汉防己甲素增加乳腺癌细胞放射敏感性的实验研究

Study of potentiation of radiosensitivity by tetrandrine and its mechanism

目的:研究汉防己甲素(tetrandrine,Tet)是否增加人乳腺癌细胞对γ射线敏感性,并研究其作用机制及p53基因的作用。方法:采用克隆形成分析法检测Tet和γ射线对人乳腺癌细胞的作用,使用流式细胞术检测细胞在Tet及γ射线作用后各周期的分布情况,Westernblotting检测Tet及γ射线对CyclinB1和Cdc2的影响,应用分裂指数法检测细胞进入有丝分裂期的情况。结果:突变型MCF7ADR细胞在受到γ射线照射后,明显阻滞于G2期;Tet可以降低这种阻滞,显著增加γ射线的杀伤作用,其增敏比为1.51。p53野生型MCF7细胞在γ射线照射后,阻滞于G1期和G2期;加入Tet,阻滞作用降低不明显,增敏比为1.10,表明细胞受照射后诱导的周期阻滞与p53基因功能有密切关系,Tet增加γ射线的杀伤作用与p53基因功能有关;进一步研究显示,细胞在受到γ射线照射后,其CyclinB1与Cdc2蛋白表达水平明显降低,分裂指数也明显降低;在用γ射线处理后,CyclinB1与Cdc2蛋白的表达水平明显增高,分裂指数明显增高。结论:Tet是一种G2期阻滞清除剂,能显著增加γ射线对人乳腺癌细胞的杀伤作用,这种作用与细胞的p53基因功能有关。

Objective To investigate the radiosensitizing effect and the mechanism of action of tetrandrine(Tet) in human breast cancer MCF 7/ADR and MCF 7 cells. Methods The effect of Tet on the cytotoxicity of γ-ray was determined by clonogenic assay. Cell cycle arrest induced by γ irradiation was studied in the two cell lines by using flow cytometry, Western blotting was preformed to indicate the changes of Cyclin B1 and Cdc2 protein levels. Results Tet sensitized MCF 7/ADR cell line with mutant p53 to γ-ray by clonogenic assay. Flow cytometry assay showed that exposure of MCF 7/ADR cells to γ-ray caused cells arrest in G_2 phase. The SER of Tet was 1.51 in MCF 7/Adr cells. However, in MCF 7 cell line with wild p53, the SER of Tet was 1.10, the numbers of arrest G_2 phase cells were less than those in MCF 7/ADR cell line. It indicated that the sensitizing effect was lower than that in the p53 mutant cancer cells. The addition of Tet after irradiation resulted in a dose-dependent reduction of G_2 phase arrest induced by γ-ray. Furthermore, the results showed that Tet blocked decrease of Cyclin B1 and Cdc2 expression induced by γ-ray, while mitotic index measurement indicated that γ-ray-irradiated cells treated with Tet entered mitosis. Conclusion Tet is a potent G_2_ checkpoint abrogator and markedly enhances the cytotoxicity of γ irradiation in the p53 mutant cancer cells.