Objective To investigate the influence of pertussis toxin(PTX)on G protein-coupled estrogen receptor(GPER)-mediated activation of phosphatidylinositol 3-kinase(PI3K)/protein kinase B (Akt)signaling activated by 17 β-estradiol(17β-E2)in endometrial carcinoma cells.Methods Expressions of GPER protein were detected by immunohistochemical SP method in Ishikawa and HEC-1A cells.Changes of levels of GPER,ERα and ERβ protein and the activation of Akt protein were observed by western blot in the two cells after they were treated by PTX for 30 minutes at different concentrations(0,0.1,0.5,1.0 μg/ml),and then co-stimulated with with 1 × 10-6 mol/L 17β-E2 respectively at different time (Ishikawa 30 minutes,HEC-1A 15 minutes).Results(1)Immunohistochemical SP method showed that GPER was positive stained in cell cytoplasm of Ishikawa and HEC-1A cell.(2)After co-treated with PTX at different concentrations(0,O.1,0.5,1.0 μg/ml)and 10-6 mol/L 17β-E2,in Ishikawa cell,the ratio of pAkt/Akt was 0.74 ±0.54,0.34 ±0.06,0.18 ±0.03,0.07 ±0.15,the gray values of GPER was 0.872 ± 0.490,0.395 ± 0.054,0.145 ± 0.014,0.034 ± 0.008,and with increasing concentration of PTX,the ratio of p-Akt/Akt and the expression of GPER decreased gradually(P ＜ 0.05),which was most obviously when the concentration was 1.0 μg/ml(F =63.729,P =0.0001;F =160.284,P =0.0001);ERα and ERβ protein had no significant change among different groups(P ＞0.05).In HEC-1A cell,the ratio of pAkt/Akt was 0.73 ±0.09,0.26 ±0.14,0.11 ±0.03,0,the Gray values of GPER is 0.927 ±0.134,0.485 ± 0.022,0.194 ± 0.004,0,and with increasing concentration of PTX,the ratio of p-Akt/Akt and the expression of GPER decreased gradually(P ＜ 0.05),which were also completely inhibited when the concentration was 1 μg/ml(F =1039.321,P =0.0001;F =109.646,P =0.0001),ERα protein had no significant differences(P ＞ 0.05)among different groups.ERβ was negatively expressed.Conclusion The results proposed that the activation of PI3K/Akt signaling in Ishikawa and HEC-1A cells could be inhibited after blocking the role of GPER by PTX.%目的 研究百日咳毒素(PTX)对G蛋白偶联ER(GPER)介导的雌激素激活的子宫内膜癌细胞系Ishikawa和HEC-1A细胞中磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路的影响.方法 采用免疫组化SP法检测Ishikawa和HEC-1A细胞中GPER蛋白的表达.将不同浓度(0、0.1、0.5、1.0 μg/ml)的PTX分别处理Ishikawa和HEC-1A细胞30 min后,再予上述浓度的PTX分别联合17β雌二醇(17β-E2；1×10-6molL)共同处理HEC-1A细胞15 min、Ishikawa细胞30 min,采用蛋白印迹法检测Ishikawa和HEC-1A细胞中GPER、ERα、ERβ蛋白的表达及Akt的活化状态[Akt的活化状态以磷酸化Akt(p-Akt)/Akt表示].结果(1)免疫组化法检测显示,在Ishikawa和HEC-1A细胞中GPER蛋白均在细胞质中呈棕黄色阳性表达.(2)蛋白印迹法检测显示,不同浓度(0、0.1、0.5、1.0μg/ml)的PTX联合17β-E2(1×10-6 mol/L)处理后,在Ishikawa细胞中,p-Akt/Akt分别为0.74±0.54、0.34 ±0.06、0.18±0.03、0.07±0.15,GPER蛋白的表达水平分别为0.872±0.490、0.395 ±0.054、0.145 ±0.014、0.034±0.008,随着PTX浓度的增加,p-Akt/Akt、GPER蛋白的表达水平均明显下降(P＜0.05),且当PTX浓度为1.0μg/ml时下降最显著(F=63.729,P=0.0001;F ＝160.284,P=0.0001)；而ERα和ERβ蛋白的表达水平均无明显变化(P＞0.05).在HEC-1A细胞中,p-Akt/Akt分别为0.73 ±0.09、0.26±0.14、0.11 ±0.03、0,GPER蛋白的表达水平分别为0.927±0.134、0.485±0.022、0.194±0.004、0,随着PTX浓度的增加,p-Akt/Akt、GPER蛋白的表达水平均明显下降(P＜0.05),且当PTX浓度为1.0 μg/ml时均降至0(F=1039.321,P=0.0001；F=109.646,P=0.0001)；而ERα蛋白的表达水平无明显变化(P＞0.05),ERβ蛋白呈阴性表达.结论 在子宫内膜癌HEC-1A和Ishikawa细胞中,FTX阻断GPER后,可抑制雌激素对子宫内膜癌细胞PI3K/Akt信号通路的活化作用.
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