Involvement of mitochondrial pathway in NCTD-induced cytotoxicity in human hepG2 cells

Shi-Quan Liu,Jun Luo,Juan-Juan Mei,Cheng Chang,You-Qing Zhu

doi:10.1186/1756-9966-29-145

Abstract

BackgroundNorcantharidin, the demethylated analog of cantharidin derived from a traditional Chinese medicine, Mylabris, has been used in the treatment of anti-cancer effects. However, the detailed mechanisms underlying this process are generally unclear. The aim of this study was to investigate the mechanism of NCTD-induced apoptosis in HepG2 cells.MethodsThe cytotoxicity was measured by MTT assay for cellular viability and by flow cytometry. The mitochondrial membrane potential and reactive oxygen species production was evaluated by flow cytometry analysis. The role of caspase activities were assayed using caspase apoptosis detection kit . Western blot analysis was used to evaluate the level of Cyto-C, Bcl-2, Bax, Bid, caspase 3, -9, -8 and PARP expressionResultsAfter treatment with NCTD, a decrease in the viability of HepG2 cells and increase in apoptosis were observed. NCTD-induced apoptosis was accompanied by an increase in ROS production, loss of mitochondrial membrane potential and release of cytochrome c(cyto-c) from the mitochondria to the cytosol and down-regulation of anti-apoptotic protein Bcl-2 levels with concurrent up-regulation in pro-apoptotic protein Bax levels. However, another pro-apoptotic molecule, Bid, showed no change in such same treatment. NCTD-increased activity of caspase 9,caspase 3 and the subsequent cleavage caspase substrate PARP were also observed. The expression levels of pro-caspase-8 were not changed after NCTD treatment.ConclusionThese results indicate that NCTD induced cytotoxicity in HepG2 cells by apoptosis, which is mediated through ROS generation and mitochondrial pathway.

Highlights

Norcantharidin, the demethylated analog of cantharidin derived from a traditional Chinese medicine, Mylabris, has been used in the treatment of anti-cancer effects
Anticancer drugs may damage the mitochondria by increasing the permeability of the outer mitochondrial membrane, which is associated with the collapse of the mitochondrial membrane potential (Δm), because a decline in Δm can disturb intracellular ATP synthesis, generation of reactive oxygen species (ROS), altered mitochondrial redox ratio, translocation of cyto c to the cytosol, and degradation of caspase-3/PARP [9,10,11,12]
The anti-apoptotic protein Bcl-2 was decreased in a dose-dependent manner (Figure 6A). These results suggest that NCTD might induce apoptosis through Bcl-2/Bax, but not Bid, -mediated mitochondrial dysfunction pathway

Summary

Introduction

Norcantharidin, the demethylated analog of cantharidin derived from a traditional Chinese medicine, Mylabris, has been used in the treatment of anti-cancer effects. Anticancer drugs may damage the mitochondria by increasing the permeability of the outer mitochondrial membrane, which is associated with the collapse of the mitochondrial membrane potential (Δm), because a decline in Δm can disturb intracellular ATP synthesis, generation of reactive oxygen species (ROS), altered mitochondrial redox ratio, translocation of cyto c to the cytosol, and degradation of caspase-3/PARP [9,10,11,12] In this regard, we have initiated experiments aimed at characterizing the mitochondrial function of NCTD on human HepG2 cells, a rapidly proliferating and malignant cell line

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