细胞外调节蛋白激酶在雌激素促乳腺癌细胞MCF-7增殖中的作用

Role of extracellular signal-regulated protein kinase in estrogen-induced proliferation of breast cancer cell line MCF-7

目的:探讨细胞外调节蛋白激酶(ERK)在雌激素促乳腺癌细胞MCF-7增殖、细胞周期转化中的作用及机制。方法:以雌激素受体阳性乳腺癌细胞MCF-7为研究对象,MTT法检测17β-雌二醇(17β-E2)对MCF细胞增殖的影响,确定实验处理用浓度;MTT法检测PD98059对17β-E2促MCF-7细胞增殖作用的影响,求取其中效抑制浓度;流式细胞术检测细胞周期;TRAP-PCR银染法检测端粒酶活性,Western印迹法检测p-ERK1/2、野生型p53蛋白水平;RT-PCR检测野生型p53 mR-NA水平。结果:ERK磷酸化抑制剂PD98059能抑制17β-E2对MCF-7细胞的促增殖作用,具有时效-量效关系,其差异具有显著统计学意义(P<0.01),各作用时间点PD98059中效抑制浓度分别为89.28μmol/L.24 h、39.81μmol/L.48 h、21.87μmol/L.72 h。应用PD98059 48 h后,能抑制17β-E2促进MCF-7细胞周期转化作用,使G1期细胞增加,S期和G2期细胞减少(P<0.01);阻抑17β-E2增强MCF-7细胞端粒酶活性的作用(P<0.05);增强野生型p53蛋白表达和p53基因转录水平(P<0.01);p-ERK1/2蛋白表达水平显著降低(P<0.01)。结论:ERK在17β-E2促乳腺癌细胞MCF-7增殖、细胞周期转化中具有重要的作用,其机制与野生型p53转录和端粒酶活性改变有关。

Objective： To investigate the role of extracellular signal-regulated protein kinase （ERK） in estrogen-induced proliferation and cell cycle transformation of breast cancer cell line MCF-7 and the related mechanisms. Methods： Estrogen receptor-positive breast cancer cell line MCF-7 was used in our study. The effects of 17β-E2 on the proliferation of MCF-7 cells was investigated by MTT assay to determine the optimal concentration of 17β-E2 for the following experiment. The effect of PD98059 on 17β-E2 induced proliferation of MCF-7 cells was measured by MTT assay to determine the intermediate concentration of PD98059. The cell cycle was analyzed by flow cytometry and telomerase activity was determined by Telomerase repeat amplification protocol PCR （TRAP PCR） silver staining. The expression of wild-type p53 and phosphorylated ERK1/2 protein was determined by Western blotting and the expression of wild type p53 mRNA was detected by RT PCR. Results： ERK phosphorylation inhibitor PD98059 inhibited the proliferation of MCF-7 cells treated with 17β-E2 in a time- and dosedependent manner（P〈0.01）. The intermediate concentrations of PD98059 were 89.28 μmol/L for 24 h,39.81 μmol/L for 48 h and 21.87 μmol/L for 72 h. Treatment with 20μmol/L PD98059 for 48 h reversed the promoting effect of 17β-estradiol on the cell cycle transformation of MCF-7, increasing the number of G1 phase cells and decreasing the number of S and M phase cells （P〈0.01） ,inhibited the enhancing effect of 17β-E2 on the telomerase activity of MCF 7 cells（P〈0.05）, increased the protein expression level and genetic transcription of wild-type p53 （P〈0. 01）, and decreased the expression of p-ERK1/2 protein（P〈 0.01）. Conclusion： ERK plays an important role in 17β-Ee induced proliferation and cell cycle transformation of breast cancer cell line MCF7, which might be related to the changes of genetic transcription of wild-type p53 and telomerase activity.