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Title: [Estradiol activates MAPK signaling pathway by estrogen induced VEGF and bFGF in endometrial cancer cells].
Author: Lu Y, Jiang S, Zhang J, Song H, Li L.
Journal: Zhonghua Fu Chan Ke Za Zhi; 2014 Dec; 49(12):925-31. PubMed ID: 25608994.
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. CONCLUSION: Estradiol inducing the production of VEGF and bFGF could activate MAPK pathway through ER-independent manner, further promote development.

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