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.

Role of 1-Deoxysphingolipids in docetaxel neurotoxicity.

A major dose-limiting side effect of docetaxel chemotherapy is peripheral neuropathy. Patients' symptoms include pain, numbness, tingling and burning sensations, and motor weakness in the extremities. The molecular mechanism is currently not understood, and there are no treatments available. Previously, we have shown an association between neuropathy symptoms of patients treated with paclitaxel and the plasma levels of neurotoxic sphingolipids, the 1-deoxysphingolipids (1-deoxySL) (Kramer et al, FASEB J, 2015). 1-DeoxySL are produced when the first enzyme of the sphingolipid biosynthetic pathway, serine palmitoyltransferase (SPT), uses L-alanine as a substrate instead of its canonical amino acid substrate, L-serine. In the current investigation, we tested whether 1-deoxySL accumulate in the nervous system following systemic docetaxel treatment in mice. In dorsal root ganglia (DRG), we observed that docetaxel (45 mg/kg cumulative dose) significantly elevated the levels of 1-deoxySL and L-serine-derived ceramides, but not sphingosine-1-phosphate (S1P). S1P is a bioactive sphingolipid and a ligand for specific G-protein-coupled receptors. In the sciatic nerve, docetaxel decreased 1-deoxySL and ceramides. Moreover, we show that in primary DRG cultures, 1-deoxysphingosine produced neurite swellings that could be reversed with S1P. Our results demonstrate that docetaxel chemotherapy up-regulates sphingolipid metabolism in sensory neurons, leading to the accumulation of neurotoxic 1-deoxySL. We suggest that the neurotoxic effects of 1-deoxySL on axons can be reversed with S1P.

Bioactive sphingolipid profile in a xenograft mouse model of head and neck squamous cell carcinoma.

The purpose of this study was to determine the profile of bioactive sphingolipids in xenograft mouse tissues of head and neck squamous cell carcinoma. We utilized UHPLC-MS/MS to determine the profile of full set of ceramides, sphingosine, and sphingosine 1-phosphate in this xenograft mouse model. The tissues isolated and investigated were from brain, lung, heart, liver, spleen, kidney, bladder, tumors and blood. With the exception of equal volume of blood plasma (100ul), all tissues were studied with the same amount of protein (800ug). Results demonstrated that brain contained the highest level of ceramide and kidney had the highest level of sphingosine, whereas sphingosine 1-phosphate and dihydrosphingosine 1-phosphate were heavily presented in the blood. Brain also comprised the highest level of phospholipids. As for the species, several ceramides, usually present in very low amounts in cultured tumor cells, showed relatively high levels in certain tissues. This study highlights levels of bioactive sphingolipids profiles in xenograft mouse model of head and neck squamous cell carcinoma, and provides resources to investigate potential therapeutic targets and biomarkers that target bioactive sphingolipids metabolism pathways.

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.

Functional antagonism of sphingosine-1-phosphate receptor 1 prevents cuprizone-induced demyelination.

Recent evidence suggests that the oral drug Fingolimod (FTY720) for relapsing-remitting multiple sclerosis (MS) may act directly on the central nervous system (CNS) and modulate disease pathogenesis and progression in experimental models of MS. However, the specific subtype of sphingosine-1-phosphate (S1P) receptors that mediates the effect of FTY720 on the CNS cells has not been fully elucidated. Here, we report that S1P receptor 1 (S1PR1) is elevated in reactive astrocytes in an autoimmunity independent mouse model of MS and that selective S1PR1 modulation is sufficient to ameliorate the loss of oligodendrocytes and demyelination. The non-selective S1PR modulator, FTY720, or a short-lived S1PR1-specific modulator, CYM5442, was administered daily to mice while on cuprizone diet. Both FTY720- and CYM5422-treated mice displayed a significant reduction in oligodendrocyte apoptosis and astrocyte and microglial activation in comparison to vehicle-treated groups, which was associated with decreased production of proinflammatory mediators and down-regulation of astrocytic S1PR1 protein. Interestingly, S1PR1 modulation during the early phase of cuprizone intoxication was required to suppress oligodendrocyte death and consequent demyelination as drug treatment from 10 days after the initiation of cuprizone feeding was no longer effective. CYM5442 treatment during the brief cuprizone exposure significantly prevented Il-1ß, Il-6, Cxcl10, and Cxcl3 induction, resulting in suppression of subsequent reactive gliosis and demyelination. Our study identifies functional antagonism of S1PR1 as a major mechanism for the protective effect of FTY720 in the cuprizone model and suggests pathogenic contributions of astrocyte S1PR1 signaling in primary demyelination and its potential as a therapeutic target for CNS inflammation.

Sphingosine kinase 1 expression enhances colon tumor growth.

BACKGROUND: Accumulating evidence suggests that sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate pathway plays a pivotal role in colon carcinogenesis. METHODS: To further support the evidence, we investigated the effects of SphK1 using three separate animal models: SphK1 knockout mice, SphK1 overexpressing transgenic mice, and SphK1 overexpression in human colon cancer xenografts. Using azoxymethane (AOM, colon carcinogen), we analyzed colon tumor development in SphK1 KO and SphK1 overexpression in intestinal epithelial cells regulated by a tet-on system. Then, we analyzed subcutaneous tumor growth using xenografts of HT-29 human colon cancer cell. Finally, immunohistochemical analyses for SphK1 and COX-2 were performed on human colon cancer tissue microarray. RESULTS: SphK1 KO mice, compared to wild-type mice, demonstrated a significant inhibition in colon cancer development induced by AOM (58.6% vs. 96.4%, respectively, P < 0.005). Tumor multiplicity (1.00 vs. 1.64 per colon, respectively, P < 0.05) and tumor volume (14.82 mm3 vs. 29.10 mm3, P < 0.05) were both significantly reduced in SphK1 KO mice compared to wild-type mice. Next, SphK1 overexpression in HT-29 enhanced tumor growth as compared to GFP control in nude mice (229.5 mm3 vs. 90.9 mm3, respectively, P < 0.05). Furthermore, overexpression of SphK1 in intestinal epithelial cells significantly enhances AOM-induced colon tumor formation (P < 0.05). Lastly, SphK1 and COX-2 intensity tended to reduce overall survival of late stage colon cancer patients. CONCLUSIONS: SphK1 expression regulates the early stage of colon carcinogenesis and tumor growth, thus inhibition of SphK1 may be an effective strategy for colon cancer chemoprevention.

Inhibiting glucosylceramide synthase exacerbates cisplatin-induced acute kidney injury.

Acute kidney injury (AKI), resulting from chemotherapeutic agents such as cisplatin, remains an obstacle in the treatment of cancer. Cisplatin-induced AKI involves apoptotic and necrotic cell death, pathways regulated by sphingolipids such as ceramide and glucosylceramide. Results from this study indicate that C57BL/6J mice treated with cisplatin had increased ceramide and hexosylceramide levels in the renal cortex 72 h following cisplatin treatment. Pretreatment of mice with inhibitors of acid sphingomyelinase and de novo ceramide synthesis (amitriptyline and myriocin, respectively) prevented accumulation of ceramides and hexosylceramide in the renal cortex and protected from cisplatin-induced AKI. To determine the role of ceramide metabolism to hexosylceramides in kidney injury, we treated mice with a potent and highly specific inhibitor of glucosylceramide synthase, the enzyme responsible for catalyzing the glycosylation of ceramides to form glucosylceramides. Inhibition of glucosylceramide synthase attenuated the accumulation of the hexosylceramides and exacerbated ceramide accumulation in the renal cortex following treatment of mice with cisplatin. Increasing ceramides and decreasing glucosylceramides in the renal cortex sensitized mice to cisplatin-induced AKI according to markers of kidney function, kidney injury, inflammation, cell stress, and apoptosis. Under conditions of high ceramide generation, data suggest that metabolism of ceramides to glucosylceramides buffers kidney ceramides and helps attenuate kidney injury.-Dupre, T. V., M. A. Doll, P. P. Shah, C. N. Sharp, D. Siow, J. Megyesi, J. Shayman, A. Bielawska, J. Bielawski, L. J. Beverly, M. Hernandez-Corbacho, C. J. Clarke, A. J. Snider, R. G. Schnellmann, L. M. Obeid, Y. A. Hannun, and L. J. Siskind. Inhibiting glucosylceramide synthase exacerbates cisplatin-induced acute kidney injury. J. Lipid Res 2017. 58: 1439-1452.

Altered Levels of Serum Ceramide, Sphingosine and Sphingomyelin Are Associated with Colorectal Cancer: A Retrospective Pilot Study.

BACKGROUND/AIM: Because patients with cancer of apparently equivalent stage often have different outcomes, it is necessary to gather additional information to complement cancer staging. Dysregulated sphingolipid metabolism contributes to carcinogenesis. In this retrospective pilot study, we tested the hypothesis that changes in serum levels of sphingolipids are associated with stage IV colorectal cancer (CRC). PATIENTS AND METHODS: We used commercially available serum samples from healthy males and patients with CRC (adenocarcinoma of the large intestine, stage IV with metastases). Blood samples were obtained immediately prior to anesthesia/surgery. We measured sphingolipid levels in sera using mass spectrometry. RESULTS: In serum of patients with CRC, the levels of C16-, C18-, C18:1-, and C24:1-ceramide, as well as those of sphingosine, were significantly higher than those of controls. In contrast, the levels of C24-sphingomyelin were significantly lower than those of controls. A global test of association showed that ceramides and sphingomyelins but not hexosylceramides were significantly associated with stage IV CRC. CONCLUSION: Sphingolipids have a potential of serving as novel, non-invasive, inexpensive, and effective blood-based biomarkers to complement CRC staging for better prognosis and more personalized medicine.

Fumonisin B1 Inhibits Endoplasmic Reticulum Stress Associated-apoptosis After FoscanPDT Combined with C6-Pyridinium Ceramide or Fenretinide.

BACKGROUND/AIM: Combining an anticancer agent fenretinide (HPR) or C6-pyridinium ceramide (LCL29) with Foscan-mediated photodynamic therapy (FoscanPDT) is expected to augment anticancer benefits of each substance. We showed that treatment with FoscanPDT+HPR enhanced accumulation of C16-dihydroceramide, and that fumonisin B1 (FB), an inhibitor of ceramide synthase, counteracted caspase-3 activation and colony-forming ability of head and neck squamous cell carcinoma (HNSCC) cells. Because cancer cells appear to be more susceptible to increased levels of the endoplasmic reticulum (ER) stress than normal cells, herein we tested the hypothesis that FoscanPDT combined with HPR or LCL29 induces FB-sensitive ER stress-associated apoptosis that affects cell survival. MATERIALS AND METHODS: Using an HNSCC cell line, we determined: cell survival by clonogenic assay, caspase-3 activity by spectrofluorometry, the expression of the ER markers BiP and CHOP by quantitative real-time polymerase chain reaction and western immunoblotting, and sphingolipid levels by mass spectrometry. RESULTS: Similar to HPR+FoscanPDT, LCL29+FoscanPDT induced enhanced loss of clonogenicity and caspase-3 activation, that were both inhibited by FB. Our additional pharmacological evidence showed that the enhanced loss of clonogenicity after the combined treatments was singlet oxygen-, ER stress- and apoptosis-dependent. The combined treatments induced enhanced, FB-sensitive, up-regulation of BiP and CHOP, as well as enhanced accumulation of sphingolipids. CONCLUSION: Our data suggest that enhanced clonogenic cell killing after the combined treatments is dependent on oxidative- and ER-stress, apoptosis, and FB-sensitive sphingolipid production, and should help develop more effective mechanism-based therapeutic strategies.

Increased Plasma Levels of Select Deoxy-ceramide and Ceramide Species are Associated with Increased Odds of Diabetic Neuropathy in Type 1 Diabetes: A Pilot Study.

Plasma deoxy-sphingoid bases are elevated in type 2 diabetes patients and correlate with the stage of diabetic distal sensorimotor polyneuropathy; however, associations between deoxy-sphingolipids (DSL) and neuropathy in type 1 diabetes have not been examined. The primary aim of this exploratory pilot study was to assess the associations between multiple sphingolipid species including DSL and free amino acids and the presence of symptomatic neuropathy in a DCCT/EDIC type 1 diabetes subcohort. Using mass spectroscopy, plasma levels of DSL and free amino acids in DCCT/EDIC type 1 diabetes participants (n = 80), with and without symptoms of neuropathy, were investigated. Patient-determined neuropathy was based on 15-item self-administered questionnaire (Michigan Neuropathy Screening Instrument) developed to assess distal symmetrical peripheral neuropathy in diabetes. Patients who scored ≥4, or reported inability to sense their feet during walking or to distinguish hot from cold water while bathing were considered neuropathic. Plasma levels of ceramide, sphingomyelin, hexosyl- and lactosylceramide species, and amino acids were measured and analyzed relative to neuropathy status in the patient. Deoxy-C24-ceramide, C24- and C26-ceramide were higher in patients with neuropathy than those without neuropathy. Cysteine was higher in patients with neuropathy. No differences in other sphingolipids or amino acids were detected. The covariate-adjusted Odds Ratios of positive patient-reported neuropathy was associated with increased levels of deoxy-C24-, and deoxy-C24:1-ceramide; C22-, C24-, and C26-ceramide; and cysteine. Plasma deoxy-ceramide and ceramide species may have potential diagnostic and prognostic significance in diabetic neuropathy.