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.
Liu
Lei
L
MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, South China Normal University, Guangzhou 510631, China.
Xing
Da
D
Chen
Wei R
WR
Chen
Tongsheng
T
Pei
Yihui
Y
Gao
Xuejuan
X
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002154:Calpain / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology*; D045304:Cytochromes c / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D000970:Antineoplastic Agents; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D034741:RNA, Small Interfering; D045304:Cytochromes c; D002154:Calpain; D020169:Caspases; D002945:Cisplatin