Caspase 3 inhibition attenuates hydrogen peroxide-induced DNA fragmentation but not cell death in neuronal PC12 cells

Exposure of neurons to H2O2 results in both necrosis and apoptosis. Caspases play a pivotal role in apoptosis, but exactly how they are involved in H2O2-mediated cell death is unknown. We examined H2O2-induced toxicity in neuronal PG12 cells and the effects of inducible overexpression of the H2O2-scavenging enzyme catalase on this process. H2O2 caused cell death in a time- and concentration-dependent manner. Cell death induced by H2O2 was found to be mediated in part through an apoptotic pathway as H2O2-treated cells exhibited cell shrinkage, nuclear condensation and marked DNA fragmentation. H2O2 also triggered activation of caspase 3. Genetic up-regulation of catalase not only significantly reduced cell death but also suppressed caspase 3 activity and DNA fragmentation. While the caspase 3 inhibitor DEVD inhibited both caspase 3 activity and DNA fragmentation induced by H2O2 it did not prevent cell death. Treatment with the general caspase inhibitor ZVAD, however, resulted in complete attenuation of H2O2-mediated cellular toxicity. These results suggest that DNA fragmentation induced by H2O2 is attributable to caspase 3 activation and that H2O2 may be critical for signaling leading to apoptosis. However, unlike inducibly increased catalase expression and general caspase inhibition both of which protect cells from cytotoxicity, caspase 3 inhibition alone did not improve cell survival suggesting that prevention of DNA fragmentation is insufficient to prevent H2O2-mediated cell death.