Calpain-mediated pathway dominates cisplatin-induced apoptosis in human lung adenocarcinoma cells as determined by real-time single cell analysis

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. (C) 2008 Wiley-Liss, Inc.