桂枝茯苓丸抑制人乳腺癌细胞MCF-7增殖机制

目的:通过观察桂枝茯苓丸对人乳腺癌细胞MCF-7增殖的影响,并检测细胞周期的变化和蛋白表达的改变,从而探讨桂枝茯苓丸对乳腺癌细胞产生抑制的作用机制。方法:体外培养人乳腺癌细胞MCF-7,以5-氟尿嘧啶(5-Fu,25 mg·L~(-1))作为阳性药,噻唑蓝(MTT)比色法检测不同剂量桂枝茯苓丸培养液和在不同作用时间对细胞的抑制作用;选用1.8,2.7 g·L~(-1)的桂枝茯苓丸培养液作用48 h,流式细胞技术检测5-Fu组和桂枝茯苓丸培养液组的周期,实时荧光定量聚合酶链式反应(Real-time PCR)检测表皮生长因子(EGFR),周期素A2(Cyclin A2)及周期素依赖性激酶2(Cdk2)mRNA表达,蛋白免疫印迹法(Western blot)检测其EGFR,Cyclin A2,Cdk2蛋白表达。结果:桂枝茯苓丸对人乳腺癌细胞MCF-7细胞有抑制作用(P0.05),且具有时间-剂量依赖性。与空白组比较,1.8,2.7 g·L~(-1)桂枝茯苓丸作用MCF-7细胞48 h后S期细胞明显增加(P0.05),EGFR,Cdk2,Cyclin A2mRNA表达明显降低(P0.01);桂枝茯苓丸各质量浓度组中EGFR,Cdk2蛋白表达均下降(P0.05),但Cyclin A2蛋白表达仅2.7 g·L~(-1)组有明显降低(P0.05)。结论:桂枝茯苓丸在S期阻滞MCF-7细胞增殖,这可能与下调Cyclin A2,Cdk2,EGFR mRNA和其蛋白的表达有关。

Mechanism of Guizhi Fuling Wan in Inhibiting Proliferation of Human Breast Cancer MCF-7 Cells

Objective: To investigate the mechanism of Guizhi Fuling Wan in inhibiting the human breast cancer MCF-7 cell by observing its effect on MCF-7 cells and detecting the changes of cell cycle and protein expressions. Method:The human breast cancer MCF-7 cells were cultured in vitro, and 5-Fluorouracil (5-Fu, 25 mg·L^-1) was used as a positive control medicine to detect the effect of Guizhi Fuling Wan culture on inhibiting the proliferation of the cells under different duration and different doses with the method of 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT). Guizhi Fuling Wan cultures (1.8, 2.7 g·L^-1) were used for treatment for 48 h. Then the cell cycles was measured by flow cytometry (FCM) in 5-fu group and Guizhi Fuling Wan culture groups; mRNA expression levels of epidermal growth factor receptor (EGFR), CyclinA₂ and cyclin-dependent kinase (Cdk2) were detected by Real-time quantitative polymerase chain reaction (Real-time PCR), and their protein expression levels were detected by Western blot. Result: Guizhi Fuling Wan can inhibit the proliferation of the human breast cancer MCF-7 cell in a time-dose dependent manner (P<0.05). As compared with blank control group, the number of S phase cells was increased significantly after treatment by 1.8, 2.7 g·L^-1 Guizhi Fuling Wan for 48 h (P<0.05); the mRNA expression levels of EGFR, Cdk2 and CyclinA₂ were significantly reduced (P<0.01); and the protein expression levels of EGFR and Cdk2 were also decreased (P<0.05); however, the expression of CyclinA₂ was only deceased in 2.7 g·L^-1 group (P<0.05). Conclusion: Guizhi Fuling Wan can inhibit the proliferation of MCF-7 cell in S phase which possiblly related to the expression of CyclinA₂, Cdk2 and EGFR mRNA and their proteins.