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Nevirapine induced mitochondrial dysfunction in HepG2 cells.

ABSTRACT: Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor frequently used in combination with other antiretroviral agents for highly active antiretroviral therapy (HAART) of patients infected with the human immunodeficiency virus type 1 (HIV-1). However NVP can cause serious, life-threatening complications. Hepatotoxicity is one of the most severe adverse effects, particularly in HIV patients with chronic hepatitis C virus co-infection as these patients can develop liver toxicity after a relatively short course of treatment. However, the mechanism of NVP-associated hepatotoxicity remains unclear. This study sought to investigate the effect of NVP on protein expression in liver cells using a proteomic approach. HepG2 cells were treated or not treated with NVP and proteins were subsequently resolved by two-dimensional gel electrophoresis. A total of 33 differentially regulated proteins were identified, of which nearly 40% (13/33) were mitochondrial proteins. While no obvious differences were observed between NVP treated and untreated cells after staining mitochondria with mitotracker, RT-PCR expression analysis of three mitochondrially encoded genes showed all were significantly up-regulated in NVP treated cells. Mitochondrial dysfunction was observed in response to treatment even with slightly sub-optimal therapeutic treatment concentrations of NVP. This study shows that NVP induces mitochondrial dysregulation in HepG2 cells.

SUBMITTER: Paemanee A

PROVIDER: S-EPMC5569014 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Nevirapine induced mitochondrial dysfunction in HepG2 cells.

Paemanee Atchara A Sornjai Wannapa W Kittisenachai Suthathip S Sirinonthanawech Naraporn N Roytrakul Sittiruk S Wongtrakul Jeerang J Smith Duncan R DR

Scientific reports 20170823 1

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor frequently used in combination with other antiretroviral agents for highly active antiretroviral therapy (HAART) of patients infected with the human immunodeficiency virus type 1 (HIV-1). However NVP can cause serious, life-threatening complications. Hepatotoxicity is one of the most severe adverse effects, particularly in HIV patients with chronic hepatitis C virus co-infection as these patients can develop liver toxicity afte ...[more]

PMID: 28835669

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Project description:Peroxiredoxin 6 (PRDX6) has been associated with tumor progression and cancer metastasis. Its acting on phospholipid hydroperoxides and its phospholipase-A2 activity are unique among the peroxiredoxin family and add complexity to its action mechanisms. As a first step towards the study of PRDX6 involvement in cancer, we have constructed a human hepatocarcinoma HepG2PRDX6-/- cell line using the CRISPR/Cas9 technique and have characterized the cellular response to lack of PRDX6. Applying quantitative global and redox proteomics, flow cytometry, in vivo extracellular flow analysis, Western blot and electron microscopy, we have detected diminished respiratory capacity, downregulation of mitochondrial proteins and altered mitochondrial morphology. Autophagic vesicles were abundant while the unfolded protein response (UPR), HIF1A and NRF2 transcription factors were not activated, despite increased levels of p62/SQSTM1 and reactive oxygen species (ROS). Insulin receptor (INSR), 3-phosphoinositide-dependent protein kinase 1 (PDPK1), uptake of glucose and hexokinase-2 (HK2) decreased markedly while nucleotide biosynthesis, lipogenesis and synthesis of long chain polyunsaturated fatty acids (LC-PUFA) increased. 254 Cys-peptides belonging to 202 proteins underwent significant redox changes. PRDX6 knockout had an antiproliferative effect due to cell cycle arrest at G2/M transition, without signs of apoptosis. Loss of PLA2 may affect the levels of specific lipids altering lipid signaling pathways, while loss of peroxidase activity could induce redox changes at critical sensitive cysteine residues in key proteins. Oxidation of specific cysteines in Proliferating Cell Nuclear Antigen (PCNA) could interfere with entry into mitosis. The GSH/Glutaredoxin system was downregulated likely contributing to these redox changes. Altogether the data demonstrate that loss of PRDX6 slows down cell division and alters metabolism and mitochondrial function, so that cell survival depends on glycolysis to lactate for ATP production and on AMPK-independent autophagy to obtain building blocks for biosynthesis. PRDX6 is an important link in the chain of elements connecting redox homeostasis and proliferation.

Project description:Activity of the oxidative phosphorylation system (OXPHOS) is decreased in humans and mice with nonalcoholic steatohepatitis. Nitro-oxidative stress seems to be involved in its pathogenesis. The aim of this study was to determine whether fatty acids are implicated in the pathogenesis of this mitochondrial defect. In HepG2 cells, we analyzed the effect of saturated (palmitic and stearic acids) and monounsaturated (oleic acid) fatty acids on: OXPHOS activity; levels of protein expression of OXPHOS complexes and their subunits; gene expression and half-life of OXPHOS complexes; nitro-oxidative stress; and NADPH oxidase gene expression and activity. We also studied the effects of inhibiting or silencing NADPH oxidase on the palmitic-acid-induced nitro-oxidative stress and subsequent OXPHOS inhibition. Exposure of cultured HepG2 cells to saturated fatty acids resulted in a significant decrease in the OXPHOS activity. This effect was prevented in the presence of a mimic of manganese superoxide dismutase. Palmitic acid reduced the amount of both fully-assembled OXPHOS complexes and of complex subunits. This reduction was due mainly to an accelerated degradation of these subunits, which was associated with a 3-tyrosine nitration of mitochondrial proteins. Pretreatment of cells with uric acid, an antiperoxynitrite agent, prevented protein degradation induced by palmitic acid. A reduced gene expression also contributed to decrease mitochondrial DNA (mtDNA)-encoded subunits. Saturated fatty acids induced oxidative stress and caused mtDNA oxidative damage. This effect was prevented by inhibiting NADPH oxidase. These acids activated NADPH oxidase gene expression and increased NADPH oxidase activity. Silencing this oxidase abrogated totally the inhibitory effect of palmitic acid on OXPHOS complex activity. We conclude that saturated fatty acids caused nitro-oxidative stress, reduced OXPHOS complex half-life and activity, and decreased gene expression of mtDNA-encoded subunits. These effects were mediated by activation of NADPH oxidase. That is, these acids reproduced mitochondrial dysfunction found in humans and animals with nonalcoholic steatohepatitis.

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Nevirapine induced mitochondrial dysfunction in HepG2 cells.

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Nevirapine induced mitochondrial dysfunction in HepG2 cells.

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