黄连素调控PTEN/Akt/FOXO通路诱导非小细胞肺癌凋亡的作用机制研究

Targeted cancer theray is one of the best strategies for the treatment of advanced non-small cell lung cancer (NSCLC). Akt plays a pivotal role in the initiation and progression of NSCLC and was identified as one of the driver mutations in NSCLC,so recent studies have highlighted the ability of oncogenic AKT. Bererine (BBR) is an isoquinoline derivative alkaloid isolated from many medicinal herbs, such as Rhizoma coptidis and Cortex phellodendri. It is widely used in traditional Chinese medicine for anti-tumor acitivity, but its efficacy in the treatment of NSCLC and the regulation on Akt signaling pathway have not been reported. Based on our previous studies, we found that BBR could induce apoptosis in Akt activation/PTEN deficiency A549 cells concomitance with Forkhead (FOXO) inhibition. In this study, we try to demonstrate that BBR could induce apoptosis and inhibition of Akt pathway in Akt activation NSCLC lines, such as A549, H1299, PC9, SPC-A1, H460, Calu-3, et al. In vitro, we used transfection and siRNA interference of Akt/FOXO gene to dectect the apoptosis on BBR. And we constructed Akt overexpression transgenic mices to further comfirm the correlation between BBR and Akt pathway in vivo. We expect to provide a new strategy for NSCLC treatment by targeting PTEN/Akt pathway with a low-toxic, efficient and natural traditional Chinese drug.

靶向治疗是晚期非小细胞肺癌(NSCLC)最佳治疗策略，2011年研究发现Akt是NSCLC驱动基因之一，针对该途径的靶向治疗一直是研究热点。黄连素(bererine,BBR)是从植物中提取的单体异喹啉类生物碱，有抗肿瘤药理活性。BBR对信号通路Akt的调控作用尚未见报道。本课题组前期研究发现BBR可诱导Akt过表达/PTEN缺失的A549细胞凋亡，对Akt下游叉头转录因子(Forkhead/FOXO)有明显抑制作用。本课题拟进一步观察BBR对多种Akt高磷酸化的NSCLC细胞株诱导细胞凋亡的作用以及Akt下游底物磷酸化水平；探讨激活型Akt/FOXO基因转染对BBR诱导凋亡影响；siRNA干扰Akt/FOXO阐明BBR作用靶点；构建Akt过表达转基因小鼠，体内验证BBR抗肿瘤活性与作用机制，为使其开发成针对NSCLC PTEN/Akt途径的低毒、高效、天然靶向治疗药物提供重要的实验室依据。

肺癌是世界上发病率和死亡率最高的恶性肿瘤，非小细胞肺癌占肺癌的80%，约70%的非小细胞肺癌患者确诊时已发生局部或远处转移，预后很差。从天然化合物中寻找安全、有效、低毒的抗肿瘤药物已成为抗肿瘤药物的重要发展战略之一。黄连素（Bererine, BBR）是从植物中提取的单体异喹啉类生物碱，有抗肿瘤药理活性，但BBR抑制肺癌增殖、转移的潜在作用及相关机制尚未见报道。我们本课题组前期研究发现BBR可诱导Akt过表达/PTEN缺失的A549细胞凋亡，抑制细胞增殖, TGF-β1能诱导上皮间叶转化（Epithelial-to-mesenchymal transition, EMT）的发生，促进肺癌细胞的迁移和侵袭。我们进一步通过Transwell实验测定BBR对肺癌细胞的迁移和侵袭能力，QRT-PCR和Western blotting方法检测BBR对肺癌细胞中EMT相关蛋白表达的影响，动物实验检测BBR对肺癌异体移植瘤的作用。结果发现BBR能通过抑制E-cadherin，促进Vimentin、Slug、Snail的表达来抑制EMT的发生，从而抑制了肺癌细胞的迁移和侵袭，且体内实验发现BBR能抑制肺癌异体移植瘤的生长。因此，BBR能有效抑制肺癌的增殖、诱导其凋亡，并可能作用于EMT抑制肺癌的迁移和侵袭。