[百日咳毒素对17β-雌二醇诱导的子宫内膜癌细胞中G蛋白偶联雌激素受体介导的磷脂酰肌醇3激酶/蛋白激酶B信号通路激活的影响]

[Influence of pertussis toxin on GPER-mediated activation of phosphatidylinositol 3-kinase/protein kinase B signaling induced by 17β-estradiol in endometrial carcinoma cells].

作者信息

Guo Rui-xia, Lei Jia, Wang Xin-yan, Ge Xin, Hu Dong-mei, Ma Xiu-ying, Li Liu-xia, Qiao Yu-huan

机构信息

Department of Obstetrics and Gynecology, Zhengzhou University, Zhengzhou, China.

出版信息

Zhonghua Fu Chan Ke Za Zhi. 2013 Feb;48(2):129-33.

PMID:23544495

Abstract

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 17β-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, 0.1, 0.5, 1.0 µg/ml) and 10(-6) mol/L 17β-E2, in Ishikawa cell, the ratio of p-Akt/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 p-Akt/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.

摘要

目的

探讨百日咳毒素（PTX）对G蛋白偶联雌激素受体（GPER）介导的17β-雌二醇（17β-E2）激活子宫内膜癌细胞中磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）信号通路的影响。

方法

采用免疫组织化学SP法检测Ishikawa细胞和HEC-1A细胞中GPER蛋白的表达。将两种细胞分别用不同浓度（0、0.1、0.5、1.0μg/ml）的PTX处理30分钟，然后分别与1×10⁻⁶mol/L的17β-E2共刺激不同时间（Ishikawa细胞30分钟，HEC-1A细胞15分钟），采用蛋白质免疫印迹法观察两种细胞中GPER、雌激素受体α（ERα）和雌激素受体β（ERβ）蛋白水平的变化以及Akt蛋白的激活情况。

结果

（1）免疫组织化学SP法显示，GPER在Ishikawa细胞和HEC-1A细胞的细胞质中呈阳性染色。（2）不同浓度（0、0.1、0.5、1.0μg/ml）的PTX与10⁻⁶mol/L的17β-E2共同处理后，在Ishikawa细胞中，磷酸化Akt（p-Akt）与Akt的比值分别为0.74±0.54、0.34±0.06、0.18±0.03、0.07±0.15，GPER的灰度值分别为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比值和GPER表达逐渐降低（P<0.05），当浓度为1.0μg/ml时最明显（F=63.729，P=0.0001；F=160.284，P=0.0001）；不同组间ERα和ERβ蛋白无明显变化（P>0.05）。在HEC-1A细胞中，p-Akt/Akt比值分别为0.73±0.09、0.26±0.14、0.11±0.03、0，GPER的灰度值分别为0.927±0.134、0.485±0.022、0.194±0.004、0，随着PTX浓度的增加，p-Akt/Akt比值和GPER表达逐渐降低（P<0.05），当浓度为1μg/ml时也完全被抑制（F=1039.321，P=0.0001；F=109.646，P=0.0001），不同组间ERα蛋白无明显差异（P>0.05），ERβ呈阴性表达。