thermozymocidin and Neuroblastoma

The sphingolipid ceramide has been recognized as an important mediator in the apoptotic machinery, and its efficient conversion to glucosylceramide has been associated with multidrug resistance. Therefore, inhibitors of glucosylceramide synthase are explored as tools for treatment of cancer. In this study, we used D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol to sensitize Neuro-2a murine neuroblastoma cells to the microtubule-stabilizing agent paclitaxel. This treatment resulted in a synergistic inhibition of viable cell number increase, which was based on a novel mechanism: (a) After a transient mitotic arrest, cells proceeded through an aberrant cell cycle resulting in hyperploidy. Apoptosis also occurred but to a very limited extent. (b) Hyperploidy was not abrogated by blocking de novo sphingolipid biosynthesis using ISP-1, ruling out involvement of ceramide as a mediator. (c) Cyclin-dependent kinase 1 and 2 activities were synergistically decreased on treatment. In conclusion, instead of inducing apoptosis through ceramide accumulation, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol by itself affects cell cycle-related proteins in paclitaxel-arrested Neuro-2a cells resulting in aberrant cell cycle progression leading to hyperploidy.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; CDC2 Protein Kinase; Cell Cycle; Ceramides; Cyclin-Dependent Kinase 2; Down-Regulation; Drug Resistance, Neoplasm; Fatty Acids, Monounsaturated; Lipid Metabolism; Mice; Morpholines; Neuroblastoma; Paclitaxel; Polyploidy; Sphingolipids

Ethanol induces apoptosis in cultured neurons. To assess the involvement of sphingolipids and neutral lipids in the apoptotic process, ethanol-induced alterations in lipid content and metabolism were examined by using primary cultured rat cerebellar granule neurons (CGNs), human neuroblastoma SK-N-SH cells, and mouse neuroblastoma Neuro2a cells. Ethanol treatment conditions that induced apoptosis in CGNs and SK-N-SH cells but not in Neuro2a cells were used for these experiments.. Cultured neurons were treated with and without 100 mM ethanol for one to three days, and the amounts of cellular sphingolipids [ceramide, glucosylceramide (GlcCer), and sphingomyelin] and neutral lipids [cholesterol, triglyceride (TG), and cholesterol ester (ChE)] were analyzed by high-performance thin-layer chromatography, using a Coomassie brilliant blue staining method. The incorporation of [C] acetate into each lipid fraction was measured in CGNs treated with and without ethanol. Also, the effect of delipidated serum, sterols, myriocin (a serine-palmitoyltransferase inhibitor), and desipramine (an acid sphingomyelinase inhibitor) on ethanol-induced lipid changes was studied by using Neuro2a cells.. The most prominent change common to CGN, SK-N-SH, and Neuro2a cells was ethanol-induced TG accumulation. Higher incorporation of radioactivity into TG was also observed in ethanol-treated cultures when cellular lipids were metabolically labeled with [C] acetate in CGNs. In addition, ethanol elevated ceramide levels in all these neurons. However, ethanol induced decreases in GlcCer along with the reduction of cell viability in SK-N-SH cells and CGNs, whereas it increased GlcCer in Neuro2a cells that remained viable. Myriocin, which reduced ceramide levels, attenuated ethanol-induced cell death in SK-N-SH cells. Ethanol-induced accumulation of TG was sterol-independent, whereas changes in ceramide and GlcCer were affected in Neuro2a cells by the presence of sterols in the medium. Staurosporine, which induced cell death in SK-N-SH cells, increased levels of TG, ChE, and ceramides and reduced the level of GlcCer.. The results showing that ethanol induces accumulation of TG and ceramide in cultured neurons suggest that ethanol enhances lipogenesis and/or reduces fatty acid degradation in neurons, as previously observed in other cell types. Further, ethanol-induced changes in lipid metabolism, specifically those of ceramide and GlcCer, may be related to the ethanol-induced apoptotic pathway.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cells, Cultured; Ceramides; Cerebellum; Desipramine; Enzyme Inhibitors; Ethanol; Fatty Acids, Monounsaturated; Glucosylceramides; Humans; Mice; Neuroblastoma; Neurons; Rats; Rats, Sprague-Dawley; Serine C-Palmitoyltransferase; Sphingomyelin Phosphodiesterase; Triglycerides

In this study, we examined the role of endogenous ceramide in the inhibition of telomerase and induction of morphologic differentiation in response to all-trans-retinoic acid (ATRA) in the SK-N-SH and SK-N-AS human neuroblastoma cell lines. The results showed that ATRA inhibited the activity of telomerase significantly in a time- and dose-dependent manner, as determined by telomere repeat amplification protocol (TRAP). The inhibition of telomerase by ATRA was maximum (about 50-80% of untreated controls) at 5-10 microM for 4-8 days. Treatment of cells with ATRA (5 microM) also resulted in the inhibition of growth by about 30-70% after 4 and 8 days of treatment, respectively, which was measured by trypan blue exclusion method. Measurement of accumulation of endogenous ceramide by high pressure liquid chromatography coupled with mass spectroscopy (LC/MS) showed that treatment of cells with ATRA resulted in increased levels of mainly C24:0 and C24:1 ceramides at days 2, 4, and 8, respectively. Also, treatment of cells with ATRA in the presence of myriocin blocked the accumulation of ceramide significantly, and more importantly, presence of myriocin partially prevented the inhibition of telomerase. Mechanistically, inhibition of telomerase by endogenous ceramide in response to ATRA treatment involves, at least in part, down-regulation of the expression of telomerase reverse transcriptase (hTERT) mRNA, as determined by semi-quantitative RT-PCR, in these cells. In addition, the modulation of telomerase activity by ATRA correlated with the induction of morphologic differentiation, which was also blocked by myriocin, as determined by extension of neurites using phase-contrast microscopy. These results, therefore, reveal an important effect of ATRA on telomerase inhibition and induction of morphologic differentiation in human neuroblastoma cells. These data also demonstrate that endogenous ceramide is one of the upstream regulators of telomerase activity in human neuroblastoma cells in response to ATRA.

Topics: Cell Differentiation; Cell Line, Tumor; Ceramides; Dose-Response Relationship, Drug; Enzyme Activation; Fatty Acids, Monounsaturated; Humans; Neuroblastoma; Telomerase; Tretinoin