Sawada M, Nakashima S, Banno Y, Yamakawa H, Hayashi K, Takenaka K, Nishimura Y, Sakai N, Nozawa Y
Department of Neurosurgery, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500-8705, Japan.
Cell Death Differ. 2000 Sep;7(9):761-72. doi: 10.1038/sj.cdd.4400711.
Etoposide (VP-16) a topoisomerase II inhibitor induces apoptosis of tumor cells. The present study was designed to elucidate the mechanisms of etoposide-induced apoptosis in C6 glioma cells. Etoposide induced increased formation of ceramide from sphingomyelin and release of mitochondrial cytochrome c followed by activation of caspase-9 and caspase-3, but not caspase-1. In addition, exposure of cells to etoposide resulted in decreased expression of Bcl-2 with reciprocal increase in Bax protein. z-VAD.FMK, a broad spectrum caspase inhibitor, failed to suppress the etoposide-induced ceramide formation and change of the Bax/Bcl-2 ratio, although it did inhibit etoposide-induced death of C6 cells. Reduced glutathione or N-acetylcysteine, which could reduce ceramide formation by inhibiting sphingomyelinase activity, prevented C6 cells from etoposide-induced apoptosis through blockage of caspase-3 activation and change of the Bax/Bcl-2 ratio. In contrast, the increase in ceramide level by an inhibitor of ceramide glucosyltransferase-1, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol caused elevation of the Bax/Bcl-2 ratio and potentiation of caspase-3 activation, thereby resulting in enhancement of etoposide-induced apoptosis. Furthermore, cell-permeable exogenous ceramides (C2- and C6-ceramide) induced downregulation of Bcl-2, leading to an increase in the Bax/Bcl-2 ratio and subsequent activation of caspases-9 and -3. Taken together, these results suggest that ceramide may function as a mediator of etoposide-induced apoptosis of C6 glioma cells, which induces increase in the Bax/Bcl-2 ratio followed by release of cytochrome c leading to caspases-9 and -3 activation.
依托泊苷（VP - 16）是一种拓扑异构酶II抑制剂，可诱导肿瘤细胞凋亡。本研究旨在阐明依托泊苷诱导C6胶质瘤细胞凋亡的机制。依托泊苷可诱导鞘磷脂生成神经酰胺增加，线粒体细胞色素c释放，随后激活半胱天冬酶 - 9和半胱天冬酶 - 3，但不激活半胱天冬酶 - 1。此外，细胞暴露于依托泊苷会导致Bcl - 2表达降低，同时Bax蛋白表达相应增加。广谱半胱天冬酶抑制剂z - VAD.FMK虽然能抑制依托泊苷诱导的C6细胞死亡，但未能抑制依托泊苷诱导的神经酰胺生成以及Bax/Bcl - 2比值的变化。还原型谷胱甘肽或N - 乙酰半胱氨酸可通过抑制鞘磷脂酶活性减少神经酰胺生成，通过阻断半胱天冬酶 - 3激活以及改变Bax/Bcl - 2比值，防止C6细胞发生依托泊苷诱导的凋亡。相反，神经酰胺葡糖基转移酶 - 1抑制剂D - 苏式 - 1 - 苯基 - 2 - 癸酰氨基 - 3 - 吗啉代 - 1 - 丙醇使神经酰胺水平升高，导致Bax/Bcl - 2比值升高以及半胱天冬酶 - 3激活增强，从而增强依托泊苷诱导的凋亡。此外，细胞可渗透的外源性神经酰胺（C2 - 和C6 - 神经酰胺）诱导Bcl - 2下调，导致Bax/Bcl - 2比值增加，随后激活半胱天冬酶 - 9和 - 3。综上所述，这些结果表明神经酰胺可能作为依托泊苷诱导C6胶质瘤细胞凋亡的介质，诱导Bax/Bcl - 2比值增加，随后细胞色素c释放，导致半胱天冬酶 - 9和 - 3激活。
