Role of ceramide during cisplatin-induced apoptosis in C6 glioma cells

Cisplatin is commonly used for the treatment of malignant brain tumors. How ever, the mechanisms of cell death by cisplatin are not fully understood. T herefore, the present study was designed to elucidate the apoptotic signali ng pathway(s) activated by cisplatin in a C6 rat glioma cell line. C6 cells were treated with various concentrations of cisplatin (0.2-10 mug/ml) for 24-72 h. At 10 mug/ml cisplatin, over 90% of the cells became dead at 72 h. Apoptotic death was confirmed by condensation and fragmentation of nuclei, and DNA laddering. Even in cells treated with 1.5 mug/ml cisplatin, typica l apoptotic cells were observed at 72 h. The intracellular level of ceramid e, measured Escherichia coli diacylglycerol kinase markedly increased durin g 24-72 h after the addition of 10 mug/ml cisplatin. The activity of caspas e-3(-like) proteases increased and reached a peak at 48 h. Inhibitors of ca spases reduced the number of apoptotic cells. Pretreatment of C6 cells with glutathione or N-acetyl-cysteine, which are known to block the activation of neutral magnesium-dependent sphingomyelinase, inhibited ceramide formati on, leading to suppression of both activation of caspase-3(-like) proteases and apoptosis by cisplatin. In contrast, pretreatment of the cells with N- oleoylethanolamine (OE), a ceramidase inhibitor, potentiated apoptosis indu ced by cisplatin. Furthermore, OE enhanced sensitivity of the cisplatin-res istant cells to cisplatin. These results suggest that ceramide is closely i mplicated in apoptosis of glioma cells by cisplatin through activation of c aspase-3(-like) proteases.