基于p53调控糖酵解探索臭椿抗胃癌的药效物质基础与作用机制

Gastric cancer is one of the malignant tumors in China. Most of chemotherapy drugs used in clinic have toxicity and side effects. Therefore, it is imperative to seek active molecules with high efficacies and low toxicities from traditional Chinese medicine and clarify its anti-cancer mechanism. It was demonstrated that compounds isolated from Ailanthus altissima had anticancer effect. However, most studies of Ailanthus altissima focus on its chemical component and it remains unknown the underlying anti-cancer mechanism. Previously, we extracted the ingredient of it and found crude extracts could increase p53 protein level, disturb glycolysis pathway and decrease the cell viability of MKN45 cells. However, it remains unclear that the material foundation of Ailanthus altissima and the underlying regulation mechanism of active compound on p53 and p53 on glycolysis process. Based on our previous data, we intend to establish a screening model based on metabolism disturbance and elevated p53 level to screen the extracts isolated from Ailanthus altissima and confirm the active ones. Next, the compounds are systematic isolated from the active extracts by the guidance of their activities. A variety of chemical reactions and spectroscopy are used to identify the structure of these purified compounds, thus illuminating the material foundation of Ailanthus altissima in treating gastric cancers. Moreover, the glucose and lactic acid levels are determined by kit assays. The gene and protein levels involved in glycolysis pathway are also detected through PCR and western blot method respectively, verifying the regulation effect of the active compound on glycolysis process. At last, the molecular mechanism that the active compound exerts its anti-gastric cancer effect through p53 mediated glycolysis is elucidated through small interfering RNA, ChIP and some other experiments.

胃癌是我国恶性肿瘤之一，现临床使用的化疗药物大多存在毒副作用。从中药中寻找高效低毒的抗胃癌活性分子并阐明其作用机制迫在眉睫。文献研究表明臭椿有较好的抗肿瘤作用，但目前对臭椿的研究主要集中在化学成分上，对其作用机制尚不清楚。前期对臭椿成分进行提取，发现提取物可以升高p53水平，扰动糖酵解，抑制胃癌细胞增殖。但是臭椿抗胃癌的物质基础和臭椿对p53以及p53对糖酵解的调控机制未知。本项目拟在此基础上，以扰动代谢和升高p53水平为评价指标建立筛选模型，确证臭椿中的活性部位；采用活性导向分离追踪臭椿抗胃癌的活性化合物，利用多种化学、谱学手段鉴定其结构，以阐明臭椿抗胃癌的药效物质；利用试剂盒测定葡萄糖和乳酸水平，PCR和western blot检测糖酵解通路中基因和蛋白水平，验证活性化合物对糖酵解通路的影响；进一步通过小干扰RNA、ChIP等实验深入阐明活性化合物通过p53调控糖酵解的抗胃癌分子机制。

胃癌严重威胁人类健康，目前临床常用的药物往往毒副作用较强而且容易产生耐药性，因此开发新的候选药物尤为重要。臭椿具有包括抗肿瘤在内的多种药理活性，因此本项目拟提取分离臭椿中的成分并探索其抗胃癌的作用及机制。本研究主要完成以下工作：.（1）对臭椿进行系统的分离纯化，鉴定出16个单体化合物，其中包括2个甾体类化合物、2个木脂素类化合物、2个香豆素类化合物、2个苦木素类化合物和6个三萜类化合物；.（2）对提取出来的化合物进行活性筛选，结果表明臭椿酮活性最好，接着我们从体内外两个方面证明其对胃癌的增殖抑制作用。MTT、单克隆形成和Edu实验表明臭椿酮能剂量依赖性的降低胃癌MGC-803和HGC-27细胞的增殖；.（3）接下来我们探索了臭椿酮抑制细胞增殖的原因，通过流式分析表明臭椿酮能够诱导细胞周期阻滞在G1/S期和线粒体损伤介导的凋亡；.（4）另外我们考察了臭椿酮对糖酵解通路的影响，结果表明臭椿酮能显著降低糖酵解相关蛋白表达，另外臭椿酮也能增加ROS表达，提示糖酵解通路抑制后对ROS水平有一定影响；.（5）最后我们考察了臭椿酮对p53的影响，结果表明臭椿酮可以导致p53蛋白和mRNA水平都升高，而且p53蛋白水平升高介导了臭椿酮对糖酵解的抑制作用和促进凋亡的作用。另外我们也证明p53蛋白水平的积累不是由于臭椿酮抑制了其泛素化降解，因此我们进一步考察了组蛋白去甲基化酶LSD1对p53表达的影响，结果表明臭椿酮可以抑制LSD1表达，诱导其底物蛋白H3K4的单甲基化、双甲基化水平升高，并且促进LSD1蛋白与p53基因的相互结合。. 此项目研究表明臭椿酮具有显著的抗胃癌活性，可以为其开发成治疗胃癌的药物提供理论依据和科学支撑。