title: Calcium Contributes to the Cytotoxic Interaction Between Diclofenac and Cytokines.
abstract: Diclofenac (DCLF) is a widely used non-steroidal anti-inflammatory drug that is associated with idiosyncratic, drug-induced liver injury (IDILI) in humans. The mechanisms of DCLF-induced liver injury are unknown; however, patients with certain inflammatory diseases have an increased risk of developing IDILI, which raises the possibility that immune mediators play a role in the pathogenesis. DCLF synergizes with the cytokines tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN) to cause hepatocellular apoptosis in vitro by a mechanism that involves activation of the endoplasmic reticulum (ER) stress response pathway and of the mitogen-activated protein kinases, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK). DCLF also causes an increase in intracellular calcium (Ca(++)) in hepatocytes, but the role of this in the cytotoxic synergy between DCLF and cytokines is unknown. We tested the hypothesis that Ca(++) contributes to DCLF/cytokine-induced cytotoxic synergy. Treatment of HepG2 cells with DCLF led to an increase in intracellular Ca(++) at 6 and 12 h, and this response was augmented in the presence of TNF and IFN at 12 h. The intracellular Ca(++) chelator BAPTA/AM reduced cytotoxicity and caspase-3 activation caused by DCLF/cytokine cotreatment. BAPTA/AM also significantly reduced DCLF-induced activation of the ER stress sensor, protein kinase RNA-like ER kinase (PERK), as well as activation of JNK and ERK. Treatment of cells with an inositol trisphosphate receptor antagonist almost completely eliminated DCLF/cytokine-induced cytotoxicity and decreased DCLF-induced activation of PERK, JNK, and ERK. These findings indicate that Ca(++) contributes to DCLF/cytokine-induced cytotoxic synergy by promoting activation of the ER stress-response pathway and JNK and ERK.
authors.affiliation: Department of Pharmacology and Toxicology, Institute for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824 rothr@msu.edu.
authors.forename: Ashley R
authors.initials: AR
authors.lastname: Maiuri
authors.affiliation: Department of Pharmacology and Toxicology, Institute for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Anna B
authors.initials: AB
authors.lastname: Breier
authors.affiliation: Department of Pharmacology and Toxicology, Institute for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Turkus
authors.affiliation: Department of Pharmacology and Toxicology, Institute for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Patricia E
authors.initials: PE
authors.lastname: Ganey
authors.affiliation: Department of Pharmacology and Toxicology, Institute for Integrative Toxicology, Michigan State University, East Lansing, Michigan 48824 rothr@msu.edu.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Roth
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000633:toxicity*; D002118:Calcium / Q000502:physiology*; D056486:Chemical and Drug Induced Liver Injury / Q000209:etiology*; D016207:Cytokines / Q000494:pharmacology*; D004008:Diclofenac / Q000633:toxicity*; D059865:Endoplasmic Reticulum Stress; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000037:antagonists & inhibitors / Q000502:physiology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D050794:STAT1 Transcription Factor / Q000502:physiology
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D016207:Cytokines; D053496:Inositol 1,4,5-Trisphosphate Receptors; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D004008:Diclofenac; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D002118:Calcium
keywords: BAPTA/AM; ER stress; MAPK; calcium; caspase; diclofenac; idiosyncratic drug-induced liver injury; interferon-gamma; non-steroidal anti-inflammatory drugs; tumor necrosis factor