title: [Estradiol activates MAPK signaling pathway by estrogen induced VEGF and bFGF in endometrial cancer cells].
abstract: OBJECTIVE
  To explore the effects of mitogen-activated protein kinase (MAPK) pathway by estradiol induced vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in endometrial cancer Ishikawa cells.
  
  
  METHODS
  The experiments were divided into 4 groups: E2 group (Ishikawa cells treated with 1 µmol/L estradiol for 30 minutes); inhibitor group: including Ishikawa cells treated with 10 µmol/L Bibf1120 (Bibf1120 group), or treated with 2.5 µmol/L Ponatinib (Ponatinib group), or treated with 10 µmol/L U0126 (U0126 group) for 60 minutes; inhibitor + E2 group: including Ishikawa cells treated with 10 µmol/L Bibf1120 (Bibf1120 + E2 group), or treated with 2.5 µmol/L Ponatinib (Ponatinib + E2 group), or treated with 10 µmol/L U0126 (U0126 + E2 group) for 60 minutes following incubation with 1 µmol/L estradiol for 30 minutes;control group: only adding the culture medium without serum DMEM. (1) Western blot analysis was used to detect phosphorylation extracellular signal-regulated kinase 1/2(p-ERK1/2) protein expression with stimulation in different concentrations of estradiol (0.01,0.1, 1, 10, 100 µmol/L). (2)Quantitative fluorescent reverse transcription (qRT)-PCR and western blot analysis was used to test the level of mRNA and protein of VEGF, bFGF, MAPK kinase 1/2 (MEK1/2), extracellular signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2 and phosphorylation MEK1/2(p-MEK1/2). Flow cytometry were used to examine the cell cycle, and transwell chamber assay were used to detect the cell migration in different groups.
  
  
  RESULTS
  The expression of the p-ERK1/2 protein at 0.01,0.1, 1, 10, 100 µmol/L were 0.16±0.03, 0.10±0.03, 0.41±0.04, 0.19±0.03, 0.19±0.03, there were significantly higher than that in control group (0.05±0.00, P < 0.05), and which was more obvious at the concentration of 1 µmol/L estradiol. The expression level of VEGF, bFGF mRNA and protein in E2 group were higher than those in the control group(P < 0.05). VEGF mRNA and protein in Bibf1120+E2 group were higher than those in E2 group. The expression of MEK1/2, ERK1/2 mRNA protein in E2 group were higher than those in control group (P < 0.05). The expression of MEK1/2, ERK1/2 mRNA or p-MEK1/2, p-ERK1/2 protein in Bibf1120 + E2 group, Ponatinib+E2 group or U0126+E2 group were lower than those in E2 group (all P < 0.05). Percentage of G1 phase ([53.6±3.2)%] and S phase ([ 29.2±4.2)%] in E2 group was significantly different with those in control group respectively(P < 0.05). Percentage of G1 phase [(66.8±2.6)%, (63.1±2.6)% and (63.3±0.4)%] and S phase [(25.4±1.9)%, (25.0±3.8)% and (23.8±0.5)%] in U0126+E2 group, Bibf1120+E2 group or Ponatinib +E2 group was also significantly different with those in control group(all P < 0.05); percentage of G1 phase and S phase in U0126+E2 group was significant difference with those in Bibf1120+E2 group or ponatinib+E2 group (P < 0.05). The number of cell colony in E2 group (110±17) was more than those in control group(65±8);the number of cell colony in U0126+E2 group (28±4), Bibf1120+E2 group (38±5) or Ponatinib+E2 group (42±6) were significant different with those in E2 group (P < 0.05), the number of cell colony in U0126+E2 group was significant difference with those in Bibf1120+E2 group or Ponatinib+E2 group (all P < 0.05). The results shown that the abilities of proliferation and cell migration were significantly increased in cells after estradiol stimulation.
  
  
  CONCLUSIONS
  Estradiol inducing the production of VEGF and bFGF could activate MAPK pathway through ER-independent manner, further promote development.
authors.affiliation: Department of Gynecologic Oncology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, China.
authors.forename: Yanqiong
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Department of Gynecologic Oncology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, China.
authors.forename: Si
authors.initials: S
authors.lastname: Jiang
authors.affiliation: Department of Gynecologic Oncology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, China. Email: zjq201008@hotmail.com.
authors.forename: Jieqing
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Gynecologic Oncology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, China.
authors.forename: Honglin
authors.initials: H
authors.lastname: Song
authors.affiliation: Department of Gynecologic Oncology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning 530021, China.
authors.forename: Li
authors.initials: L
authors.lastname: Li
mesh_terms: D000970:Antineoplastic Agents; D015153:Blotting, Western; D002453:Cell Cycle; D045744:Cell Line, Tumor; D016889:Endometrial Neoplasms / Q000378:metabolism* / Q000473:pathology; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology*; D004967:Estrogens; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D007093:Imidazoles; D048369:MAP Kinase Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011724:Pyridazines; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D004967:Estrogens; D007093:Imidazoles; D047428:Protein Kinase Inhibitors; D011724:Pyridazines; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; C545373:ponatinib; D004958:Estradiol; D019869:Phosphatidylinositol 3-Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human