Activation of caspase-9 and -3 during H2O2-induced apoptosis of PC12 cells independent of ceramide formation

Abstract

The treatment of PCI2 cells with H₂0₂ (100-500 µM) resulted in typical apoptotic changes including fragmentation and condensation of nuclei, and DINA fragmentation observed as DNA ladder. H₂O₂-induced apoptosis was associated with activation of caspase-3 as assessed by cleavage of specific fluorogenic substrate peptide and processing of procaspase-3 and poly(ADP-ribose) polymerase. However, formation of ceramide, which often locates upstream of caspase-3, was not observed. The inhibitory peptide relatively specific for caspase-3, z-DEVD-FMK and non-selective caspase inhibitor z-VAD-FMK inhibited activation of caspase-3 and apoptotic cell death. However, the relatively specific inhibitors, Ac-YVKD for caspase-1 and Ac-IETD for caspase-8/6, did not affect the occurrence of apoptotic cell death. As an upstream activation of caspase-3, induction of cytochrome c release followed by processing of procaspase-9 was observed by Western blotting, although the formation of intracellular ceramide was not observed. On the other hand, in PCI2 cells overexpressing Bcl-2, the number of apoptotic cells was markedly decreased and activation of both caspases-9 and -3 was prevented. These results suggest that cytochrome c and caspase-9 initiate the activation of executor caspase-3 in H₂O₂-treated PCI2 cells, and that Bcl-2 inhibits H₂O₂-induced release of cytochrome c from mitochondria and then proteolytic processing of procaspase-9. [Neurol Res 2000; 22: 556-564]