[Estradiol activates MAPK signaling pathway by estrogen induced VEGF and bFGF in endometrial cancer cells].

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.

Effects of estradiol on VEGF and bFGF by Akt in endometrial cancer cells are mediated through the NF-κB pathway.

Endometrial carcinogenesis may be related to the long-term effects of estradiol with no antagonism. However, how estradiol regulates cell proliferation is unknown. In the present study, through investigating the molecular events involved in estradiol induced angiogenics factors VEGF and bFGF, we found that estradiol induced endometrial cancer cell division, proliferation, migratory and invasive capacity in vitro and upregulated mRNA expression and protein synthesis of VEGF and bFGF. The estradiol-dependent induction of the expression of VEGF and bFGF was blocked by ER inhibitor, AKT inhibitor and NF-κB inhibitor (PDTC) in estrogen receptor positive Ishikawa cells and blocked by AKT inhibitor, NF-κB inhibitor (PDTC) in estrogen receptor negative HEC-1A cells. Moreover, estradiol activation of AKT was also blocked by AKT antagonist. NF-κB activation was restricted by estradiol concentration and time. Estradiol leading to VEGF and bFGF induction was also confirmed by the development of xenograft tumors in vivo. Taken together, our data suggest that estradiol induces the production of angiogenic factors via a mechanism involving AKT-mediated NF-κB activation partly in non-genomic manner without the estrogen receptor.