| Summary: | 博士 === 高雄醫學大學 === 醫學研究所 === 97 === Betel quid chewing is a risk factor of liver cirrhosis and hepatocellular carcinoma, especially in Taiwan. However, the pathogenic mechanism remains unclear. Arecoline is the most abundant alkaloid in betel quid and is cytotoxic, genotoxic, mutagenic and tumorigenic to various kinds of cells. In this study, we examined the effects of arecoline on cell growth, cell cycle regulators and DNA damage in clone-9 cells (rat hepatocytes). Comet assay showed that arecoline-induced DNA damage in clone-9 cells. In this study, we found that arecoline dose and time-dependently inhibited cell growth in clone-9 cells. We also found that arecoline induced G0/G1 cell cycle arrest and increased p21WAF1 protein while inhibiting cyclin D1 protein expression. Higher dose arecoline also induced apoptosis in clone-9 cells. Arecoline activated caspase-3 and caspase-7, which induced PARP cleavage. In addition, we found that arecoline increased TGF-β1 promoter transcriptional activity and TGF-β1 mRNA expression in clone-9 cells. Moreover, neutralizing anti-TGF-β1 antibody attenuated arecoline-induced growth inhibiton. In another study, arecoline-induced DNA damage in clone-9 cells is mediated by activing ATM/ATR-chk1/2 kinase and the up-regulation of the downstream p53 and p21 WAF1 protein expression. Arecoline increased phosphorylation levels of p-AktSer473 and p-mTORSer2448 at 30–60 min. Arecoline increased the p21WAF1 promoter transactivation and p21WAF1 protein expression whereas LY294002 (PI3kinase inhibitor), PFT-α (p53 inhibitor) reversed this effect. ATM shRNA attenuated arecoline-induced p-p53 and p21WAF1 protein expression and pifithrin-α attenuated arecoline- inhibited cell growth. We conclude that arecoline activates the TGF-β1、ATM/ATR-p53-p21 WAF1 and PI3K/Akt-mTOR-p53 pathways in clone-9 cells.
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