Liu Lei, Xing Da, Chen Wei R, Chen Tongsheng, Pei Yihui, Gao Xuejuan
MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China.
Int J Cancer. 2008 May 15;122(10):2210-22. doi: 10.1002/ijc.23378.
Cisplatin is an efficient anticancer agent. Cisplatin-based chemotherapy is believed to involve different signal transduction pathways, among which calpain activation has been proposed as an important factor in the induced apoptosis. In our study, based on real-time single cell analysis, we investigated the molecular involvement of calpain in cisplatin-induced apoptosis in living human lung adenocarcinoma cells. After cisplatin treatment, calpain was activated, resulting in Bid cleavage at 4-5 hr, followed by Bid translocation and cytochrome c release, leading to cell death. Calpeptin and PD150606, specific inhibitors of calpain, blocked Bid activation completely; however, cytochrome c release was delayed by more than 2 hr, which was associated with the delay of caspase-3 activation and cell death. Remarkably, calpain-mediated release of cytochrome c and cell death was significantly compromised in the Bid knockdown cells. Z-IETD-fmk and Z-VDVAD-fmk were used to block the activation of caspase-8 and caspase-2, respectively; however, the progression of apoptosis were not affected, suggesting that caspase-8 and caspase-2 were not involved in this experimental model. Taken together, the data demonstrate that calpain mediated cisplatin-induced apoptosis in human lung adenocarcinoma cells through activating Bid, which then regulated the mitochondrial apoptotic pathway. The delays of cytochrome c release, caspase-3 activation and subsequent cell death by inactivating calpain or silencing Bid exclude other earlier or parallel pathways, strongly suggesting that the calpain-mediated pathway is the kinetically earliest one, which dominates the cisplatin-induced apoptosis.
顺铂是一种有效的抗癌药物。基于顺铂的化疗被认为涉及不同的信号转导途径，其中钙蛋白酶激活被认为是诱导细胞凋亡的一个重要因素。在我们的研究中，基于实时单细胞分析，我们研究了钙蛋白酶在顺铂诱导的人肺腺癌细胞凋亡中的分子作用。顺铂处理后，钙蛋白酶被激活，导致Bid在4-5小时被切割，随后Bid易位和细胞色素c释放，导致细胞死亡。钙蛋白酶的特异性抑制剂Calpeptin和PD150606完全阻断了Bid的激活；然而，细胞色素c的释放延迟了超过2小时，这与caspase-3激活和细胞死亡的延迟有关。值得注意的是，在Bid基因敲低的细胞中，钙蛋白酶介导的细胞色素c释放和细胞死亡明显受损。分别使用Z-IETD-fmk和Z-VDVAD-fmk阻断caspase-8和caspase-2的激活；然而，细胞凋亡的进程没有受到影响，这表明caspase-8和caspase-2不参与这个实验模型。综上所述，数据表明钙蛋白酶通过激活Bid介导顺铂诱导的人肺腺癌细胞凋亡，然后调节线粒体凋亡途径。通过使钙蛋白酶失活或沉默Bid来延迟细胞色素c释放、caspase-3激活和随后的细胞死亡排除了其他更早或平行的途径，强烈表明钙蛋白酶介导的途径是动力学上最早的途径，它主导了顺铂诱导的细胞凋亡。
