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Cisplatin‐Induced Apoptosis in Melanoma Cells
Author(s) -
BELLO BARBARA,
VALENTINI MARTA A.,
COMPORTI MARIO,
MAELLARO EMILIA
Publication year - 2003
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1196/annals.1299.034
Subject(s) - apoptosis , poly adp ribose polymerase , caspase , cytochrome c , caspase 9 , apoptosome , microbiology and biotechnology , caspase 3 , biology , caspase 7 , caspase 8 , programmed cell death , nlrp1 , caspase 2 , cytosol , polymerase , biochemistry , enzyme
A bstract : 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 ≊ 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.