Chou Wen-Wen, Guh Jinn-Yuh, Tsai Jung-Fa, Hwang Chi-Ching, Chen Hung-Chun, Huang Jau-Shyang, Yang Yu-Lin, Hung Wen-Chun, Chuang Lea-Yea
Graduate Institute of Medicine, Faculty of Medicine, Kaohsiung Medical University, Taiwan.
Toxicology. 2008 Jan 14;243(1-2):1-10. doi: 10.1016/j.tox.2007.09.003. Epub 2007 Sep 8.
Betel-quid use is associated with the risk of liver cirrhosis and hepatocellular carcinoma and arecoline, the major alkaloid of betel-quid, is hepatotoxic in mice. Therefore, we studied the cytotoxic and genotoxic effects of arecoline in normal rat hepatocytes (Clone-9 cells). Arecoline dose-dependently (0.1-1mM) decreased cell cycle-dependent proliferation while inducing DNA damage at 24h. Moreover, arecoline (1mM)-induced apoptosis and necrosis at 24h. Arecoline dose-dependently (0.1-0.5mM) increased transforming growth factor-beta (TGF-beta) mRNA, gene transcription and bioactivity and neutralizing TGF-beta antibody attenuated arecoline (0.5mM)-inhibited cell proliferation at 24h. Arecoline (0.5mM) also increased p21(WAF1) protein expression and p21(WAF1) gene transcription. Moreover, arecoline (0.5mM) time-dependently (8-24h) increased p53 serine 15 phosphorylation. Pifithrin-alpha (p53 inhibitor) and the loss of the two p53-binding elements in the p21(WAF1) gene promoter attenuated arecoline-induced p21(WAF1) gene transcription at 24h. Pifithrin-alpha also attenuated arecoline (0.5mM)-inhibited cell proliferation at 24h. We concluded that arecoline induces cytotoxicity, DNA damage, G(0)/G(1) cell cycle arrest, TGF-beta1, p21(WAF1) and activates p53 in Clone-9 cells. Moreover, arecoline-induced p21(WAF1) is dependent on p53 while arecoline-inhibited growth is dependent on both TGF-beta and p53.
嚼食槟榔与肝硬化和肝细胞癌风险相关，槟榔的主要生物碱槟榔碱对小鼠具有肝毒性。因此，我们研究了槟榔碱对正常大鼠肝细胞（Clone-9细胞）的细胞毒性和遗传毒性作用。槟榔碱在0.1 - 1mM剂量范围内呈剂量依赖性降低细胞周期依赖性增殖，同时在24小时时诱导DNA损伤。此外，槟榔碱（1mM）在24小时时诱导细胞凋亡和坏死。槟榔碱在0.1 - 0.5mM剂量范围内呈剂量依赖性增加转化生长因子-β（TGF-β）mRNA、基因转录和生物活性，中和TGF-β抗体可减弱槟榔碱（0.5mM）在24小时时对细胞增殖的抑制作用。槟榔碱（0.5mM）还增加p21（WAF1）蛋白表达和p21（WAF1）基因转录。此外，槟榔碱（0.5mM）在8 - 24小时内呈时间依赖性增加p53丝氨酸15磷酸化。Pifithrin-α（p53抑制剂）以及p21（WAF1）基因启动子中两个p53结合元件的缺失可减弱槟榔碱在24小时时诱导的p21（WAF1）基因转录。Pifithrin-α也可减弱槟榔碱（0.5mM）在24小时时对细胞增殖的抑制作用。我们得出结论，槟榔碱在Clone-9细胞中诱导细胞毒性、DNA损伤、G(0)/G(1)细胞周期阻滞、TGF-β1、p21（WAF1）并激活p53。此外，槟榔碱诱导的p21（WAF1）依赖于p53，而槟榔碱抑制的生长依赖于TGF-β和p53。
