Zhang Tao, Tan Yan, Zhao Rui, Liu Zhaoyang
Central Research Laboratory, the First Affiliated Hospital of Jilin University, Changchun, China ; School of Basic Medical Sciences, Jiamusi University, No. 148 Xuefu Street, Jiamusi City, Heilongjiang Province, China.
Contemp Oncol (Pozn). 2013;17(1):38-44. doi: 10.5114/wo.2013.33772. Epub 2013 Mar 15.
To study the mechanisms of inhibition of cell growth and induction of DNA damage in oridonin-treated MCF-7 human breast cancer cells.
The cytotoxicity of oridonin-treated MCF-7 cells was measured by MTT assay. Cell cycle phase distribution was analyzed by flow cytometry. P-ATM, P-CHK2, γH2AX and P-P53 protein expressions were detected by Western blot analysis. The expression of r-h2ax and P-ATM was also detected by immunofluorescence staining. The degree of cellular damage of oridonin-induced MCF-7 human breast cancer cells was confirmed by the comet assay analysis of DNA fragmentation.
Oridonin inhibited cell growth in a time- and dose-dependent manner. The IC50 values at 48 and 72 hours were 78.3 and 31.62 µmol/l, respectively. Oridonin induced G2/M phase arrest in MCF-7 cells. MCF-7 cells treated with oridonin showed significant DNA damage as shown by an increase in olive tail moment (OTM). The protein expression levels of P-ATM, P-CHK2, γH2AX and P-P53 were increased significantly in a dose-dependent manner.
DNA damage provokes p53-mediated G2/M cell cycle arrest in oridonin-induced MCF-7 cells through the mechanism of CHK2 activation by activated ATM protein kinase, which is induced by oridonin.
研究冬凌草甲素处理的MCF-7人乳腺癌细胞中细胞生长抑制及DNA损伤诱导的机制。
采用MTT法检测冬凌草甲素处理的MCF-7细胞的细胞毒性。通过流式细胞术分析细胞周期阶段分布。采用蛋白质免疫印迹法检测P-ATM、P-CHK2、γH2AX和P-P53蛋白表达。通过免疫荧光染色检测r-h2ax和P-ATM的表达。通过彗星试验分析DNA片段化来确认冬凌草甲素诱导的MCF-7人乳腺癌细胞的细胞损伤程度。
冬凌草甲素以时间和剂量依赖性方式抑制细胞生长。48小时和72小时的IC50值分别为78.3和31.62µmol/L。冬凌草甲素诱导MCF-7细胞发生G2/M期阻滞。如橄榄尾矩（OTM）增加所示，用冬凌草甲素处理的MCF-7细胞显示出明显的DNA损伤。P-ATM、P-CHK2、γH2AX和P-P53的蛋白表达水平以剂量依赖性方式显著增加。
DNA损伤通过冬凌草甲素诱导的活化ATM蛋白激酶激活CHK2的机制，在冬凌草甲素诱导的MCF-7细胞中引发p53介导的G2/M细胞周期阻滞。
