Cisplatin-induced apoptosis in melanoma cells: role of caspase-3 and caspase-7 in Apaf-1 proteolytic cleavage and in execution of the degradative phases.
Apoptosis protease-activating factor-1 (Apaf-1), which plays a central role in the formation of the apoptosome, is absent or poorly expressed (because of a transcriptional silencing by methylation) in a substantial percentage of metastatic melanomas and melanoma cell lines, which are unable to activate caspase-9 and execute the mitochondrial pathway of apoptosis. We studied cisplatin-induced apoptosis of the Apaf-1-positive human metastatic Me665/2/21 melanoma cells. Our results indicate that caspase-7 is already processed in still-adhering cells and such activation, contrary to the common view, precedes caspase-3 processing. As expected by the cytochrome c release into the cytosol, caspase-9 is processed to active forms (p37 and p35), along with a yet-unidentified p28. Interestingly, we also demonstrate a remarkable loss of Apaf-1 protein, along with the appearance of a related immunoreactive fragment of approximate, equals 26 kDa; such proteolytic degradation proves to be a caspase-3/-7-mediated event. Our data also indicate that the inhibition afforded by ac-DEVD-CHO on several components (i.e., caspase-3/-7 and caspase-9 activities), and Apaf-1 proteolytic degradation, does not significantly abrogate either the apoptotic morphology or the cleavage of canonical targets, such as poly(ADP-ribose) polymerase (PARP) and lamin B. These results suggest that caspase-3 and caspase-7 are dispensable for the execution of apoptosis and, in our cellular model, the point of no return could be out of the mitochondrial cascade.
Del Bello
Barbara
B
Department of Pathophysiology and Experimental Medicine, University of Siena, via A. Moro, 53100 Siena, Italy.
Valentini
Marta A
MA
Comporti
Mario
M
Maellaro
Emilia
E
D017209:Apoptosis / Q000187:drug effects*; D053477:Apoptotic Protease-Activating Factor 1; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases / Q000378:metabolism*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000633:toxicity*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006801:Humans; D008545:Melanoma / Q000473:pathology; D009362:Neoplasm Metastasis; D011506:Proteins / Q000378:metabolism*
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D015853:Cysteine Proteinase Inhibitors; D011506:Proteins; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; D002945:Cisplatin