Hu Yan-Wei, Liu Chun-Yu, Du Chong-Min, Zhang Jian, Wu Wen-Qian, Gu Zhen-Lun
Department of Pharmacology, Medical College of Soochow University, 199 RenAi Road, Suzhou 215123, PR China.
J Ethnopharmacol. 2009 Jun 22;123(2):293-301. doi: 10.1016/j.jep.2009.03.016. Epub 2009 Mar 24.
Flavonoids extracted from the seeds of Astragalus complanatus R.Br. reduce the proliferation of many cancer cells. The present study was carried out to evaluate the effects of these flavonoids from Astragalus complanatus (FAC) on human hepatocarcinoma cell viability and apoptosis and to investigate its mechanisms of action in SMMC-7721 cells.
Cell viability was measured using the MTT assay. To detect apoptotic cells, SMMC-7721 cells treated with FAC were stained with Hoechst 33258 and subjected to agarose gel electrophoresis. Quantitative detection of apoptotic cells was performed by flow cytometry. The effects of FAC on apoptosis and cell cycle regulatory genes and proteins in SMMC-7721 cells were examined using an S series apoptosis and cell cycle gene array and Western blot analysis.
The growth of SMMC-7721 and HepG2 cells was inhibited by treatment with FAC. Cell death induced by FAC was characterized by nuclear condensation and DNA fragmentation. Moreover, the cell cycle was arrested in the G0/G1 and S phases in FAC-treated SMMC-7721 cells. A sub-G1 peak with reduced DNA content was also formed. The activity of caspase-3 was significantly increased following FAC treatment. Microarray data indicated that the expression levels of 76 genes were changed in SMMC-7721 cells treated with FAC: 35 genes were up-regulated and 41 were down-regulated. Western blot analysis showed that caspase-3, caspase-8, Bax, P21, and P27 protein levels in SMMC-7721 cells were increased after 48 h of FAC treatment, while cyclinB1, cyclinD1, CDK1, and CDK4 protein levels were decreased.
These results suggest that FAC may play an important role in tumor growth suppression by inducing apoptosis in human hepatocarcinoma cells via mitochondria-dependent and death receptor-dependent apoptotic pathways.
从扁茎黄芪种子中提取的黄酮类化合物可减少多种癌细胞的增殖。本研究旨在评估扁茎黄芪黄酮类化合物（FAC）对人肝癌细胞活力和凋亡的影响，并探讨其在SMMC - 7721细胞中的作用机制。
采用MTT法检测细胞活力。为检测凋亡细胞，用FAC处理的SMMC - 7721细胞用Hoechst 33258染色并进行琼脂糖凝胶电泳。通过流式细胞术对凋亡细胞进行定量检测。使用S系列凋亡和细胞周期基因芯片以及蛋白质免疫印迹分析来检测FAC对SMMC - 7721细胞凋亡及细胞周期调控基因和蛋白质的影响。
FAC处理可抑制SMMC - 7721和HepG2细胞的生长。FAC诱导的细胞死亡表现为核浓缩和DNA片段化。此外，在FAC处理的SMMC - 7721细胞中，细胞周期停滞在G0/G1期和S期。还形成了DNA含量降低的亚G1峰。FAC处理后caspase - 3的活性显著增加。基因芯片数据表明，用FAC处理的SMMC - 7721细胞中有76个基因的表达水平发生了变化：35个基因上调，41个基因下调。蛋白质免疫印迹分析显示，FAC处理48小时后，SMMC - 7721细胞中caspase - 3、caspase - 8、Bax、P21和P27蛋白水平升高，而细胞周期蛋白B1、细胞周期蛋白D1、细胞周期蛋白依赖性激酶1（CDK1）和细胞周期蛋白依赖性激酶（CDK4）蛋白水平降低。
这些结果表明，FAC可能通过线粒体依赖性和死亡受体依赖性凋亡途径诱导人肝癌细胞凋亡，从而在肿瘤生长抑制中发挥重要作用。
