Apoptosis induced in HepG2 cells by short exposure to millimolar concentrations of ethanol involves the Fas-receptor pathway.
OBJECTIVE
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.
Castañeda
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F
Max-Planck-Institut für molekulare Physiologie, Abteilung Epithelphysiologie. franisco.castaneda@mpi-dortmund.mpg.de
Kinne
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D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D000431:Ethanol / Q000494:pharmacology*; D053222:Fas Ligand Protein; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects; D009994:Osmolar Concentration; D010539:Permeability / Q000187:drug effects; D013997:Time Factors; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000187:drug effects* / Q000378:metabolism*
D000970:Antineoplastic Agents; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D008562:Membrane Glycoproteins; D019014:fas Receptor; D000431:Ethanol; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases