| Background Globally,breast cancer is one of the most commonly diagnosed cancers in women.Lung cancer is the leading cause of female cancer deaths,followed by breast cancer and then colorectal cancer.The main cause of death in breast cancer patients is not the tumor in situ but the distant metastasis of the tumor.Estrogen is an important steroid hormone and is essential for glucose homeostasis,immune stability,bone health,cardiovascular health,fertility and neurological function in vivo.At the same time,estrogen also plays a role in the development and malignant progression of various hormone-sensitive cancers,including breast cancer.Although there are many related studies,the specific molecular mechanism of estrogen affecting breast cancer metastasis has not been elucidated.Therefore,revealing its regulatory mechanism will provide new guidance for the clinical treatment of breast cancer.For the treatment of breast cancer,in addition to traditional methods such as surgery,radiotherapy and chemotherapy,targeted therapy has become a hot topic because of its specificity,low side effects,and minimal damage to normal tissues.Recent studies have confirmed that hydrogen sulfide,as a gaseous transmitter,is involved in the regulation of many important physiological functions in the body.As one of the key enzymes in the synthesis of hydrogen sulfide,cystathionine-β-synthase(CBS)is overexpressed in various tumor tissues and has been confirmed to play an important role in tumor cell proliferation,migration,and anti-apoptosis.In view of the above,we have some scientific questions: 1.Whether could estrogen affect the expression of CBS or not;2.If it affects,what is the specific molecular mechanism;3.What role does CBS play in estrogen affecting breast cancer cell migration.Our study uses CBS as an entry point to explore its role in breast cancer cell migration.This work may provide a basis for the use of tissue-specific CBS inhibitors as adjuvant therapy for breast cancer.we hypothesized that estrogen may promote the migration of breast cancer cells by up-regulating CBS protein expression.Objectives To investigate the molecular mechanism of 17β-estradiol(E2)modulating CBS,and to clarify the effect of E2 through CBS/H2 S on breast cancer cell migration.Methods and Results1 Effect of E2 on CBS protein expression and H2 S release in MCF-7 breast cancer cells1.1 E2 upregulates CBS protein expression in MCF-7 breast cancer cells MCF-7 cells were treated with different concentrations of E2(10-9-10-6 M)for24 h.Proteins were isolated for Western Blot analysis of CBS.The results showed that compared with the con group,different concentrations of E2 treatment could upregulate the expression of CBS protein in MCF-7 cells(**P<0.01 vs.con),increased by(44±9)%,(87±11)%,(91±21)%,(75±13)%,respectively.Among them,E2(10-8 M)was more effective(**P<0.01 vs.con),and within the range of E2 physiological concentration.So we chose 10-8 M as the treatment concentration of E2.MCF-7 cells were treated with 10-8 M of E2 over 48 h.Proteins were isolated for Western Blot analysis of CBS.The results showed that compared with the con group,E2 treatment upregulated CBS protein expression in MCF-7 cells for 3 h (**P<0.01 vs.con),increased by(35±22)%;and the effect of E2 continued to 48 h,CBS protein expression were increased by(62±20)%,(54±9)%,(52±8)%,(54±18)%,respectively(**P<0.01 vs.con).It is suggested that E2 can upregulate the expression of CBS protein in MCF-7cells in a concentration-and time-dependent manner.1.2 E2 increases H2 S levels in MCF-7 breast cancer cells MCF-7 cells were treated with different concentrations of E2(10-9-10-6 M)for24 h.H2 S levels were detected by free radical electrodes.The results showed that E2 at concentrations of 10-9,10-8,and 10-7 M increased the level of H2 S in MCF-7 cells compared with the con group(*P<0.05 vs.con,**P<0.01 vs.con),increased by(84±41)%,(79±15)%,(67±22)%,respectively;E2 at 10-6 M concentration also increased H2 S level,increased by(38±24)%,but with no statistical difference compared with the con group(P>0.05 vs.con).MCF-7 cells were treated with 10-8 M of E2 over 48 h.H2 S levels were detected by free radical electrodes.The results showed that compared with the con group,E2(10-8M)treatment increased the H2 S level in MCF-7 cells for 6 h(**P<0.01 vs.con)and increased by(38±15)%;the effect of E2 continued to 48 h(**P<0.01 vs.con),H2 S levels were increased by(61±13)%,(69±30)%,(71±27)%,respectively.It is suggested that E2 can increase H2 S level in MCF-7 cells in a concentration-and time-dependent manner.2 E2 promotes MCF-7 breast cancer cells migration through CBS protein To clarify the role of CBS protein in E2-promoting MCF-7 breast cancer cells migration,scrambled si RNA(40 n M)or CBS si RNA(40 n M)were transfected for12 h,and E2(10-8 M)was treated for 36 h.The cell migration was detected by RTCA.The results showed that after transfection with scrambled si RNA,E2 treatment enhanced cell migration(**P<0.01 vs.con),increased by(85±38)%;after transfected with CBS si RNA,the effect of E2 was significantly inhibited(#P<0.01 vs.E2),cell migration was decreased by(65±6)%.It is suggested that E2 promotes MCF-7 breast cancer cells migration through CBS.3 The molecular mechanism of E2 upregulation of CBS protein expression in MCF-7 cells3.1 ER mediates E2 upregulation of CBS protein expression in MCF-7 cells To clarify the role of ER in this process.MCF-7 cells were pretreated with the estrogen receptor antagonist ICI(10-7 M)or ERα agonist PPT(10-9 M)or ERβagonist DPN(10-9 M)for half an hour and then treated with E2(10-8 M)for 24 h.Proteins were isolated for Western Blot analysis of CBS.The results showed that compared with the con group,the CBS protein expression in the E2 and agonist treated groups were significantly increased(**P<0.01 vs.con),the E2 group was increased by(71±15)%,the PPT group was increased by(80±22)%,the DPN group was increased by(49±17)%.The ICI pretreatment group,compared with the E2 group,E2 upregulated CBS protein expression was inhibited(##P<0.01 vs.E2),CBS protein expression was decreased by(32±8)%.It is suggested that estrogen receptors ERα and ERβ mediate E2 up-regulation of CBS protein expression in MCF-7 cells.3.2 NF-κB pathway mediates E2 up-regulation of CBS protein expression in MCF-7 cells In order to screen out the signaling pathways that mediate the E2 upregulation of CBS protein expression in MCF-7 cells.Pretreated MCF-7 cells with MAPK inhibitor PD98059(PD,5 μM),PI3 K inhibitor LY294002(LY,1 μM),NF-κB inhibitor PDTC(5 μM)and SP1 inhibitor MA(1 μM)for 30 min,respectively.Then treated with E2(10-8 M)for 24 h.Proteins were isolated for Western Blot analysis of CBS.The results showed that pretreated with PDTC,compared with the E2 group,the expression of CBS protein was decreased(#P<0.05 vs.E2)and decreased by(52±8)%.Pretreated with PD,LY,and MA,the expression of CBS protein had no significant difference compared with the E2 group(P>0.05).It is suggested that NF-κB pathway mediates E2 upregulation of CBS protein expression in MCF-7 cells.3.3 The effect of E2 on phosphorylation of p65 protein To determine the effect of E2 on NF-κB activity,MCF-7 cells were treated with E2(10-8 M)over 180 min.Proteins were isolated for Western Blot analysis of p-p65 and p65.The results showed that compared with the con group,E2(10-8 M)treated for 60 min,p65 protein phosphorylation was increased(*P<0.05 vs.con)and increased by(48±28)%.The effect of E2 continued to 180 min(*P<0.05 vs.con,**P<0.01 vs.con),p-p65 protein expression was increased by(48±21)% and(65±19)%,respectively.E2 treatment had no effect on the expression of total p65 protein.It is suggested that E2 promotes phosphorylation of p65 protein without affecting the expression of p65 total protein.3.4 Effect of silencing p65 on E2 upregulation of CBS protein expression and H2 S levels in MCF-7 cells To clarify the role of p65,MCF-7 cells were transfected with scrambled si RNA(20 n M)or p65 si RNA(20 n M)for 24 h,then treated with E2(10-8 M)for 24 h.Proteins were isolated for Western Blot analysis of CBS.The results showed that after transfection of p65 si RNA,compared with the E2 group,the effect of E2 was significantly inhibited(##P<0.01 vs.scrambled si RNA+E2),the expression of CBS protein was decreased by(30±6)%.After treatment as above,the H2 S levels were detected by free radical electrode.The results showed that after transfection of p65 si RNA,compared with the E2 group,the effect of E2 increased H2 S level was significantly inhibited(##P<0.01 vs.scrambled si RNA+E2),H2 S level were reduced by(34±11)%.It is suggested that after transfected with p65 si RNA,the effect of E2 upregulating CBS protein expression and increasing H2 S level in MCF-7 cells was significantly inhibited.Conclusions1.E2 upregulates CBS protein expression and increases H2 S levels,which in turn promotes MCF-7 breast cancer cells migration.2.E2 activates NF-κB/p65 protein signaling pathway through ERα and ERβ. |
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