| Objective:Breast cancer is a highly prevalent type of tumor that threatens women seriously.Genistein can be take into body as phytoestrogen from daily diet,but whether it could promote the growth of breast cancer is still controversial.The cancer testis-specific protease 50(TSP50)is an oncogene discovered recently.It was found to be closely related to the recurrence of human breast cancer.About 70%of breast cancer cases are ER+type,while it was not clear that whether the expression of TSP50 in those ER+breast cancer cells were regulated by ER mediated signals.The focus of this study was to investigate the cell growth inhibition effect induced by different concentration of Gen on ER+ type and ER-type breast cancers cells cultured in vitro,thus to establish a model that Gen stimulating the growth on ER+type breast cancer cells.Using this model,the expression of TSP50 will be investigate after cells are treated by Gen,and whether the effect of Gen on ER+breast cancer cells are under the control of ER mediated non-genomic signals.In this study,it will be also investigated that whether procyanidin B1(PB1)has antagonistic effect against up-regulation of TSP50 and the spring of CD44+CD24-tumor stem cells induced by Gen.Methods:ER+,ER-breast cancer cells and mammary epithelial cells(MCF-7,MDA-MB-23 1 and MCF-10A)were cultured in vitro to analyze the growth inhibition effect induced by different concentration of Gen by MTT reduction assay.To measure the changes of cell cycle,apoptosis,and the expression of CD24,CD44 and CD326 on cell surface by flow cytometry(FCM),after the cells were treated by Gen with the concentration of 5,25 and 100μmol/L.Those concentrations are the dose that results in 20%growth-stimulating,20%growth-inhibiting concentration and 50%growth-inhibiting in ER+ breast cancer cells.The percentage of CD44+CD24-tumor stem cell population would be also counted after cells are detected by FCM.Western blot was used to analyze the change of TSP50,MMP-2 and key molecules of ER mediated non-genomic signals.After cells were treated by Gen with a inhibitor of MEK/Erk(U0126)PB1,the antagonistic effect of them would be also studied by measuring the expression of TSP50 and the percentage of CD44+CD24-tumor stem cell.Result:It was found the sensitivity of breast cancer cells to Gen induced proliferation inhibition were different.Among them,MCF-10A was most sensitive to Gen-induced apoptosis or growth inhibition,while in MCF-7 cells,low concentration of Gen(2.5-10 μmol/L)could promoted cell growth,only higher concentration of Gen could inhibit the cell growth.It could be found that the 20% growth-stimulating concentration,20%growth-inhibiting concentration,and 50%growth-inhibiting concentration of Gen on MCF-7 were approximately 5 μmol/L,25 μmol/L,and 100μmol/L,respectively.So,different types of cells were treated by Gen with the concentration of 5 μmol/L,25 μmol/L and 100 μmol/L to measure the change of cell cycles.In the cells from control group,and 5μmol/L,25μmol/L,and 100μmol/L of Gen,the percentages of G0G1 phase cells were 67.60%,64.31%,64.64%,and 72.76%respectively;while the percentages of S phase cells were 14.67%,15.64%,15.64%,and 11.74%;the percentages of G2M phase cells were 14.84%,17.71%,16.21%,11.57%.The results indicated that treated by 5 μmol/L Gen resulted in an increase of S phase and G2M phase cells in MCF-7 cells;while treating by 25 μmol/L Gen resulted in an increase of G2M phase cells,and treating by 100 μmol/L Gen resulted in the blocking in G0G1 phase.It is interesting that 5 μmol/L Gen and 25 μmol/L Gen also resulted in an increase in the percentage of S phase and G2M phase cells in MCF-10A cells.FCM was used to analyze cell surface molecules.Compared with the control group,the expression of CD24,CD44,and CD326 increased in the cells treated by Gen.It was reported that CD44+CD24" cell populations had stem cell-like features in breast cancer.For ER+breast cancer cells,the rates of the stem cell subsets in the groups of control,5 μmol/L Gen,25μmol/L Gen and 100 μmol/L Gen were 7.37±0.43%,11.57±1.13%,12.05±0.96%and 6.04±0.22%respectively.That indicated lower concentration of Gen resulted in stem cells population increasing.Western blot was used to analyze the change of cell signal molecules in MCF-7 cells treated by different concentration of Gen.Compared with the control group,the phosphorylation of P70S6K1 was slightly increased after treatment with 5 μmol/L Gen and 25 μmol/L Gen,and the phosphorylation of Erkl was significantly increased also.Compared with the control group,the expression of MMP-2 and TSP50 protein in MCF-7 cells increased at a concentration of 5 μmol/L,and decreased at the concentration of 100 μmol/L Gen.In order to study the mechanism of TSP50 expression changing,MCF-7 cells were treated by Gen(5μmol/L)with or without Erk inhibitor U0126(10μmol/L).The results showed that phosphorylation of Erk was significantly reduced by U0126,and the MMP-2 and TSP50 protein were also reduced by U0126.That indicated the proliferation and TSP50 expression of MCF-7 cell may be regulated by MEK/Erk signaling pathway.PB1 is a polyphenol component from grape seed.MCF-7 cells were treated with PB1(10 μmol/L)and Gen(5μmol/L),and the changes of relevant signaling molecules and TSP50 were analyzed.It was found that PB1 could significantly reduced the phosphorylation of Erkl and decrease the Erkl phosphorylation and TSP50 expression that promoted by Gen.The phenotype(membrane molecules)affected by PB1 and Gen was analyzed by FCM.The results indicated that PB1 could reduced the percentage of CD44+CD24-cells pr0moted by Gen.Conclusion:Low concentrations of Gen had weak growth stimulating effects on ER+breast cancer and could increased the percentage of CD24-CD44+ stem cell subsets.Cancer testis antigen TSP50 would be increased as downstream molecule of non-genomic effect-related signals.MEK/Erk inhibitors or PB1 could affected the expression of TSP50 and reduced the stem cell population in ER+ breast cancer. |
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