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-induced
necrosis. Apoptosis coincided with the release of
cytochrome c from mitochondria, activation of
caspases, cleavage of cellular
proteins, and also involved nuclear condensation and DNA fragmentation.
Necrosis was not accompanied by DNA fragmentation,
caspase activation or cleavage of
caspase target
proteins, despite
cytochrome c release from mitochondria. In fact, the addition of
menadione to cells undergoing CD95-mediated apoptosis blocked their
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, cells were either co-incubated with
antioxidant enzymes or their
ATP level was manipulated to maintain it at a relatively high level during the experiments. Co-incubation with
catalase, but not
Cu/Zn superoxide dismutase, substantially reduced the levels of ROS and reversed the inhibitory effect of
menadione on
caspase activity. In contrast, increasing cellular
ATP level had little effect on restoring
caspase activity. These data suggest that
menadione inhibits
caspase activity by the generation of
hydrogen peroxide through redox cycling and that
caspase inactivation by this mechanism may prevent cell death by apoptosis in this oxidative-stress model.