Molecular Mechanisms of Apoptosis in HepaRG Cell Line Induced by Polyphyllin VI via the Fas Death Pathway and Mitochondrial-Dependent Pathway.

Yi Liu,Chunjing Yang,Longtai You,Xin Leng,Xiaoxv Dong,Wenping Wang,Na Sai,Jian Ni,Xingbin Yin

doi:10.3390/toxins10050201

Yi Liu, Chunjing Yang + Show 7 more

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https://doi.org/10.3390/toxins10050201

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Abstract

Polyphyllin VI, which is an active saponin, is mainly isolated from traditional medicinal plant Paris polyphylla, which causes liver damage in rats. In the present study, we aimed to explore the potential cytotoxicity of polyphyllin VI on the growth of HepaRG cells and to determine the molecular mechanism. The results revealed that polyphyllin VI changed cell morphology and induced apoptosis in HepaRG cells. Flow cytometric assay displayed that polyphyllin VI promoted the generation of reactive oxygen species (ROS), depolarized the mitochondrial membrane potential (MMP), and induced S phase cell cycle arrest by decreasing the expression of cyclin A2 and CDK2, while significantly increasing the expression of p21 protein. Polyphyllin VI induced the release of cytochrome c from the mitochondria to the cytosol and activated Fas, caspase-3, -8, -9, and PARP proteins. Pretreatment with NAC and Z-VAD-FMK (ROS scavenger and caspase inhibitor, respectively) on HepaRG cells increased the percentage of viable cells, which indicated that polyphyllin VI induced cell apoptosis through mitochondrial pathway by the generation of ROS and Fas death-dependent pathway. All of the effects are in dose- and time-dependent manners. Taken together, these findings emphasize the necessity of risk assessment to polyphyllin VI and offer an insight into polyphyllin VI-induced apoptosis of HepaRG cells.

Highlights

HepaRG cells, which were separated from a human hepatocellular carcinoma, exhibited several unique characteristics [1]
To study the hepatotoxicity and its underlying mechanism of the main component-polyphyllin assays showed that polyphyllin VI effectively induced the apoptosis of HepaRG cells in a time- and VI
DAPI-staining studies further proved that polyphyllin VI impaired cell the apoptosis of HepaRG cells in a time- and dose-dependent manner

Summary

Introduction

HepaRG cells, which were separated from a human hepatocellular carcinoma, exhibited several unique characteristics [1]. The cell expresses the similar metabolic characteristics when compared to human primary and the stronger growth capacity in vitro. According to these advantages, HepaRG plays a vital role in the assessment of drug-induced hepatotoxicity as a reliable surrogate to primary human hepatocytes for inquiring the drug-metabolism with activators of hepatic receptors (CAR, AhR, and PXR) [2,3]. 22 of most of the major metabolizing cytochrome P450s were expressed high functional levels in HepaRG the cytochrome. P450s were expressed highlosing functional levels in HepaRG cells [4]. Inmetabolizing addition, HepaRG can be cryopreserved without the specific biology function of

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