title: [Influence of pertussis toxin on GPER-mediated activation of phosphatidylinositol 3-kinase/protein kinase B signaling induced by 17β-estradiol in endometrial carcinoma cells].
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Obstetrics and Gynecology, Zhengzhou University, Zhengzhou, China. grx0322@yahoo.com.cn
authors.forename: Rui-xia
authors.initials: RX
authors.lastname: Guo
authors.forename: Jia
authors.initials: J
authors.lastname: Lei
authors.forename: Xin-yan
authors.initials: XY
authors.lastname: Wang
authors.forename: Xin
authors.initials: X
authors.lastname: Ge
authors.forename: Dong-mei
authors.initials: DM
authors.lastname: Hu
authors.forename: Xiu-ying
authors.initials: XY
authors.lastname: Ma
authors.forename: Liu-xia
authors.initials: LX
authors.lastname: Li
authors.forename: Yu-huan
authors.initials: YH
authors.lastname: Qiao
mesh_terms: D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D016889:Endometrial Neoplasms / Q000378:metabolism* / Q000473:pathology; D004789:Enzyme Activation / Q000187:drug effects; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000378:metabolism; D047629:Estrogen Receptor beta / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D037342:Pertussis Toxin / Q000494:pharmacology*; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000037:antagonists & inhibitors / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; C109584:GPER1 protein, human; D011960:Receptors, Estrogen; D043562:Receptors, G-Protein-Coupled; D004958:Estradiol; D037342:Pertussis Toxin; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt