[Influence of estrogen and progestin on nm23-H1 expression in epithelial ovarian cancer cell lines via activation of phosphorylation signaling]

Abstract

Objective: To assess the estrogen and progestin's effect on protein expression of metastasis repression gene nm23-H1 via regulation of phosphorylation signaling in epithelial ovarian cancer cell line ES-2.

Methods: Ovarian clear cell adenocarcinom cell line ES-2 was treated by different doses of 17beta-estradiol (estrogen), medroxyprogestogen (progestin) and dimethyl sulfoxide (control group), and then the following experiments were conducted. (1) Change of the cell migration capacity after treatment with estrogen and progestin for 24 and 48 hours was measured by in vitro wound healing assay. (2) Transwell experiments were used to detect the ability of cell invasion, which was also used to inhibit the phosphorylation of protein kinase B (AKT) pathway with estrogen and progestin. (3) Change of nm23-H1, AKT and phosphorylated protein kinase B (pAKT) protein level of ES-2 cells after treated with estrogen and progestin was detected by western blot. (4) Cells were transfected with the small interfering RNA (siRNA) expression vector targeting AKT. The efficiency of cells transfected was calculated according to the number of cells with green fluorescent produced by cells transfected. Change of nm23-H1 expression was assessed.

Results: ES-2 cells treated with estrogen, progestin or vehicle all migrated into the wound surface after 24 and 48 hours. The migration of the cells treated with estrogen was (1.39 +/- 0.08) mm, significantly elevated (P = 0.029), and that of the cells treated with progestin was (1.96 +/- 0.07) mm, significantly decreased compared with cells treated with vehicle (P = 0.014). The cells were cultured in transwell after 24 hours. The invasion of the cells treated with estrogen was 119 +/- 13, significantly elevated (P = 0.015), and that of the cells treated with progestin was 78 +/- 8, significantly decreased compared with cells treated with vehicle (P = 0.006). Western blot results showed that 17beta-estradiol decreased nm23-H1 expression, and both effects were dose and time dependent (P = 0.020, P = 0.001). Progestin increased nm23-H1 expression in ES-2 cells, and both effects were dose and time dependent (P = 0.003, P = 0.002). 17beta-Estradiol elevated the expression of pAKT, which was also dose and time dependent (P = 0.001, P = 0.007), while progestin repressed pAKT expression which was also dose and time dependent (P = 0.012, P = 0.039). When cells were transfected with the siRNA expression vector targeting AKT, the effects of estrogen and progestin on nm23-H1 expression were both attenuated.

Conclusions: Estrogen downregulates the expression of nm23-H1 via activation of the phosphorylation signaling, thus participated in the regulation of invasion and metastasis of epithelial ovarian cancer cells. Progestin upregulates the expression of nm23-H1, and might repress invasion and metastasis of tumor cells.