VDAC-dependent permeabilization of the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release

Enhanced formation of reactive oxygen species (ROS), superoxide (O₂^·−), and hydrogen peroxide (H₂O₂) may result in either apoptosis or other forms of cell death. Here, we studied the mechanisms underlying activation of the apoptotic machinery by ROS. Exposure of permeabilized HepG2 cells to O₂^·− elicited rapid and massive cytochrome c release (CCR), whereas H₂O₂ failed to induce any release. Both O₂^·− and H₂O₂ promoted activation of the mitochondrial permeability transition pore by Ca²+, but Ca²+-dependent pore opening was not required for O₂^·−-induced CCR. Furthermore, O₂^·− alone evoked CCR without damage of the inner mitochondrial membrane barrier, as mitochondrial membrane potential was sustained in the presence of extramitochondrial ATP. Strikingly, pretreatment of the cells with drugs or an antibody, which block the voltage-dependent anion channel (VDAC), prevented O₂^·−-induced CCR. Furthermore, VDAC-reconstituted liposomes permeated cytochrome c after O₂^·− exposure, and this release was prevented by VDAC blocker. The proapoptotic protein, Bak, was not detected in HepG2 cells and O₂^·−-induced CCR did not depend on Bax translocation to mitochondria. O₂^·−-induced CCR was followed by caspase activation and execution of apoptosis. Thus, O₂^·− triggers apoptosis via VDAC-dependent permeabilization of the mitochondrial outer membrane without apparent contribution of proapoptotic Bcl-2 family proteins.