淫羊藿总黄酮及其代谢产物调控JAK2/STAT3通路逆转非小细胞肺癌侵袭转移的机制研究

Non-small cell lung cancer (NSCLC) is characteristic with strong invasion and early metastasis. JAK2/STAT3 signal pathway is important during the invasion and metastasis in NSCLC. The pathway occupies the focus point of multiple signaling pathways. It induced tumor invasion and metastasis through promoting the extracellular matrix degradation and angiogenesis and is expected to become a new target for the treatment of lung cancer. The anticancer drugs of traditional Chinese medicine Epimedium has entered Phase II clinical test. It can block metastasis of lung cancer, and prolong the survival time of tumor-bearing mice significantly, but its mechanism of action and material basis is unknown. The project focused on the JAK2/STAT3 pathway and major aspects during NSCLC invasion and metastasis. Lung adenocarcinoma cell line A549 and highly metastatic human lung cancer cell line 95D were applied to explore the mechanism of Epimedium Flavonoids and its metabolites to inhibit cell invasion migration. A computer docking technology will forecast the target protein and the in vivo imaging of mouse lung cancer xenograft model will reveal the material basis. The project will help to further understand the JAK2/STAT3 pathway in invasion in NSCLC, and to provide a scientific basis and theoretical guidance for the clinical application of Epimedium Flavonoids.

非小细胞肺癌(NSCLC)具有侵袭力强，极易发生早期转移的特点，JAK2/STAT3信号通路的失调控是NSCLC侵袭转移的重要调节机制，该通路处于多种信号途径的聚焦点，通过促进胞外基质降解以及血管生成诱导肿瘤侵袭转移，有望成为肺癌治疗的新靶标。淫羊藿中活性单体成分淫羊藿素抗乳腺癌的药物研究已进入二期临床，淫羊藿总黄酮能有效阻滞肺癌的转移，显著延长荷瘤小鼠的生存期，但是其作用机制和物质基础不明。本项目以JAK2/STAT3通路为切入点，针对肺癌转移的主要环节，以肺腺癌细胞株A549和人高转移肺癌细胞株95D为模型，探讨淫羊藿总黄酮和其代谢产物体外抑制细胞侵袭迁移的作用机制，结合计算机对接技术预测作用靶蛋白；建立活体成像的小鼠肺癌移植瘤模型，揭示其发挥药效的活性代谢产物。本项目将进一步了解JAK2/STAT3通路在NSCLC转移过程中的作用，并为淫羊藿抗肺癌的临床应用提供科学依据和理论指导。

上皮间质细胞转化（EMT）是肿瘤转移的关键步骤，以细胞粘附、极性、上皮性标记物损失为特征同时伴随间充质细胞运动性增加。炎症微环境诱导的转移被认为是癌症治疗期间的重大挑战，炎症微环境可以通过促进EMT进程来促进肿瘤的转移。本课题研究的宝藿苷I（Baohuoside I）是中药淫羊藿的有效单体成分之一，也是淫羊藿黄酮在体内的主要代谢产物之一，动物体内实验结果表明宝藿苷I具有一定的抗肿瘤侵袭的活性，进一步研究发现宝藿苷I可调控Akt/NFκB通路，在不引起明显细胞凋亡的剂量即可抑制炎症微环境下非小细胞肺癌细胞的迁移和EMT。本课题主要是应用数据库靶点预测、Western blotting、siRNA、EMSA、免疫共沉淀、激光共聚焦、质粒转染等方法，验证了炎症微环境条件下宝藿苷I对EMT标志物钙粘蛋白、神经钙粘蛋白、波形蛋白、锌指转录因子Snail及其关键通路的影响。宝藿苷I阻遏了炎症因子TNF-α诱导的NF-κB活化和IκBα的磷酸化，并且抑制NF-κB的DNA结合活性。此外，我们设计了自组装混合胶束（TPGS-Solutol HS 15复合物）来包载宝藿苷I，以提高其溶解性并抑制外排，从而提高其生物利用度和抗肿瘤的效果。在裸鼠肺部肿瘤模型和皮下移植瘤模型中，免疫组化和活体成像实验证实宝藿苷I混合胶束明显提高了宝藿苷I抑制肿瘤的效果。本课题阐明了宝藿苷I在Akt/NFκB调节肿瘤细胞EMT平衡中的作用，揭示了宝藿苷I抗肿瘤作用的新机制。