Role of mitochondria in aspirin-induced apoptosis in human gastric epithelial cells.
This study was undertaken to determine whether the Bcl-2 family proteins and Smac are regulators of aspirin-mediated apoptosis in a gastric mucosal cell line known as AGS cells. Cells were incubated with varying concentrations of acetylsalicylic acid (ASA; 2-40 mM), with or without preincubation of caspase inhibitors. Apoptosis was characterized by Hoechst staining and DNA-histone-associated complex formation. Antiapoptotic Bcl-2, proapoptotic Bax and Bid, Smac, and cytochrome-c oxidase (COX IV) were analyzed by Western blot analyses from cytosol and mitochondrial fractions. ASA downregulated Bcl-2 protein expression and induced Bax translocation into the mitochondria and cleavage of Bid. In contrast, expression of Smac was significantly decreased in mitochondrial fractions of ASA-treated cells. Bax and Bid involvement in apoptosis regulation was dependent on caspase activation, because caspase-8 inhibition suppressed Bax translocation and Bid processing. Caspase-9 inhibition prevented Smac release from mitochondria. Additionally, increased expression of the oxidative phosphorylation enzyme COX IV was observed in mitochondrial fractions exposed to ASA at concentrations >5 mM. Although caspase-8 inhibition had no effect on aspirin-induced apoptosis and DNA-histone complex formation, caspase-9 inhibition significantly decreased both of these events. We conclude that Bcl-2 protein family members and Smac regulate the apoptotic pathway in a caspase-dependent manner. Our results indicate also that mitochondrial integration and oxidative phosphorylation play a critical role in the pathogenesis of apoptosis in human gastric epithelial cells.
Dept. of Surgery, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298-0568, USA.
Maria J
MJ
Redlak
Jacinda J
JJ
Power
Thomas A
TA
Miller
D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D001241:Aspirin / Q000494:pharmacology*; D051023:BH3 Interacting Domain Death Agonist Protein; D015153:Blotting, Western; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D053181:Caspase 8; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000096:biosynthesis / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects*; D005753:Gastric Mucosa / Q000187:drug effects*; D015870:Gene Expression / Q000187:drug effects; D019254:Genes, bcl-2 / Q000187:drug effects; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D024101:Mitochondrial Proteins / Q000096:biosynthesis / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D051028:bcl-2-Associated X Protein
D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D061945:Caspase Inhibitors; C412403:DIABLO protein, human; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D003576:Electron Transport Complex IV; C505530:CASP8 protein, human; D053181:Caspase 8; D020169:Caspases; D001241:Aspirin