Metformin Inhibits Ovarian Cancer Via Decreasing H3K27 Trimethylation

Part 1 Metformin inhibits the malignant progression of ovarian cancerObjective To investigate the effects of metformin on the proliferation,apoptosis and migration of ovarian cancer cells.Methods 1.Established normoglycemic(5.5 m M)and hyperglycemic(25 m M)culture model in the ovarian cancer cell lines SKOV3,A2780,and ES2.2.Ovarian cancer cells were treated with 10 m M metformin for 24 h.Ed U assay to detect cell proliferation,Annexin V-FITC/PI flow cytometry to detect apoptosis,scratch wound healing and Transwell migration assay to detect cell migration capacity.3.Ovarian cancer cells were treated with 10 m M metformin for 24 h.The m RNA levels of MMP 11,Caspase 3,Caspase 7,Caspase 9,Bax and bcl-2 were determined by PCR.Results 1.Metfrormin inhibits proliferation of ovarian cancer cells.1)In normoglycemic medium,compared to the control cells,the proliferation rates in SKOV3,A2780,and ES2 were decreased by 9.1%(PSKOV3=0.0023),33.37%(PA2780<0.0001),and 34.7%(PES2=0.0010)after metformin treatment,respectively.2)In hyperglycemic medium,compared to the control cells,the proliferation rates in SKOV3,A2780 and ES2 cells were decreased by 6.55%(PSKOV3=0.0043),38.72%(PA2780<0.0001),and 33.96%(PES2=0.0004)after metformin treatment,respectively.3)Intriguingly,the cell proliferation rates in the normoglycemic cells were significantly lower than those in the hyperglycemic cells after exposure to metformin(PSKOV3=0.0020,PA2780=0.0009,PES2=0.0467).2.Metfrormin promotes apoptosis of ovarian cancer cells.1)In normoglycemic medium,compared to the control cells,the apoptosis rate of SKOV3,ES2 were significantly increased after metformin treatment(PSKOV3=0.0152,PES2=0.0051).2)All three ovarian cancer cell lines undergoing metformin treatment in normoglycemic condition showed a higher apoptosis rate(PSKOV3=0.0051,PA2780=0.0245,PES2=0.0062),compared with those treated with metformin in hyperglycemic condition.3.Metfrormin inhibits migration of ovarian cancer cells.1)Compared to the control cells,the wound healing ability of ovarian cancer cells SKOV3,A2780,and ES2 was significantly reduced(PSKOV3=0.0334,PA2780=0.0024,PES2=0.0004),and the number of ovarian cancer cells migrated to the lower chamber was significantly reduced(PSKOV3=0.0044,PA2780=0.0001,PES2=0.0001)after administration of metformin under normoglycemic condition.2)Compared to the control cells,the wound healing ability of SKOV3 and ES2 cells decreased(PSKOV3=0.0052,PES2=0.0120),and the number of SKOV3 and ES2 cells that migrated to the lower chamber decreased(PSKOV3=0.009,PES2=0.0006)after treatment with metformin in hyperglycemic condition.3)Compared with the hyperglycemic group,the three ovarian cancer cell lines treated with metformin exhibited lower scratch healing ability(PSKOV3=0.0207,PA2780=0.005,PES2=0.0263),and cell number migrating to the lower chamber was significantly reduced(PSKOV3=0.0480,PA2780<0.0001,PES2=0.0002).4.Under normoglycemic condition,compared to the control cells,the apoptosis of SKOV3,ES2 did not have significantly change(PSKOV3=0.5252,S2=0.4476);but the ability of wound healing of ovarian cancer cells was significantly reduced(PSKOV3=0.0193,PES2=0.0349)after administration of 2.5 mmol/L metformin.5.The m RNA expression of MMP11 was decreased(P=0.0245),Caspase 7,Caspase 9 and Bax was increased(PMMP11=0.0245,PCaspase 7=0.0486,PCaspase 9=0.0225,PBax =0.0366)in SKOV3 under normoglycemic condition.The m RNA expression of caspase 3 increased more than 8 times,but P=0.6172,the difference was not has statistically significant.Conclusion Metformin could inhibit the proliferation and migration,and promote apoptosis of ovarian cancer cells.And reducing glucose concentration enhanced the antitumor effect of metformin.Part 2 Metformin inhibits ovarian cancer via decreasing H3K27 trimethylationObjective To investigate whether metformin affects the malignant progression of ovarian cancer via regulating H3K27me3.Methods 1.0,2.5,5,and 10 m M metformin was administered to ovarian cnacer cells SKOV3,A2780 and ES2,the protein levels of H3K27me3 and three main components of the PRC2(i.e.Enhancer of zeste homolog 2(EZH2),Suppressor of zeste 12(SUZ12),and Embryonic ectoderm development(EED))were detected by western blot,the m RNA levels of EZH2,SUZ12,and EED was detected by PCR.2.A recombinant lentivirus harboring EZH2 DNAwas transfected into SKOV3 and ES2 cells,and monoclones were obtained to establish EZH2 overexpression stable cell lines and corresponding empty vector cells lines.3.5 m M metformin was administered to the stably transfected cells and the corresponding empty vector cells for 24 h,Ed U assay o detect cell proliferation,Annexin V-PE/7-AADflow cytometry to detect apoptosis,scratch wound healing and Transwell migration assay to detect cell migration capacity.Results 1.Metformin suppresses H3K27me3 level in ovarian cancer cells.1)Metformin suppressed H3K27me3 level in ovarian cancer cells in a dose-dependent manner under normoglycemic culture conditions.2)H3K27me3 decreased slightly in SKOV3 after given metformin under hyperglycemic culture conditions.2.Metformin suppresses EZH2,SUZ12,and EED levels in ovarian cancer cells.1)The protein and m RNA expression of EZH2,SUZ12,and EED were decreased to varying extents after exposure to metformin under normoglycemic condition in all three cell lines,while the suppression was rather modest in the cells under hyperglycemic condition.2)Under hyperglycemic condition,the EED m RNA level was decreased,but there was no significant inhibition in protien level,and the protein level of SUZ12 was reduced,but there was no significant inhibition in m RNA level in SKOV3,which may be due to post-transcriptional modifications.4.Metformin inhibits ovarian cancer through decreasing H3K27me3.1)Compared with the cells transfected with empty lentivirus,the protein expression of H3K27me3 was significantly increased in ovarian cancer cells SKOV3 and ES2 transfected with EZH2 DNA lentivirus.2)Compared to the control cells,the cell proliferation rate were decreased(PSKOV3=0.0004,PES2=0.0009),scratch healing ability was inhibited(PSKOV3 = 0.0028),cell number migrating to the lower chamber were reduced(PSKOV3 = 0.0109,PES2=0.0102),apoptosis rate were increased(PSKOV3 = 0.0350,PES2=0.0286)after treatment with metformin in cells transfected with empty lentivirus.3)Compared with the cells transfected with empty lentivirus,metformin did not significantly affect the cell proliferation,apoptosis,or migration of cells transfected with EZH2 DNA lentivirus(P > 0.05).Part 3 Metformin inhibits H3K27me3 through AMPK pathwayObjective To investigate whether metformin reduces H3K27me3 through the adenosine monophosphate activated protein kinase(AMPK)pathway.Methods 1.0,2.5,5,and 10 m M metformin was administered to ovarian cnacer cells SKOV3,A2780 and ES2,western blot was used to detect the protein levels of AMPK and P-AMPK.2.2-DG(25 m M),an AMPK activator,was administered to ovarian cancer cells SKOV3 and ES2 for 24 h.The protein levels of AMPK,P-AMPK,H3K27me3,and PRC2 were detected by western blot.3.Pretreated with 20 μM Compound C for 2 h,then given 5 m M metformin for 24 h in SKOV3 and ES2 cells.The protein levels of AMPK,P-AMPK,H3K27me3,and PRC2 were detected by western blot.Results 1.Metformin activates AMPK of ovarian cancer cells.1)Compared to the control cells,the ratio of p-AMPKα/AMPKα was dramatically increased after metformin administration in the ovarian cancer cells cultured in normoglycemic medium.2)Compared to the control cells,the ratio of p-AMPKα/AMPKα was slight increased in ovarian cancer cells cultured in hyperglycemic medium.2.Compared to the control cells,2-DG could elevate the p-AMPK/AMPK ratio,and repress the expression of H3K27me3,EZH2,SUZ12,and EED.3.In the cells pretreated with Compound C,metformin was not able to elevate the p-AMPK/AMPK ratio,or inhibit EZH2,SUZ12,EED,or H3K27me3.Conclusion Metformin inhibits H3K27me3 through AMPK pathway.