新型汉防己甲素衍生物H1对耐5-氟尿嘧啶肝癌的抗耐药作用及其机理研究

5-fluorouracil (5-FU) is remain the first-line chemotherupeutic drug for treamtment of hepatocellular carcinoma. But when the hepatocellular carcinoma cells frequently exposure to 5-FU, it will lead to cancer drug resistance, and affect the therapeutic efficacy of chemotherapy. It is also the main obstractle of 5-FU in clinical application. Further studying on the mechanisms of hepatocellular carcinoma cells resistant to 5-FU and looking for novel compounds with overcoming hepatocellular carcinoma resistance activity may be contribute to solve this issue. Therefore, from gene expression profiles to exlore the mechanisms of hepatocellular carcinoma cells resistant to 5-FU by using gene expression chip. We firstly found that the abnormal activated Fyn-p38MAPK and JAK-STAT pathways may be associated with 5-FU resistance. Then, we will focus on the biological function of these pathways in Bel7402/5-FU cells by RNAi ,specific inhibitors or transfecting vectors. H1 is a novel derivatives of Tet. We will evaule the anti-MDR effect of H1 by establishing Bel7402/5-FU cells in vitro model and 5-FU resistant in vivo model, expolre its targeting molecular on the apoptoic sensitivity, cellular survival pathway, Fyn-p38MAPK, JAK-STAT pathways. However, these studies will contribute to understand the mechanisms of hepatocarcinoma resistant to 5-FU and will provide novel idea and therapeutic target for overcoming hepatocarcinoma resistance.

5-FU仍是肝癌化疗的一线药物，但长期应用存在耐药性问题，已成为影响其化疗疗效的最大障碍。深入研究肝癌耐5-FU机制并寻找具有抗肝癌耐药活性的先导化合物以期解决肝癌耐药性问题。因此，本项目首先采用基因表达芯片技术从基因组表达框架探讨Bel7402/5-FU细胞耐药机制，首次发现Fyn-p38MAPK、JAK-STAT异常活化可能参与了5-FU耐药过程。采用RNA干扰、特异性抑制剂或表达载体研究Fyn-p38MAPK、JAK-STAT信号通路在肝癌耐5-FU中的作用。以Bel7402/5-FU细胞及建立体内耐5-FU裸鼠移植瘤模型评价新型汉防己甲素衍生物H1抗肝癌耐5-FU作用，围绕细胞凋亡敏感性、细胞存活通路及Fyn-p38MAPK、JAK-STAT通路等，探讨H1发挥抗肝癌耐药作用的潜在靶点与信号通路。本课题为揭示肝癌耐5-FU的分子机制、寻找新型抗肝癌耐药药物提供新思路与新靶点。

本课题采用基因表达芯片技术结合RT-PCR及蛋白质免疫印记技术确证p-STAT3在耐药性肝癌BEL7402/5-FU细胞呈高表达状态。采用特异性小分子干扰RNA敲除STAT3基因后能部分恢复BEL7402/5-FU细胞对5-FU的敏感性。在此，我们首次发现STAT3分子的异常活化参与了BEL7402/5-FU细胞耐药性的产生。. 继而采用肝癌耐5-FU模型评价了新型汉防己甲素衍生物H1的抗耐药作用。MTT结果显示BEL7402/5-FU细胞耐药因子为161.1，具有良好的耐药表型。H1不仅能抑制BEL7402细胞增殖还能有效抑BEL7402/5-FU细胞增殖，IC50分别为0.89±0.31(µM) 和5.26±1.09(µM)， 耐药倍数仅为6.0，表现出良好的抗耐药活性。H1处理耐药肝癌细胞BEL7402/5-FU及其亲本BEL7402细胞24小时后, 能够有效启动耐药表型肝癌细胞BEL7402/5-FU的线粒体凋亡通路，进而诱导耐药细胞发生不可逆性凋亡，直接杀伤耐药表型的肝癌细胞。此外，我们也发现H1能显著抑制肿瘤细胞的存活信号通路的关键介导分子，如p-AKT, p-ERK。. 值得注意的是，本研究首次发现汉防己甲素衍生物H1能时间与浓度依赖性的诱导PUMA蛋白表达。无论是在敏感性细胞还是耐药性细胞H1均可显著上调PUMA蛋白表达，进而启动肿瘤细胞线粒体凋亡通路。调控机制研究揭示，H1调控PUMA分子机制可能与下调AKT/FoxO3a轴信号转导通路有关。可见，线粒体凋亡调控分子PUMA可能在H1发挥抗耐药过程中具有重要作用。此外，研究还发现H1显著抑制NF-KB与STAT3活性，这些因素也是H1发挥抗耐药作用的原因之一。