Messmer U K, Pereda-Fernandez C, Manderscheid M, Pfeilschifter J
Klinikum der Johann Wolfgang Goethe-Universität, (pharmazentrum frankfurt), Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany.
Br J Pharmacol. 2001 Jun;133(4):467-76. doi: 10.1038/sj.bjp.0704093.
Exposure of human mammary carcinoma cell line MCF-7 to TNF-alpha leads to apoptotic cell death within 24 h. In search for apoptosis-preventing signals, we identified glucocorticoids as potent death-preventing compounds. Ten nM dexamethasone provided a significant protective effect whereas 100 nM dexamethasone roughly blocked 80 - 90% of TNF-alpha-induced apoptosis. Surprisingly, dexamethasone exerted a protective effect even when supplied several hours after TNF-alpha. This points to a powerful inhibition of even advanced apoptotic processes by dexamethasone. To further pinpoint the anti-apoptotic glucocorticoid action, we investigated the expression levels of several members of the inhibitors of apoptosis (IAPs) family of proteins in response to TNF-alpha and dexamethasone. IAP proteins directly block caspase protease activities including caspase-3, caspase-7, and caspase-9. Exposure of MCF-7 cells to TNF caused an extensive downregulation of cIAP1, cIAP2, and XIAP protein levels. The decline of the IAP protein levels temporally paralleled the appearance of apoptotic DNA fragments which started 12 - 14 h following TNF-alpha addition and maximal effects were seen within 24 h. Coincubation of cells with TNF-alpha and dexamethasone potently blocked cIAP1, cIAP2, and XIAP downregulation. TNF-alpha-mediated IAP protein downregulation was not affected by proteasome inhibitors like lactacystin, ALLN or ALLM, whereas it was blocked by the broad-spectrum caspase inhibitor Z-VAD-fmk which also prevented TNF-alpha-induced apoptotic cell death. These data suggest that inhibition of IAP downregulation mediated by a caspase proteolytic activity constitutes the anti-apoptotic action of glucocorticoids in MCF-7 carcinoma cells.
将人乳腺癌细胞系MCF-7暴露于肿瘤坏死因子-α（TNF-α）会导致其在24小时内发生凋亡性细胞死亡。为了寻找防止凋亡的信号，我们确定糖皮质激素是有效的防止细胞死亡的化合物。10 nM地塞米松具有显著的保护作用，而100 nM地塞米松大致可阻断80 - 90%的TNF-α诱导的凋亡。令人惊讶的是，即使在TNF-α作用数小时后再给予地塞米松，它仍能发挥保护作用。这表明地塞米松对甚至晚期凋亡过程也有强大的抑制作用。为了进一步明确糖皮质激素的抗凋亡作用，我们研究了凋亡抑制蛋白（IAPs）家族中几个成员在TNF-α和地塞米松作用下的表达水平。IAP蛋白直接阻断包括半胱天冬酶-3、半胱天冬酶-7和半胱天冬酶-9在内的半胱天冬酶蛋白酶活性。将MCF-7细胞暴露于TNF会导致细胞凋亡抑制蛋白1（cIAP1）、细胞凋亡抑制蛋白2（cIAP2）和X连锁凋亡抑制蛋白（XIAP）的蛋白水平大幅下调。IAP蛋白水平的下降在时间上与凋亡DNA片段的出现平行，凋亡DNA片段在添加TNF-α后12 - 14小时开始出现，并在24小时内达到最大效应。将细胞与TNF-α和地塞米松共同孵育可有效阻断cIAP1、cIAP2和XIAP的下调。TNF-α介导的IAP蛋白下调不受蛋白酶体抑制剂如乳胞素、N-乙酰-亮氨酰-亮氨酰-缬氨醛（ALLN）或N-乙酰-亮氨酰-亮氨酰-亮氨醛（ALLM）的影响，而被广谱半胱天冬酶抑制剂Z-VAD-fmk阻断，Z-VAD-fmk也可防止TNF-α诱导的凋亡性细胞死亡。这些数据表明，由半胱天冬酶蛋白水解活性介导的IAP下调的抑制构成了糖皮质激素在MCF-7癌细胞中的抗凋亡作用。
