1 induced by several stimuli. Interestingly, the ES18 transcript was differently regulated in the mutually antagonistic process, between dexamethasone- and A23187-induced cell death of T-cells. Moreover, the message level of ES18 was selectively enhanced by staurosporine, a broad protein kinase inhibitor, but not by other protein kinase inhibitors such as GF109203X and H89. In addition, ES18 transcript was induced by C2-ceramide, which is a mediator of both dexamethasone- and staurosporine-induced apoptotic signaling. We further showed that transient overexpression of ES18 in mouse T-cell lymphoma increased the apoptotic cell death. These data suggest that ES18 may be selectively involved in specific apoptotic processes in mouse T-cells. (+info)
Signal transduction triggered by lipid A-like molecules in 70Z/3 pre-B lymphocyte tumor cells.
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The lipid A (endotoxin) moiety of lipopolysaccharide (LPS) elicits rapid cellular responses from many cell types, including macrophages, lymphocytes, and monocytes. In CD14 transfected 70Z/3 pre-B lymphocyte tumor cells, these responses include activation of the MAP kinase homolog, p38, activation of NF-kappaB, and transcription of kappa light chains, leading to the assembly of surface IgM. In this work, we explored the specificity of the response with regard to lipid structure, and the requirement for p38 kinase activity prior to NF-kappaB activation in control and CD14 transfected 70Z/3 (CD14-70Z/3) cells. A p38-specific inhibitor, SB203580, was used to block p38 kinase activity in cells. CD14-70Z/3 cells were incubated with 1-50 microM SB203580, and then stimulated with LPS. Nuclear extracts were prepared, and NF-kappaB activation was measured using an electrophoretic mobility shift assay. SB203580 did not inhibit LPS induced NF-kappaB activation. In addition, LPS failed to activate p38 tyrosine phosphorylation in 70Z/3 cells lacking CD14, in spite of rapid NF-kappaB activation and robust surface IgM production with appropriate higher doses of LPS. LPS stimulation of p38 phosphorylation, NF-kappaB activation, and surface IgM expression were all blocked completely by lipid A-like endotoxin antagonists whether or not CD14 was present. Acidic glycerophospholipids and ceramides did not mimic lipid A-like molecules either as agonists or antagonists in this system. Our data support the hypothesis that lipid A-mediated activation of cells requires stimulation of a putative lipid A sensor that is downstream of CD14, but upstream of p38 and NF-kappaB. (+info)
Nitric oxide donors induce stress signaling via ceramide formation in rat renal mesangial cells.
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Exogenous NO is able to trigger apoptosis of renal mesangial cells, and thus may contribute to acute lytic phases as well as to resolution of glomerulonephritis. However, the mechanism involved in these events is still unclear. We report here that chronic exposure of renal mesangial cells for 24 h to compounds releasing NO, including spermine-NO, (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1, 2-diolate (MAHMA-NO), S-nitrosoglutathione (GS-NO), and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) results in a potent and dose-dependent increase in the lipid signaling molecule ceramide. Time courses reveal that significant effects occur after 2-4 h of stimulation with NO donors and reach maximal levels after 24 h of stimulation. No acute (within minutes) ceramide production can be detected. When cells were stimulated with NO donors in the presence of phorbol ester, a direct activator of protein kinase C, both ceramide production and DNA fragmentation are completely abolished. Furthermore, addition of exogenous ceramide partially reversed the inhibitory effect of phorbol ester on apoptosis, thus suggesting a negative regulation of protein kinase C on ceramide formation and apoptosis. In contrast to exogenous NO, tumor necrosis factor (TNF)-alpha stimulates a very rapid and transient increase in ceramide levels within minutes but fails to induce the late-phase ceramide formation. Moreover, TNF fails to induce apoptosis in mesangial cells. Interestingly, NO and TNFalpha cause a chronic activation of acidic and neutral sphingomyelinases, the ceramide-generating enzymes, whereas acidic and neutral ceramidases, the ceramide-metabolizing enzymes, are inhibited by NO, but potently stimulated by TNFalpha. Furthermore, in the presence of an acidic ceramidase inhibitor, N-oleoylethanolamine, TNFalpha leads to a sustained accumulation of ceramide and in parallel induces DNA fragmentation. In summary, our data demonstrate that exogenous NO causes a chronic up-regulation of ceramide levels in mesangial cells by activating sphingomyelinases and concomitantly inhibiting ceramidases, and that particularly the late-phase of ceramide generation may be responsible for the further processing of a proapoptotic signal. (+info)
TNF-alpha increases ceramide without inducing apoptosis in alveolar type II epithelial cells.
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Ceramide is a bioactive lipid mediator that has been observed to induce apoptosis in vitro. The purpose of this study was to determine whether endogenous ceramide, generated in response to in vivo administration of tumor necrosis factor-alpha (TNF-alpha), increases apoptosis in primary rat alveolar type II epithelial cells. Intratracheal instillation of TNF-alpha (5 microgram) produced a decrease in sphingomyelin and activation of a neutral sphingomyelinase. These changes were associated with a significant increase in lung ceramide content. TNF-alpha concomitantly activated the p42/44 extracellular signal-related kinases and induced nuclear factor-kappaB activation in the lung. Hypodiploid nuclei studies revealed that intratracheal TNF-alpha did not increase type II cell apoptosis compared with that in control cells after isolation. A novel observation from separate in vitro studies demonstrated that type II cells undergo a gradual increase in apoptosis after time in culture, a process that was accelerated by exposure of cells to ultraviolet light. However, culture of cells with a cell-permeable ceramide, TNF-alpha, or a related ligand, anti-CD95, did not increase apoptosis above the control level. The results suggest that ceramide resulting from TNF-alpha activation of sphingomyelin hydrolysis might activate the mitogen-activated protein kinase and nuclear factor-kappaB pathways without increasing programmed cell death in type II cells. (+info)