Ascorbic acid protects from activation of multiple caspases and of cytochrome c release induced by oxidative stress in human leukemia cells

We previously observed inhibition by ascorbic acid (AA) of apoptosis induced by oxidative stress in myeloid leukemia cells. In the present study, three complementary techniques were utilized to follow caspase activation during exposure of HL-60 cells to H2O2, and the effect of AA loading on caspase activation. Protease activity that cleaved DEVD-AMC increased in cells exposed to H2O2, the increase was time and dose dependent. Fluorescence studies and immunoblotting revealed a dose dependent protective effect of AA against procaspase-3 cleavage. Double staining with polyclonal antibody (Ab) (which recognizes the p18 subunit of cleaved caspase-3) and Hoechst 33258 dye has shown that a high percentage of cells exposed to H2O2 stained positively with the Ab and showed nuclear fragmentation. Upon loading with AA a protective effect was observed. Exposure of HL-60 cells to H2O2 resulted also in the processing of procaspase -8 and -9 (immunoblot analysis). AA had a dose dependent protective effect against procaspase-8 and -9 processing induced by H2O2. We measured cytochrome c release to the cytosol and the protective effect of AA upon exposure of the cells to H2O2, by two complementary techniques. Immunoblotting and immunostaining studies revealed that cytochrome c release was detectable already at 2h following exposure of the cells to H2O2 and that AA loading had a protective effect. Our data demonstrate that AA can inhibit activation of multiple caspases and the release of cytochrome c in myeloid leukemia cells, induced by oxidative stress.