比卡鲁胺通过下调PKC的表达水平抑制三阴性乳腺癌BT549细胞的增殖和侵袭

Bicalutamide inhibits proliferation and invasion of triple negative breast cancer BT549 cells by down-regulation PKC expression

目的:探讨雄激素受体(androgen receptor,AR)拮抗剂比卡鲁胺(bicalutamide)对AR阳性的三阴性乳腺癌(triple-negative breast cancer,TNBC)型BT549细胞的生物学功能的影响及可能的分子机制。方法:采用蛋白质印迹法筛选AR高表达的TNBC细胞株;CCK-8法检测不同浓度比卡鲁胺对BT549细胞增殖的抑制作用,并计算半数抑制浓度(half maximal inhibitory concentration,IC50),以等体积的DMSO处理BT549细胞作为对照组。采用比卡鲁胺(150μmol/L)处理BT549细胞后,蛋白质印迹法检测比卡鲁胺对BT549细胞中AR表达水平的影响;FCM法检测对BT549细胞凋亡和细胞周期的影响,Transwell小室法检测对BT549细胞迁移及侵袭能力的影响;最后,再用蛋白印迹法检测细胞中E-上皮钙黏蛋白(E-cadherin)、波形蛋白(Vimentin)、蛋白激酶C(protein kinase C,PKC)、基质金属蛋白酶3(matrix metalloproteinases 3,MMP3)、细胞外信号调节激酶1/2(extracellular signal-regulated kinase 1/2,ERK1/2)及其磷酸化ERK1/2(phospho-ERK1/2,p-ERK1/2)蛋白的表达水平。结果:筛选AR高表达的TNBC细胞株BT549为研究对象;不同浓度比卡鲁胺(20、40、80和160μmol/L)均能抑制BT549细胞的增殖(P值均<0.01),比卡鲁胺对BT549细胞的IC50值为143.9μmol/L。用比卡鲁胺(150μmol/L)处理BT549细胞24 h后,与对照组相比,比卡鲁胺能明显抑制BT549细胞中AR蛋白的表达水平(P<0.01),并诱导BT549细胞发生凋亡(P<0.01),但对细胞周期无明显影响。比卡鲁胺明显抑制BT549细胞的迁移及侵袭能力(P<0.001和P<0.01);上调E-cadherin蛋白的表达水平,下调Vimentin、PKC、MMP3、ERK1/2和p-ERK1/2蛋白的表达水平(P值均<0.05)。结论:AR拮抗剂比卡鲁胺可抑制AR高表达TNBC型BT549细胞的增殖、迁移和侵袭能力,并诱导其凋亡。PKC可能是比卡鲁胺调控BT549细胞增殖和侵袭的关键分子。

Objective:To investigate the effect of androgen receptor(AR)antagonist bicalutamide on the biological functions of AR-positive triple-negative breast cancer(TNBC)BT549 cells,and its possible molecular mechanism.Methods:The TNBC cell lines with high AR expression were screened by Western blotting.The ability of cell proliferation was detected using cell counting kit-8(CCK-8)assay in BT549 cells treated with different concentrations of bicalutamide and calculated the half maximal inhibitory concentration(IC50),BT549 cells were treated with the equal volume of DMSO as the control group.After BT549 cells treatment with 150μmol/L bicalutamide,the effect of bicalutamide on AR expression in BT549 cells was detected by Western blotting.The apoptotic rate and cell cycle distribution were evaluated using FCM,and the migration and invasion capabilities were analyzed using Transwell assay.And the expression of E-cadherin,Vimentin,protein kinase C(PKC),matrix metalloproteinases 3(MMP3),extracellular signal-regulated kinase 1/2(ERK1/2)and phospho-ERK1/2(p-ERK1/2)were detected by Western blotting.Results:TNBC BT549 cells which its high AR expression were selected as cell model.The ability of cell proliferation was inhibited which the BT549 cells treated at the different concentrations of bicalutamide(20,40,80 and 160μmol/L),The IC50 value of bicalutamide on BT549 cells was 143.9μmol/L.After BT549 cells treatment with 150μmol/L bicalutamide for 24 h,compared with the control group,bicalutamide significantly inhibited the expression level of AR protein in BT549 cells(P<0.01),and significantly induced BT549 cells apoptosis rate(P<0.01),but it had no effect on cell cycle.Bicalutamide observably inhibited the abilities of invasion and migration of BT549 cells(P<0.01 and P<0.001).Bicalutamide could increase the expression level of E-cadherin,while decrease the expression levels of Vimentin,PKC,MMP3,ERK1/2,and p-ERK1/2(all P<0.05).Conclusion:AR antagonist bicalutamide inhibits the proliferation,migration,invasion abilities,and induces apoptosis of AR-postive TNBC BT549 cells.PKC may be a key molecule in the regulation of BT549 cell proliferation and invasion by bicalutamide.