Toxicarioside G 诱导自噬调控p53表达影响结直肠癌细胞干性机制研究

Cancer stem cells (CSCs) which play an important role in tumor recurrence and chemotherapy tolerance are an important target in the treatment of cancer. Previous studies found that Toxicarioside G (TCG) decreased the number of CD133+ cells in colorectal cancer cells. It is further showed that the decrease of CD133+ cells was associated with the downregulation of p53 by TCG. Furthermore, it is showed that TCG can induce autophagy in colorectal cancer cells; after inhibition of autophagy, the expression of p53 is restored, suggesting that TCG regulates p53 expression by inducing autophagy, and then affecting the stemness of colorectal cancer cells. In this project, we will use molecular biology, cell biology and molecular bioinformatics and other technologies to further clarify the regulatory effect of TCG on the stemness of colorectal cancer cells; to further confirm that the downregulation of p53 expression by TCG was associated with the stemness of colorectal cancer cells; to clarify both TCG-induced autophagy in colorectal cancer cells and the role of autophagy in the regulation of p53 expression, and finally clarifying the molecular mechanism of TCG regulating p53 by autophagy. This project will elucidate the mechanism of TCG in inhibiting the stemness of colorectal cancer cells, providing the foundation for the development of TCG in treatment of tumor.

肿瘤干细胞(CSCs)是肿瘤复发和产生化疗耐受的主要原因，已成为肿瘤治疗中的一个重要靶点。我们前期研究发现，Toxicarioside G(TCG)减少结直肠癌细胞中的CD133+细胞数量，进一步研究发现TCG通过抑制p53表达降低CD133+细胞数量；我们研究还发现，TCG诱导细胞自噬，抑制自噬后p53表达水平得到恢复，提示TCG通过自噬调控p53表达进而影响肿瘤细胞干性的作用。本项目拟在此基础上，应用分子生物学、细胞生物学和生物信息学等技术，进一步验证TCG对结直肠癌细胞干性的调控作用，明确p53在TCG调控结直肠癌细胞干性中的作用；进一步验证TCG诱导细胞自噬，明确自噬对p53表达的调控作用，阐明TCG通过自噬调控p53表达的分子机制。本项目通过阐明TCG抑制结直肠癌细胞干性的作用机制，为TCG开发为潜在的抗肿瘤药物奠定基础。

目的: 天然化合物 toxicarioside G（TCG），toxicarioside O（TCO）和calotropin（CTP）都具有抗肿瘤作用，但其作用机制还没有完全阐明。方法: MTT实验，BrdU实验,克隆形成实验和LDH释放试验检测细胞活力和增殖；WB和流式细胞仪检测细胞凋亡；Wound healing实验和Transwell 实验用来检测细胞的迁移能力；小鼠肿瘤模型中观察药物在体内的抗肿瘤活性；RT-PCR，WB，免疫荧光染色以及siRNA干扰技术等主要用来探讨这些药物抗肿瘤机制。结果: 研究结果表明 TCG能够抑制结肠癌细胞的生长。TCG主要通过诱导致死性的细胞自噬导致抑制细胞生长，但同时激活具有保护作用的YAP信号通路。TCO体内外也能够抑制肺癌细胞的生长。机理研究表明TCO下调Trop2表达，进而抑制PI3K/Akt通路，从而抑制肿瘤细胞的EMT和迁移，进一步抑制肺癌细胞的增殖，诱导肺癌细胞凋亡。另外，研究也表明CTP在体内外都具有抗结肠癌细胞的作用；同样CTP在抑制结肠癌细胞生长的同时也激活细胞保护机制-YAP信号通路。结论：我们研究表明TCG，TCO和CTP都具有抗肿瘤作用并揭示其作用机理，为这些药物开发成为抗肿瘤的天然药物奠定基础。