Involvement of Lysosomal Cysteine Proteases in Hydrogen Peroxide-induced Apoptosis in HL-60 Cells

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Abstract
Hydrogen peroxide is a well-known mediator of apoptosis. As a mechanism for H202-induced apoptosis, both a mitochondrial Cyt.c-dependent pathway and a lysosome-mediated pathway have been suggested. However, the relative roles of and the relation between these two pathways in H2O2-induced apoptosis remain to be discovered. In this study, to find the relative roles of the lysosomal and mitochondrial pathways, the effects of E-64-d, a cell-permeable inhibitor of lysosomal cysteine proteases, on apoptosis caused by H2O2 in HL-60 cells were investigated. It was found that the concentration of H2O2 strongly affected the inhibitory effect of E-64-d on the apoptosis in HL-60 cells: dose-dependent inhibition (up to 40%) of both DNA fragmentation and caspase-3 activation was observed when a high concentration of H2O2 (50 microM) was used to induce apoptosis, but no inhibitory effect was detected when a low concentration (10 microM) was used. Consistent with these observations, apparent lysosomal destabilization was observed only with 50 microM H2O2. The release of mitochondrial Cyt.c, in contrast, was observed at both 10 microM and 50 microM. These results indicated that the mitochondrial Cyt.c-mediated pathway predominates in the H202-induced apoptosis in HL-60 cells and the lysosomal mediated pathway is partially involved when high concentrations of H2O2 are used to induce apoptosis.