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 a (TNF-a). HepG2 cells were treated with diclofenac followed by TNF-a challenge and subsequent evaluation of necrosis and apoptosis. Diclofenac caused a mild apoptosis of HepG2 cells, which was strongly potentiated by TNF-a. A focused apoptosis machinery short interference RNA (siRNA) library screen identified that this TNF-a-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-a-induced apoptosis. Live cell imaging of GFPp65/RelA showed that diclofenac dampened the TNF-a-mediated nuclear factor kappaB (NF-jB) translocation oscillation in association with reduced NF-jB transcriptional activity. This was associated with inhibition by diclofenac of the TNF-ainduced phosphorylation of the inhibitor of NF-jB alpha (IjBa). Finally, inhibition of IjB kinase b (IKKb) with BMS-345541 as well as stable lentiviral short hairpin RNA (shRNA)-based knockdown of p65/RelA sensitized hepatocytes towards diclofenac/TNFa-induced cytotoxicity. Conclusion: Together, our data suggest a model whereby diclofenac-mediated stress signaling suppresses TNF-a-induced survival signaling routes and sensitizes cells to apoptosis. (HEPATOLOGY 2011;53:2027-2041 A dverse drug reactions are an important cause of morbidity and mortality in humans and druginduced liver injuries (DILIs) are the leading cause of acute liver failure. 1 In addition, DILI accounts for most of the drug attritions 2 and more than 10% of the occurring liver failures happen due to idiosyncratic DILIs. 1 We propose that the crosstalk between drug reactive metabolite-mediated stress responses and cytokine-mediated pro-and antiapoptotic signaling is an important component in the pathophysiology of DILI.Abbreviations: AIF, apoptosis inducing factor; AnxV, annexin V; APAF1, apoptotic protease activating factor 1; c-FLIP, cellular FLICE-like inhibitory protein; DILI, drug-induced liver injury; GFP, green fluorescent protein; IjBa, inhibitor of NF-jB a; IKKb, IjB kinase b; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; MAPK, mitogen activated protein kinase; NF-jB, nuclear factor-jB; NSAID, nonsteroidal antiinflammatory drug; PUMA, p53 up-regulated modulator of apoptosis; ROS, reactive oxygen species; RAIDD, RIP-associated protein with a death domain, siRNA, short interfering RNA; TNFR-1, TNF receptor-1; TNF-a, tumor necrosis factor a.From the

Ado-Trastuzumab Emtansine Targets Hepatocytes Via Human Epidermal Growth Factor Receptor 2 to Induce Hepatotoxicity

“…Cytokine-mediated proapoptotic signaling is an important component in the pathophysiology of drug-induced liver injury (43,44). It has been reported that TNFa severely enhances liver damage caused by various xenobiotics (43,(45)(46)(47) and is the major cytokine to be excreted by the liver stationary macrophages (Kupffer cells) in response to hepatocyte damage (33). It is possible that T-DM1-mediated secretion of TNFa, which activates proapoptotic signaling pathway, significantly enhances the liver damage that is initially caused by DM1-mediated intracellular stress.…”

Aberrant Hepatic MicroRNA Expression in Nonalcoholic Fatty Liver Disease

“…Specifically, cells were seeded in normal growth medium at 60,000 cells/ml, with 2.0 ml/well in 6-well culture plates, and grown for 2 days. On day 3, the cells were washed with serum-free DMEM and then incubated in serumfree DMEM for an additional 24 h. Subsequently, the cells were washed and then incubated with DMEM containing 10 ng/ml TNF-α [21], 25 ng/ml IL-6 [22], or both of these compounds for 0, 4, 8 or 24 h. All of the cell experiments were performed in triplicate.…”

“…HepG2 cells were cultured for 24 h in complete medium containing FFAs and then cultured for an additional 24 h with the proinflammatory factors TNF-α (10 ng/ml) [21] and IL-6 (25 ng/ml) [22]. The cells were collected after 0, 4, 8, and 24 h to determine the expression levels of the six selected miRNAs.…”

Involvement of RNA Polymerase III in Immune Responses

“…NF-B regulates the transcription of a wide variety of target genes, including inflammation-related genes (e.g., those encoding cytokines and chemokines, Cox-2, and those encoding tumor necrosis factor alpha [TNF-␣]) (10). NF-B is activated by a variety of inflammatory stimuli, such as interleukin 1 (IL-1) and TNF-␣, and inhibited by IL-10 and nonsteroidal anti-inflammatory drugs (9,11,12). Moreover, aberrant NF-B activity is frequently involved in diseases, and the function of RelA is altered in many cancers (13).…”