Abstract
Diclofenac, a widely used nonsteroidal anti-inflammatory drug, has been associated with rare but severe cases of clinical hepatotoxicity. Diclofenac causes concentration-dependent cell death in human hepatocytes (after 24-48 h) by mitochondrial permeabilization via poorly defined mechanisms. To explore whether the cyclophilin D (CyD)-dependent mitochondrial permeability transition (mPT) and/or the mitochondrial outer membrane permeabilization (MOMP) was primarily involved in mediating cell death, we exposed immortalized human hepatocytes (HC-04) to apoptogenic concentrations of diclofenac (>500 microM) in the presence or absence of inhibitors of upstream mediators. The CyD inhibitor, cyclosporin A (CsA, 2 microM) fully inhibited diclofenac-induced cell injury, suggesting that mPT was involved. However, CyD gene silencing using siRNA left the cells susceptible to diclofenac toxicity, and CsA still protected the CyD-negative cells from lethal injury. Diclofenac induced early (9 h) activation of Bax and Bak and caused mitochondrial translocation of Bax, indicating that MOMP was involved in cell death. Inhibition of Bax protein expression by using siRNA significantly protected HC-04 from diclofenac-induced cell injury. Diclofenac also induced early Bid activation (tBid formation, 6 h), which is an upstream mechanism that initiates Bax activation and mitochondrial translocation. Bid activation was sensitive to the Ca2+ chelator, BAPTA. In conclusion, we found that Bax/Bak-mediated MOMP is a key mechanism of diclofenac-induced lethal cell injury in human hepatocytes, and that CsA can prevent MOMP through inhibition of Bax activation. These data support our concept that the Ca2+-Bid-Bax-MOMP axis is a critical pathway in diclofenac (metabolite)-induced hepatocyte injury.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Anti-Inflammatory Agents, Non-Steroidal / antagonists & inhibitors
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Anti-Inflammatory Agents, Non-Steroidal / toxicity*
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Apoptosis
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BH3 Interacting Domain Death Agonist Protein / antagonists & inhibitors
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BH3 Interacting Domain Death Agonist Protein / metabolism
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Calcium / metabolism
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Cell Line
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Chelating Agents / pharmacology
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Cyclophilins / antagonists & inhibitors
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Cyclophilins / genetics
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Cyclosporine / pharmacology*
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Cytoprotection*
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Diclofenac / antagonists & inhibitors
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Diclofenac / toxicity*
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Egtazic Acid / analogs & derivatives
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Egtazic Acid / pharmacology
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Hepatocytes / drug effects*
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Hepatocytes / ultrastructure
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Humans
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MAP Kinase Kinase Kinase 5 / metabolism
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Mitochondria, Liver / drug effects
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Mitochondria, Liver / metabolism
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Mitochondrial Membranes / drug effects*
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Mitochondrial Membranes / metabolism
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Mitochondrial Proteins / metabolism
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Permeability / drug effects
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RNA, Small Interfering / pharmacology
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Thioredoxins / metabolism
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bcl-2 Homologous Antagonist-Killer Protein / antagonists & inhibitors
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bcl-2 Homologous Antagonist-Killer Protein / genetics
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bcl-2 Homologous Antagonist-Killer Protein / metabolism
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bcl-2-Associated X Protein / antagonists & inhibitors
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bcl-2-Associated X Protein / genetics
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bcl-2-Associated X Protein / metabolism*
Substances
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Anti-Inflammatory Agents, Non-Steroidal
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BAK1 protein, human
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BH3 Interacting Domain Death Agonist Protein
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BID protein, human
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Chelating Agents
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Mitochondrial Proteins
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RNA, Small Interfering
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TXN2 protein, human
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bcl-2 Homologous Antagonist-Killer Protein
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bcl-2-Associated X Protein
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Diclofenac
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Thioredoxins
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Egtazic Acid
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Cyclosporine
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MAP Kinase Kinase Kinase 5
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MAP3K5 protein, human
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Cyclophilins
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PPID protein, human
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1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
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Calcium