海芋粗提物对人肝癌细胞的细胞毒和凋亡诱导作用

The Cytotoxic and Apoptotic Effect of Crude Extract of Alocasia Macrorrhiza on Human Hepatocellular Carcinoma Cells

目的:探讨海芋粗提物对人肝癌细胞的细胞毒和凋亡诱导作用及其作用机制。方法:用海芋粗提物处理人正常肝细胞(ID2)及肝癌细胞(SMMC-7721),用细胞增殖试验(MTT法)、克隆形成试验、EdU掺入试验研究海芋粗提物对人正常肝细胞和肝癌细胞的增殖抑制作用;吖啶橙/溴乙啶(A0/EB)染色检测细胞凋亡;流式细胞术检测海芋粗提物对细胞周期的影响;RT-PcR检测细胞周期及凋亡相关基因PPARγ、cyclin D1、Rb、P21、Bax、Bcl-2基因mRNA表达。结果:用不同浓度海芋粗提物处理后,肿瘤细胞增殖活性降低(P<0.05),呈时间-剂量-效应关系;克隆形成下降(P<0.05);EdU标记指数降低(P<0.05);AO/EB染色凋亡细胞增多(P<0.01);细胞周期分布改变,凋亡指数显著增加(P<0.01),G_0/G_1期细胞增加(P<0.05),S期和G_2/M期细胞减少(P<0.05);RT-PCR结果显示,海芋粗提物(400μg/ml)处理后,PPARγ表达增加46.0%(P<0.01)、Cyclin D1降低43.7%(P<0.01)、Rb增加283.3%(P<0.01)、Bax增加77.1%(P<0.01)、Bcl-2降低24.8%(P<0.05)。结论:海芋粗提物对肝癌细胞具有细胞毒和诱导凋亡作用,其作用机理可能与激活PPARγ,上调Rb、Bax基因表达,下调Cyclin D1、Bcl-2基因表达有关。

Objective： To investigate the cytotoxic and apoptotic effect of crude extract of alocasia macrorrhiza on human hepatocellular carcinoma cells and to elucidate its mechanism of activity. Methods： Human normal liver cells （1.02） and hepatocellular carcinoma cells （SMMC-7721） were tested in vitro for cytotoxicity, colony formation inhibition, EdU incorporation, AO/EB staining apoptotic cells, and cell cycle distribution through flow cytometry after treated with crude extract of aloeasia macrorrhiza. The expression of PPARγ, Cyclin D1, Rb, P21, Bax, Bel-2 mRNA were detected through RT-PCR. Results： After treated by crude extract of alocasia macrorrhiza, the proliferation （ IC50 = 414 μg/ml） and colony formation of cancer cells decreased （ P 〈 0. 05 ） in a dose-time- dependent manner; EdU labeling index of cancer cells decreased （P 〈 0. 05 ） ; and AO/EB staining apoptotic cells increased （P 〈 0. 01 ）. The apoptotic index increased （ P 〈 0. 01 ） with the cells in G0/G1 phase increased （P 〈 0. 05 ） and decreased in S and G2/M phase （ P 〈 0.05 ） through FCM analysis ; RT-RCR results showed that the crude extract of aloeasia macrorrhiza treatment significantly increased the mRNA expression levels of PPARγ （P 〈0. 01 ）, Rb （P 〈0. 01 ） and Bax（ P 〈0. 01 ） genes, and decreased expression levels of Cyclin D1 （P 〈 0. 01 ）, and Bcl-2 （P 〈0. 05 ） genes. Conclusion： The findings in this experimental study suggest that the crude extract of aloc＆sia maerorrhiza has potential eytotoxic and apoptotic effect on human hepatocellular carcinoma cells, its mechanism of activity might be associated with the inhibition of DNA synthesis, cell cycle （ G0/G1 ） arrest, apoptosis induction and up-regu-lation the expressions of PPARγ, Rb, and Bax genes and downregulation of the expressions of Cyclin D1 and Bcl-2 genes.