Pajak Beata, Turowska Agnieszka, Orzechowski Arkadiusz, Gajkowska Barbara
Department of Cell Ultrastructure, Medical Research Center, Polish Academy of Sciences, Pawinskiego 5, 02-106, Warsaw, Poland.
Apoptosis. 2008 Apr;13(4):509-22. doi: 10.1007/s10495-008-0194-9.
Human COLO 205 colon adenocarcinoma cells are immune to extrinsic apoptosis induced by immunomodulatory cytokines. Among the antiapoptotic mechanisms responsible for the immune escape, the overexpression of the cFLIP protein seems to be critical. cFLIP appears to inhibit the TNF-alpha-induced death receptor signal. The application of the metabolic inhibitor bisindolylmaleimide IX (Bis-IX), known as a potent PKC repressor, sensitized COLO 205 cells to TNF-alpha-mediated apoptosis. The Western-blot analysis revealed that the susceptibility of human COLO 205 cells to apoptogenic stimuli resulted from time-dependent reduction in cFLIP(L) and TRADD protein levels. At the same time, the level of FADD protein was up-regulated. Additionally, the combined TNF-alpha and Bis-IX treatment caused cleavages of Bid and procaspase-9, as well as cytochrome c release. Thus, the evidence of this study indicates that Bis-IX facilitates the death receptor signal mediated by TNF-R1. Moreover, Bis-IX alone initiated intrinsic apoptosis, which could be abolished by Bcl-2 delivery. It heralds the involvement of mitochondria in caspase-8-independent intrinsic apoptosis. In turn, the treatment with bisindolylmaleimide III (Bis-III) did not assist TNF-alpha-dependent apoptosis.
人COLO 205结肠腺癌细胞对免疫调节细胞因子诱导的外源性凋亡具有抗性。在导致免疫逃逸的抗凋亡机制中，cFLIP蛋白的过表达似乎至关重要。cFLIP似乎能抑制肿瘤坏死因子-α（TNF-α）诱导的死亡受体信号。代谢抑制剂双吲哚马来酰亚胺IX（Bis-IX）作为一种有效的蛋白激酶C（PKC）抑制剂，可使COLO 205细胞对TNF-α介导的凋亡敏感。蛋白质免疫印迹分析显示，人COLO 205细胞对凋亡刺激的敏感性源于cFLIP(L)和肿瘤坏死因子受体相关死亡结构域蛋白（TRADD）水平随时间的降低。同时，Fas相关死亡结构域蛋白（FADD）的水平上调。此外，联合使用TNF-α和Bis-IX处理会导致Bid和procaspase-9的裂解以及细胞色素c的释放。因此，本研究的证据表明，Bis-IX促进了由肿瘤坏死因子受体1（TNF-R1）介导的死亡受体信号。此外，单独使用Bis-IX可引发内源性凋亡，而这种凋亡可通过导入Bcl-2来消除。这预示着线粒体参与了不依赖于半胱天冬酶-8的内源性凋亡。相反，双吲哚马来酰亚胺III（Bis-III）处理并不能促进TNF-α依赖性凋亡。
