Synthesis of erythro- B13 enantiomers and stereospecific action of full set of B13-isomers in MCF7 breast carcinoma cells: Cellular metabolism and effects on sphingolipids.

B13 is an acid ceramidase (ACDase) inhibitor. The two chiral centers of this aromatic amido alcohol lead to four stereoisomers, yet we have little knowledge about its erythro- enantiomers, (1R, 2S) and (1S, 2R). In this paper, for the first time, the synthesis of two erythro- enantiomers is described, and the compounds are evaluated along with two threo- enantiomers, (1R, 2R) and (1S, 2S). The key metabolites and sphingolipid (SL) profile of the full set of B13 stereoisomers in MCF7 breast carcinoma cells are presented. The results demonstrated that the erythro- enantiomers were more effective than the threo- enantiomers on growth inhibition in MCF7 cells, although there were no statistically significant differences within the threo- and erythro- series. Measurement of intracellular levels of the compounds indicated that the erythro- seemed a little more cell permeable than the threo- enantiomers; also, the (1R, 2S) isomer with the same stereo structure as natural ceramide (Cer) could be hydrolyzed and phosphorylated in MCF7 cells. Furthermore, we also observed the formation of C16 homologs from the full set of B13 isomers within the cells, indicating the occurrence of de-acylation and re-acylation of the amino group of the aromatic alcohol. Moreover, the decrease in the Cer/Sph ratio suggests that the growth inhibition from (1R, 2S) isomer is not because of the inhibition of ceramidases. Taken together, (1R, 2S) could be developed as a substitute of natural Cer.

Sphingolipids as mediators of inflammation and novel therapeutic target in inflammatory bowel disease.

Morbidity of inflammatory gastrointestinal (GI) diseases continues to grow resulting in worsen quality of life and increased burden on public medical systems. Complex and heterogenous illnesses, inflammatory bowel diseases (IBDs) encompass several inflammation -associated pathologies including Crohn's disease and ulcerative colitis. IBD is often initiated by a complex interplay between host genetic and environmental factors, lifestyle and diet, and intestinal bacterial components. IBD inflammatory signature was linked to the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) signaling pathway that is currently targeted by IBD therapies. Sphingolipid signaling was identified as one of the key mediators and regulators of pro-inflammatory conditions, and, specifically, TNF-α related signaling. All GI tissues and circulating immune/blood cells contain activated sphingolipid-metabolizing enzymes, including sphingosine kinases (SphK1 and SphK2) that generate sphingosine-1-phosphate (S1P), a bioactive lipid and ligand for five G-protein coupled membrane S1P receptors (S1PRs). Numerous normal and pathogenic inflammatory responses are mediated by SphK/S1P/S1PRs signaling axis including lymphocyte trafficking and activation of cytokine signaling machinery. SphK1/S1P/S1PRs axis has recently been defined as a target for the treatment of GI diseases including IBD/colitis. Several SphK1 inhibitors and S1PRs antagonists have been developed as novel anti-inflammatory agents. In this review, we discuss the mechanisms of SphK/S1P signaling in inflammation-linked GI disorders. The potential role of SphK/S1PRs inhibitors in the prevention and treatment of IBD/colitis is critically evaluated.

Tamoxifen acts on Trypanosoma cruzi sphingolipid pathway triggering an apoptotic death process.

This study shows the effects of tamoxifen, a known estrogen receptor antagonist used in the treatment of breast cancer, on the sphingolipid pathway of Trypanosoma cruzi, searching for potential chemotherapeutic targets. A dose-dependent epimastigote growth inhibition at increasing concentration of tamoxifen was determined. In blood trypomastigotes, treatment with 10 µM showed 90% lysis, while 86% inhibition of intracellular amastigote development was obtained using 50 µM. Lipid extracts from treated and non-treated metabolically labelled epimastigotes evidenced by thin layer chromatography different levels of sphingolipids and MALDI-TOF mass spectrometry analysis assured the identity of the labelled species. Comparison by HPLC-ESI mass spectrometry of lipids, notably exhibited a dramatic increase in the level of ceramide in tamoxifen-treated parasites and a restrained increase of ceramide-1P and sphingosine, indicating that the drug is acting on the enzymes involved in the final breakdown of ceramide. The ultrastructural analysis of treated parasites revealed characteristic morphology of cells undergoing an apoptotic-like death process. Flow cytometry confirmed cell death by an apoptotic-like machinery indicating that tamoxifen triggers this process by acting on the parasitic sphingolipid pathway.

Multiple actions of doxorubicin on the sphingolipid network revealed by flux analysis.

Sphingolipids (SLs) have been implicated in numerous important cellular biologies; however, their study has been hindered by the complexities of SL metabolism. Furthermore, enzymes of SL metabolism represent a dynamic and interconnected network in which one metabolite can be transformed into other bioactive SLs through further metabolism, resulting in diverse cellular responses. Here we explore the effects of both lethal and sublethal doses of doxorubicin (Dox) in MCF-7 cells. The two concentrations of Dox resulted in the regulation of SLs, including accumulations in sphingosine, sphingosine-1-phosphate, dihydroceramide, and ceramide, as well as reduced levels of hexosylceramide. To further define the effects of Dox on SLs, metabolic flux experiments utilizing a d17 dihydrosphingosine probe were conducted. Results indicated the regulation of ceramidases and sphingomyelin synthase components specifically in response to the cytostatic dose. The results also unexpectedly demonstrated dose-dependent inhibition of dihydroceramide desaturase and glucosylceramide synthase in response to Dox. Taken together, this study uncovers novel targets in the SL network for the action of Dox, and the results reveal the significant complexity of SL response to even a single agent. This approach helps to define the role of specific SL enzymes, their metabolic products, and the resulting biologies in response to chemotherapeutics and other stimuli.

Dose dependent actions of LCL521 on acid ceramidase and key sphingolipid metabolites.

The function of acid ceramidase (ACDase), whose congenital deficiency leads to Farber disease, has been recognized to be vital to tumor cell biology, and inhibition of its activity may be beneficial in cancer therapy. Therefore, manipulation of the activity of this enzyme may have significant effect, especially on cancer cells. LCL521, Di-DMG-B13, is a lysosomotropic inhibitor of ACDase. Here we define complexities in the actions of LCL521 on ACDase. Systematic studies in MCF7 cells showed dose and time divergent action of LCL521 on ACDase protein expression and sphingolipid levels. Low dose of LCL521 (1 µM) effectively inhibited ACDase in cells, but the effects were transient. A higher dose of LCL521 (10 µM) caused a profound decrease of sphingosine and increase of ceramide, but additionally affected the processing and regeneration of the ACDase protein, with biphasic and reversible effects on the expression of ACDase, which paralleled the long term changes of cellular sphingosine and ceramide. Finally, the higher concentrations of LCL521 also inhibited Dihydroceramide desaturase (DES-1). In summary, LCL521 exhibits significant effects on ACDase in a dose and time dependent manner, but dose range and treatment time need to be paid attention to specify its future exploration on ACDase targeted cancer treatment.

Combining Deep Sequencing, Proteomics, Phosphoproteomics, and Functional Screens To Discover Novel Regulators of Sphingolipid Homeostasis.

Sphingolipids (SLs) are essential components of cell membranes and are broad-range bioactive signaling molecules. SL levels must be tightly regulated as imbalances affect cellular function and contribute to pathologies ranging from neurodegenerative and metabolic disorders to cancer and aging. Deciphering how SL homeostasis is maintained and uncovering new regulators is required for understanding lipid biology and for identifying new targets for therapeutic interventions. Here we combine omics technologies to identify the changes of the transcriptome, proteome, and phosphoproteome in the yeast Saccharomyces cerevisiae upon SL depletion induced by myriocin. Surprisingly, while SL depletion triggers important changes in the expression of regulatory proteins involved in SL homeostasis, the most dramatic regulation occurs at the level of the phosphoproteome, suggesting that maintaining SL homeostasis demands rapid responses. To discover which of the phosphoproteomic changes are required for the cell's first-line response to SL depletion, we overlaid our omics results with systematic growth screens for genes required during growth in myriocin. By following the rate of SL biosynthesis in those candidates that are both affecting growth and are phosphorylated in response to the drug, we uncovered Atg9, Stp4, and Gvp36 as putative new regulators of SL homeostasis.

Sng1 associates with Nce102 to regulate the yeast Pkh-Ypk signalling module in response to sphingolipid status.

All cells are delimited by biological membranes, which are consequently a primary target of stress-induced damage. Cold alters membrane functionality by decreasing lipid fluidity and the activity of membrane proteins. In Saccharomyces cerevisiae, evidence links sphingolipid homeostasis and membrane phospholipid asymmetry to the activity of the Ypk1/2 proteins, the yeast orthologous of the mammalian SGK1-3 kinases. Their regulation is mediated by different protein kinases, including the PDK1 orthologous Pkh1/2p, and requires the function of protein effectors, among them Nce102p, a component of the sphingolipid sensor machinery. Nevertheless, the mechanisms and the actors involved in Pkh/Ypk regulation remain poorly defined. Here, we demonstrate that Sng1, a transmembrane protein, is an effector of the Pkh/Ypk module and identify the phospholipid asymmetry as key for yeast cold adaptation. Overexpression of SNG1 impairs phospholipid flipping, reduces reactive oxygen species (ROS) and improves, in a Pkh-dependent manner, yeast growth in myriocin-treated cells, suggesting that excess Sng1p stimulates the Pkh/Ypk signalling. Furthermore, we link these effects to the association of Sng1p with Nce102p. Indeed, we found that Sng1p interacts with Nce102p both physically and genetically. Moreover, mutant nce102∆ sng1∆ cells show features of impaired Pkh/Ypk signalling, including increased ROS accumulation, reduced life span and defects in Pkh/Ypk-controlled regulatory pathways. Finally, myriocin-induced hyperphosphorylation of Ypk1p and Orm2p, which controls sphingolipid homeostasis, does not occur in nce102∆ sng1∆ cells. Hence, both Nce102p and Sng1p participate in a regulatory circuit that controls the activity of the Pkh/Ypk module and their function is required in response to sphingolipid status.

Modification of the Host Cell Lipid Metabolism Induced by Hypolipidemic Drugs Targeting the Acetyl Coenzyme A Carboxylase Impairs West Nile Virus Replication.

West Nile virus (WNV) is a neurotropic flavivirus transmitted by the bite of mosquitoes that causes meningitis and encephalitis in humans, horses, and birds. Several studies have highlighted that flavivirus infection is highly dependent on cellular lipids for virus replication and infectious particle biogenesis. The first steps of lipid synthesis involve the carboxylation of acetyl coenzyme A (acetyl-CoA) to malonyl-CoA that is catalyzed by the acetyl-CoA carboxylase (ACC). This makes ACC a key enzyme of lipid synthesis that is currently being evaluated as a therapeutic target for different disorders, including cancers, obesity, diabetes, and viral infections. We have analyzed the effect of the ACC inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) on infection by WNV. Lipidomic analysis of TOFA-treated cells confirmed that this drug reduced the cellular content of multiple lipids, including those directly implicated in the flavivirus life cycle (glycerophospholipids, sphingolipids, and cholesterol). Treatment with TOFA significantly inhibited the multiplication of WNV in a dose-dependent manner. Further analysis of the antiviral effect of this drug showed that the inhibitory effect was related to a reduction of viral replication. Furthermore, treatment with another ACC inhibitor, 3,3,14,14-tetramethylhexadecanedioic acid (MEDICA 16), also inhibited WNV infection. Interestingly, TOFA and MEDICA 16 also reduced the multiplication of Usutu virus (USUV), a WNV-related flavivirus. These results point to the ACC as a druggable cellular target suitable for antiviral development against WNV and other flaviviruses.

Identification of a New Class of Antifungals Targeting the Synthesis of Fungal Sphingolipids.

UNLABELLED: Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N'-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N'-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. IMPORTANCE: Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.

Novel agents targeting bioactive sphingolipids for the treatment of cancer.

Sphingolipids are a class of lipids that have important functions in a variety of cellular processes such as, differentiation, proliferation, senescence, apoptosis and chemotherapeutic resistance. The most widely studied bioactive shingolipids include ceramides, dihydroceramide (dhCer), ceramide-1-phosphate (C1P), glucosyl-ceramide (GluCer), sphingosine and sphingosine-1-phosphate (S1P). Although the length of fatty acid chain affects the physiological role, ceramides and sphingosine are known to induce apoptosis whereas C1P, S1P and GluCer induce proliferation of cells, which causes the development of chemoresistance. Previous studies have implicated the significance of bioactive shingolipids in oncogenesis, cancer progression and drug- and radiation-resistance. Therefore, targeting the elements of sphingolipid metabolism appears important for the development of novel therapeutics or to increase the effectiveness of the current treatment strategies. Some approaches involve the development of synthetic ceramide analogs, small molecule inhibitors of enzymes such as sphingosine kinase, acid ceramidase or ceramide synthase that catalyze ceramide catabolism or its conversion to various molecular species and S1P receptor antagonists. These approaches mainly aim to up-regulate the levels of apoptotic shingolipids while the proliferative ones are down-regulated, or to directly deliver cytotoxic sphingolipids like short-chain ceramide analogs to tumor cells. It is suggested that a combination therapy with conventional cytotoxic approaches while preventing the conversion of ceramide to S1P and consequently increasing the ceramide levels would be more beneficial. This review compiles the current knowledge about sphingolipids, and mainly focuses on novel agents modulating sphingolipid pathways that represent recent therapeutic strategies for the treatment of cancer.

Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation.

Membrane rafts are thought to be sphingolipid- and cholesterol-dependent lateral assemblies involved in diverse cellular functions. Their biological roles and even their existence, however, remain controversial. Using an original fluorescence correlation spectroscopy strategy that recently enabled us to identify nanoscale membrane organizations in live cells, we report here that highly dynamic nanodomains exist in both the outer and inner leaflets of the plasma membrane. Through specific inhibition of biosynthesis, we show that sphingolipids and cholesterol are essential and act in concert for formation of nanodomains, thus corroborating their raft nature. Moreover, we find that nanodomains play a crucial role in triggering the phosphatidylinositol-3 kinase/Akt signaling pathway, by facilitating Akt recruitment and activation upon phosphatidylinositol-3,4,5-triphosphate accumulation in the plasma membrane. Thus, through direct monitoring and controlled alterations of rafts in living cells, we demonstrate that rafts are critically involved in the activation of a signaling axis that is essential for cell physiology.

A sphingolipid inhibitor induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia.

Sphingolipid biosynthesis pathways have recently emerged as a promising target for therapeutic intervention against pathogens, including parasites. A key step in the synthesis of complex sphingolipids is the glucosylation of ceramide, mediated by glucosylceramide (GlcCer) synthase, whose activity can be inhibited by PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). In this study, we investigated whether PPMP inhibits the proliferation and differentiation of the pathogenic parasite Giardia lamblia, the major cause of parasite-induced diarrhea worldwide. PPMP was found to block in vitro parasite replication in a dose-dependent manner, with a 50% inhibitory concentration of 3.5 muM. The inhibition of parasite replication was irreversible at 10 muM PPMP, a concentration that did not affect mammalian cell metabolism. Importantly, PPMP inhibited the completion of cell division at a specific stage in late cytokinesis. Microscopic analysis of cells incubated with PPMP revealed the aberrant accumulation of cellular membranes belonging to the endoplasmic reticulum network in the caudal area of the parasites. Finally, PPMP induced a 90% reduction in G. lamblia differentiation into cysts, the parasite stage responsible for the transmission of the disease. These results show that PPMP is a powerful inhibitor of G. lamblia in vitro and that as-yet-uncharacterized sphingolipid biosynthetic pathways are potential targets for the development of anti-G. lamblia agents.

Sphingolipid signaling pathways as potential therapeutic targets in gliomas.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor. The interconvertible bioactive sphingolipids sphingosine-1-phosphate (S1P) and ceramide have profound effects on GBM cells, with ceramide causing cell death and S1P leading to cell survival, proliferation and invasion. This review will examine the effects of ceramide and S1P on glioma cells and the therapeutic potential of these pathways.

Overcoming resistance to gamma-rays in squamous carcinoma cells by poly-drug elevation of ceramide levels.

Recent strategies to sensitize radioresistant tumours are based on combining gamma-irradiation with inducers of apoptosis. We report that the combination of three inhibitors of sphingolipid metabolism, DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCl(DL-PDMP)+imipramine +/- D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-MAPP), with 10-Gy irradiation triggers both mitotic and apoptotic killing in radioresistant SQ20B squamous carcinoma cells. In these cells, apoptosis is defective due to a lack of ceramide generation upstream, which cannot be explained by sphingomyelinase (neutral and acidic) deficiency or rapid derivation to the sphingolipid pathway. We present evidence of a functional transduction death pathway when ceramide generation is restored, which involves the mitochondrial-mediated pathway coupled to alterations in redox status and to executive caspases activation. The poly-drug treatment restored apoptosis to levels similar to those observed in radiosensitive SCC61 squamous carcinoma cells. Simultaneous exposure to gamma-irradiation and poly-drug treatment acted synergistically in SQ20B cells to produce a marked increase in both mitochondrial dysfunction and caspase cleavage, which led to a 7.8-fold increase in apoptosis within 48 h, relative to irradiated cells. Moreover, the results suggest that the ceramide released by irradiation or poly-drug treatment converges upon common cellular targets. Modulation of endogenous ceramide levels by inhibitors of sphingolipid metabolism may represent a new cellular target for the sensitization of radioresistant tumours to gamma-ray therapy.

Microbial natural products as a source of antifungals.

The vast number and variety of chemotherapeutic agents isolated from microbial natural products and used to treat bacterial infections have greatly contributed to the improvement of human health during the past century. However, only a limited number of antifungal agents (polyenes and azoles, plus the recently introduced caspofungin acetate) are currently available for the treatment of life-threatening fungal infections. Furthermore, the prevalence of systemic fungal infections has increased significantly during the past decade. For this reason, the development of new antifungal agents, preferably with novel mechanisms of action, is an urgent medical need. A selection of antifungal agents in early stages of development, produced by micro-organisms, is summarized in this review. The compounds are classified according to their mechanisms of action, covering inhibitors of the synthesis of cell wall components (glucan, chitin and mannoproteins), of sphingolipid synthesis (serine palmitoyltransferase, ceramide synthase, inositol phosphoceramide synthase and fatty acid elongation) and of protein synthesis (sordarins). In addition, some considerations related to the chemotaxonomy of the producing organisms and some issues relevant to antifungal drug discovery are also discussed.

Enzymes of sphingosine metabolism as potential pharmacological targets for therapeutic intervention in cancer.

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.

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.

Inhibition of sphingolipid biosynthesis in rat primary hepatocyte cultures by fumonisin B1 and other structurally related compounds.

The fumonisins and toxins produced by Alternaria alternata f. sp. lycopersici (AAL toxins) are structurally related mycotoxins that disrupt sphingolipid biosynthesis by inhibiting the rate-limiting enzyme, ceramide synthase. Rat primary hepatocytes were exposed to fumonisin B1 (FB1), its N-acetyl analogue, FA1, its fully hydrolysed analogue, AP1 and the AAL toxins (TA and TB) at concentrations of 1 microM for 40 hr in culture. The extent to which these compounds disrupt sphingolipid biosynthesis in hepatocytes in vitro was investigated by analysing the sphingosine (So) and sphinganine (Sa) levels by HPLC. The inhibition of ceramide synthase was irreversible as the Sa:So ratio was maximally increased by FB1 after 24 hr of exposure and the subsequent removal of FB1 had no effect on the ratio as compared with the 40-hr incubation period in the presence of FB1. The Sa concentration was significantly (P < 0.01) increased in all the cultures treated with the different structurally related compounds, while only AP1 increased the So concentration significantly (P < 0.05) above the control. As AP1 was found to be less effective in disrupting sphingolipid biosynthesis it would appear that the tricarballylic (TCA) moiety is required for maximal inhibition of ceramide synthase. The presence of an amino group appears not to be a requisite for activity, since FA1 increased the Sa:So ratio to the same extent as FB1. The AAL toxins TA and TB increased the Sa concentration significantly (P < 0.01) above that of FB1 and FA1, while the Sa:So ratios were altered to the same extent. The structural requirements for the induction of cytotoxicity differ from those required for ceramide synthase inhibition as TA and TB were significantly (P < 0.05 to P < 0.01) less toxic to primary hepatocytes than FB1 at all the concentrations tested.

Inhibition of sphingolipid induced apoptosis by caspase inhibitors indicates that sphingosine acts in an earlier part of the apoptotic pathway than ceramide.

Caspases are specific proteases involved in apoptosis, and their inhibition by specific peptide inhibitors can inhibit apoptosis. With these inhibitors we examined the relationship of caspases and sphingolipids involved in the induction of apoptosis of human leukemic HL60 cells. We have previously shown that sphingosine (Sph) and its methylated derivative dimethylsphingosine (DMS) effectively induce apoptosis in HL60 cells. Using these lipids as well as ceramide analogues we found both similarities and differences in the caspase involvement in apoptosis induced by the two distinct lipid types. The wide-spectrum caspase inhibitor Z-VAD-FMK and Z-DEVD-FMK, an inhibitor of the downstream caspases 3 (CPP32, Yama) and 7, both inhibited apoptosis induced by all the lipids tested. Z-AAD-FMK which inhibits the serine protease Granzyme B, inhibited Sph/DMS induced apoptosis, but little or no effect on ceramide induced apoptosis. Granzyme B shares a substrate sequence preference with upstream caspases capable of activating themselves and other caspases downstream. Z-IETD-FMK, which inhibits caspase 8/FLICE also inhibited Sph/DMS induced apoptosis with no inhibition of apoptosis induced by either ceramide. Together, these data indicate that Sph/DMS act independently from ceramide in the apoptosis pathway and further suggest that Sph/DMS act earlier in the pathway than ceramide and are involved upstream of even the early proteases, whereas the point of action for ceramide is downstream of the early proteases but upstream from the late caspases.

Fumonisins and human health.

Fumonisins are mycotoxins produced by Fusarium moniliforme that are prevalent in corn, sorghum, millet and other agricultural products. It is possible that fumonisins are aetiological agents in human oesophageal cancers. The International Agency for Research on Cancer have designated toxins derived from F. moniliforme as group 2B (possibly carcinogenic to humans). Fumonisins are hepatotoxic, nephrotoxic, atherogenic, immunosuppressive and embryotoxic in experimental animal systems. Methods of detoxifying fumonisin-contaminated foods are required. Fumonisins have potent, apparently specific, inhibitory effects on sphingolipid biosynthesis and as such are valuable in studies of the complex biochemical events involved in sphingolipid metabolism and function. Fumonisins may serve as templates for therapeutic agents for treating diseases related to sphingolipid turnover (lysosomal storage disease), such as Farber's disease.