Apoptosis induced in HepG2 cells by short exposure to millimolar concentrations of ethanol involves the Fas-receptor pathway

Purpose: We previously found that ethanol-induced apoptosis is associated with an activation of caspase-3. However, the initial triggering of this process is yet unknown. Therefore, the present study was designed to determine whether the Fas-receptor pathway plays a role in the initiation by ethanol of human hepatocellular carcinoma (HepG2) cell apoptosis. Methods: HepG2 cells were incubated with or without 1 mM ethanol for 24 h. Apoptosis was assessed by DNA fragmentation and caspase-8 activity. Selective inhibitors of caspase-8 and caspase-9 were used to analyze the role of both caspases on apoptosis. Soluble human Fas ligand (Fas-L) was determined by enzyme-linked immunosorbent assay (ELISA). A fluorescent dye was used to investigate the permeability of the mitochondrial outer membrane. A recombinant Fas fusion protein was used to inhibit the activation of Fas receptors. Human anti-Fas-L antibody was employed to neutralize Fas-L released from the cells. Results: Caspase-8 activity increased significantly threefold (P < 0.005) after 12 h incubation of HepG2 cells with 1 mM ethanol whereas no change was observed in control cells. Incubation with caspase-8 inhibitor completely prevented apoptosis induced by ethanol (P < 0.001). In contrast, a caspase-9 inhibitor did not significantly reduce apoptosis. The permeability of the outer mitochondrial membrane was not altered. Neutralization of Fas-receptors by Fas fusion proteins completely attenuated ethanol-induced apoptosis in HepG2 cells treated with ethanol. Conclusions: These findings show that apoptosis induced by low concentrations of ethanol in human HepG2 cells is associated with Fas-receptor activation and subsequent caspase-8 activation. Triggering of apoptosis through Fas-receptors represents a mechanism of action different from that observed with high concentrations of ethanol. Received: 22 August 2000 / Accepted: 10 November 2000