Opposite roles of ceramide and sphingosine-1-phosphate in autophagic-mediated death of human glioma cells

The sphingoid molecules ceramide and sphingosine-1-phosphate (S1P) can act as mediators in fundamental biological processes, with specific links to cancer pathogenesis. Despite ceramide and S1P metabolism is strictly interconnected, these molecules exert opposing functional roles on cell properties and fate. In fact, ceramide is involved in cell growth arrest and apoptosis, whereas S1P stimulates proliferation and survival. The opposing directions of ceramide- and S1P- mediated signaling gave birth to the concept of the ceramide/S1P rheostat, and the assumption that the balance between these two sphingolipids will determine the cell fate. A decrease of ceramide/S1P ratio appears to favor cellular escape from drug-induced programmed cell death, and has been implicated in drug resistance too. Some studies have shown that ceramide and S1P act as effectors also in gliomas, the most common, aggressive and treatment-refractory human brain tumors. The alkylating agent temozolomide has recently emerged as effective drug for treating these neoplasias. Notwithstanding, intrinsic or acquired chemoresistance limits its therapeutic benefits. The aim of this study was to evaluate the possible involvement of ceramide and S1P in the toxicity of, and/or resistance to, temozolomide in human glioblastoma cells. In T98G cells, microscopic analyses performed after acridine orange staining showed that temozolomide induces the accumulation of acidic vesicular organelles, thus suggesting an autophagic mechanism of cell death. This mechanism was confirmed by the increase of the microtubule-associated protein 1 light-chain 3B-II, an autophagosome marker, and by the evidence that the inhibitor of autophagy 3-methyladenine significantly reduces temozolomide cytotoxicity. Further studies demonstrated that the administration of toxic doses of temozolomide to T98G cells results in the increase of ceramide levels. Of note, short-chain, cell permeable ceramide analogues are able to mimic temozolomide toxicity, by inducing glioma cell autophagy. Taken together these results suggest that ceramide acts as an intracellular messenger of temozolomide-induced cell death. A temozolomide resistant cell line was then established from T98G cells by selection with gradually increasing drug concentration. In temozolomide-resistant cells, no significant variation in ceramide content was found, but S1P level was about two-fold higher than in sensitive cells. In addition, western blot analysis of sphingosine kinase (SK) -1 and -2, the enzymes responsible for S1P production, showed that SK-1 level is about 2-fold higher in temozolomide-resistant cells than in T98G. Furthermore, immunofluorescence studies revealed that SK-1 preferentially localizes to the cytoplasm in T98G cells, whereas a significant increase of SK-1 at the plasma membrane is characteristic of resistant cells. In parallel, no difference was observed in both expression and localization of SK-2, this enzyme being mainly localized in the cytosol of both sensitive and resistant cells. Interestingly, the exposure of resistant cells to sphingosine kinase inhibitors restored cell sensitivity to temozolomide-induced autophagy. In addition, when S1P was administrated to T98G cells, ceramide-induced autophagic death was significantly reduced. Altogether, these data indicate that ceramide is involved as intracellular mediator of temozolomide-induced autophagy, and demonstrate that the up-regulation of SK-1/S1P signaling characterizes and favors temozolomide resistance in human T98G glioma cells.