The Influence Of Estrogen And Progestin On Metastasis And Their Regulatory Effect On PI3K Signaling In Epithelial Ovarian Cancer Cell

I Clinical researchObjective.To explore the relationships between the expressions of ER, PR, nm23-H1, E-cadherin, AKT and pAKT were correlated with the prognosis and biological behaviour of epithelial ovarian tumor. Methods.ER, PR, nm23-Hl, E-cadherin, AKT and pAKT expressions were measured by using of immunohistochemical staining in ovarian clear cell adenocarcinoma, ovarian serous adenocarcinoma, ovarian borderline and benign serous cystadenoma paraffin-embedded specimen. Results.The results showed that weak or negative ER, PR, nm23-Hl and E-cadherin expressions and high or positively AKT and pAKT expressions in ovarian serous cystadenoma, and ovarian clear cell adenocarcinom. were negatively correlated with stage I - II, grade 1, negatively correlated with lymph node metastasis, higher survival rates specimen Conclusions.Weak or negatively ER, PR ,nm23-Hl and E-cadherin expression, high or positively AKT and pAKT expression showed unfavourable prognosis in ovarian clear cell adenocarcinoma, ovarian serous adenocarcinoma.II Foundation researchPart 1 The Regulatory Effect of Estrogen and Progestin on Migration and Metastasis in Ovarian Clear Cell and Serous AdenocarcinomaObjective.To investigation estrogen and progestin's effect on migration and metastasis of ovarian clear cell and serous adenocarcinoma, and their impact on expression of metastasisrepression gene nm23-H1.Methods.Change of nm23-Hl protein level of ES-2 and SKOV3 cells after treated with estrogen and progestin was detected by WesternBlot. And the relationship between nm23-H1 protein expression and dose and duration of estrogen and progestin treatment was also investigated. Change of the cell migration capacity after treatment with estrogen and progestin for 24h and 48h was measured by in vitro wound healing assay. Invasion assay was performed using transwell chambers. The cells that crossed the membrane were counted under an inverted microscope to evaluate invasion of the cells.Results.1.The migration and invasion of ES-2 and SKOV3 cells treated with 17β-Estradiol was significantly elevated, and that of the cells treated with Medroxyprogesterone was significantly decreased comparing with cells treated with vehicle.2.17β-Estradiol decreased nm23-H1 expression, and that Medroxyprogesterone increased nm23-H1 expression in ES-2 and SKOV3 cells. And both effects were dose and time dependent.Conclusions.Estrogen downregulated the expression of nm23-H1 in ES-2 and SKOV3 cells, thus promoted metastasis and invasion of the tumor cells. Progestin upregulated the expression of nm23-H1 in ES-2 and SKOV3 cells, and might repress invasion and metastasis of the tumor cells..Part 2 The Regulation of Estrogen and Progestin on Migration and Metastasis of Ovarian Clear Cell and Serous Adenocarcinoma is mediated by Signal Transduction PathwayObjective.To assess the role of signal transduction pathway in estrogen and progestin's regulation on migration, invasion and nm23-H1 expression in epithelial ovarian cancer cell line ES-2 and SKOV3. Methods.Change of AKT and pAKT protein level of ES-2 and SKOV3 cells after treated with estrogen and progestin was detected by WesternBlot. And the relationship between AKT and pAKT protein expression and dose and duration of estrogen and progestin treatmentwas also investigated. Cells were transfected with the small interfering RNA(siRNA) expression vector targeting AKT, and change of nm23-H1 protein level of ES-2 and SKOV3 cells after treated with estrogen and progestin was detected by WesternBlot. Then cells were treated with AKT and MAPK inhibitor, and change of nm23-H1 protein level of ES-2 and SKOV3 cells treated with estrogen and progestin was detected by WesternBlot. Invasion assay was performed using transwell chambers. The cells that crossed the membrane were counted under an inverted microscope to evaluate invasion of the cells transfected with siRNA expression vector targeting AKT or treated with AKT and MAPK inhibitor.Results.1.17β-Estradiol increased pAKT expression, and Medroxyprogesterone decreased pAKT expression in ES-2 and SKOV3 cells. And both effects were dose and time dependent.2.17β-Estradiol downregulated the expression of metastasis repression gene nm23-H1 via activation of the AKT phosphorylation signaling, and progestin upregulated the expression of nm23-H1 via repression of the AKT phosphorylation signaling3. When cells were treated with both estrogen and PI3K/AKT inhibitor or AKT siRNA expressing vector at the same time, the expression of nm23-H1 in ES-2 and SKOV3 cells were not decreased, and the invasion and migration of the cells were not increased. When cells were treated with both progestin and PI3K inhibitor or AKT siRNA expressing vector, the expression of nm23-H1 in ES-2 and SKOV3 cells were not increased, and the invasion and migration of the cells were not decreased. While MAPK signaling inhibitor had no effect on steroid's regulation on nm23-H1 expression or invasion and migration of the cells.Conclusions.PI3K/AKT signaling was an upstream signal in steroid's regulation on nm23-H1 expression, while MAPK signaling was not involved in steroid's effect on nm23-H1 expression.Part 3 The Regulation of Estrogen and Progestin on HIF-1α Expression in Ovarian Clear Cell and Serous AdenocarcinomaObjective.To explore the regulation of estrogen and progestin on HIF-1α expression in ovarianclear cell and serous adenocarcinoma, and the relationship between HIF-1α and nm23-H1 expression. Methods.Change of HIF-1α protein level of ES-2 and SKOV3 cells after treated with estrogen and progestin was detected by WesternBlot. And the relationship between HIF-1α protein expression and dose and duration of estrogen and progestin treatment was also investigated. Cells were transfected with the siRNA expression vector targeting AKT, and change of HIF-1α protein level of ES-2 and SKOV3 cells after treated with estrogen and progestin was detected by WesternBlot. Then cells were treated with AKT and MAPK inhibitor, and change of HIF-1α protein level of ES-2 and SKOV3 cells after treated with estrogen and progestin was detected by WesternBlot. The cells were then transfected with the siRNA expression vector targeting HIF-1α or HIF-1α overexpression vector, and change of nm23H-l protein level of ES-2 and SKOV3 cells after treated with estrogen and progestin was detected by WesternBlot. Results.1.17β-Estradiol increased HIF-1α expression, and Medroxyprogesterone decreased HIF-1α expression in ES-2 and SKOV3 cells. And both effects were dose and time dependent.2. 17β-Estradiol upregulated the expression of HIF-1α via activation of the AKT phosphorylation signaling, and Medroxyprogesterone downregulated the expression of HIF-1α via repression of the AKT phosphorylation signaling.3.When cells were treated with both estrogen and P13K/AKT inhibitor, the expression of HIF-1α in ES-2 and SKOV3 cells were not increased, and when cells were treated with both Medroxyprogesterone and PI3K/AKT inhibitor, the expression of HIF-1α in ES-2 and SKOV3 cells were not decreased. While MAPK signaling inhibitor had no effect on steroid's regulation on HIF-1α expression.4.When HIF-1α was overexpressed, estrogen's downregulation and progestin's upregulation on nm23-H1 expression was reinforced. When cells was transfected with siRNA targeting HIF-1α, estrogen's downregulation and progestin's upregulation on nm23-H1 expression was repressed. Conclusions.PI3K/AKT signaling was an upstream signal in steroid's regulation on HIF-1α expression, and HIF-1α was the upstream signal of nm23-H1. While MAPK signaling was not involved in steroid's regulation on HIF-1α expression.Summary:. Weak or negatively ER,PR ,nrn23-H1 and E-cadherin expression, high or positively AKT and pAKT expression showed unfavourable prognosis in ovarian clear cell adenocarcinoma, ovarian serous adenocarcinoma.Estrogen upregulated. the expression of HIF-1α and downregulated the expression of metastasis repression gene nm23-H1 via activation of the AKT phosphorylation signaling in ES-2 and SKOV3 cells, thus participated in the regulation of metastasis of ovarian adenocarcinoma.