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Diclofenac impairs autophagic flux via oxidative stress and lysosomal dysfunction: Implications for hepatotoxicity.

ABSTRACT: Treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with various side effects, including cardiovascular and hepatic disorders. Studies suggest that mitochondrial damage and oxidative stress are important mediators of toxicity, yet the underlying mechanisms are poorly understood. In this study, we identified that some NSAIDs, including diclofenac, inhibit autophagic flux in hepatocytes. Further detailed studies demonstrated that diclofenac induced a reactive oxygen species (ROS)-dependent increase in lysosomal pH, attenuated cathepsin activity and blocked autophagosome-lysosome fusion. The reactivation of lysosomal function by treatment with clioquinol or transfection with the transcription factor EB restored lysosomal pH and thus autophagic flux. The production of mitochondrial ROS is critical for this process since scavenging ROS reversed lysosomal dysfunction and activated autophagic flux. The compromised lysosomal activity induced by diclofenac also inhibited the fusion with and degradation of mitochondria by mitophagy. Diclofenac-induced cell death and hepatotoxicity were effectively protected by rapamycin. Thus, we demonstrated that diclofenac induces the intracellular ROS production and lysosomal dysfunction that lead to the suppression of autophagy. Impaired autophagy fails to maintain mitochondrial integrity and aggravates the cellular ROS burden, which leads to diclofenac-induced hepatotoxicity.

SUBMITTER: Jung SH

PROVIDER: S-EPMC7575798 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Diclofenac impairs autophagic flux via oxidative stress and lysosomal dysfunction: Implications for hepatotoxicity.

Jung Seung-Hwan SH Lee Wonseok W Park Seung-Hyun SH Lee Kang-Yo KY Choi You-Jin YJ Choi Soohee S Kang Dongmin D Kim Sinri S Chang Tong-Shin TS Hong Soon-Sun SS Lee Byung-Hoon BH

Redox biology 20201012

Treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with various side effects, including cardiovascular and hepatic disorders. Studies suggest that mitochondrial damage and oxidative stress are important mediators of toxicity, yet the underlying mechanisms are poorly understood. In this study, we identified that some NSAIDs, including diclofenac, inhibit autophagic flux in hepatocytes. Further detailed studies demonstrated that diclofenac induced a reactive oxygen species ...[more]

PMID: 33080439

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Project description:Background & aimsHepatitis B surface antigen (HBsAg) drives hepatocarcinogenesis. Factors and mechanisms involved in this progression remain poorly defined, hindering the development of effective therapeutic strategies. Therefore, the mechanisms involved in the HBsAg-induced transformation of normal liver into hepatocellular carcinoma (HCC) were investigated.MethodsHemizygous Tg(Alb1HBV)44Bri/J mice were examined for HBsAg-induced carcinogenic events. Gene set-enrichment analysis identified significant signatures in HBsAg-transgenic mice that correlated with endoplasmic reticulum (ER) stress, unfolded protein response, autophagy and proliferation. These events were investigated by western blotting, immunohistochemical and immunocytochemical staining in 2-, 8- and 12-month-old HBsAg-transgenic mice. The results were verified in HBsAg-overexpressing Hepa1-6 cells and validated in human HBV-related HCC samples.ResultsIncreased BiP expression in HBsAg-transgenic mice indicated induction of the unfolded protein response. In addition, early-phase autophagy was enhanced (increased BECN1 and LC3B) and late-phase autophagy blocked (increased p62) in HBsAg-transgenic mice. Finally, HBsAg altered lysosomal acidification via ATF4- and ATF6-mediated downregulation of lysosome-associated membrane protein 2 (LAMP2) expression. In patients, HBV-related HCC and adjacent tissues showed increased BiP, p62 and downregulated LAMP2 compared to uninfected controls. In vitro, the use of ER stress inhibitors reversed the HBsAg-related suppression of LAMP2. Furthermore, HBsAg promoted hepatocellular proliferation as indicated by Ki67, cleaved caspase-3 and AFP staining in paraffin-embedded liver sections from HBsAg-transgenic mice. These results were further verified by colony formation assays in HBsAg-expressing Hepa1-6 cells. Interestingly, inhibition of ER stress in HBsAg-overexpressing Hepa1-6 cells suppressed HBsAg-mediated cell proliferation.ConclusionsThese data showed that HBsAg directly induces ER stress, impairs autophagy and promotes proliferation, thereby driving hepatocarcinogenesis. In addition, this study expanded the understanding of HBsAg-mediated intracellular events in carcinogenesis.Impact and implicationsFactors and mechanisms involved in hepatocarcinogenesis driven by hepatitis B surface antigen (HBsAg) are poorly defined, hindering the development of effective therapeutic strategies. This study showed that HBsAg-induced endoplasmic reticulum stress suppressed LAMP2, thereby mediating autophagic injury. The present data suggest that restoring LAMP2 function in chronic HBV infection may have both antiviral and anti-cancer effects. This study has provided insights into the role of HBsAg-mediated intracellular events in carcinogenesis and thereby has relevance for future drug development.

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Diclofenac impairs autophagic flux via oxidative stress and lysosomal dysfunction: Implications for hepatotoxicity.

Publications

Diclofenac impairs autophagic flux via oxidative stress and lysosomal dysfunction: Implications for hepatotoxicity.

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