Activation of caspase-3 by lysosomal cysteine proteases and its role in 2,2 '-azobis-(2-amidinopropane)dihydrochloride (AAPH)-induced apoptosis in HL-60 cells

We previously reported that in addition to mitochondrial cytochrome c depen dent activation, lysosomal cysteine proteases were also involved in the act ivation of caspase-3, in this study, we have separately obtained the lysoso mal and mitochondrial caspase-3 activating factors in a crude mitochondrial fraction and characterized their ability to activate pro-caspase-3 in the in vitro assay system. When a rat liver crude mitochondrial fraction contai ning lysosomes (ML) was treated with a low concentration of digitonin, lyso somal factors were selectively released without the release of a mitochondr ial factor (cytochrome c, Cyt,c), Treatment of ML with Ca2+ in the presence of inorganic phosphate (P-i), in contrast, released mitochondrial Cyt,c wi thout the release of lysosomal factors. The obtained lysosomal and mitochon drial factors activated caspase-3 in different manners; caspase-3 activatio n by lysosomal and mitochondrial factors was specifically suppressed by E-6 4, a cysteine protease inhibitor, and caspase-9 inhibitor, respectively. Th us, the activation of caspase-3 by lysosomal factors was found to be distin ct from the activation by mitochondrial Cyt.c dependent formation of the Ap af-1/caspase-9 complex. To further determine whether or not the activation of caspase-3 by lysosomal cysteine proteases is involved in cellular apopto sis, the effect of E-64-d, a cell-permeable inhibitor of cysteine protease, on 2,2'-azobis-(2-amidinopropane)dihydrochloride (AAPH)-induced apoptosis in HL-60 cells was investigated. As a result, DNA fragmentation induced by AAPH was found to be remarkably (up to 50%) reduced by pretreatment with E- 64-d, indicating the participation of lysosomal cysteine proteases in AAPH- induced apoptosis in HL-60 cells.