基于Wnt通路虎杖苷对乳腺癌细胞放疗增敏的作用机制研究

Study on mechanism of polydatin for radiosensitization of breast cancer cells based on Wnt pathway

目的探讨虎杖苷(polydatin,PD)对乳腺癌细胞放疗增敏的影响及可能作用机制。方法MTT法检测不同浓度PD(5、10、20、40和80μmol/L)对人乳腺癌MCF-7细胞增殖的影响,克隆形成实验检测细胞放疗敏感性。将MCF-7细胞随机分为对照组(常规培养)、放疗组(2 Gy放射线)、LiCl组(2 Gy放射线+200μmol/L LiCl)、PD组(2 Gy放射线+20μmol/L PD)和PD+LiCl组(2 Gy放射线+20μmol/L PD+200μmol/L LiCl)。流式细胞术检测细胞凋亡率及细胞周期变化,Western blot法检测细胞中β-catenin、细胞周期素D1(cyclinD1)、B细胞淋巴瘤-2(B cell lymphoma-2,Bcl-2)和Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)的表达水平。结果不同浓度PD培养24、48和72 h对MCF-7细胞增殖均有抑制作用(均P<0.05)。20μmol/L的PD作用48 h可降低放疗(2、4、6和8 Gy)后细胞克隆形成率(plating efficiency,PE;均P<0.05)。与放疗组比较,PD组细胞凋亡率、G_(2)/M期细胞比例和Bax蛋白表达均升高,G_(0)/G_(1)期细胞比例与β-catenin、cyclinD1和Bcl-2蛋白表达均降低(均P<0.05);而PD+LiCl组可逆转PD改善的上述指标(均P<0.05)。结论PD可将MCF-7细胞阻滞在G_(2)/M期,促进细胞凋亡,提高放疗敏感性,可能与抑制Wnt/β-catenin信号通路有关。

Objective To investigate the effect of polydatin(PD)on the radiosensitization of breast cancer cells and its possible mechanism.Methods MTT method was used to detect the effect of different concentrations of PD(5,10,20,40 and 80μmol/L)on the proliferation of human breast cancer MCF-7 cells.The clone formation experiment was used to detect the sensitivity of the cells to radiotherapy.MCF-7 cells were randomly divided into the control group(conventional culture),radiotherapy group(2 Gy radiation),LiCl group(2 Gy radiation+200μmol/L LiCl),PD group(2 Gy radiation+20μmol/L PD),and PD+LiCl group(2 Gy radiation+20μmol/L PD+200μmol/L LiCl).Flow cytometry was used to detect the apoptosis rate and cell cycle changes.Western blot was used to detectβ-catenin,cyclin D1,B cell lymphoma-2(Bcl-2),and Bcl-2 associated X(Bax)protein expression in the cells.Results The proliferation of MCF-7 cells was inhibited by PD at all concentrations for 24,48 and 72 h(all P<0.05).Treatment with 20μmol/L PD for 48 h decreased the cell plating efficiency(PE)after radiotherapy(2,4,6 and 8 Gy;all P<0.05).Compared with the radiotherapy group,the apoptosis rate,G_(2)/M phase cell ratio and Bax protein expression were all increased,while the G_(0)/G_(1) phase cell ratio,β-catenin,cyclin D1,and Bcl-2 protein expression were all decreased in the PD group(all P<0.05).The above indicators of PD improvement were all reversed in the PD+LiCl group(all P<0.05).Conclusions PD can block MCF-7 cells in G_(2)/M phase,promote cell apoptosis,and improve radiosensitivity,which may be related to the inhibition of Wnt/β-catenin signaling pathway.