Molecular Mechanisms Of Megestrol Acetate In Anti-endometrial Carcinoma Via Interacting With Progesterone Receptor B

BACKGROUND AND OBJECTIVESMegestrol acetate(megestrol acetate,MA),one of the 17α-hydroxy progesterone derivatives,has high progesterone activity,and is one of the crucial choices for conservative treatment in patients with endometrial carcinoma.Previous studies reported that MA could play the role of anti-tumor by inhibiting the activity of estrogen.However,the exact role and molecular mechanism of MA in anti-endometrial carcinoma are still unclear.It is known that progesterone receptors(PR) are the main mediators of progesterone activity,and progesterone could regulate the physiological changes of cells by interacting with related receptors.However,the expression of progesterone receptors in normal and endometrial cancer tissues is unclear.Dose it involved in the anti-endometrial carcinoma of MA?And which specific subtype of receptors is responsible for its role?Whether is the molecular mechanism corelated to the alteration of functional activity of endometrial cancer cells?Answers to the above questions are of great significance to further understanding the pathogenesis of endometrial cancer and in-depth analysis of the anti-tumor effect of MA in the treatment of endometrial cancer.We constructed the mouse model and cell model of endometrial carcinoma to investigate the expression of different progesterone receptors under normal and pathological conditions,to confirm the anti-endometrial carcinoma of MA in vivo and in vitro,and to determine the involved potential cellular and molecular mechanisms.The aim is to provide experimental basis for further understanding of the mechanism of MA against endometrial cancer.METHODSThe Cancer Genome Atlas(TCGA) and GTEX(Genotype-Tissue Expression) data were obtained and analyzed through GEPIA database using bioinformatics method.The expression level of progesterone receptor(PR) in normal tissues and tissues of uterus cervical endometrial cancer(EC) patients,as well as in tissues of different stages of EC tumors was analyzed.Kaplan Meier method was used to analyze the relationship between PR level and the overall survival and progression-free survival of EC patients.20 EC and normal uterus cervical endometrial tissues were collected,human endometrial cancer cell lines Ishikawa and HHUA cells and human normal endometrial epithelial cell line HEEC were cultured,and the expressions of PR subtypes PR-A and PR-B in each cell line were detected by western blotting.In addition,Ishikawa cells were injected subcutanically into the back of nude mice to establish a tumor-bearing xenograft model.The expressions of PR-A and PR-B and the ratio of PR-A/PR-B were detected by western blotting.Endometrial cancer cells Ishikawa and HHUA were treated with MA at different concentrations(1 nM,10 nM and 100 nM) .Cell viability of each group at different time points was evaluated using CCK8 kit.PR-A and PR-B siRNA or their overexpression lentivirus vectors were constructed and transfected/infected into Ishikawa and HHUA cells,and then cells were treated with 10 nM MA and cell viability in each group was detected by CCK8.A mouse model of endometrial cancer was treated with different concentrations of MA to detect the changes of tumor volume and weight.At the same time,PR-A and PR-B overexpression lentivirus vectors were constructed and transfected into Ishikawa cells to establish stable interfering cell lines.The cells were inoculated subcutaneously in nude mice.After intragastric administration of MA,the changes of tumor volume and weight in each group were determined.Ishikawa and HHUA cells were treated with MA at 10 nM for 72 h and 96 h,respectively.Flow cytometry was used to detect the apoptosis rate and cell cycle changes,SAβ-Gal staining were used to detected cell senescence,and western blotting was used to detect the expression of Cyclin D1,p16 and p21.Immunohistochemical analysis was used to detect the expression of p21 in EC samples.In addition,PR-B siRNA was transfected and 10 nM MA was added to the cells,and SAβ-Gal staining was performed.Bioinformatics method was used to obtain and analyze the correlation between PR and FOXO1 expression in endometrial cancer tissues of the patients through GEPIA database,as well as the expression level of FOXO1 in normal tissues and tissues of endometrial cancer patients.Kaplan Meier method was used to analyze the relationship between FOXO1 expression and overall survival and progressive-free survival of patients.Ishikawa and HHUA cells were treated with MA,and RT-PCR and western blotting were uded to detect the expressions of FoxO1 mRNA and protein.The expression and localization of FOXO1 were observed by immunofluorescence staining.MA was into Ishikawa established tumor-bearing models were intragastrically administrated with MA,and the expression and localization of FOXO1 in tumors were observed by immunohistochemical staining.The positive expression of FOXO1 in EC samples was detected using immunohistochemical analysis.Ishikawa and HHUA endometrial cancer cells were pretreated with specific FOXO1 inhibitor AS 184285(AS) for 1 h,or transfected with PR-B siRNA and OE-PR-B,and then treated with 10 nM MA for 96 h.Cell viability of each treatment group was detected by CCK8,and cell senescence and the related proteins were detected by SAβ-gal staining and western blotting.The effect of FOXO1 signaling pathway in MA against endometrial cancer was analyzed.RESULTSThe differences in gene levels of progesterone receptor PR in normal endometrial tissues and endometrial cancer tissues were analyzed by GEPIA and Linkedomics in the TCGA-UCEC data set.The data showed that the median expression of PR in normal tissues was 35.05,and that in tumor tissues was 3.235,indicating a statistically significant difference(P<0.001) .There was a statistical difference in the expression of PR in UCEC patients of different pathological stages(F=6.81,P<0.001),and the expression of PR gradually decreased with the increase of clinical stages.The patients were divided to high and low PR groups according to the median value of PR expression.Kaplan Meier analysis showed a 10-year overall survival(OS) of about 70%in high PR group,and a OS of approximately 20%in low PR group(Logrank P=0.0042) .Similarly,DFS surial in patients with high PR group is signidicantly higher than that of low PR group(Logrank P=0.0028) .Two subtypes of PR,PR-A and PR-B,were expressed in normal endometrial epithelial cells and endometrial cancer cells.The expression of PR-A and PR-B were similar in hEEC cells.However,PR-A decreased in Ishikawa and HHUA cells,while PR-B increased in the two cells,and the ratio of PR-A/PR-B decreased.Both PR-A and PR-B proteins were expressed in normal endometrial tissues and transplanted tumor tissues,and the expression levels of the two proteins were similar in normal tissues.Compared with normal tissue,the expression of PR-A decreased gradually with the prolonging of tumor formation time,while the expression of PR-B increased,and the ratio of PR-A/PR-B decreased significantly(P<0.05) .The CCK8 assay showed that there was no significant change in cell viability of the two cells after 1 nM MA treatment compared with the normal control group.However,the viability of Ishikawa and HHUA cells treated with 10 nM and 100 nM MA significantly decreased(P<0.05) .Transfection with PR-A siRNA and PR-B siRNA did not significantly change the viability of Ishikawa and HHUA cells.Then,10 nM MA was added after transfection of PR-A siRNA,PR-B siRNA and corresponding control siRNA.The inhibitory effect of MA on cell growth was eliminated after the silencing of PR-A or PR-B,and the cell growth activity of PR-A and PR-B siRNA transfection groups was similar to that of control siRNA group.Tumor bearing mice were treated with MA.The results showed that compared with control group,tumor volume and weight of mice treated with 50mg/kg MA had no significant changes.Tumor volume and weight significantly decreased after 100mg/kg and 200mg/kg MA treatment(P<0.05) .The tumor xenograft experiments in nude mice showed that the volume and weight of the xenografts in the OE-PR-A and OE-PR-B groups were not significantly different from those in the model group infected with control shRNA lentivirus vector.However,in OE-PR-B group combined with the treatment of MA,the tumor size and weight were significantly decreased compared with the mice infected with control vector and treated with MA,while OE-PR-A groups combined with treatment of MA showed no obvious changes in the growth of the transplanted tumor.Flow cytometry results showed that the apoptosis rate of Ishkawa control group was(1.51±0.11) %,and that of HHUA control group was(1.23±0.09) %.After 10 nM MA treatment,the apoptosis rate increased but no statistically significant difference was indicated.Compared with control group,Ishikawa and HHUA cell cycle arrest occurred after MA treatment for 72 h and 96 h,and the proportion of cells in G1 phase was significantly increased(P<0.05) .After 10 nM MA treatment,the expressions of Cyclin D1 in Ishikawa and HHUA cells were significantly down-regulated compared with those in the control group(P<0.05) .Treatment with 10 nM MA for 96 h,the number of SAβ-gal positive cells in Ishikawa and HHUA cells was significantly increased(P<0.01),and the expression levels of cell senescence markers p21 and p16 were significantly increased compared with the control group(P<0.05) .In vitro,PR-B silencing combined with MA was used to treat endometrial cancer cells.It was found that the inducible effect of cell senescence by MA on endometrial cancer cells was weakened by PR-B siRNA.Bioinformatics analysis showed that there was a strong correlation between the levels of PR and FOXO1 in the tissues of endometrial cancer patients(R=0.4,P<0.05) .Kaplan-Meier analysis showed that the 10-year DFS of patients in the high FOXO1 group and the low FOXO1 group were 63%and 45%,respectively(LogRank P=0.017),while the OS of the two groups was similar(LogRank P=0.34) .Immunohistochemical results showed a significant decrease in FOXO1 positive rate in EC tissues.After MA treatment,the levels of FOXO1 mRNA and protein in endometrial cancer cells were significantly higher than those in the control group.Inhibition or overexpression of PR-B affects the inhibitory effect of MA on EC cells and FOXO1 induction.The expression of FOXO1 was decreased when treated with a FOXO1 inhibitor combined with MA.After treated with AS,the inhibitory effect of MA on Ishikawa and HHUA cell proliferation was weakened by AS(P<0.05) .At the same time,compared with MA treatment group,the number of SAβ-gal positive cells decreased and the p21 and p16 proteins decreased after the addition of AS.CONCLUSIONMetesterone acetate can regulate the activation of intracellular FOXO1 signaling pathway via PR-B,thereby inhibiting the cycle progression and inducing cell senescence of endometrial cancer cells,thus playing a role in the treatment of endometrial cancer.