title: Aspirin-induced mucosal cell death in human gastric cells: role of a caspase-independent mechanism.
abstract: Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used for the treatment of pain and inflammation. Their use may result in gastroduodenal side effects, such as gastric irritation and ulcer formation. Although various strategies have been employed to minimize these adverse effects induced by NSAIDs, effective therapeutic targeting of this problem has been prevented by an incomplete understanding of the mechanisms underlying their pathogenesis. This study was undertaken to determine the role that non-caspase-mediated apoptosis plays in inducing cellular injury and death in gastric mucosa exposed to aspirin. We proposed that the responsible mechanism was through mitochondrial failure, increased mitochondrial membrane permeability, and translocation of the intramitochondrial protein apoptosis-inducing factor (AIF). Human gastric adenocarcinoma mucosal cells (AGS cells) received no pretreatment or were preincubated with caspase inhibitors for 30 min. Cells were then treated with 40 mM aspirin for 2-4 h. Apoptosis was assessed by measuring the DNA-histone complex formation. Cell viability was determined by an acridine orange-ethidium bromide (EtBr) assay. The activation of AIF was evaluated by both Western blotting of the cytosol and mitochondrial extracts as well as by visualization and staining using fluorescence microscopy. Results showed that caspase inhibitor preincubation decreased DNA-histone complex formation when compared to aspirin treatment alone. Based on light microscope visualization, however, we determined that caspase inhibitor preincubation was unable to prevent AGS cell damage and death. These findings were confirmed by the acridine orange-EtBr test, which showed decreased cell viability with caspase inhibitor preincubation and aspirin treatment. We then tested whether non-caspase-mediated cell death occurred through an AIF mitochondrial pathway using Western blotting and fluorescence microscopy to determine AIF activation. The results showed that untreated cells had AIF localized to the mitochondria and cytosol. With 40 mM ASA at 4 h, translocation of AIF from the mitochondria to the nucleus occurred, showing activation. Caspase inhibition with z-VAD was unable to prevent AIF localization to the nucleus and subsequently unable to prevent cell death. Our results indicate that ASA in the presence of caspase inhibitors causes gastric mucosal cell death through a caspase-independent pathway suggestive of apoptosis-like programmed cell death. Effective therapeutic targeting of aspirin-induced apoptosis likely requires inhibition of both mitochondrial and caspase-mediated pathways.
authors.affiliation: Department of Surgery, Medical College of Virginia Campus of Virginia Commonwealth University, P.O. Box 980645, Richmond, Virginia 23298-0568, USA.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Leung
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Redlak
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Miller
mesh_terms: D000230:Adenocarcinoma / Q000473:pathology; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000009:adverse effects / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D001241:Aspirin / Q000009:adverse effects / Q000494:pharmacology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000502:physiology*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003600:Cytosol / Q000378:metabolism; D053938:DNA Fragmentation; D005753:Gastric Mucosa / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013274:Stomach Neoplasms / Q000473:pathology
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D051033:Apoptosis Inducing Factor; D061945:Caspase Inhibitors; D020169:Caspases; D001241:Aspirin