中药槐耳清膏通过EphB4调控PI3K/AKT/mTOR信号通路介导乳腺癌EMT机制的研究

Distant metastasis of breast cancer is the major factor affecting prognosis, epithelial-mesenchymal transition(EMT) is key step of metastasis of breast cancer. Qi-blood deficiency and blood stasis are two main syndromes of advanced breast cancer in traditional Chinese medicine, so Fuzheng Guben should be the main way for the treatment of breast cancer. Coincidentally, the function of Huaier is known as Huoxue Xiaozheng, Fuzheng Guben, and has been verified clinically. Although Huaier is used in clinical medicine wildly, it is still not clear about the acting constituent in Huaier which obstruct EMT of breast cancer and the main mechanism. The preliminary study of our research group found that ①Huaier can inhibit proliferation of BCCs; ②the over-expression of protein EphB4 can promote the EMT of breast cancer by activating the pathway of PI3K/AKT/mTOR. According to previous researches, we speculate that Huaier can inhibit the activation of PI3K/AKT/mTOR pathway by down-regulating the expression of EphB4 to suppress EMT of breast cancer, in order to control the metastasis of it. To identify the hypothesis, we design this experiment which depends on the integrative theory of traditional Chinese medicine together with western medicine system. In which, we protocol to use breast carcinoma cell lines and mice models of breast cancer metastases, so as to discuss the regulatory mechanism of Huaier for inhibits the EMT of breast cancer, reveal the scientific connotation about how Huaier against breast cancer EMT and analyze the effective constituent of it. The aim of the experiment is to find out new target site and new drug which may act to reduce the metastasis of breast cancer, consequently to provide a new way for prevention and treatment of breast cancer by traditional Chinese medicine.

乳腺癌远处转移是影响其预后的主要原因，上皮-间质转化(EMT)是导致转移的关键环节。中医认为气血两虚、瘀血阻滞是乳腺癌的主要症型，治疗上应以扶正活血为主。槐耳清膏具有活血消癥、扶正固本之功效，并已应用于乳腺癌临床治疗，但其活性成分，对乳腺癌转移的影响及具体调控机制不明。课题组前期研究发现：①槐耳清膏抑制乳腺癌细胞(BCCs)增殖，控制乳腺癌EMT；②EphB4蛋白过表达可激活PI3K/AKT/mTOR信号通路促进乳腺癌EMT。结合文献和前期研究基础，我们推测：槐耳清膏可能通过下调EphB4蛋白表达抑制PI3K/AKT/mTOR信号通路激活，从而抑制乳腺癌EMT，控制乳腺癌转移。为验证假说，本项目拟在乳腺癌细胞系和小鼠乳腺癌移植瘤模型上，探讨槐耳清膏对乳腺癌EMT的调控机制，揭示槐耳清膏抗乳腺癌EMT的科学内涵及其有效成分，以寻求抑制乳腺癌转移的新靶点和药物，为中医药防治乳腺癌提供新思路。

为验证槐耳清膏抑制乳腺癌EMT的作用，我们检测槐耳清膏血清培养的不同乳腺癌细胞系（MCF-7、MDA-MB-231）与正常血清培养的细胞系中EphB4、PI3K信号通路蛋白及EMT相关标志物的表达情况，同时检测BCCs 侵袭和迁移能力的变化。结果显示口服槐耳颗粒的患者，术后肿瘤组织中上皮标志物表达增加，间质细胞标志物表达降低。随着槐耳清膏工作液浓度的增加、处理时间的延长，其对乳腺癌细胞系的抑制率逐渐增强。同时我们应用美国TCGA数据库中将近1000名乳癌患者的基因表达数据进行深入挖掘，发现EphB4在乳腺癌HER2阳性亚型中，表达明显增高。接下来，我们利用大连医科大学附属第二医院乳腺外科及乳腺肿瘤科收集的肿瘤标本库及组织芯片技术检测208例乳腺癌患者癌症组织中的EphB4蛋白表达水平（应用免疫组织化学方法），得到与TCGA数据库相似的结论，均发现EphB4在乳腺癌HER2阳性亚型中表达水平较高，并具有统计学差异。通过我们科室的长期随访，比较EphB4高表达组与低表达组DFS和OS生存数据，发现低表达EphB4乳腺癌患者DFS和OS均明显延长。为了深入研究EphB4所产生的预后影响，我们采用乳腺癌细胞系MDA-MB-231 和MCF-7为模型，进行深入的机制研究。通过敲低EphB4（siRNA），发现乳腺癌细胞系增殖能力受到抑制，迁移能力下降。乳腺癌细胞中pAKT及m-TOR水平均较对照组明显降低，预示下调EphB4表达可抑制PI3K/AKT/mTOR信号通路。对槐耳清膏及顺铂对三阴性乳腺癌细胞系生物学行为的研究结果显示：槐耳清膏和顺铂对三阴性乳腺癌细胞系具有协同作用。较低浓度的槐耳清膏和顺铂联合应用可以协同抑制TNBC细胞系MDA-MB-231和BT-549细胞系的增殖、侵袭和迁移、使细胞周期停滞在G2/M期以及促进TNBC细胞系的细胞凋亡及DNA损伤修复。应用网络药理学探索槐耳清膏活性成分的作用靶点。以上研究结果为探讨槐耳清膏对乳腺癌EMT的调控机制，揭示槐耳清膏抗乳腺癌EMT的科学内涵及其有效成分，引领了中医药防治乳腺癌新方向。