AZD-8055与自噬抑制剂联合应用促进胆管癌细胞凋亡及抑制转移的机制

Cholangioccarcinoma (CCA) is a malignant neoplasm, that derive from bile duct epithelium with a high degree of malignancy and poor prognosis. The PI3K/AKT/mTOR signaling pathway which regulates normal cellular function, including cell proliferation, differentiation, apoptosis and autophagy, plays critical roles in variety of tumor. Previous studies has shown that the overexpression of mTOR may involve in tumor metastasis . Thus,the mTOR inhibitor may be works as an useful therapeutic strategy in CCA. At present, as one of mTORC1 inhibitor in clinic application , Rapamycin can only repress the expression level of mTORC1. What's more, it is easy to cause drug resistance due to the feedback induction of AKT ser473 phosphorylation.However, AZD-8055 which is a novel multitarget inhibitor of PI3K and mTORC1/C2 can suppresses the phosphorylation of Akt to reduce drug resistance.Our previous studies also demonstrated that multitarget mTOR inhibitor(NVP-BEZ235) can induce autophagy. Autophagy can induce the development of malignant, and also works as one way for malignant cells to escape from death.Therefore, In this study we use the AZD-8055 with autophagy inhibitor chloroquine combination therapy to treat human cholangiocarcinoma cell lines and animal models of cholangiocarcinoma. To explore the potential mechanism of autophagy inhibition and apoptosis promoting by molecular biology method and animal model experiments, and give more trail for further study.

胆管癌是来源于胆管上皮细胞的肿瘤，其恶性程度高、预后差。研究表明，PI3K/AKT/mTOR 信号通路介导细胞增殖、分化、凋亡及自噬，在多种恶性肿瘤中起关键的调控作用。胆管癌中mTOR过表达，并与癌的恶性程度及转移相关，提示mTOR的抑制剂在胆管癌治疗中可能起积极作用。目前用于临床的雷帕霉素只能抑制mTORC1，而mTORC2引起AKT的负反馈性磷酸化。新型的mTOR多靶向抑制剂AZD-8055可抑制AKT的磷酸化，减少药物抵抗性。我们前期研究发现， mTOR多靶向抑制剂可诱导胆管上皮细胞自噬。自噬是恶性肿瘤细胞逃避死亡的一种方式,是促肿瘤因素。因此本项目采用AZD-8055与自噬抑制剂chloroquine联合用药，通过多种分子生物学方法及动物实验，探讨其对人胆管癌细胞株及胆管癌动物模型的抑制自噬、促进凋亡作用，阐明其分子机制，为胆管癌的临床治疗提供新的理论基础和实验依据。

项目的背景.胆管癌是具有不良预后的肝胆的恶性肿瘤。手术切除和肝移植是早期胆管癌的最佳治疗方式，但手术后常伴发局部复发及转移，其5年生存率不足30%。AZD-8055是一种新型的mTOR激酶的抑制剂，可同时抑制mTORC1和mTORC2复合物，有效抑制PI3K/AKT/mTOR信号通路。总所周知，氯喹是一种比较安全的抗疟疾药物，具有弱化溶酶体内酸性环境，稳定溶酶体膜的功能，阻滞自噬流等功能。他能增强多种化疗药物对肿瘤细胞的杀伤作用。截至目前为止尚无关于mTOR通路的抑制剂与自噬抑制剂在胆管癌研究方面的报道。..主要研究内容.本项目主要研究AZD-8055与氯喹联合应用对胆管癌细胞增殖及迁移的机制研究，通过体内外实验观察两种药物联合应用对胆管癌细胞的增殖、转移、EMT进程以及体内肿瘤形成的抑制作用，并阐明其分子机制。..主要结果.实验结果显示，AZD-8055与氯喹联合用药相比单独用药组对人胆管癌HuCCT1、RBE细胞的增殖活力、克隆形成能力、横向和纵向迁移能力、EMT进程以及核瘤的形成均具有更明显的抑制作用。联合用药组比单独用药组相比细胞中凋亡小体明显增多。而自噬相关蛋白Beclin 1、ULK1蛋白表达下降，而LC3Ⅱ、p-62、p-ULK的表达上调。蛋白印迹实验结果显示，p-akt、p-4EBP1蛋白表达在AZD-8055和CQ联合用药后明显降低，而其总蛋白表达变化不明显。..关键数据及其科学意义.1.AZD8055与氯喹联合应用可以抑制胆管癌细胞的增殖与迁移及上皮间质转化的进程，其机制与抑制PI3K/Akt信号通路相关；2. AZD-8055与氯喹联合应用抑制细胞增殖与阻滞自噬流，增加胆管癌细胞的凋亡水平相关。.阐明联合用药对胆管癌增殖、迁移及EMT进程的抑制作用及其分子机制，为胆管癌的临床治疗提供新的用药选择。