A comparative study of apoptosis and necrosis in HepG2 cells: Oxidant-induced caspase inactivation leads to necrosis

Apoptosis and necrosis are two distinct forms of cell death that can occur in response to various agents. In the present study the HepG2 cell line was used for a comparative study of CD95-mediated apoptosis and menadione-indu ced necrosis. Apoptosis coincided with the release of cytochrome c from mit ochondria, activation of caspases, cleavage of cellular proteins, and also involved nuclear condensation and DNA fragmentation. Necrosis was not accom panied by DNA fragmentation, caspase activation or cleavage of caspase targ et proteins, despite cytochrome c release from mitochondria. In fact, the a ddition of menadione to cells undergoing CD95-mediated apoptosis blocked th eir caspase activity. Inhibition of caspases coincided with an accumulation of reactive oxygen species (ROS) and ATP depletion. In order to determine the predominance of either of these events in the inhibition of caspase, ce lls were either co-incubated with antioxidant enzymes or their ATP level wa s manipulated to maintain it at a relatively high level during the experime nts. Go-incubation with catalase, but not Cu/Zn superoxide dismutase, subst antially reduced the levels of ROS and reversed the inhibitory effect of me nadione on caspase activity. In contrast, increasing cellular ATP level had little effect on restoring caspase activity. These data suggest that menad ione inhibits caspase activity by the generation of hydrogen peroxide throu gh redox cycling and that caspase inactivation by this mechanism may preven t cell death by apoptosis in this oxidative-stress model. (C) 1999 Academic Press.