Role of caspases-3 and -7 in Apaf-1 proteolytic cleavage and degradation events during cisplatin-induced apoptosis in melanoma cells.
Apoptosis protease-activating factor-1 (Apaf-1), the central element in the mitochondrial pathway of apoptosis, is frequently absent or poorly expressed in metastatic melanomas, a tumor type showing a low degree of spontaneous apoptosis and a poor response to conventional therapies. In the present study, we used the Apaf-1-positive Me665/2/21 melanoma cell line to investigate the fate of Apaf-1 during cisplatin-induced apoptosis. As novel findings described for the first time in melanoma cells, we observed that Apaf-1 was markedly decreased during apoptosis, already at early stages of cell damage; concurrently, an immunoreactive N-terminal fragment of congruent with 26 kDa was evident. In spite of the remarkable decrease of Apaf-1 in apoptotic cells, caspase-9 was found to be processed and enzymatically active. Both Apaf-1 depletion and its proteolytic cleavage were markedly prevented in presence of the caspase-3/-7 inhibitor ac-DEVD-CHO. In presence of ac-DEVD-CHO, caspase-9 activity was also inhibited, along with a partially different pattern of caspase-9 processing forms. Unexpectedly, the inhibition afforded by ac-DEVD-CHO on several components, that is, caspase-3/-7 and caspase-9 activities, and Apaf-1 proteolytic degradation, did not abrogate the apoptotic morphology and cell detachment, nor the proteolytic degradation of crucial targets, such as poly(ADP-ribose) polymerase (PARP) and lamin B. Together, our results suggest that caspase-3 and -7, proved to be dispensable for the above apoptosis-associated events, play a role on Apaf-1 handling and possibly on apoptosome function.
Del Bello
Barbara
B
Department of Pathophysiology, Experimental Medicine and Public Health, University of Siena, via A. Moro, 53100 Siena, Italy.
Valentini
Marta A
MA
Mangiavacchi
Paola
P
Comporti
Mario
M
Maellaro
Emilia
E
D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D053477:Apoptotic Protease-Activating Factor 1; D053148:Caspase 3; D053179:Caspase 7; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology*; D045304:Cytochromes c / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D019008:Drug Resistance, Neoplasm / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D034921:Lamin Type B / Q000378:metabolism; D046911:Macromolecular Substances; D008545:Melanoma / Q000188:drug therapy / Q000201:enzymology*; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000187:drug effects / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D011506:Proteins / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D034921:Lamin Type B; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011506:Proteins; D045304:Cytochromes c; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D010447:Peptide Hydrolases; C505407:CASP3 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053179:Caspase 7; D020169:Caspases; D002945:Cisplatin