基于PI3K/AKT与ER信号通路的串话研究益气小复方逆转乳腺癌Tamoxifen耐药的作用机制

Tamoxifen (TAM) resistance in breast cancer cells was closely related with ER and PI3K/AKT signaling pathways. In our previous work we already demonstrated that Yiqi formula and its effective components resisted the proliferation and migration of breast cancer cells by modulating the PI3K/AKT signaling pathway. In preliminary experiment, we has found that Yiqi formula improved ER expression and promoted cell apoptosis in TAM resistant breast cancer cells. Based on the above understanding, we hypothesize that Yiqi formula may restore TAM resistance breast cancer cell to dependence on ER signaling pathway, through regulating the PI3K/AKT signaling pathway, to reverse TAM resistance in breast cancer cell. In this study, MCF-7/TAM resistant breast cancer cells will be treated with Yiqi formula, then the cell proliferation, apoptosis and migration of breast cancer will be observed. The expression of ER, PI3K and AKT proteins expression will be detected by using western blot, and cell cycle-, apoptosis- and migration-related gene expressions will be detected by using real-time PCR. The target point of Yiqi formula will be verified by siRNA in vitro. And in vivo breast cancer model will be used to confirm the effect of Yiqi formula on reversal of TAM resistance. The results of this study which may preliminary clarify the effect and mechanism of Yiqi formula on reversal of TAM resistance in breast cancer, and provide certain theoretical guidance and experimental basis for the treatment of breast cancer.

乳腺癌他莫昔芬（TAM）耐药与ER和PI3K/AKT等通路密切相关。前期证实，益气小复方及其有效组分可调控PI3K/AKT通路抑制乳腺癌细胞的增殖和迁移。预实验发现TAM耐药的乳腺癌细胞经益气小复方干预后，ER表达上调，且细胞凋亡增加。因此，本项目组提出“益气小复方可通过调控PI3K/AKT通路，恢复TAM耐药乳腺癌细胞对ER信号的依赖，从而逆转其耐药”的科学假说；拟采用益气小复方干预MCF-7/TAM耐药细胞，观察细胞的增殖、凋亡及迁移改变，采用western blot检测ER和PI3K信号关键蛋白的表达，PCR检测细胞周期、凋亡及迁移相关基因的表达水平；在明确作用靶点后，进一步采用siRNA技术，验证益气小复方的作用靶点；及采用乳腺脂肪垫注射细胞株动物模型，从体内验证益气小复方逆转TAM耐药的作用；初步阐明益气小复方逆转乳腺癌TAM耐药的作用机制，为临床提供一定的理论指导和实验依据。

他莫昔芬是治疗ER阳性乳腺癌最有效且应用时间最长的一线内分泌治疗药物，能够有效降低ER阳性乳腺癌患者的死亡率和复发率。然而，TAM耐药的出现不仅不利于患者的预后，还会增加药物的不良反应。中医药，特别是益气类药物在改善乳腺癌患者预后的临床疗效已得到业界认可，但其具体的分子机制还不甚明了。本实验采用高浓度短时间4-羟tamoxifen冲击法建立了人乳腺癌MCF-7-TamR耐药株，通过MTT实验、流式细胞分析、迁移实验分析益气小复方对乳腺癌MCF-7-TamR耐药株的增殖和迁移影响。实验结果表明，益气小复方可抑制TAM耐药的乳腺癌细胞PI3K、AKT、mTOR的磷酸化，减少抗凋亡蛋白BCX-XL的表达，增加促凋亡蛋白BAX、Cleaved Parp的表达，增加TAM耐药乳腺癌细胞的凋亡，抑制TAM耐药乳腺癌细胞的增殖、迁移及体内生长。初步阐明益气小复方通过调控PI3K/AKT/mTOR信号通路逆转乳腺癌TAM耐药的作用机制，明确益气小复方的作用靶点，为临床采用益气扶正法预防乳腺癌TAM耐药提供一定实验依据。