Abstract: The sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) pathway has been associated with cancer promotion, progression and resistance to treatments in a number of cancers, including prostate adenocarcinoma. Here we provide the first evidence that dietary agents, namely epigallocatechin gallate (EGCg, IC50≈75µM), resveratrol (IC50≈40µM) or a mixture of polyphenols from green tea (Polyphenon E, PPE, IC50≈70µM) or grapevine extract (vineatrol, IC50≈30µM) impede prostate cancer cell growth in vitro and in vivo, by inhibiting SphK1/S1P pathway. We establish that SphK1 is a downstream effector of the ERK/Phospholipase D (PLD) pathway, which is inhibited by green tea and wine polyphenols. Enforced expression of SphK1 impaired the ability of green tea and wine polyphenols, and pharmacological inhibitors of PLD and ERK activities, to induce apoptosis in PC-3 and C4-2B cells. The therapeutic efficacy of these polyphenols on tumor growth and SphK1/S1P pathway were confirmed in animals using an heterotopic PC-3 tumor in place model. PC-3/SphK1 cells implanted in animals developed larger tumors and resistance to treatment with polyphenols. Furthermore, using an orthotopic PC-3/GFP model, the chemopreventive effect of EGCg or PPE diet was associated with SphK1 inhibition, decrease in primary tumor volume, and occurrence and number of metastases. These results provide the first demonstration that the prosurvival, anti-apoptotic SphK1/S1P pathway represents a target of dietary green tea and wine polyphenols in cancer.
Abstract: Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.
Abstract: Sphingolipid metabolites are critical to the regulation of a number of fundamental biological processes including cancer. Whereas ceramide and sphingosine mediate and trigger apoptosis or cell growth arrest, sphingosine 1-phosphate promotes proliferation, cell survival and angiogenesis. The delicate equilibrium between the intracellular levels of each of these sphingolipids is controlled by the enzymes that either produce or degrade these metabolites. Sphingosine kinase-1 is a crucial regulator of this two-pan balance, because its produces the pro-survival and pro-angiogenic sphingosine 1-phosphate and decreases the amount of both ceramide and sphingosine, the pro-apoptotic sphingolipids. Moreover, its gene is oncogenic, its mRNA is overproduced in several solid tumors, its overexpression protects cells from apoptosis, and its activity is down-regulated by anti-cancer treatments. Therefore, the sphingosine kinase-1/sphingosine 1-phosphate signaling pathway appears to be a target of interest for therapeutic manipulation.
Abstract: It has recently been shown that docetaxel chemotherapy is effective in prolonging life in patients with prostate cancer (PCa). We have investigated potential ways of increasing the effectiveness of chemotherapy in this disease. We have previously reported that sphingosine kinase 1 (SphK1) inhibition is a key step in docetaxel-induced apoptosis in the PC-3 PCa cell line and that pharmacologicalSphK1 inhibition is chemosensitizing in the docetaxel-resistant PCa LNCaP cell line. In this study we have addressed the mechanism of docetaxel-induced apoptosis of PC-3 cells and identified SphK1-dependent and -independent components. We have shown that SphK1 inhibition by docetaxel is a two-step process involving an initial loss of enzyme activity followed by a decrease in SphK1 gene expression. Using hormoneresistant PC-3 and DU145 PCa cells we have demonstrated that both pharmacological and siRNA-mediated SphK1 inhibition leads to a four-fold decrease in the docetaxel IC50 dose. This work points out to potential ways of increasing the effectiveness of chemotherapy for PCa by SphK1 inhibition. (c) 2009 UICC.
Abstract: BACKGROUND: Sphingosine kinase-1 (SphK1) is an oncogenic lipid kinase notably involved in response to anticancer therapies in prostate cancer. Androgens regulate prostate cancer cell proliferation, and androgen deprivation therapy is the standard of care in the management of patients with advanced disease. Here, we explored the role of SphK1 in the regulation of androgen-dependent prostate cancer cell growth and survival. METHODOLOGY/PRINCIPAL FINDINGS: Short-term androgen removal induced a rapid and transient SphK1 inhibition associated with a reduced cell growth in vitro and in vivo, an event that was not observed in the hormono-insensitive PC-3 cells. Supporting the critical role of SphK1 inhibition in the rapid effect of androgen depletion, its overexpression could impair the cell growth decrease. Similarly, the addition of dihydrotestosterone (DHT) to androgen-deprived LNCaP cells re-established cell proliferation, through an androgen receptor/PI3K/Akt dependent stimulation of SphK1, and inhibition of SphK1 could markedly impede the effects of DHT. Conversely, long-term removal of androgen support in LNCaP and C4-2B cells resulted in a progressive increase in SphK1 expression and activity throughout the progression to androgen-independence state, which was characterized by the acquisition of a neuroendocrine (NE)-like cell phenotype. Importantly, inhibition of the PI3K/Akt pathway--by negatively impacting SphK1 activity--could prevent NE differentiation in both cell models, an event that could be mimicked by SphK1 inhibitors. Fascinatingly, the reversability of the NE phenotype by exposure to normal medium was linked with a pronounced inhibition of SphK1 activity. CONCLUSIONS/SIGNIFICANCE: We report the first evidence that androgen deprivation induces a differential effect on SphK1 activity in hormone-sensitive prostate cancer cell models. These results also suggest that SphK1 activation upon chronic androgen deprivation may serve as a compensatory mechanism allowing prostate cancer cells to survive in androgen-depleted environment, giving support to its inhibition as a potential therapeutic strategy to delay/prevent the transition to androgen-independent prostate cancer.
Abstract: Defeating pancreatic cancer resistance to the chemotherapeutic drug gemcitabine remains a challenge to treat this deadly cancer. Targeting the sphingolipid metabolism for improving tumor chemosensitivity has recently emerged as a promising strategy. The fine balance between intracellular levels of the prosurvival sphingosine-1-phosphate (S1P) and the proapoptotic ceramide sphingolipids determines cell fate. Among enzymes that control this metabolism, sphingosine kinase-1 (SphK1), a tumor-associated protein overexpressed in many cancers, favors survival through S1P production, and inhibitors of SphK1 are used in ongoing clinical trials to sensitize epithelial ovarian and prostate cancer cells to various chemotherapeutic drugs. We here report that the cellular ceramide/S1P ratio is a critical biosensor for predicting pancreatic cancer cell sensitivity to gemcitabine. A low level of the ceramide/S1P ratio, associated with a high SphK1 activity, correlates with a robust intrinsic pancreatic cancer cell chemoresistance toward gemcitabine. Strikingly, increasing the ceramide/S1P ratio, by using pharmacologic (SphK1 inhibitor or ceramide analogue) or small interfering RNA-based approaches to up-regulate intracellular ceramide levels or reduce SphK1 activity, sensitized pancreatic cancer cells to gemcitabine. Conversely, decreasing the ceramide/S1P ratio, by up-regulating SphK1 activity, promoted gemcitabine resistance in these cells. Development of novel pharmacologic strategies targeting the sphingolipid metabolism might therefore represent an interesting promising approach, when combined with gemcitabine, to defeat pancreatic cancer chemoresistance to this drug.
Abstract: The reduction in the normal level of tissue oxygen tension or hypoxia is a characteristic of solid tumors that triggers the activation of signaling pathways promoting neovascularization, metastasis, increased tumor growth, and resistance to treatments. The activation of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha) has been identified as the master mechanism of adaptation to hypoxia. In a recent study, we identified the sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) pathway, which elicits various cellular processes including cell proliferation, cell survival, or angiogenesis, as a new modulator of HIF-1alpha activity under hypoxic conditions. Here, we consider how the SphK1/S1P signaling pathway could represent a very important target for therapeutic intervention in cancer.
Abstract: BACKGROUND: The sphingolipids ceramide and sphingosine 1-phosphate (S1P) are key regulators of cell death and proliferation. The subtle balance between their intracellular levels is governed mainly by sphingosine kinase-1, which produces the pro-survival S1P. Sphingosine kinase-1 is an oncogene; is overexpressed in many tumors; protects cancer cells from apoptosis in vitro and in vivo; and its activity is decreased by anticancer therapies. Hence, sphingosine kinase-1 appears to be a target of interest for therapeutic manipulation. OBJECTIVE: This review considers recent developments regarding the involvement of sphingosine kinase-1 as a therapeutic target for cancer, and describes the pharmacological tools currently available. RESULTS/CONCLUSION: The studies described provide strong evidence that strategies to kill cancer cells via sphingosine kinase-1 inhibition are valid and could have a favorable therapeutic index.
Abstract: We examined the involvement of sphingosine kinase-1 (SphK1), which governs the ceramide/sphingosine-1-phosphate balance, in susceptibility to imatinib of either sensitive or resistant chronic myeloid leukemia cells. Imatinib-sensitive LAMA84-s displayed marked SphK1 inhibition coupled with increased content of ceramide and decreased pro-survival sphingosine-1-phosphate. Conversely, no changes in the sphingolipid metabolism were observed in LAMA84-r treated with imatinib. Overcoming imatinib resistance in LAMA84-r with farnesyltransferase or MEK/ERK inhibitors as well as with cytosine arabinoside led to SphK1 inhibition. Overexpression of SphK1 in LAMA84-s cells impaired apoptosis and inhibited the effects of imatinib on caspase-3 activation, cytochrome c and Smac release from mitochondria through modulation of Bim, Bcl-xL and Mcl-1 expression. Pharmacological inhibition of SphK1 with F-12509a or its silencing by siRNA induced apoptosis of both imatinib-sensitive and -resistant cells, suggesting that SphK1 inhibition was critical for apoptosis signaling. We also show that imatinib-sensitive and -resistant primary cells from chronic myeloid leukemia patients can be successfully killed in vitro by the F-12509a inhibitor. These results uncover the involvement of SphK1 in regulating imatinib-induced apoptosis and establish that SphK1 is a downstream effector of the Bcr-Abl/Ras/ERK pathway inhibited by imatinib but upstream regulator of Bcl-2 family members.
Abstract: Here, we provide the first evidence that sphingosine kinase 1 (SphK1), an oncogenic lipid kinase balancing the intracellular level of key signaling sphingolipids, modulates the transcription factor hypoxia inducible factor 1alpha (HIF-1alpha), master regulator of hypoxia. SphK1 activity is stimulated under low oxygen conditions and regulated by reactive oxygen species. The SphK1-dependent stabilization of HIF-1alpha levels is mediated by the Akt/glycogen synthase kinase-3beta signaling pathway that prevents its von Hippel-Lindau protein-mediated degradation by the proteasome. The pharmacologic and RNA silencing inhibition of SphK1 activity prevents the accumulation of HIF-1alpha and its transcriptional activity in several human cancer cell lineages (prostate, brain, breast, kidney, and lung), suggesting a canonical pathway. Therefore, we propose that SphK1 can act as a master regulator for hypoxia, giving support to its inhibition as a valid strategy to control tumor hypoxia and its molecular consequences.
Abstract: We have previously reported that, in prostate cancer, inhibition of the oncogenic sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) pathway is a key element in chemotherapy-induced apoptosis. Here, we show that selective pharmacologic inhibition of SphK1 triggers apoptosis in LNCaP and PC-3 prostate cancer cells, an effect that is reversed by SphK1 enforced expression. More importantly, we show for the first time that the up-regulation of the SphK1/S1P pathway plays a crucial role in the resistance of prostate cancer cells to chemotherapy. Importantly, pharmacologic SphK1 inhibition with the B-5354c compound sensitizes LNCaP and PC-3 cells to docetaxel and camptothecin, respectively. In vivo, camptothecin and B-5354c alone display a limited effect on tumor growth in PC-3 cells, whereas in combination there is a synergy of effect on tumor size with a significant increase in the ceramide to S1P sphingolipid ratio. To conclude, our study highlights the notion that drugs specifically designed to inhibit SphK1 could provide a means of enhancing the effects of conventional treatment through the prosurvival antiapoptotic SphK1/S1P pathway.
Abstract: Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.
Abstract: We examined the role of sphingosine kinase-1 (SphK1), a critical regulator of the ceramide/sphingosine 1-phosphate (S1P) biostat, in the regulation of death and survival of SH-SY5Y neuroblastoma cells in response to amyloid beta (Abeta) peptide (25-35). Upon incubation with Abeta, SH-SY5Y cells displayed a marked down-regulation of SphK1 activity coupled with an increase in the ceramide/S1P ratio followed by cell death. This mechanism was redox-sensitive; N-acetylcysteine totally abrogated the down-regulation of SphK1 activity and strongly inhibited Abeta-induced cell death. SphK1 overexpression impaired the cytotoxicity of Abeta, whereas SphK1 silencing by RNA interference mimicked Abeta-induced cell death, thereby establishing a critical role for SphK1. We further demonstrated that SphK1 could mediate the well established cytoprotective action of insulin-like growth factor (IGF-I) against Abeta toxicity. A dominant-negative form of SphK1 or its pharmacological inhibition not only abrogated IGF-I-triggered stimulation of SphK1 but also hampered IGF-I protective effect. Similarly to IGF-I, the neuroprotective action of TGF-beta1 was also dependent on SphK1 activity; activation of SphK1 as well as cell survival were impeded by a dominant-negative form of SphK1. Taken together, these results provide the first illustration of SphK1 role as a critical regulator of death and survival of Abeta-treated cells.
Abstract: The mitochondrial enzyme monoamine oxidase (MAO), its isoform MAO-A, plays a major role in reactive oxygen species-dependent cardiomyocyte apoptosis and postischemic cardiac damage. In the current study, we investigated whether sphingolipid metabolism can account for mediating MAO-A- and reactive oxygen species-dependent cardiomyocyte apoptosis. In H9c2 cardiomyoblasts, MAO-A-dependent reactive oxygen species generation led to mitochondria-mediated apoptosis, along with sphingosine kinase-1 (SphK1) inhibition. These phenomena were associated with generation of proapoptotic ceramide and decrease in prosurvival sphingosine 1-phosphate. These events were mimicked by inhibition of SphK1 with either pharmacological inhibitor or small interfering RNA, as well as by extracellular addition of C(2)-ceramide or H(2)O(2). In contrast, enforced expression of SphK1 protected H9c2 cells from serotonin- or H(2)O(2)-induced apoptosis. Analysis of cardiac tissues from wild-type mice subjected to ischemia/reperfusion revealed significant upregulation of ceramide and inhibition of SphK1. It is noteworthy that SphK1 inhibition, ceramide accumulation, and concomitantly infarct size and cardiomyocyte apoptosis were significantly decreased in MAO-A-deficient animals. In conclusion, we show for the first time that the upregulation of ceramide/sphingosine 1-phosphate ratio is a critical event in MAO-A-mediated cardiac cell apoptosis. In addition, we provide the first evidence linking generation of reactive oxygen species with SphK1 inhibition. Finally, we propose sphingolipid metabolites as key mediators of postischemic/reperfusion cardiac injury.
Abstract: Sphingolipid metabolites play critical functions in the regulation of a number of fundamental biological processes including cancer. Whereas ceramide and sphingosine mediate and trigger apoptosis or cell growth arrest, sphingosine 1-phosphate promotes proliferation and cell survival. The delicate equilibrium between the intracellular levels of each of these sphingolipids is controlled by the enzymes that either produce or degrade these metabolites. Sphingosine kinase-1 is a crucial regulator of this two-pan balance, because it produces the prosurvival sphingosine 1-phosphate, and reduces the content of both ceramide and sphingosine, the proapoptotic sphingolipids. Sphingosine kinase-1 controls the levels of sphingolipids having opposite effects on cell survival/death, its gene was found to be of oncogenic nature, its mRNA is overexpressed in many solid tumors, its overexpression protects cells from apoptosis and its activity is decreased during anticancer treatments. Therefore, sphingosine kinase-1 appears to be a target of interest for therapeutic manipulation via its pharmacological inhibition. Strategies to kill tumor cells by increasing their ceramide and/or sphingosine content while blocking sphingosine 1-phosphate generation should have a favorable therapeutic index.
Abstract: Herein we report that the prosurvival sphingolipid sphingosine 1-phosphate regulates the activities of both Bad and Bax during apoptosis of Jurkat cells. First, sphingosine 1-phosphate treatment results in Bad inactivation via the ERK/Rsk-1 pathway. Second, sphingosine 1-phosphate blocks the translocation of Bax to the mitochondria induced by Fas ligation. MEK inhibition by PD98059 or U0126 not only abrogates sphingosine 1-phosphate-induced Bad phosphorylation, but also its cytoprotective effect. Furthermore, inhibition of both mitochondrial cytochrome c efflux and Bax translocation to the mitochondria by sphingosine 1-phosphate could be overcome by PD98059 or U0126. Hence, the MEK/ERK pathway seems to be crucial for the survival effects initiated by sphingosine 1-phosphate.
Abstract: We examined the involvement of sphingosine kinase-1, a critical regulator of the sphingolipid balance, in susceptibility to antineoplastic agents of either sensitive or multidrug-resistant acute myeloid leukemia cells. Contrary to parental HL-60 cells, doxorubicin and etoposide failed to trigger apoptosis in chemoresistant HL-60/Doxo and HL-60NP16 cells overexpressing MRP1 and MDR1, respectively. Chemosensitive HL-60 cells displayed sphingosine kinase-1 inhibition coupled with ceramide generation. In contrast, chemoresistant HL-60/ Doxo and HL-60/VP16 had sustained sphingosine kinase-1 activity and did not produce ceramide during treatment. Enforced expression of sphingosine kinase-1 in chemosensitive HL-60 cells resulted in marked inhibition of apoptosis that was mediated by blockade of mitochondrial cytochrome c efflux hence suggesting a control of apoptosis at the pre-mitochondrial level. Incubation with cell-permeable ceramide of chemoresistant cells led to a sphingosine kinase-1 inhibition and apoptosis both prevented by sphingosine kinase-1 over-expression. Furthermore, F-12509a, a new sphingosine kinase inhibitor, led to ceramide accumulation, decrease in sphingosine 1-phosphate content and caused apoptosis equally in chemosensitive and chemoresistant cell lines that is inhibited by adding sphingosine 1-phosphate or overexpressing sphingosine kinase-1. F-12509a induced classical apoptosis hallmarks namely nuclear fragmentation, caspase-3 cleavage as well as downregulation of antiapoptotic XIAP, and release of cytochrome c and SMAC/Diablo.
Abstract: Systemic chemotherapy was considered of modest efficacy in prostate cancer until the recent introduction of taxanes. We took advantage of the known differential effect of camptothecin and docetaxel on human PC-3 and LNCaP prostate cancer cells to determine their effect on sphingosine kinase-1 (SphK1) activity and subsequent ceramide/sphingosine 1-phosphate (S1P) balance in relation with cell survival. In vitro, docetaxel and camptothecin induced strong inhibition of SphK1 and elevation of the ceramide/S1P ratio only in cell lines sensitive to these drugs. SphK1 overexpression in both cell lines impaired the efficacy of chemotherapy by decreasing the ceramide/S1P ratio. Alternatively, silencing SphK1 by RNA interference or pharmacologic inhibition induced apoptosis coupled with ceramide elevation and loss of S1P. The differential effect of both chemotherapeutics was confirmed in an orthotopic PC-3/green fluorescent protein model established in nude mice. Docetaxel induced a stronger SphK1 inhibition and ceramide/S1P ratio elevation than camptothecin. This was accompanied by a smaller tumor volume and the reduced occurrence and number of metastases. SphK1-overexpressing PC-3 cells implanted in animals developed remarkably larger tumors and resistance to docetaxel treatment. These results provide the first in vivo demonstration of SphK1 as a sensor of chemotherapy.
Abstract: In contrast to caspase-8, controversy exists as to the ability of caspase-10 to mediate apoptosis in response to FasL. Herein, we have shown activation of caspase-10, -3, and -7 as well as B cell lymphoma-2-interacting domain (Bid) cleavage and cytochrome c release in caspase-8-deficient Jurkat (I9-2) cells treated with FasL. Apoptosis was clearly induced as illustrated by nuclear and DNA fragmentation. These events were inhibited by benzyloxycarbonyl-VAD-fluoromethyl ketone, a broad spectrum caspase inhibitor, indicating that caspases were functionally and actively involved. Benzyloxycarbonyl-AEVD-fluoromethyl ketone, a caspase-10 inhibitor, had a comparable effect. FasL-induced cell death was not completely abolished by caspase inhibitors in agreement with the existence of a cytotoxic caspase-independent pathway. In subpopulations of I9-2 cells displaying distinct caspase-10 expression levels, cell sensitivity to FasL correlated with caspase-10 expression. A robust caspase activation, Bid cleavage, and DNA fragmentation were observed in cells with high caspase-10 levels but not in those with low levels. In vitro, caspase-10, as well as caspase-8, could cleave Bid to generate active truncated Bid (p15). Altogether, our data strongly suggest that caspase-10 can serve as an initiator caspase in Fas signaling leading to Bid processing, caspase cascade activation, and apoptosis.
Abstract: Conversion of ceramide, a putative mediator of anticancer drug-induced apoptosis, into glucosylceramide, by the action of glucosylceramide synthase (GCS), has been implicated in drug resistance. Herein, we compared GM95 mouse melanoma cells deficient in GCS activity, with cells stably transfected with a vector encoding GCS (GM95/GCS). Enzymatic and metabolic analysis demonstrated that GM95/GCS cells expressed a fully functional enzyme, resulting in normal ceramide glycosylation. However, cytotoxicity assays, as well as caspase activation and cytochrome c release studies, did not reveal any difference between the two cell lines with respect to their sensitivity toward doxorubicin, vinblastine, paclitaxel, cytosine arabinoside, or short-chain ceramide analogs. Administration of doxorubicin resulted in ceramide accumulation in both cell lines, with similar kinetics and amplitude. Although glucosylceramide formation was detected in doxorubicin-treated GM95/GCS cells, metabolism of drug-induced ceramide did not appear to be instrumental in cell survival. Furthermore, N-(n-butyl)deoxynojirimycin, a potent and non-toxic GCS inhibitor, had no chemosensitizing effect on wild-type melanoma cells. Altogether, both genetic and pharmacological alterations of the cellular ceramide glycosylation capacity failed to sensitize melanoma cells to anticancer drugs, therefore moderating the importance of ceramide glucosylation in drug-resistance mechanisms.
Abstract: Various sphingolipids are being viewed as bioactive molecules and/or second messengers. Among them, ceramide (or N-acylsphingosine) and sphingosine generally behave as pro-apoptotic mediators. Indeed, ceramide mediates the death signal initiated by numerous stress agents which either stimulate its de novo synthesis or activate sphingomyelinases that release ceramide from sphingomyelin. For instance, the early generation of ceramide promoted by TNF is mediated by a neutral sphingomyelinase the activity of which is regulated by the FAN adaptor protein, thereby controlling caspase activation and the cell death programme. In addition, the activity of this neutral sphingomyelinase is negatively modulated by caveolin, a major constituent of some membrane microdomains. The enzyme sphingosine kinase also plays a crucial role in apoptosis signalling by regulating the intracellular levels of two sphingolipids having opposite effects, namely the pro-apoptotic sphingosine and the anti-apoptotic sphingosine 1-phosphate molecule. Ceramide and sphingosine metabolism therefore appears as a pivotal regulatory pathway in the determination of cell fate.
Abstract: Whereas some sphingolipids such as sphingoid bases and ceramide can mediate and induce cell killing, other sphingolipids such as sphingosine 1-phosphate promote cell survival or proliferation. The tight equilibrium between the intracellular levels of each of these biomodulators is controlled by the various enzymes that either produce or degrade these lipid molecules. Herein, the effects of sphingoid bases and their derivatives on the regulation of (cancer) cell growth and death are reviewed. In addition, the consequences of pharmacological manipulation of the enzymes that govern sphingoid base metabolism on in vitro and in vivo tumor cell growth are presented. Further development of pharmacological tools aimed at interfering with the metabolism of sphingolipids is expected to provide new avenues in the treatment of cancers as well as other diseases.
Abstract: Somatostatin receptor subtype 2 (sst2) gene expression is lost in 90% of human pancreatic adenocarcinomas. We previously demonstrated that stable sst2 transfection of human pancreatic BxPC-3 cells, which do not endogenously express sst2, inhibits cell proliferation, tumorigenicity, and metastasis. These sst2 effects occur as a consequence of an autocrine sst2-dependent loop, whereby sst2 induces expression of its own ligand, somatostatin. Here we investigated whether sst2 induces apoptosis in sst2-transfected BxPC-3 cells. Expression of sst2 induced a 4.4- +/- 0.05-fold stimulation of apoptosis in BxPC-3 through the activation of tyrosine phosphatase SHP-1. sst2 also sensitized these cells to apoptosis induced by tumor necrosis factor alpha (TNFalpha), enhancing it 4.1- +/- 1.5-fold. Apoptosis in BxPC-3 cells mediated by TNF-related apoptosis-inducing ligand (TRAIL) and CD95L was likewise increased 2.3- +/- 0.5-fold and 7.4- +/- 2.5-fold, respectively. sst2-dependent activation and cell sensitization to death ligand-induced apoptosis involved activation of the executioner caspases, key factors in both death ligand- or mitochondria-mediated apoptosis. sst2 affected both pathways: first, by up-regulating expression of TRAIL and TNFalpha receptors, DR4 and TNFRI, respectively, and sensitizing the cells to death ligand-induced initiator capase-8 activation, and, second, by down-regulating expression of the antiapoptotic mitochondrial Bcl-2 protein. These results are of interest for the clinical management of chemoresistant pancreatic adenocarcinoma by using a combined gene therapy based on the cotransfer of genes for both the sst2 and a nontoxic death ligand.
Abstract: Various lipidic molecules serve as second messengers for transducing signals from the cell surface to the cell interior and trigger specific cellular responses. Sphingolipids represent a complex group of lipids that have recently emerged as new transducers in eukaryotic cells. Several sphingolipid molecules are able to modulate cell growth, differentiation and death. This review summarises current knowledge of the signalling functions of sphingolipids, especially in the regulation of tumour necrosis factor [alpha] (TNF-[alpha])-mediated cytotoxic effects. TNF-[alpha] is a multifaceted cytokine that controls a wide range of immune responses in mammals, including induction of programmed cell death (also called apoptosis). On the basis of recent observations, a working model is proposed for the molecular mechanisms underlying regulation of sphingolipid generation following TNF-[alpha] receptor 1 activation. The implications of these findings for the development of future pharmacological strategies to prevent the cytotoxic TNF-[alpha] response and subsequent cellular dysfunctions (as seen in various human diseases) are discussed.
Abstract: Through the induction of apoptosis, CD95 plays a crucial role in the immune response and the elimination of cancer cells. Ligation of CD95 receptor activates a complex signaling network that appears to implicate the generation of reactive oxygen species (ROS). This study investigated the place of ROS production in CD95-mediated apoptosis and the role of the antioxidant enzyme glutathione peroxidase-1 (GPx1). Anti-CD95 antibodies triggered an early generation of ROS in human breast cancer T47D cells that was blocked by overexpression of GPx1 and inhibition of initiator caspase activation. Enforced expression of GPx1 also resulted in inhibition of CD95-induced effector caspase activation, DNA fragmentation, and apoptotic cell death. Resistance to CD95-mediated apoptosis was not due to an increased expression of anti-apoptotic molecules and could be reversed by glutathione-depleting agents. In addition, whereas the anti-apoptotic protein Bcl-xL prevented CD95-induced apoptosis in MCF-7 cells, it did not inhibit the early ROS production. Moreover, Bcl-xL but not GPx1 overexpression could suppress the staurosporine-induced late generation of ROS and subsequent cell death. Altogether, these findings suggest that GPx1 functions upstream of the mitochondrial events to inhibit the early ROS production and apoptosis induced by CD95 ligation. Finally, transgenic mice overexpressing GPx1 were partially protected from the lethal effect of anti-CD95, underlying the importance of peroxide formation (and GPx1) in CD95-triggered apoptosis.
Abstract: The sphingolipid ceramide has recently emerged as a new transducer or modulator of apoptotic cell death. This function, however, has recently been challenged. Here, in the light of recent observations, the role of ceramide in apoptosis signaling is discussed.
Abstract: The sphingolipid metabolites ceramide, sphingosine, and sphingosine 1-phosphate contribute to controlling cell proliferation and apoptosis. Ceramide and its catabolite sphingosine act as negative regulators of cell proliferation and promote apoptosis. Conversely, sphingosine 1-phosphate, formed by phosphorylation of sphingosine by a sphingosine kinase, has been involved in stimulating cell growth and inhibiting apoptosis. As the phosphorylation of sphingosine diminishes apoptosis, while dephosphorylation of sphingosine 1-phosphate potentiates it, the role of sphingosine as a messenger of apoptosis is of importance. Herein, the effects of sphingosine on diverse signaling pathways implicated in the apoptotic process are reviewed.
Abstract: Acetaminophen is a widely used analgesic and antipyretic drug that exhibits toxicity at high doses to the liver and kidneys. This toxicity has been attributed to cytochrome P-450-generated metabolites which covalently modify target proteins. Recently, acetaminophen, in its unmetabolized form, has been shown to affect a variety of cells and tissues, for instance, testicular and lymphoid tissues and lymphocyte cell lines. The effects on cell viability of acetaminophen at a concentration comparable to that achieved in plasma during acetaminophen toxicity have now been examined with a hepatoma cell line SK-Hep1, primary human peripheral blood lymphocytes and human Jurkat T cells. Acetaminophen reduced cell viability in a time-dependent manner. Staining of cells with annexin-V also revealed that acetaminophen induced, after 8 hr of treatment, a loss of the asymmetry of membrane phospholipids, which is an early event associated with apoptosis. Acetaminophen triggered the release of cytochrome c from mitochondria into the cytosol, activation of caspase-3, 8, and 9, cleavage of poly(ADP-ribose) polymerase, and degradation of lamin B1 and DNA. Whereas cleavage of DNA into internucleosomal fragments was apparent in acetaminophen treated SK-Hep1 and primary lymphocytes, DNA was only degraded to 50-kb fragments in treated Jurkat cells. Overexpression of the antiapoptotic protein Bcl-XL prevented these various apoptotic events induced by acetaminophen in Jurkat cells. Caspase-8 activation was a postmictochondrial event and occurred in a Fas-independent manner. These results demonstrate that acetaminophen induces caspases-dependent apoptosis with mitochondria as a primary target. These results also reiterate the potential role of apoptosis in acetaminophen hepatic and extrahepatic toxicity.
Abstract: Treatment of human breast carcinoma MCF7 cells with doxorubicin, one of the most active antineoplastic agents used in clinical oncology, induces apoptosis and leads to increases in sphingosine levels. The transient generation of this sphingolipid mediator preceded cytochrome c release from the mitochondria and activation of the executioner caspase-7 in MCF7 cells which do not express caspase-3. Bcl-x(L) overexpression did not affect sphingosine generation whereas it reduced apoptosis triggered by doxorubicin and completely blocked apoptosis triggered by sphingosine. Exogenous sphingosine-induced apoptosis was also accompanied by cytochrome c release and activation of caspase-7 in a Bcl-x(L)-sensitive manner. Furthermore, neither doxorubicin nor sphingosine treatment affected expression of Fas ligand or induced activation of the apical caspase-8, indicating a Fas/Fas ligand-independent mechanism. Our results suggest that a further metabolite of ceramide, sphingosine, may also be involved in mitochondria-mediated apoptotic signaling induced by doxorubicin in human breast cancer cells.
Abstract: Stress-induced activation of an acidic sphingomyelinase leading to generation of ceramide, an important lipid mediator, has been associated with apoptosis; however, the implication of this hydrolase has been questioned. The present study aimed at re-evaluating the role of this lysosomal enzyme in apoptosis initiated by different apoptotic inducers. The sensitivity of a series of acid sphingomyelinase-deficient cell lines derived from Niemann-Pick disease patients to stress-induced apoptosis was investigated. We have now shown that stress stimuli, such as anthracyclines, ionizing radiation, and Fas ligation trigger similar apoptotic hallmarks in normal and acid sphingomyelinase-deficient cell lines. Retrovirus-mediated gene correction of enzyme deficiency in Niemann-Pick cells does not modify response to apoptosis. Ceramide production is comparable in normal and Niemann-Pick cells, and increased activity of neutral sphingomyelinase is observed. Thus, our findings cast serious doubts that lysosomal sphingomyelinase activation is responsible for stress-induced apoptosis of cultured cells.
Abstract: TNF-alpha is a pleiotropic cytokine activating several signaling pathways initiated at distinct intracellular domains of the TNF receptors. Although the C-terminal region is believed to be responsible for apoptosis induction, the functions of more membrane-proximal domains, including the domain that couples to neutral sphingomyelinase activation, are not yet fully elucidated. The roles of this region and of the associated adapter protein FAN (factor associated with neutral SMase activation) in the cytotoxic response to TNF have been investigated. We have now shown that stable expression in human fibroblasts of a dominant negative form of FAN abrogates TNF-induced ceramide generation from sphingomyelin hydrolysis and reduces caspase processing, thus markedly inhibiting TNF-triggered apoptosis. However, the cytotoxic responses to daunorubicin and exogenous ceramide remain unaltered, as do the TNF-induced p42/p44 MAPK activation and CD54 expression. Fibroblasts from FAN-knockout mice also proved to be resistant to TNF toxicity. These findings highlight the previously unrecognized role of the adapter protein FAN in signaling cell death induction by TNF.
Abstract: Sphingosine-1-phosphate (SPP), a bioactive sphingolipid metabolite, suppresses apoptosis of many types of cells, including rat pheochromocytoma PC12 cells. Elucidating the molecular mechanism of action of SPP is complicated by many factors, including uptake and metabolism, as well as activation of specific G-protein-coupled SPP receptors, known as the endothelial differentiation gene-1 (EDG-1) family. In this study, we overexpressed type 1 sphingosine kinase (SPHK1), the enzyme that converts sphingosine to SPP, in order to examine more directly the role of intracellularly generated SPP in neuronal survival. Enforced expression of SPHK1 in PC12 cells resulted in significant increases in kinase activity, with corresponding increases in intracellular SPP levels and concomitant decreases in both sphingosine and ceramide, and marked suppression of apoptosis induced by trophic factor withdrawal or by C(2)-ceramide. NGF, which protects PC12 cells from serum withdrawal-induced apoptosis, also stimulated SPHK1 activity. Surprisingly, overexpression of SPHK1 had no effect on activation of two known NGF-stimulated survival pathways, extracellular signal regulated kinase ERK 1/2 and Akt. However, trophic withdrawal-induced activation of the stress activated protein kinase, c-Jun amino terminal kinase (SAPK/JNK), and activation of the executionary caspases 2, 3 and 7, were markedly suppressed. Moreover, this abrogation of caspase activation, which was prevented by the SPHK inhibitor N,N-dimethylsphingosine, was not affected by pertussis toxin treatment, indicating that the cytoprotective effect was likely not mediated by binding of SPP to cell surface G(i)-coupled SPP receptors. In agreement, there was no detectable release of SPP into the culture medium, even after substantially increasing cellular SPP levels by NGF or sphingosine treatment. In contrast to PC12 cells, C6 astroglioma cells secreted SPP, suggesting that SPP might be one of a multitude of known neurotrophic factors produced and secreted by glial cells. Collectively, our results indicate that SPHK/SPP may play an important role in neuronal survival by regulating activation of SAPKs and caspases.
Abstract: Sphingosine 1-phosphate (S-1P) has been implicated as a second messenger preventing apoptosis by counteracting activation of executioner caspases. Here it is reported that S-1P prevents apoptosis and executioner caspase-3 activation by inhibiting the translocation of cytochrome c and Smac/DIABLO from mitochondria to the cytosol induced by anti-Fas, tumor necrosis factor-alpha (TNF-alpha), serum deprivation, and cell-permeable ceramides in the human acute leukemia Jurkat, U937, and HL-60 cell lines. Furthermore, the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate, which stimulates sphingosine kinase, the enzyme responsible for S-1P production, also inhibits cytochrome c and Smac/DIABLO release. In contrast, dimethylsphingosine (DMS), a specific inhibitor of sphingosine kinase, sensitizes cells to cytochrome c and Smac/DIABLO release triggered by anti-Fas, TNF-alpha, serum deprivation, or ceramide. DMS-induced mitochondrial apoptogenic factor leakage can likewise be overcome by S-1P cotreatment. Hence, S-1P, likely generated through a protein kinase C- mediated activation of sphingosine kinase, inhibits the apoptotic cascade upstream of the release of the mitochondrial apoptogenic factors, cytochrome c, and Smac/DIABLO in human acute leukemia cells.
Abstract: Sphingolipids have emerged as a new class of lipid mediators. In response to various extracellular stimuli, sphingolipid turnover can be stimulated in vascular cells and cardiac myocytes. Subsequent generation of sphingolipid molecules such as ceramide, sphingosine, and sphingosine-1-phosphate, is followed by regulation of ion fluxes and activation of various signaling pathways leading to smooth muscle cell proliferation, endothelial cell differentiation or apoptotic cell death, cell contraction, retraction, or migration. The importance of sphingolipids in cardiovascular signaling is illustrated by recent observations implicating them in physiological processes such as vasculogenesis as well as in frequent pathological conditions, including atherosclerosis and its complications.
Abstract: Exposure to anti-Fas antibody in Jurkat cells (type II cells), which are characterized by a weak caspase-8 activation at the death-inducing signaling complex (DISC), induced a biphasic increase in ceramide levels. The early generation of ceramide preceded transient activation of acidic ceramidase and subsequent production of sphingosine, followed by cytochrome c release, activation of caspases-2, -3, -6, -7, -8, and -9, Bid cleavage, and a later sustained ceramide accumulation. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone inhibited early increases of ceramide and sphingosine, whereas overexpression of Bcl-x(L) had no effect, and both prevented the later sustained ceramide accumulation. Exogenous sphingosine, as well as cell-permeable C(2)-ceramide, induced cytochrome c release from mitochondria in a caspase-independent fashion leading to activation of caspase-9 and executioner caspases and, surprisingly, activation of the initiator caspase-8 and processing of its substrate Bid. These effects were also completely abolished by Bcl-x(L) overexpression. Our results suggest that sphingosine might also be involved in the mitochondria-mediated pathway of Fas-induced cell death in type II cells.
Abstract: Ceramide has been implicated as an important component of radiation-induced apoptosis of human prostate cancer cells. We examined the role of the sphingolipid metabolites--ceramide, sphingosine, and sphingosine-1-phosphate--in susceptibility to radiation-induced apoptosis in prostate cancer cell lines with different sensitivities to gamma-irradiation. Exposure of radiation-sensitive TSU-Pr1 cells to 8-Gy irradiation led to a sustained increase in ceramide, beginning after 12 h of treatment and increasing to 2.5- to 3-fold within 48 h. Moreover, irradiation of TSU-Pr1 cells also produced a marked and rapid 50% decrease in the activity of sphingosine kinase, the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate. In contrast, the radiation-insensitive cell line, LNCaP, had sustained sphingosine kinase activity and did not produce elevated ceramide levels on 8-Gy irradiation. Although LNCaP cells are highly resistant to gamma-irradiation-induced apoptosis, they are sensitive to the death-inducing effects of tumor necrosis factor alpha, which also increases ceramide levels in these cells (K. Kimura et al., Cancer Res., 59: 1606-1614, 1999). Moreover, we found that although irradiation alone did not increase sphingosine levels in LNCaP cells, tumor necrosis factor alpha plus irradiation induced significantly higher sphingosine levels and markedly reduced intracellular levels of sphingosine-1-phosphate. The elevation of sphingosine levels either by exogenous sphingosine or by treatment with the sphingosine kinase inhibitor N,N-dimethylsphingosine induced apoptosis and also sensitized LNCaP cells to gamma-irradiation-induced apoptosis. Our data suggest that the relative levels of sphingolipid metabolites may play a role in determining the radiosensitivity of prostate cancer cells, and that the enhancement of ceramide and sphingosine generation could be of therapeutic value.
Abstract: ELL-12, a liposome formulation of the ether-lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH(3)), is a nonmyelosuppressive antiproliferative agent that is more effective and less toxic than the ether lipid itself in tumor model systems. We found that ELL-12 induced apoptosis in Jurkat, H9, and U-937 cells that was preceded by activation of executioner caspases. In addition, ELL-12 triggered release of cytochrome c from mitochondria to the cytoplasm before caspase-9 activation. Apoptosis, activation of caspases, and cytochrome c release were blocked by Bcl-x(L) overexpression in Jurkat T cells, suggesting a critical role for mitochondria in ELL-12-triggered cell death. Furthermore, ELL-12 had no effect on expression of CD95 ligand, and inhibition of the Fas signaling pathway with antagonistic anti-CD95 antibody did not affect apoptosis induced by ELL-12. Hence, ELL-12 could be a promising adjunct for the treatment of tumors in addition to myelosuppressive chemotherapeutic drugs and/or those that use the CD95-ligand/receptor system to trigger apoptosis.
Abstract: Sphingosine-1-phosphate (SPP) is a bioactive lipid that has recently been identified as the ligand for the EDG family of G protein-coupled cell surface receptors. However, the mitogenic and survival effects of exogenous SPP may not correlate with binding to cell-surface receptors (Van Brocklyn, J.R., M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier, D.M. Thomas, P.J.P. Coopman, S. Thangada, T. Hla, and S. Spiegel. 1998. J. Cell Biol. 142:229-240). The recent cloning of sphingosine kinase, a unique lipid kinase responsible for the formation of SPP, has provided a new tool to investigate the role of intracellular SPP. Expression of sphingosine kinase markedly increased SPP levels in NIH 3T3 fibroblasts and HEK293 cells, but no detectable secretion of SPP into the medium was observed. The increased sphingosine kinase activity in NIH 3T3 fibroblasts was sufficient to promote growth in low- serum media, expedite the G(1)/S transition, and increase DNA synthesis and the proportion of cells in the S phase of the cell cycle with a concomitant increase in cell numbers. Transient or stable overexpression of sphingosine kinase in NIH 3T3 fibroblasts or HEK293 cells protected against apoptosis induced by serum deprivation or ceramide elevation. N,N-Dimethylsphingosine, a competitive inhibitor of sphingosine kinase, blocked the effects of sphingosine kinase overexpression on cell proliferation and suppression of apoptosis. In contrast, pertussis toxin did not abrogate these biological responses. In Jurkat T cells, overexpression of sphingosine kinase also suppressed serum deprivation- and ceramide-induced apoptosis and, to a lesser extent, Fas-induced apoptosis, which correlated with inhibition of DEVDase activity, as well as inhibition of the executionary caspase-3. Taken together with ample evidence showing that growth and survival factors activate sphingosine kinase, our results indicate that SPP functions as a second messenger important for growth and survival of cells. Hence, SPP belongs to a novel class of lipid mediators that can function inside and outside cells.
Abstract: Incorporation of ET-18-OCH3 into well-characterized liposomes known as ELL-12 has eliminated its gastrointestinal and hemolytic toxicity without loss of growth inhibiting activity. ET-18-OCH3, but not ELL-12, blunted the increase in membrane protein kinase C (PKC) activity induced by 12-O-tetradecanoylphorbol 13-myristate (TPA) and markedly reduced levels of PKC alpha in NIH 3T3 fibroblasts. Furthermore, prolonged treatment with ELL-12 neither inhibited TPA-induced translocations of PKC alpha and PKC delta to the particulate fraction nor caused down-regulation, and did not affect the cellular distribution of TPA-insensitive PKC zeta. In Jurkat T cells, where ELL-12 markedly induced apoptosis that was blocked by an inhibitor of caspase-3-like activities, it had no effect on PKC activity or translocation induced by TPA. Thus, it seems unlikely that PKC is involved in the therapeutic effects of ELL-12.
Abstract: Recent evidence suggests that branching pathways of sphingolipid metabolism may mediate either apoptotic or mitogenic responses depending on the cell type and the nature of the stimulus. While ceramide has been shown to be an important regulatory component of apoptosis induced by tumor necrosis factor alpha and Fas ligand, sphingosine-1-phosphate (SPP), a further metabolite of ceramide, has been implicated as a second messenger in cellular proliferation and survival induced by platelet-derived growth factor, nerve growth factor, and serum. SPP protects cells from apoptosis resulting from elevations of ceramide. Inflammatory cytokines stimulate sphingomyelinase, but not ceramidase, leading to accumulation of ceramide, whereas growth signals also leading to accumulation of ceramide, whereas growth signals also stimulate ceramidase and sphingosine kinase leading to increased SPP levels. We propose that the dynamic balance between levels of sphingolipid metabolites, ceramide, and SPP, and consequent regulation of different family members of mitogen-activated protein kinases (JNK versus ERK), is an important factor that determines whether a cell survives or dies.
Abstract: Ceramide, a sphingolipid generated by the hydrolysis of membrane-associated sphingomyelin, appears to play a role as a gauge of apoptosis. A further metabolite of ceramide, sphingosine 1-phosphate (SPP), prevents ceramide-mediated apoptosis, and it has been suggested that the balance between intracellular ceramide and SPP levels may determine the cell fate (Cuvillier, O., Pirianov, G, Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., and Spiegel, S. (1996) Nature 381, 800-803). Here, we investigated the role of SPP and the protein kinase C activator, phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), in the caspase cascade leading to the proteolysis of poly(ADP-ribose) polymerase (PARP) and lamins. In Jurkat T cells, Fas ligation or addition of exogenous C2-ceramide induced activations of caspase-3/CPP32 and caspase-7/Mch3 followed by PARP cleavage, effects that can be blocked either by SPP or TPA. Furthermore, both SPP and TPA inhibit the activation of caspase-6/Mch2 and subsequent lamin B cleavage. Ceramide, in contrast to Fas ligation, did not induce activation of caspase-8/FLICE and neither SPP nor TPA were able to prevent this activation. Thus, SPP, likely generated via protein kinase C-mediated activation of sphingosine kinase, suppresses the apoptotic pathway downstream of FLICE but upstream of the executioner caspases, caspase-3, -6, and -7.
Abstract: Sphingolipid breakdown products [ceramide, sphingosine, and sphingosine-1-phosphate (SPP)] are emerging as a new class of bioactive molecules. In agreement with previous studies, treatment of human promyelocytic leukemia HL-60 cells with 1-alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] induced a transient increase of ceramide levels within 2 h, which then returned to basal levels within 8 h. In contrast, sphingosine kinase activity increased more slowly and reached maximal levels only after 20 h of exposure, leading to a concomitant increase in SPP level. Unlike treatments with cell-permeable ceramide analogues or sphingomyelinase, which induce apoptosis, 1,25-(OH)2D3 did not induce apoptosis, despite the early formation of ceramide. Moreover, prolonged treatment of HL-60 cells with 1,25-(OH)2D3 suppressed ceramide-induced apoptosis. There was a correlation between the time course and dose response of the activation of sphingosine kinase by 1,25-(OH)2D3 and the protection against apoptosis. In contrast, treatment with all-trans-retinoic acid neither stimulated sphingosine kinase activity nor protected cells from ceramide-induced apoptosis. Treatment with SPP protected HL-60 cells from ceramide-induced apoptosis, and N,N-dimethylsphingosine (DMS), a competitive inhibitor of sphingosine kinase, prevented the survival effect of 1,25-(OH)2D3. The effect of DMS was counteracted by SPP, suggesting that SPP is a critical component of the cytoprotective effect of 1,25-(OH)2D3. Chelerythrine chloride, an inhibitor of protein kinase C, markedly reduced sphingosine kinase activity and the apoptosis-sparing effect of 1,25-(OH)2D3, and conversely, the tumor promoter 12-O-tetradecanoylphorhol-13-acetate not only suppressed ceramide-induced apoptosis but also stimulated sphingosine kinase activity. Moreover, the protective effect of 12-O-tetradecanoylphorbol-13-acetate was blocked by DMS. Collectively, our observations indicate that the cytoprotective effect of 1,25-(OH)2D3 is mediated by SPP, which is formed as a consequence of activation of sphingosine kinase.
Abstract: Sphingosine-1-phosphate (SPP), a bioactive lipid, acts both intracellularly and extracellularly to cause pleiotropic biological responses. Recently, we identified SPP as a ligand for the G protein-coupled receptor Edg-1 (Lee, M.-J., J.R. Van Brocklyn, S. Thangada, C.H. Liu, A.R. Hand, R. Menzeleev, S. Spiegel, and T. Hla. 1998. Science. 279:1552-1555). Edg-1 binds SPP with remarkable specificity as only sphinganine-1-phosphate displaced radiolabeled SPP, while other sphingolipids did not. Binding of SPP to Edg-1 resulted in inhibition of forskolin-stimulated cAMP accumulation, in a pertussis toxin-sensitive manner. In contrast, two well-characterized biological responses of SPP, mitogenesis and prevention of apoptosis, were clearly unrelated to binding to Edg-1 and correlated with intracellular uptake. SPP also stimulated signal transduction pathways, including calcium mobilization, activation of phospholipase D, and tyrosine phosphorylation of p125(FAK), independently of edg-1 expression. Moreover, DNA synthesis in Swiss 3T3 fibroblasts was significantly and specifically increased by microinjection of SPP. Finally, SPP suppresses apoptosis of HL-60 and pheochromocytoma PC12 cells, which do not have specific SPP binding or expression of Edg-1 mRNA. Conversely, sphinganine-1-phosphate, which binds to and signals via Edg-1, does not have any significant cytoprotective effect. Thus, SPP is a prototype for a novel class of lipid mediators that act both extracellularly as ligands for cell surface receptors and intracellularly as second messengers.
Abstract: Sphingosine 1-phosphate (SPP), a lipid second messenger formed by the action of sphingosine kinase, has been implicated in regulating diverse biological processes, including growth, survival, and differentiation. N,N-Dimethylsphingosine (DMS) inhibits sphingosine kinase and has been used to investigate the biological roles of SPP; however, little is known of the mechanism of inhibition of sphingosine kinase by DMS. In addition, DMS has been shown to inhibit protein kinase C in vitro. Here we report that DMS is a competitive inhibitor of sphingosine kinase from U937 monoblastic leukemia cells, Swiss 3T3 fibroblasts, and PC12 pheochromocytoma cells. DMS decreases basal levels of SPP and prevents increases in SPP in response to physiological stimuli known to activate sphingosine kinase. DMS also effectively increases cellular levels of ceramide in a variety of cell types, and resetting of the ceramide/SPP rheostat may account for the pro-apoptotic effects of DMS. Moreover, DMS, at concentrations which effectively inhibit sphingosine kinase, has no effect on protein kinase C activity or its membrane translocation. Thus, DMS acts as a specific competitive inhibitor of sphingosine kinase in diverse cell types and is a useful tool to elucidate the role of SPP as an intracellular second messenger.
Abstract: Recent evidence suggests that branching pathways of sphingolipid metabolism may mediate either apoptotic or mitogenic responses depending on the cell type and the nature of the stimulus. While ceramide has been shown to be an important regulatory component of apoptosis induced by tumor necrosis factor alpha and the Fas ligand, sphingosine-1-phosphate (SPP), a further metabolite of ceramide, has been implicated as a second messenger in cellular proliferation and survival induced by platelet-derived growth factor, neuronal growth factor, and serum. SPP protects cells from apoptosis resulting from elevations of ceramide. Inflammatory cytokines stimulate sphingomyelinase, but not ceramidase, leading to accumulation of ceramide, whereas growth signals also stimulate ceramidase and sphingosine kinase leading to increased SPP levels. We propose that the dynamic balance between levels of sphingolipid metabolites, ceramide, and SPP and consequent regulation of different members of the mitogen-activated protein kinases (JNK versus ERK) family is an important factor that determines whether a cell survives or dies.
Abstract: Ceramide is an important regulatory participant of programmed cell death (apoptosis) induced by tumour-necrosis factor (TNF)-alpha and Fas ligand, members of the TNF superfamily. Conversely, sphingosine and sphingosine-1-phosphate, which are metabolites of ceramide, induce mitogenesis and have been implicated as second messengers in cellular proliferation induced by platelet-derived growth factor and serum. Here we report that sphingosine-1-phosphate prevents the appearance of the key features of apoptosis, namely intranucleosomal DNA fragmentation and morphological changes, which result from increased concentrations of ceramide. Furthermore, inhibition of ceramide-mediated apoptosis by activation of protein kinase C results from stimulation of sphingosine kinase and the concomitant increase in intracellular sphingosine-1-phosphate. Finally sphingosine-1-phosphate not only stimulates the extracellular signal-regulated kinase (ERK) pathway, it counteracts the ceramide-induced activation of stress-activated protein kinase (SAPK/JNK). Thus, the balance between the intracellular levels of ceramide and sphingosine-1-phosphate and their regulatory effects on different family members of mitogen-activated protein kinases determines the fate of the cell.
Abstract: During a systematic study of carbohydrate material present in human meconium, in addition to the previously described mucins, glycolipids and free oligosaccharides, we have now characterized a significant quantity of free glycoasparagines. These glycoasparagines have been isolated from human meconium by a combination of ion-exchange, concanavalin A (ConA)-affinity and high-performance liquid (HPLC) chromatographies. Their structures have been established by 400 MHz 1H-NMR spectroscopy. These compounds are related to N-acetyllactosaminic type structures and are based on the common core: [formula: see text] These glycoasparagines are probably derived from both protease and partial exoglycosidase hydrolysis of fetal gastrointestinal N-glycosyl proteins. Their structures are discussed in the context of the known catabolic pathways of N-glycans.
Abstract: Four hundred MHz 1H-NMR and 100 MHz 13C-NMR spectra of thirteen sialylated oligosaccharide-alditols isolated from hen ovomucin and swallow nests (Collocalia mucin) were studied. The resonance assignments were determined by combining multiple-relayed coherence-transfer chemical-shift-correlated spectroscopy (multiple-Relay-Cosy) and 1H/13C chemical-shift-correlated 2-D experiments.
Abstract: Sphingosine kinase-1 is a crucial regulator of the endogenous levels of ceramide and sphingosine 1-phosphate, two sphingolipid metabolites that have opposite functions on cell proliferation and survival. Ceramide is an anti-growth, pro-apoptotic mediator while sphingosine 1-phosphate is a pro-growth, anti-apoptotic molecule. Sphingosine kinase-1 hence represents a key enzyme in the sphingolipid metabolism because it produces sphingosine 1-phosphate and decreases the level of ceramide, counterbalancing its effects. Sphingosine kinase-1, which has been found to be oncogenic and overexpressed in various solid tumors, can therefore be regarded as a possible target for anti-cancer therapies. In this review, we will discuss the implication of sphingosine kinase-1 in regulating cancer cell survival in response to anti-cancer treatments and the potential use of sphingosine kinase inhibitors in cancer therapy.
Abstract: Sphingosine-1-phosphate, a metabolite of membrane sphingolipids, is a second messenger promoting cellular proliferation. Many growth factors increase sphingosine-1-phosphate intracellular levels by stimulation of sphingosine kinase, the enzyme responsible for its generation. Moreover, sphingosine-1-phosphate promotes cell survival by counteracting the consequences of elevated levels of ceramide occuring during apoptotic cell death. Accordingly, the dynamic balance between ceramide and sphingosine-1-phosphate is thought to play a critical role in decisions as to whether a cell survives or dies. Sphingosine-1-phosphate also appears to inhibit cell motility and influence cell morphology by acting extracellularly through G protein-coupled receptors belonging to the emerging Edg family. Hence, sphingosine-1-phosphate could represent the prototype for a novel class of lipid messengers acting both extracellularly as ligands for cell surface receptors, and intracellularly as second messengers to accomplish pleiotropic effects.
