中药新药人参皂苷次级苷ALK诱导结直肠癌细胞线粒体自噬及其分子机制

The serious side effects of chemotherapy and its resistant to drug are still unresolved problem in the treatment of colorectal cancer. In our previously study, The secondary generation of panax ginsenosides ALK as new Chinese patent medicine was screened and identified, which was obvious effective for anti-tumor and leukemia. The specific elements and content of ALK were clear and definite, mainly composed of five monomers of F4, Rh4 and S-Rg3, etc. The invention patent of ALK applied has been authorized. For human colorectal cancer, our results showed that ALK significantly inhibited proliferation, migration and invasion of human colorectal cancer cells, furthermore, suppressed growth of tumor tissue in vivo of mouse models. Mitophagy is closely related to the development of tumor. Our preliminary experimental results showed that mitophagy was mediated by ALK in colorectal cancer HT29 cells. The induced mitophagy by ALK is associated with the activation of PINK1-PARK2 signaling pathway, suggesting that ALK-caused mitophagy is involved in inhibition of colorectal cancer cells. But the underlying molecular mechanism is still needs to study. Based on the results, we will further explore the regulation of PINK1-PARK2 signaling pathway by ALK; determine the translocation of autophagy-associated proteins in cancer cells, and the changes of autophagy. Intracellular translocation will be observed by transfection of YFP-PARK2 plasmid into the cells. Construction of mice model with colorectal cancer using Parkin-/- HT29 cells will be used to reveal the role of mitophagy induced by ALK in the treatment of colorectal cancer. Taken together, results from this study will provide novel evidence for the further study of effect target induced by ALK for anti-tumor, also may provide experimental basis for ALK in the treatment of colorectal cancer in the future.

结直肠癌化疗的毒副反应及耐药性仍是尚未解决的难题，前期工作筛选并确定人参皂苷次级苷ALK抗肿瘤和白血病的作用明显，成分含量明确主含F4、Rh4和S-Rg3等5个单体，发明专利被授权。ALK有效地抑制肠癌细胞增殖、迁移和侵袭，并抑制小鼠模型体内瘤体生长。线粒体自噬与肿瘤的发生发展密切相关，预实验显示ALK诱导肠癌HT29细胞线粒体自噬，与PINK1-PARK2信号通路活化有关，提示ALK通过调控线粒体自噬而抑制结直肠癌细胞增殖，但具体机制有待深入研究。在此基础上，本课题拟探讨ALK调控PINK1-PARK2信号通路机制，检测自噬相关蛋白在癌细胞线粒体的转位及自噬流的改变。转染YFP-PARK2质粒，观察其在细胞内的转位。采用Parkin-/- HT29细胞，建立结直肠癌小鼠模型，揭示线粒体自噬在ALK抗结直肠癌中的作用，为深入研究ALK作用靶点提供思路，可为将来治疗结直肠癌提供实验依据。

中药新药人参皂苷次级苷ALK能抑制肠癌细胞生长增殖、降低它们侵袭和迁移，并显著抑制体内成瘤，但相关作用机制并不清楚。本课题研究结果显示ALK引起肠癌细胞线粒体损伤、膜电位下降，同时伴随细胞自噬水平升高，提示线粒体自噬发生。利用激光共聚焦显微镜和电镜技术，我们进一步观察到ALK促进肠癌细胞线粒体自噬泡形成，以及与溶酶体融合。相反，干扰自噬则减弱了ALK降解线粒体膜蛋白和抑制肠癌细胞增殖的能力，提示线粒体自噬具有抗肿瘤功能。进一步深入研究表明ALK活化肠癌细胞PINK1-Parkin信号通路，募集Parkin蛋白到细胞线粒体上并引起线粒体膜蛋白泛素化。利用蛋白组学技术筛选Parkin靶蛋白，GO和KEGG分析显示Parkin靶向结合大量线粒体蛋白分子，进而影响细胞线粒体功能。其中，包括细胞能量代谢关键酶GAPDH，它参与细胞糖酵解过程，为细胞生长提供能量。免疫共沉淀法和激光共聚焦显微术均证实Parkin和GAPDH存在相互作用，并引起GAPDH泛素化。更为重要的是GAPDH参与ALK活化的线粒体自噬，并介导Parkin向线粒体转位。功能上，ALK通过调控GAPDH的非代谢功能而抑制肠癌细胞生长增殖，可能与细胞氧化应激有关。上述结果揭示了GAPDH泛素化是Parkin依赖的线粒体自噬发生的重要分子机制，且与ALK抗肠癌功能密切相关，将有可能发展为一种治疗肠癌的新策略，并为ALK潜在的临床应用提供客观依据。