摘要
本文主要探讨了穿心莲内酯在体外抑制肝癌细胞生长的作用及其机制。实验中采用MTT法检测穿心莲内酯(0～50μmol·L·1)对L-02、Hep3B和HepG2细胞存活率的影响,通过软琼脂克隆形成实验检测穿心莲内酯(0～30μmol·L·1)与Hep3B和HepG2细胞孵育14d后对其克隆形成率的影响,通过流式细胞技术检测穿心莲内酯对Hep3B细胞周期的影响。同时采用蛋白印迹方法观察穿心莲内酯(50μmol·L·1)作用不同时间后对细胞周期相关蛋白如Cdc-2、磷酸化Cdc-2、Cyclin B和Cyclin D1表达的影响。MTT检测结果显示,穿心莲内酯能明显抑制肝癌细胞株(Hep3B和HepG2细胞)的生长并呈剂量依赖性关系,而对人正常肝细胞L-02无明显作用。同时,穿心莲内酯能够剂量依赖性地降低肝癌细胞株克隆形成率。流式细胞仪检测到穿心莲内酯(50μmol·L·1)作用Hep3B细胞后,G0-G1期细胞比例减少,G2-M期细胞增多,提示穿心莲内酯将细胞阻滞于G2-M期。蛋白印迹实验发现,穿心莲内酯(50μmol·L·1)作用Hep3B细胞不同时间后,细胞内周期调控相关蛋白Cdc-2、磷酸化Cdc-2、Cyclin B和Cyclin D1的表达下调。研究结果表明,穿心莲内酯特异性地剂量依赖性地抑制肝癌细胞生长,并诱导Hep3B细胞G2-M期阻滞,其机制可能与下调Cyclin D1和Cdc2/Cyclin B等周期相关蛋白有关。
The present study is aimed to investigate the toxic effects of andrographolide （Andro） on hepatoma cells and elucidate its preliminary mechanisms. After cells were treated with different concentrations of Andro （0-50μmol·L^-1） for 24 h, cell viability was evaluated with 3-（4,5-dimethylthiazol-2-yl） 2,5- diphenyltetrazolium bromide （MTT） assay. Furthermore, after hepatoma cells （Hep3B and HepG2） were treated with different concentrations of Andro （0-30 μmol·L^-1） for 14 d, the number of colony formation was accounted under microscope. Cell cycle related proteins such as Cdc-2, phosphorylated-Cdc-2, Cyclin B and Cyclin D1 were detected with Western blotting assay and the cell cycle was analyzed by flow cytometry using propidium iodide staining. MTT results showed that Andro induced growth inhibition of hepatoma ceils in a concentra- tion-dependent manner but had no significant effects on human normal liver L-02 cells. Andro dramatically decreased the colony formation of hepatoma cells in the concentration-dependent manner. Moreover, Andro induced a decrease of Hep3B cells at the G0-G1 phase and a concomitant accumulation of cells at G2-M phase. At the molecular level, Western blotting results showed that Andro decreased the expression of Cdc-2, phosphorylated-Cdc-2, Cyclin D1 and Cyclin B proteins in a time-dependent manner, which are all cell cycle related proteins. Taken together, the results demonstrated that Andro specifically inhibited the growth of hepatoma cells and cellular cell cycle related proteins were possibly involved in this process.
出处
《药学学报》
CAS
CSCD
北大核心
2009年第9期973-979,共7页
Acta Pharmaceutica Sinica
基金
supported by Shanghai Science and Technology Committee Grants (07ZR14107, 08JC1418500)
