解毒祛瘀方通过抑制磷脂酰肌醇-3-激酶/蛋白激酶B信号通路逆转乳腺癌细胞他莫昔芬耐药的机制研究

Effect of Jiedu Quyu recipe on tamoxifen-resistance reversal of breast cancer cells by inhibiting phosphatidylinositol 3 kinase/protein kinase B pathway

目的探讨解毒祛瘀方通过抑制磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)信号通路对乳腺癌细胞他莫昔芬耐药的逆转作用及机制。方法采用低浓度逐步加量诱导法建立乳腺癌他莫昔芬耐药细胞株MCF-7C,并分为空白对照组、解毒祛瘀方(2.5 g/L)组、他莫昔芬(10μmol/L)组、他莫昔芬(10μmol/L)+解毒祛瘀方(2.5 g/L)组、胰岛素样生长因子1(IGF-1)(10μg/L)+他莫昔芬(10μmol/L)+解毒祛瘀方(2.5 g/L)组。采用噻唑蓝法验证MCF-7C细胞的耐药性、解毒祛瘀方的细胞毒性以及对MCF-7C细胞他莫昔芬耐药性的影响。利用流式细胞术检测细胞凋亡率。利用蛋白质印迹法检测各组MCF-7C细胞中PI3K/Akt/雷帕霉素靶蛋白(m TOR)信号通路相关蛋白的表达。结果 MCF-7C细胞对他莫昔芬的耐药指数为7.20;而经解毒祛瘀方(2.5 g/L)协同作用后,MCF-7C细胞对他莫昔芬的耐药指数为2.02。他莫昔芬+解毒祛瘀方组MCF-7C细胞凋亡率明显高于他莫昔芬组[(56.72±7.25)%比(13.24±0.76)%],同时p-PI3K[(0.37±0.06)比(0.68±0.07)]、p-Akt[(0.17±0.02)比(0.51±0.05)]、p-mTOR[(0.10±0.02)比(0.39±0.02)]蛋白表达量也明显低于他莫昔芬组(均P <0.05)。而加入PI2K/Akt通路激活剂IGF-1后,IGF-1+他莫昔芬+解毒祛瘀方组MCF-7C细胞增殖抑制率和凋亡率较他莫昔芬+解毒祛瘀方组均明显降低(均P <0.05)。结论解毒祛瘀方可逆转乳腺癌细胞他莫昔芬的耐药性,其作用机制可能与抑制PI3K/Akt/m TOR信号通路活化有关。

Objective To explore the reversal effect and mechanism of Jiedu Quyu recipe on tamoxifen resistance in breast cancer cells by inhibiting phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt)signal pathway.Methods The tamoxifen-resistant MCF-7C was established by exposure of parental MCF-7 cells and increasing tamoxifen concentrations.MCF-7C cells were divided into control group,TAM(tamoxifen10μmol/L)group,TCM(traditional Chinese medicine,Jiedu Quyu recipe 2.5 g/L)group,TAM+TCM group and IGF-1(insulin growth factor-1,10μg/L)+TAM+TCM group.The tamoxifen-resistance of MCF-7C cells,cytotoxicity of Jiedu Quyu recipe and influence of Jiedu Quyu recipe on tamoxifen-resistance of MCF-7C cells were examined by Methyl thiazolyl tetrazolium(MTT).Flow cytometry was used to detect the apoptosis ratio.In addition,the expression of PI3K/Akt/mammalian target of rapamycin(m TOR)pathway related proteins were analyzed by western blot.Results The resistance index(RI)of MCF-7C cells on tamoxifen was 7.20.Under the synergism of Jiedu Quyu recipe,RI of MCF-7C cells on tamoxifen was 2.02.The apoptosis rate of MCF-7C cells in TAM+TCM group was higher than that in TAM group[(56.72±7.25)%vs(13.24±0.76)%](P<0.05);the levels of p-PI3K[(0.37±0.06)vs(0.68±0.07)],p-Akt[(0.17±0.02)vs(0.51±0.05)],p-mTOR[(0.10±0.02)vs(0.39±0.02)]in TAM+TCM group were all lower than those in TAM group(all P<0.05).The proliferation inhibition rate and apoptosis rate of MCF-7C cells in IGF-1+TAM+TCM group were lower than those in TAM+TCM group(both P<0.05).Conclusion Jiedu Quyu recipe can reverse the resistance to tamoxifen of MCF-7C cells,which may be related with the inhibition of PI3K/Akt/m TOR signal pathway.