Dataset Information

Enhanced oxidative stress and increased mitochondrial mass during efavirenz-induced apoptosis in human hepatic cells.

ABSTRACT:

Background and purpose

Efavirenz (EFV) is widely used in the treatment of HIV-1 infection. Though highly efficient, there is growing concern about EFV-related side effects, the molecular basis of which remains elusive.

Experimental approach

In vitro studies were performed to address the effect of clinically relevant concentrations of EFV (10, 25 and 50 microM) on human hepatic cells.

Key results

Cellular proliferation and viability were reduced in a concentration-dependent manner. Analyses of the cell cycle and several cell death parameters (chromatin condensation, phosphatidylserine exteriorization, mitochondrial proapoptotic protein translocation and caspase activation) revealed that EFV triggered apoptosis via the intrinsic pathway. In addition, EFV directly affected mitochondrial function in a reversible manner, inducing a decrease in mitochondrial membrane potential and an increase in mitochondrial superoxide production, followed by a reduction in cellular glutathione content. The rapidity of these actions rules out any involvement of mitochondrial DNA replication, which, until now, was thought to be the main mechanism of mitochondrial toxicity of antiretroviral drugs. Importantly, we also observed an increase in mitochondrial mass, manifested as an elevated cardiolipin content and enhanced expression of mitochondrial proteins, which was not paralleled by an increase in the mtDNA/nuclear DNA copy number ratio. The toxic effect of EFV was partially reversed by antioxidant pretreatment, which suggests ROS generation is involved in this effect.

Conclusion and implications

Clinically relevant concentrations of EFV were shown to be mitotoxic in human hepatic cells in vitro, which may be pertinent to the understanding of the hepatotoxicity associated with this drug.

SUBMITTER: Apostolova N

PROVIDER: S-EPMC2958650 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Publications

Enhanced oxidative stress and increased mitochondrial mass during efavirenz-induced apoptosis in human hepatic cells.

Apostolova N N Gomez-Sucerquia L J LJ Moran A A Alvarez A A Blas-Garcia A A Esplugues J V JV

British journal of pharmacology 20100801 8

<h4>Background and purpose</h4>Efavirenz (EFV) is widely used in the treatment of HIV-1 infection. Though highly efficient, there is growing concern about EFV-related side effects, the molecular basis of which remains elusive.<h4>Experimental approach</h4>In vitro studies were performed to address the effect of clinically relevant concentrations of EFV (10, 25 and 50 microM) on human hepatic cells.<h4>Key results</h4>Cellular proliferation and viability were reduced in a concentration-dependent ...[more]

PMID: 20649602

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Project description:Background/aimsThe role of VDAC1, the most abundant mitochondrial outer membrane protein, in cell death depends on cell types and stimuli. Both silencing and upregulation of VDAC1 in various type of cancer cell lines can stimulate apoptosis. In contrast, in mouse embryonic stem (MES) cells and mouse embryonic fibroblasts (MEFs), the roles of VDAC1 knockout (VDAC1-/-) in apoptotic cell death are contradictory. The contribution and underlying mechanism of VDAC1-/- in oxidative stress-induced cell death in cardiac cells has not been established. We hypothesized that VDAC1 is an essential regulator of oxidative stress-induced cell death in H9c2 cells.MethodsWe knocked out VDAC1 in this rat cardiomyoblast cell line with CRISPR-Cas9 genome editing technique to produce VDAC1-/- H9c2 cells, and determined if VDAC1 is critical in promoting cell death via oxidative stress induced by tert-butylhydroperoxide (tBHP), an organic peroxide, or rotenone (ROT), an inhibitor of mitochondrial complex I by measuring cell viability with MTT assay, cell death with TUNEL stain and LDH release. The mitochondrial and glycolytic stress were examined by measuring O2 consumption rate (OCR) and extracellular acidification rate (ECAR) with a Seahorse XFp analyzer.ResultsWe found that under control conditions, VDAC1-/- did not affect H9c2 cell proliferation or mitochondrial respiration. However, compared to the wildtype (WT) cells, exposure to either tBHP or ROT enhanced the production of ROS, ECAR, and the proton (H+) production rate (PPR) from glycolysis, as well as promoted apoptotic cell death in VDAC1-/- H9c2 cells. VDAC1-/- H9c2 cells also exhibited markedly reduced mitochondria-bound hexokinase II (HKII) and Bax. Restoration of VDAC1 in VDAC1-/- H9c2 cells reinstated mitochondria-bound HKII and concomitantly decreased tBHP and ROT-induced ROS production and cell death. Interestingly, mitochondrial respiration remained the same after tBHP treatment in VDAC1-/- and WT H9c2 cells.ConclusionOur results suggest that VDAC1-/- in H9c2 cells enhances oxidative stress-mediated cell apoptosis that is directly linked to the reduction of mitochondria-bound HKII and concomitantly associated with enhanced ROS production, ECAR, and PPR.

Project description:In this study, cell death induced by the oxidant tert-butylhydroperoxide (tBH) was observed in U2OS cells; this phenotype was rescued by Syntaxin 17 (STX17) knockout (KO) but the mechanism is unknown. STX17 plays dual roles in autophagosome-lysosome fusion and mitochondrial fission. However, the contribution of the two functions of STX17 to apoptosis has not been extensively studied. Here, we sought to dissect the dual roles of STX17 in oxidative-stress-induced apoptosis by taking advantage of STX17 knockout cells and an autophagosome-lysosome fusion defective mutant of STX17. We generated STX17 knockout U2OS cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and the STX17 knockout cells were reconstituted with wild-type STX17 and its autophagosome-lysosome fusion defective mutant. Autophagy was assessed by autophagic flux assay, Monomer red fluorescent protein (mRFP)-GFP-LC3 assay and protease protection assay. Golgi, endoplasmic reticulum (ER)/ER-Golgi intermediate compartment (ERGIC) and mitochondrial dynamics were examined by staining the different indicator proteins. Apoptosis was evaluated by caspase cleavage assay. The general reactive oxygen species (ROS) were detected by flow cytometry. In STX17 complete knockout cells, sealed autophagosomes were efficiently formed but their fusion with lysosomes was less defective. The fusion defect was rescued by wild-type STX17 but not the autophagosome-lysosome fusion defective mutant. No obvious defects in Golgi, ERGIC or ER dynamics were observed. Mitochondria were significantly elongated, supporting a role of STX17 in mitochondria fission and the elongation caused by STX17 KO was reversed by the autophagosome-lysosome fusion defective mutant. The clearance of protein aggregation was compromised, correlating with the autophagy defect but not with mitochondrial dynamics. This study revealed a mixed role of STX17 in autophagy, mitochondrial dynamics and oxidative stress response. STX17 knockout cells were highly resistant to oxidative stress, largely due to the function of STX17 in mitochondrial fission rather than autophagy.

Dataset Information

Enhanced oxidative stress and increased mitochondrial mass during efavirenz-induced apoptosis in human hepatic cells.

Background and purpose

Experimental approach

Key results

Conclusion and implications

Publications

Enhanced oxidative stress and increased mitochondrial mass during efavirenz-induced apoptosis in human hepatic cells.

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