Caspases and calpain are independent mediators of cisplatin-induced endothelial cell necrosis

The role of caspases and calpain in cisplatin-induced endothelial cell death is unknown. Thus we investigated whether caspases and calpain are mediators of cisplatin-induced apoptosis and necrosis in endothelial cells. Cultured pancreatic microvascular endothelial (MS1) cells were exposed to 10 and 50 mu M cisplatin. Apoptosis or necrosis was determined by Hoechst 33342 and propidium iodide ( PI) nuclear staining. Cells treated with 10 mu M cisplatin had normal ATP levels, increased caspase-3-like activity, excluded PI and demonstrated morphological characteristics of apoptosis at 24 h. Cells treated with 50 mu M cisplatin had severe ATP depletion, increased caspase-3-like activity, and displayed extensive PI staining indicative of necrosis at 24 h. There was a dose-dependent increase in caspase-2-like activity and Smac/DIABLO protein. Calpain activity increased significantly with 50 mu M, but not 10 mu M cisplatin at 24 h. With 50 mu M cisplatin, ATP levels were significantly reduced starting at 18 h, caspase-2- and caspase-3-like activities were significantly increased starting at 18 h, and LDH release started at 8 h with maximum increase at 18-24 h. Calpain activity was not increased before 24 h. The increase in LDH release and the nuclear PI staining with 50 mu M cisplatin at 24 h was reduced by either the pancaspase inhibitor, Q-VD-OPH, or the calpain inhibitor, PD-150606. Calpain inhibitor had no effect on caspase-3-like activity. In conclusion, in cisplatin-treated endothelial cells, caspases, the major mediators of apoptosis, can also cause necrosis. A calpain inhibitor protects against necrosis without affecting caspase-3- like activity suggesting that calpain-mediated necrosis is independent of caspase-3.