title: Diclofenac inhibits tumor necrosis factor-α-induced nuclear factor-κB activation causing synergistic hepatocyte apoptosis.
abstract: Drug-induced liver injury (DILI) is an important clinical problem. It involves crosstalk between drug toxicity and the immune system, but the exact mechanism at the cellular hepatocyte level is not well understood. Here we studied the mechanism of crosstalk in hepatocyte apoptosis caused by diclofenac and the proinflammatory cytokine tumor necrosis factor α (TNF-α). HepG2 cells were treated with diclofenac followed by TNF-α challenge and subsequent evaluation of necrosis and apoptosis. Diclofenac caused a mild apoptosis of HepG2 cells, which was strongly potentiated by TNF-α. A focused apoptosis machinery short interference RNA (siRNA) library screen identified that this TNF-α-mediated enhancement involved activation of caspase-3 through a caspase-8/Bid/APAF1 pathway. Diclofenac itself induced sustained activation of c-Jun N-terminal kinase (JNK) and inhibition of JNK decreased both diclofenac and diclofenac/TNF-α-induced apoptosis. Live cell imaging of GFPp65/RelA showed that diclofenac dampened the TNF-α-mediated nuclear factor kappaB (NF-κB) translocation oscillation in association with reduced NF-κB transcriptional activity. This was associated with inhibition by diclofenac of the TNF-α-induced phosphorylation of the inhibitor of NF-κB alpha (IκBα). Finally, inhibition of IκB kinase β (IKKβ) with BMS-345541 as well as stable lentiviral short hairpin RNA (shRNA)-based knockdown of p65/RelA sensitized hepatocytes towards diclofenac/TNF-α-induced cytotoxicity.
  
  
  CONCLUSIONS
  Together, our data suggest a model whereby diclofenac-mediated stress signaling suppresses TNF-α-induced survival signaling routes and sensitizes cells to apoptosis.
authors: Division of Toxicology, Leiden/Amsterdam Centre for Drug Research, Leiden University, The Netherlands.
authors: Lisa
authors: L
authors: Fredriksson
authors: Bram
authors: B
authors: Herpers
authors: Giulia
authors: G
authors: Benedetti
authors: Quraisha
authors: Q
authors: Matadin
authors: Jordi C
authors: JC
authors: Puigvert
authors: Hans
authors: H
authors: de Bont
authors: Sanja
authors: S
authors: Dragovic
authors: Nico P E
authors: NP
authors: Vermeulen
authors: Jan N M
authors: JN
authors: Commandeur
authors: Erik
authors: E
authors: Danen
authors: Marjo
authors: M
authors: de Graauw
authors: Bob
authors: B
authors: van de Water
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D053181:Caspase 8 / Q000378:metabolism; D045744:Cell Line, Tumor; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004008:Diclofenac / Q000494:pharmacology*; D004357:Drug Synergism; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D016861:Cyclooxygenase Inhibitors; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D004008:Diclofenac; D048670:MAP Kinase Kinase 4; D053181:Caspase 8