木犀草素调控结直肠癌细胞自噬对化疗的减毒增敏作用机理研究

Protective autophagy is a new mechanism for colorectal cancer chemotherapy failure. The most prominent problem of synthetic chemoprevention agents is the poor selectivity and side effect. With the support by the NSFC (81374018), our group found that luteolin enhanced the effect of 5-Fu-induced apoptosis in several colon cancer cell lines. This result suggested that targeting autophagy inhibition could be a promising strategy during 5-Fu chemotherapy in CRC. However, since the autophagy is related with the activation of extranuclear p53, but p53 mutation have been found to exist in more than fifty percent in CRC patient. In this study, combined with p53 knock out cell line、RNAi、and apoptosis assay to further investigate the inhibition effect of luteolin on autophagy. Furthermore, xenografts in nude mice will be used to confirm the role of autophagy in vivo. Taken together, this study will not only show for the first time that the new effect of luteolin, but also provides a theoretical basis for the combination of Chinese and Western medicine in clinic.

保护性自噬是结直肠癌化疗失败的新机制，临床研究证实合成类自噬抑制剂可明显提高化疗疗效，但该类化合物选择性差、毒副作用大。我们前期及国家自然基金小额资助项目（81374018）研究发现，木犀草素在荷瘤裸鼠模型上可以明显增强5-Fu化疗疗效，低浓度的木犀草素可显著促进5-Fu诱导耐药的结直肠癌细胞凋亡，初步的机理研究证实其机制可能与抑制自噬有关。5-Fu诱导自噬与核外p53激活有关，但50%结直肠癌细胞存在p53基因突变，为进一步探讨和明确木犀草素新的作用机理，本课题拟应用p53基因敲除细胞模型、EMSA、细胞凋亡检测、RNAi、信号通路阻断实验等技术研究木犀草素调节的自噬信号通路，阐述化疗增敏是否与p53存在形式有关；并在此基础上，应用荷瘤裸鼠模型进行木犀草素化疗增敏的药效学评价。本项目将为研究中药的“增效减毒”效应开创新的思路，为临床中西药的联合应用提供更多的理论依据。

自噬作为在肿瘤细胞形成之后的一种保护性机制严重影响了化疗疗效。从中药中筛选结直肠癌细胞保护性自噬抑制剂有利于克服化疗耐药，促进化疗药物敏感性，减少毒副作用。本课题组在本项目的资助下，取得了如下研究进展：发现木犀草素，5-Fu对多种人结直肠癌细胞株有增殖抑制作用，当两者联合使用时，抑制效果优于单独干预。通过FACS、Western blot分析检测发现木犀草素可促进5-Fu诱导结直肠癌细胞的凋亡。利用免疫荧光技术进一步证实木犀草素是通过抑制5-Fu的保护性自噬来协同诱导结直肠癌细胞的凋亡。运用siRNA干扰技术，证实木犀草素通过调控p53相关的自噬激活来增敏5-Fu。在此基础上，课题组应用荷人结肠癌裸鼠模型进行木犀草素化疗增敏的药效学评价，结果表明木犀草素可联合低浓度5-Fu抑制肿瘤的生长，并且能够改善小鼠生存质量。本项目拓展了木犀草素在治疗结直肠癌领域的应用，为研究中药的“增效减毒”效应开创新的思路，为临床中西药的联合应用提供更多的理论依据。