Research On The Relationship Between BAG-1 And TAM Resistance And Related Pathways In Breast Cancer

Object In our research, we test the expression of BAG-1 in ER-positive breast cancer cells and its association with ER,AKT,ERK,p-AKT,p-ERK,analysis BAG-1 gene expression inhibition by RNA interference,the influence to Tamoxifen and Perifosine’s sensitivity,to Offer a new research direction forendocrine resistance in breast cancer.Methods In this study,we chose the T47 D breast cancer cell lines,used small interfering RNA(si RNA) to knockdown the expression of BAG-1 and test the variation of ER,AKT,ERK,p-AKT,p-ERK,expression by real-time PCR and Western blot.MTT assay and FACS(flow cytometry) were used to detect if the down-regulation of BAG-1expression in breast cancer cells changed their sensitivity to Tamoxifen(estrogen receptor inhibitors) and Perifosine(Protein kinase B inhibitors) induced drug reaction, apoptosis rate changes and cell-cycle arrest.Results 1. Select the breast cancercell T47 D used si RNA to knockdown the expression of BAG-1. As a result, BAG-1 si RNA transfected cells dramatically decreased both BAG-1 m RNA and protein expression compared to the negative control si RNA-transfected cells by real-time PCR and Western blot(P<0.05). 2. Compared with the control group, the BAG-1 si RNA transfected group ER protein and m RNA levels of expression significantly reduced(P<0.05), two groups of p-AKT protein expression levels appear to rise(P<0.05),AKT,ERK,p-ERK expression wasn’t significant different in two groups. 3. To the BAG-1 Si RNA transfected cells, give different concentration of Tamoxifen(1-9u M) and Perifosine(5-25 u M).The cell survival rate of BAG-1 si RNA transfected cells is lower than the negative control cells.Along with the increased concentration of Tamoxifen,the cell survival rate decreased(P<0.05),on the different reaction of Perifosine,The cell survival rate of BAG-1 si RNA transfected cells is higher(P<0.05).4. BAG-1 si RNA interference experiments on the different reaction of Tamoxifen(10u M) in cell apoptosis( 11.0±0.7%vs. 20.3±1.0%, P<0.05) and Perifosine(25u M)in cell apoptosis cycle(7.1±1.3%vs.4.7±0.8%,P<0.05)was detected By FACS(flowcytometry). BAG-1 si RNA interference experiments on the different reaction of Tamoxifen(10u M),Perifosine(25u M),the combination of Tamoxifen(10u M) and Perifosine(25u M)in cell apoptosis(7.1±1.3% vs 9.4±1.4%vs 11.5±0.9%,P<0.05). 5.BAG-1 si RNA interference experiments on the different reaction of Tamoxifen(10u M) in cell cycle(67.4±0.8%vs.77.6±1.2%,P<0.05)was detected By flowcytometry. BAG-1 si RNA interference experiments on the different reaction of Tamoxifen(10u M),Perifosine(25u M),the combination of Tamoxifen(10u M) and Perifosine(25u M)in cell cycle(67.4±0.8% vs 61.3±1.3%vs 76.7±1.4%%,P<0.05).Conclusion 1. In breast cancer cells, when BAG-1 expression are decreased by BAG-1 si RNA,ER expression are decreased.The expression of BAG-1 may be change the expression of ER.Down-regulated BAG-1 expression by RNA interference technology could decrease cell growth inhibition and inhibit tumor cell proliferation with Tamoxifen.For the treatment of breast cancer, BAG-1 is the sensitizing potential therapeutic sensitizing target. 2. Down-regulated BAG-1 expression by RNA interference technology could change the expression of p-AKT protein.When BAG-1 expression are decreased by BAG-1 si RNA,p-AKT protein expression are increased,this could be the reason ER expression are decreased.The combination of Tamoxifen and Perifosine could increase cell growth inhibition with Tamoxifen resistance because of BAG-1 si RNA.Down-regulated BAG-1 expression,ER expression are decreased and p-AKT protein expression are increased, the sensitivity to Tamoxifen is lower.Perifosine inhibit the phosphorylation of AKT then decrease cell growth.Further research of relationship between down-regulated BAG-1 and ER,p-AKT expression changes as well as the related Mechanism is needed.