银杏酚的制备及其对肝癌细胞表皮生长因子受体活性的抑制和机制探讨

Ginkgolic acids are the active compounds with anti-cancer effect from Ginkgo biloba. The previous studies show that ginkgolic acids can be decarboxylated to give bilobols, and bilobols are stronger than ginkgolic acids on the anti-cancer activity and stability. The preliminary study on the anti-tumor mechanism displays that bilobol 17:1 can inhibit proliferation of hepatocarcinoma cells and induce their apoptosis. Moreover, bilobol inhibits the subcutaneous hepatic carcinoma tumor growth in nude mice. In addition, our previous studies revealed that bilobol 17:1 can block the signal transduction of the MAPK-ERK pathway by preventing the EGF (epithelial growth factor) -induced activation of EGFR (EGF receptor). In this project, the two isomers of the bilobol 17:1 will be separated and purified. The anti-cancer activity of the two bilobol 17:1 monomers will be compared. And then, we will examine the inhibition effect of bilobol 17:1 monomer on the EGFR mediated signal transduction pathway and dissect the molecular mechanisms. We will evaluate the inhibition effects of bilobol 17:1 monomer on different mutant EGFR backgrounds. Moreover, we will compare the tumor inhibition effect of bilobol 17:1 monomer to other EGFR inhibitors. The tumor inhibition effect of bilobol 17:1 monomer on hepatic carcinoma-burden nude mice will be examined and the immunity effect will be also evaluated. In all, the results of this project will help clarifying the mechanisms by which bilobol 17:1 monomer inhibits tumorigenesis and provide theoretical and experimental basis for the research and development of anti-cancer drug, and provide direction for the design of new EGFR inhibitors.

银杏酸是银杏中具有抗肿瘤作用的活性化合物。前期研究发现银杏酸脱羧后产生的银杏酚显示出比其更强的抗肿瘤活性和稳定性。初步的抗肿瘤机制研究显示：银杏酚17:1可抑制肝癌细胞增殖、诱导细胞凋亡，抑制小鼠的肿瘤生长；可通过抑制表皮生长因子受体（EFGR）活性，阻断MAPK（ERK）通路的信号转导，具有抗癌作用。本项目将在分离银杏酚17:1的异构体基础上，比较其单体的活性作用，进一步研究银杏酚17:1单体对EGFR介导的信号转导通路的抑制作用并探讨其分子机制；针对EGFR的多种突变，观察银杏酚17:1单体对EGFR的抑制作用，比较其与现有EGFR抑制剂作用的异同点；建立单一剂量银杏酚治愈荷瘤肝癌的小鼠模型，试验银杏酚17:1单体抗肿瘤作用及对免疫功能的影响。本研究将为阐明银杏酚的抗癌机制及抗癌药物的研发提供理论和实验依据,为新型EGFR抑制剂的设计提供方向。

本项目对银杏酚C17:1两个异构体结构和活性的确认及其对肝癌细胞表皮生长因子受体（EGFR）活性的抑制作用和机制进行了系统的研究。首先，在前期对银杏酚C17:1单体的制备、纯化、鉴定的基础上，分离其两个异构体，研究发现双键在C10的异构体具有相对较强的抗肿瘤活性，两种异构体不仅能与细胞膜上相关蛋白受体结合，还能进入细胞，并能够与细胞核内的DNA嵌入式结合。其次，研究证明银杏酚C17:1能够抑制肝癌细胞EGFR介导的多条信号通路，如MAPK-ERK/c-Jun、PI3K、JAK/STAT、IP3等在肿瘤发生发展中常见的、重要的一些信号通路，均能被银杏酚抑制；同时，银杏酚能够抑制EGFR上酪氨酸位点突变，故能够抑制癌细胞的增值、迁移、侵袭等一系列生物学活性。更为重要的是，在发现银杏酚有一定抗肿瘤作用的同时，发现其能够很好的辅助顺铂的化疗作用，特别是EGFR活化的情况下，在应用较小剂量顺铂作用的同时，两者联用明显加强了对肿瘤细胞的抑制作用，同时增强了机体的免疫能力，机制是从癌细胞自噬、凋亡的角度进行研究的。研究结果表明，银杏酚C17:1单体能够显著抑制EGFR的活性，通过阻断EGFR的激活，抑制EGFR介导的多条信号通路，进而抑制肝癌细胞的增值等生物学活性，促进细胞凋亡；并有希望发展成为顺铂抗肿瘤的辅助用药。