The Mechanism Of Progesterone And LY294002 Regulating FOXO1 Expression On Proliferation And Apoptosis Of Different PR Ovarian Cancer Cells

Objective: To explore the mechanism of progesterone(P)and phosphoinositide 3-kinase(PI3K)/protein kinase B(PKB/AKT)inhibitor LY294002 regulating FOXO1 expression in ovarian cancer cells with different progesterone receptors(PR)under single and combined intervention.To provide theoretical basis for hormone therapy and targeted therapy of ovarian cancer.Methods: The well-growing ovarian cancer cell lines HO-8910(high PR expression)and SKOV3(low PR expression)were selected as the research objects.1.CCK-8 method was used to detect the effects of different concentrations of P(0,12.5,25,50,100?mol/L)and LY(0,5,10,20,40?mol/L)on the proliferation of the two cells for 24,48 and 72 hours respectively.2.Two well-growing cells were divided into four groups: Control group(0?mol/L),experimental group: P group(25?mol/L),P+LY group(25+20?mol/L),LY group(20?mol/L),The control group was added with the same amount of drug-free culture medium.Intervention 24 h line related experimental test:(1)CCK-8 method was used to detect the proliferation of the two cells;(2)Morphological changes of two cell lines under inverted microscope;(3)Annexin V-FITC/PI double-staining flow cytometry was used to detect the apoptosis of the two cells;(4)Western Blot was used to detect the expression of p-AKTSer473,p-FOXO1 Ser256,FOXO1,PR-A,PR-Bproteins in each group.Results: 1.CCK-8 detects the proliferation of two strains of cells:(1)Different concentrations of P: except for 12.5?mol/L P for 24 h,there was no significant difference compared with the survival rate of SKOV3 cells in the control group(P>0.05),The cell viability of the other experimental groups and the control group and the experimental groups were different(P<0.05).The inhibitory effect of 12.5-100?mol/L P on HO-8910 cells was significantly higher than that of SKOV3 cells and the inhibition was dose-effect and time-effect dependent decrease.(2)Different concentrations of LY intervention: except for 5?mol/L LY for 24 h and 48 h,there was no significant difference in the proliferation inhibition ability of the two cells(P>0.05).The cell viability of the other experimental groups and the control group and each experimental group were different(P<0.05).The inhibiting effect of 5-40?mol/L LY on proliferation of HO-8910 and SKOV3 cells was similar and cell survival was dose-effect versus time-effect dependent.(3)P(25?mol/L)and LY(20?mol/L)combined for 24 h,The survival rates of HO-8910 and SKOV3 cells were 45.95�1.70% and 72.23�2.08% respectively.Compared with the drug alone,the proliferation effect of P+LY group was the most significant(P<0.05),and the proliferation inhibition effect of HO-8910 cells was significantly higher than that of SKOV3 cells.2.The morphological changes of the two cells were observed by inverted microscope: the control group HO-8910 and SKOV3 showed epithelial-like cell growth,and the round,fusiform and paving stone-like arrays were compact and adherent,and the cell boundaries were clear.In the experimental group,the cell gap of HO-8910 increased and cell debris was observed,cell adhesion decreased,adherent cells decreased,suspended cells increased,cells showed long spindle-shaped irregular changes,and the morphological changes of P+LY group were most significant under microscope;The morphological changes of P group in SKOV3 cells were not significant.The cell gap between P+LY group and LY group increased some cells were polygonal,and the boundary was unclear and there was no obvious floating cells.3.Two cell apoptosis cases:(1)Compared with the control group,the apoptosis rate of each experimental group increased(P<0.05);(2)Compared with the drug alone,P+LY group had the highest apoptotic rate(P<0.05);(3)P group and P+LY group had a significantly higher apoptotic effect on HO-8910 than SKOV3(P<0.05);(4)There was no significant difference in the induction of apoptosis between the two cells in the LY group(P>0.05).4.Western Blot results of two cell lines:(1)Compared with the control group,the expression of p-AKTSer473 and p-FOXO1Ser256 decreased and FOXO1 increased in allexperimental groups and the decrease of p-AKTSer473 and p-FOXO1Ser256 in P+LY group was the most significant,and the increase of FOXO1 was the most obvious;(2)P group and P+LY group decreased p-AKTSer473 and p-FOXO1Ser256 on HO-8910,and increased FOXO1 significantly compared with SKOV3(P<0.05);(3)The LY group has a certain time and concentration,had no significant difference in the expression of p-AKTSer473,p-FOXO1Ser256 and FOXO1 on two cell lines(P>0.05);(4)Although there was no significant difference in PR-B and PR-A expression between the two cell lines after drug intervention(P>0.05),the expression of PR-B and PR-A between the two cell lines(P<0.05),Further analysis of the ratio of PR-B/PR-A in the two cell lines showed that the proportion of PR-B in PR in HO-8910 cells was significantly higher than that in SKOV3 cells(P<0.05).Conclusion: 1.Progesterone may down-regulate PI3K/AKT activity mainly by PR-B promote the expression of FOXO1,thus affecting the proliferation and apoptosis of HO-8910 and SKOV3 ovarian cancer cells.2.LY294002 inhibits PI3K/AKT activation,and the mechanism of up-regulation of FOXO1 may not be related to PR expression.3.Progesterone combined with LY294002 has synergistic effect on inhibiting proliferation and inducing apoptosis of ovarian cancer HO-8910 and SKOV3 cells.