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 microM cisplatin. Apoptosis or necrosis was determined by Hoechst 33342 and propidium iodide (PI) nuclear staining. Cells treated with 10 microM 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 microM 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 microM, but not 10 microM cisplatin at 24 h. With 50 microM 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 microM 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.
Dursun
Belda
B
University of Colorado Health Sciences Center, Department of Medicine, Denver, CO 80262, USA.
He
Zhibin
Z
Somerset
Hilary
H
Oh
Dong-Jin
DJ
Faubel
Sarah
S
Edelstein
Charles L
CL
D000179:Acrylates / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D002154:Calpain / Q000037:antagonists & inhibitors / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D020170:Caspase 1 / Q000378:metabolism; D053143:Caspase 2; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured; D002945:Cisplatin; D003546:Cysteine Endopeptidases / Q000378:metabolism; D042783:Endothelial Cells / Q000473:pathology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D024101:Mitochondrial Proteins / Q000378:metabolism; D009336:Necrosis / Q000139:chemically induced / Q000503:physiopathology*; D011804:Quinolines / Q000494:pharmacology
D000179:Acrylates; D000590:Amino Acid Chloromethyl Ketones; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D061945:Caspase Inhibitors; C499605:Diablo protein, mouse; D024101:Mitochondrial Proteins; C100442:PD 150606; D011804:Quinolines; C468548:quinoline-val-asp(OMe)-CH2-OPH; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D002154:Calpain; C090208:Casp2 protein, mouse; C505409:Casp3 protein, mouse; D053143:Caspase 2; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D020170:Caspase 1; D002945:Cisplatin