Gliotoxin-mediated apoptosis of activated human hepatic stellate cells

Background: Activated hepatic stellate cells (HSCs) play a central role in liver fibrogenesis, and apoptosis of activated HSCs might be essential to clear HSCs from injured liver. Gliotoxin induces apoptosis of activated human and rat HSCs by an unknown mechanism. Aim: This study investigated the role of reactive oxygen species (ROS) and membrane permeability transition (MPT) in gliotoxin-induced apoptosis of activated human HSCs. Methods: Primary and immortalized human HSCs were analyzed using confocal microscopy for ROS with dichlorodihdrofluorescence diacetate (DCFH-DA) fluorophore and for the mitochondrial membrane potential (MMP) using tetramethylrhodamine methylester (TMRM). Results: Gliotoxin at higher concentrations (≥ 7.5 μM) markedly increased ROS formation, and ROS production was also evident at concentrations of gliotoxin causing necrotic cell death (≥ 32.5 μM). Gliotoxin rapidly (begins about 20 min at 1.5 μM and 10 rain at 7.5 μM) disrupts MMP at a concentration as low as 300 nM. MMP disruption was followed by cytochrome c release and caspase-3 activation. The MPT inhibitors, cyclosporine A (5 μM) plus trifluoperazine (12.5 μM), blocked depolarization of the mitochondrial membrane and release of cytochrome c, but did not block apoptosis in HSCs. Conclusions: Gliotoxin (0.3-7.5 μM) induces apoptosis of activated human HSCs with induction of MPT, cytochrome c release and caspase-3 activation, whereas at higher doses (>32.5 μM), it induces necrosis. However, gliotoxin also activates a mitochondrial independent pathway.