ABSTRACT
Apoptosis induced by nonsteroidal anti-inflammatory drugs (NSAIDs) is involved not only in the production of NSAID-induced gastric lesions but also in the antitumor activity of these drugs. The endoplasmic reticulum (ER) stress response is a cellular mechanism that aids in protecting the ER against ER stressors and is involved in ER stressor-induced apoptosis. Here, we examine the relationship between this response and NSAID-induced apoptosis in cultured guinea-pig gastric mucosal cells. Exposure of cells to indomethacin, a commonly used NSAID, induced GRP78 as well as CHOP, a transcription factor involved in apoptosis. Three factors that positively regulate CHOP expression (ATF6, ATF4 and XBP-1) were activated and/or induced by indomethacin. NSAIDs other than indomethacin (diclofenac, ibuprofen and celecoxib) also induced CHOP. Monitoring of the transcriptional activities of ATF6 and CHOP by luciferase assay revealed that both were stimulated in the presence of indomethacin. Furthermore, indomethacin-induced apoptosis was suppressed in cultured guinea-pig gastric mucosal cells by expression of the dominant-negative form of CHOP, or in peritoneal macrophages from CHOP-deficient mice. These results suggest that ER stress response-related proteins, particularly CHOP, are involved in NSAID-induced apoptosis.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Apoptosis , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/pathology , Activating Transcription Factor 4 , Animals , Blotting, Northern , Celecoxib , Cell Line , Cell Survival , Cells, Cultured , DNA-Binding Proteins/metabolism , Diclofenac/pharmacology , Dose-Response Relationship, Drug , Endoplasmic Reticulum Chaperone BiP , Enzyme Activation , Gastric Mucosa/pathology , Genes, Reporter , Guinea Pigs , Heat-Shock Proteins/metabolism , Hydrogen Peroxide/pharmacology , Ibuprofen/pharmacology , Immunoblotting , Indomethacin/pharmacology , Luciferases/metabolism , Macrophages/metabolism , Microscopy, Fluorescence , Molecular Chaperones/metabolism , Plasmids/metabolism , Pyrazoles/pharmacology , RNA, Messenger/metabolism , Regulatory Factor X Transcription Factors , Sulfonamides/pharmacology , Time Factors , Transcription Factors/metabolism , Transcription, Genetic , Transfection , X-Box Binding Protein 1ABSTRACT
A major clinical problem encountered with the use of nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin is gastropathy. In this study, we examined, using guinea pig gastric mucosal cells in primary culture, how NSAIDs damage gastric mucosal cells. The short-term treatment of cells with high concentrations of indomethacin decreased cell viability in the absence of apoptotic DNA fragmentation, chromatin condensation, or caspase activation. Cells lost membrane integrity with this short-term indomethacin treatment, suggesting that indomethacin induced necrosis under these conditions. In contrast, the long-term treatment of cells with low concentrations of indomethacin decreased cell viability and was accompanied by apoptotic DNA fragmentation, chromatin condensation, and caspase activation. Pretreatment of cells with inhibitors of caspases or protein synthesis suppressed cell death caused by long-term indomethacin treatment, suggesting that apoptosis was induced when the inhibitors were not present. These results imply that NSAIDs cause gastric mucosal damage through both necrosis and apoptosis of gastric mucosal cells.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Apoptosis/physiology , Gastric Mucosa/drug effects , Indomethacin/pharmacology , Necrosis , Amino Acid Chloromethyl Ketones/pharmacology , Animals , Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Apoptosis/drug effects , Aspirin/pharmacology , Caspase Inhibitors , Caspases/metabolism , Cell Survival/drug effects , Cells, Cultured , Cycloheximide/pharmacology , Cysteine Proteinase Inhibitors/metabolism , DNA Fragmentation , Gastric Mucosa/cytology , Gastric Mucosa/pathology , Glucosamine/metabolism , Guinea Pigs , Male , Microscopy, Fluorescence , Protein Synthesis Inhibitors/pharmacology , Time FactorsABSTRACT
Gastric mucosal cell death due to various gastric stressors can cause several types of gastric diseases, such as gastric ulcers. In this study, we examined cell death following the short-term treatment of guinea pig gastric mucosal cells in primary culture with various gastric stressors. The short-term treatment of cells with ethanol, hydrogen peroxide or hydrochloric acid caused, in a dose-dependent manner, cell death in the absence of apoptotic DNA fragmentation and chromatin condensation. Cells lost membrane integrity following the treatment with each of these gastric stressors, suggesting that necrosis was induced in gastric mucosal cells by short-term treatment of the cells with gastric stressors. Geranylgeranylacetone, an anti-ulcer drug with heat-shock protein inducing properties, protected gastric mucosal cells from the necrotic cell death caused by each of these gastric stressors. Pretreatment of cells with low concentrations of ethanol (3%), which also induced heat-shock protein, made cells resistant to the necrotic cell death caused by the gastric stressors. These results suggest that heat-shock proteins is involved in the cytoprotective effect of geranylgeranylacetone against necrotic cell death.
Subject(s)
Anti-Ulcer Agents/pharmacology , Diterpenes/pharmacology , Gastric Mucosa/pathology , Heat-Shock Proteins/antagonists & inhibitors , Heat-Shock Proteins/toxicity , Animals , Anti-Inflammatory Agents, Non-Steroidal/toxicity , Cell Death/drug effects , Cell Membrane/pathology , Cell Nucleus/drug effects , Cell Nucleus/pathology , Cell Survival/drug effects , Cells, Cultured , Chromatin/drug effects , Chromatin/pathology , DNA Fragmentation/drug effects , Gastric Mucosa/drug effects , Guinea Pigs , HSP72 Heat-Shock Proteins , Heat-Shock Proteins/metabolism , Immunoblotting , Male , NecrosisABSTRACT
One of the major side effects of nonsteroidal antiinflammatory drugs, such as indomethacin, is gastropathy. The purpose of this study was to search for a therapeutic drug to prevent this side effect in vitro. We found that geranylgeranylacetone, a unique antiulcer drug with a heat-shock protein-inducing ability, protected cultured guinea pig gastric mucosal cells from cell damage caused by indomethacin. This cytoprotective effect of geranylgeranylacetone required concentrations of more than 10(-6) M and incubation periods of longer than 2 hr. Pretreatment of cells with an inhibitor of protein synthesis completely abolished the cytoprotective effect of geranylgeranylacetone, suggesting that some proteins induced by the drug are responsible for the cytoprotection. Since pretreatment of cells with low concentrations of ethanol, which also induced the heat-shock proteins, made cells resistant to indomethacin, heat-shock proteins are candidates for the proteins that are involved in the cytoprotective effect of geranylgeranylacetone against indomethacin.
