Ethanol-induced apoptosis in hepatoma cells proceeds via intracellular Ca(2+) elevation, activation of TLCK-sensitive proteases, and cytochrome c release.
Ethanol is known to induce apoptosis in hepatocytes. However, intracellular signaling events of ethanol-induced death are still only partially understood. We studied such processes in ethanol-induced apoptosis in HepG2 cells as a model system for human liver cells. We determined the incidence of apoptosis by DNA fragmentation and tested the effects of various known inhibitors. Ethanol induces apoptosis in HepG2 cells in a dose- and time-dependent manner as well as in rat primary hepatocytes. This effect was not mediated through the death receptor CD95 and the tumor necrosis factor receptors. It was efficiently inhibited by the caspase inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zVAD-fmk), the Ca(2+) chelator EGTA, and the serine protease inhibitor N-p-tosyl-l-lysine chloromethyl ketone (TLCK). Upon ethanol treatment, the intracellular calcium ion concentration was increased and cytochrome c was released from the mitochondria, and caspases were activated. EGTA and TLCK could inhibit cytochrome c release from the mitochondria. Furthermore, overexpression of Bcl-x(L) saved cells from ethanol-induced apoptosis. These data suggest that ethanol-induced apoptosis in liver cells is initiated by the intracellular Ca(2+) elevation in the cytoplasm and activation of TLCK-sensitive serine proteases. Our data provide new insight into ethanol-induced apoptosis in liver cells and may lead to therapeutic strategies to prevent liver damage.
Nakayama
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N
Tumor Immunology Program, German Cancer Research Center, Heidelberg, Germany.
Eichhorst
S T
ST
Müller
M
M
Krammer
P H
PH
D000590:Amino Acid Chloromethyl Ketones / Q000378:metabolism; D017209:Apoptosis; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002154:Calpain / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000473:pathology*; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002469:Cell Separation; D003574:Cytochrome c Group / Q000378:metabolism*; D003720:Densitometry; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D000431:Ethanol / Q000494:pharmacology*; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D006801:Humans; D007477:Ions; D008113:Liver Neoplasms / Q000473:pathology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014107:Tosyllysine Chloromethyl Ketone / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D051020:bcl-X Protein; D019014:fas Receptor / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; C494811:BCL2L1 protein, human; D002147:Calmodulin; D003574:Cytochrome c Group; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505662:Faslg protein, rat; D007477:Ions; D008562:Membrane Glycoproteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D051020:bcl-X Protein; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D019014:fas Receptor; D014107:Tosyllysine Chloromethyl Ketone; D000431:Ethanol; D004533:Egtazic Acid; D010450:Endopeptidases; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D002154:Calpain; C505410:Casp3 protein, rat; C506435:Casp9 protein, rat; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; D002118:Calcium