title: Bax-mediated mitochondrial outer membrane permeabilization (MOMP), distinct from the mitochondrial permeability transition, is a key mechanism in diclofenac-induced hepatocyte injury: Multiple protective roles of cyclosporin A.
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.
authors.affiliation: Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117 597.
authors.forename: Woen Ping
authors.initials: WP
authors.lastname: Siu
authors.forename: Pamela Boon Li
authors.initials: PB
authors.lastname: Pun
authors.forename: Calivarathan
authors.initials: C
authors.lastname: Latchoumycandane
authors.forename: Urs A
authors.initials: UA
authors.lastname: Boelsterli
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000037:antagonists & inhibitors / Q000633:toxicity*; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein / Q000037:antagonists & inhibitors / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D021983:Cyclophilins / Q000037:antagonists & inhibitors / Q000235:genetics; D016572:Cyclosporine / Q000494:pharmacology*; D019610:Cytoprotection; D004008:Diclofenac / Q000037:antagonists & inhibitors / Q000633:toxicity*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects* / Q000648:ultrastructure; D006801:Humans; D048848:MAP Kinase Kinase Kinase 5 / Q000378:metabolism; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000187:drug effects* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism; D010539:Permeability / Q000187:drug effects; D034741:RNA, Small Interfering / Q000494:pharmacology; D013879:Thioredoxins / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000097322:Peptidyl-Prolyl Isomerase D
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D051023:BH3 Interacting Domain Death Agonist Protein; D002118:Calcium; D002614:Chelating Agents; D021983:Cyclophilins; D016572:Cyclosporine; D004008:Diclofenac; D004533:Egtazic Acid; D048848:MAP Kinase Kinase Kinase 5; D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; D013879:Thioredoxins; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; C494772:BAK1 protein, human; C494814:BID protein, human; C467002:TXN2 protein, human; C482858:MAP3K5 protein, human; C114456:PPID protein, human; D000097322:Peptidyl-Prolyl Isomerase D; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid