基于ER/PI3K-AKT-mTOR交互信号通路研究阳和化岩汤抑制乳腺癌内分泌耐药的机制

The cross talk between ER and PI3K-AKT-mTOR signaling pathways is the key mechanism of endocrine resistance in breast cancer. In the previous study on the cross talk between the PI3K-Akt and caspase, JAK-STAT and COX2-VEGF signaling pathways, it was proved that Yanghe Huayan decoction could suppress the cell proliferation, promote the cell apoptosis, and suppress the growth of vessels. In this research, we will establish an animal model of disease and syndrome. Through the tumor growth curve, the protein and mRNA expressions of ERα, ERβ, PI3K, PIP2, PIP3, AKT, mTOR, PTEN and MAPK will be determined, to verify the effect of Yanghe Huayan decoction to suppress endocrine resistance. The cell line with endocrine resistance will be established through transfection in vitro. By using MTT, flowcytometry, wound healing and soft agar tests, cell proliferation, apoptosis, migration and clone will be tested. WB and qPCR will be used to test the expression of key proteins. Through the above tests, the effect of the Yanghe Huayan decoction on the cross talk between ER/mTOR signaling pathways will be explored. After the mTOR gene is silenced, the cell biological behavior, proliferation and the expression of target proteins will be studied to determine the key target of drugs. The research results will provide a sound support for using Yanghe Huayan decoction to treat breast cancer during the endocrine treatment.

ER/PI3K-AKT-mTOR信号通路交叉对话是乳腺癌内分泌耐药的关键机制。前期研究从PI3K-Akt与caspase、STAT、VEGF交叉信号通路证实阳和化岩汤具有抑制细胞增殖、促进凋亡、抑制脉管生成等作用。本课题将阳和化岩汤用于抑制乳腺癌内分泌治疗耐药。体内实验建立病证结合动物模型，通过肿瘤生长曲线、检测ERα、ERβ、PI3K、PIP2、PIP3、AKT、mTOR、PTEN、MAPK蛋白和mRNA表达，验证阳和化岩汤抑制内分泌耐药作用。体外实验质粒转染构建内分泌耐药细胞株，通过MTT、流式细胞术、划痕实验、软琼脂实验评价细胞增殖凋亡和迁移克隆能力，WB、qPCR检测关键因子的表达，探讨该方对ER/mTOR交叉信号通路的影响；基因沉默mTOR，比较细胞生长、增殖凋亡及相关因子表达的差异，明确mTOR为关键靶点。研究结果将为阳和化岩汤在乳腺癌内分泌治疗期的临床应用提供有力依据。

乳腺癌发病率居高不下，其中激素受体阳性乳腺癌占50%以上，内分泌治疗是最有效方法，但常出现耐药问题，因此有必要同期结合中医药治疗。根据乳腺癌内分泌治疗期常见不良反应及症状表现，中医认为其主要病因病机为“肝肾不足、肝郁脾虚”，其中“肾虚”为发病之根本，这为阳和化岩汤的临床应用提供理论基础。mTOR作为PI3K-Akt通路重要下游基因，也是乳腺癌内分泌治疗耐药的关键靶基因，成为本课题研究的重点。.本研究采取裸鼠卵巢切除+乳腺癌细胞株原位移植方法建立病证结合肾虚型乳腺癌内分泌耐药动物模型，采用他莫昔芬低剂量长期诱导和基因沉默技术构建HR阳性内分泌耐药乳腺癌细胞株MCF-7R和MCF-7R/mTOR-，探讨阳和化岩汤对内分泌耐药乳腺癌的作用机制。结果显示：①阳和化岩汤联用依维莫司可有效抑制人乳腺癌耐药细胞MCF-7/TAM-R的增殖、迁移作用，并诱导细胞凋亡，细胞周期被阻滞于G1期，联合依维莫司作用优于单药；②阳和化岩汤明显影响MCF-7R细胞的增殖和克隆能力，促进细胞凋亡，下调ER、PI3K、AKT、mTOR、p-mTOR表达；对MCF-7R细胞进行mTOR基因敲除，基因敲除组与对照组对比，MCF-7R/mTOR-处于G1期和S期的细胞减少，同时基因敲除组细胞增殖减缓；联合依维莫司对mTOR mRNA及蛋白表达有明显抑制作用；③免疫荧光法和QPCR技术证实阳和化岩汤联合依维莫司对裸鼠肿瘤的血管生成抑制率高于单药，能够抑制PI3K、Akt、mTOR、ER mRNA的表达，说明中药联合依维莫司预防乳腺癌的内分泌治疗耐药有良好的疗效。.本研究通过阳和化岩汤对HR阳性乳腺癌内分泌治疗耐药的关键信号通路之一P13K-Akt-mTOR的观察,从体内实验、体外实验证实阳和化岩汤对乳腺癌内分泌耐药细胞具有干预作用，并对P13K-Akt-mTOR信号通路具有明显影响作用，mTOR成为其关键作用靶点，从而揭示阳和化岩汤抑制内分泌治疗耐药的作用机制，全方位诠释中医药的综合调理作用，对推动乳腺癌内分泌治疗同期结合中医药辅助治疗具有重要意义。