【作者】 李萍；

【导师】 张雁冰；

【作者基本信息】 郑州大学 ， 药学硕士（专业学位）， 2019， 硕士

【摘要】 微管广泛存在于真核生物的细胞中,并与肿瘤细胞的发生和发展有着非常密切的关系。它也被认为是化疗药物治疗癌症的一个有吸引力的靶点。本课题组在前期工作的基础上设计合成了一批新型吲哚类衍生物,选取活性最优化合物2803研究其对胃癌细胞MGC803的作用机制。本文的主要研究如下:1.用MTT法检测化合物2803对人食管癌细胞（EC109）、人前列腺癌细胞（PC3）和人胃癌细胞（MGC803）的抗增殖活性。结果显示,2803对MGC803的作用最为显著,其IC₅₀为1.59μmol/L。2.为了验证化合物对微管蛋白的抑制作用,我们用免疫荧光、免疫印迹和分子对接技术分别试验。免疫荧光结果发现,随着2803浓度的增大,解聚微管的能力逐渐增强,2μmol/L时纺锤体已无法形成;EBI结果显示,一定浓度下经过2803作用后可以竞争性结合秋水仙碱位点;利用MOE进行1SA0分子对接,结果表明2803能够占据秋水仙碱的结合位点,与微管蛋白形成良好的相互作用。3.进一步探索2803作用于细胞的死亡方式,流式细胞术检测结果显示,在浓度为2μmol/L时化合物诱导MGC803的凋亡率已达到65.2%。DAPI染色后出现显著的核变化,如破碎和凝结。彗星实验中,彗星的尾长出现明显变长。这些结果都表明了2803作用细胞后凋亡的存在。在Western Blot检测中,抗凋亡蛋白Bcl-2和Bcl-xl出现下调,而促凋亡蛋白Bax上调,两名执行者Caspase-3/7和其底物蛋白PARP也明显被剪切。另外,死亡受体DR5和激活的Caspase-8均出现上调。这些结果说明了2803通过外源和内源联合途径诱导细胞凋亡。4.由于DR5的积累,猜想2803作用于MGC803可能与NEDDylation有关系。在NEDD8结合测定中,发现Ubc12-NEDD8、NAE1-NEDD8和Cullins-NEDD8都被2803抑制。接着我们又检测了NEDDylation调控的周期蛋白p21的表达,结果显示它有很大程度的积累。5.P21的积聚可以诱导细胞周期的阻滞。首先从流式结果看2803确实剂量依赖地阻滞细胞周期,G2/M期的百分比从20%上升到60%。克隆形成实验结果发现,实验组细胞集落明显依次减少。但是Western Blot的结果显示,影响G2期的蛋白并没有降低,而与M期有关的蛋白磷酸化Histone H3明显上调,说明2803可以抑制细胞增殖且阻滞周期在M期。6.MAPK是参与细胞增殖的一条重要信号通路。它主要有3条途径,检测结果显示p-c-Jun不受2803调节,而ERK和p38途径均受到抑制。其中ERK途径抑制最为明显,检测ERK途径的上游蛋白发现c-Raf,MEK1/2,ERK1/2都被抑制而起始的Ras不受影响。在进一步检测中发现,2803可以直接与Ras相互作用。这说明2803是通过与Ras互作,影响Ras对Raf的磷酸化,从而抑制下游MEK1/2和ERK1/2的活化,从而导致细胞增殖抑制。综上所述,吲哚类衍生物2803可以作为潜在的微管蛋白抑制剂用于靶向的抗肿瘤研究。另外,它可以抑制MGC803的NEDDylation过程,并通过与Ras相互作用抑制MAPK的ERK途径。最终化合物2803通过诱导胃癌细胞MGC803的凋亡和抑制其增殖来发挥抗肿瘤作用。更多还原

【Abstract】 Microtubules are widely present in the cells of eukaryotes and have a very close relationship with the occurrence and development of tumor cells.It is also considered to be an attractive target for chemotherapy drugs to treat cancer.Based on the preliminary work,our research group designed and synthesized a new class of steroid derivatives,and selected the activity-optimized compound 2803 to study its mechanism of action on gastric cancer cells MGC803.The main research of this paper is as follows:1.The antiproliferative activity of compound 2803 against human esophageal cancer cells（EC109）,human prostate cancer cells（PC3）,and human gastric cancer cells（MGC803）was examined by MTT assay.The results showed that 2803 had the most significant effect on MGC803 with an IC₅₀ of 1.59μmol/L.2.To verify the inhibitory effect of compounds on tubulin,we tested them by immunofluorescence,immunoblotting and molecular docking techniques.The results of immunofluorescence showed that with the increase of 2803concentration,the ability to depolymerize microtubules gradually increased,and the spindle could not form at 2μmol/L.The EBI results showed that after a certain concentration,it could competitively bind colchicum after 2803.Base site;docking of 1SA0 molecule by MOE,the results showed that 2803 can occupy the binding site of colchicine and form a good interaction with tubulin.3.Further explore 2803 of the methods of death that act on cells,and the results of flow cytometry showed that the apoptosis rate of compound 2803induced MGC803 at concentrations of 2μmol/L had reached 65.2%.There are significant nuclear changes after DAPI staining,such as breakage and condensation.In the comet experiment,the tail length of the comet appeared significantly longer.These results all indicate the presence of apoptosis after 2803acting cells.In the Western Blot test,anti-apoptosis protein Bcl-2 and Bcl-xl appeared downwards,while the apoptosis protein Bax increased,two executors Caspase-3/7 and its substrate protein PARP were also significantly cut.In addition,both death receptor DR5 and activated Caspase-8 showed an increase.These results show that 2803 induced apoptosis of MGC803 by external source and endogenous combined pathway.4.Due to the accumulation of DR5,it is suspected that the effect of 2803 on MGC803 may be related to NEDDylation.In the NEDD8 binding assay,Ubc12-NEDD8,NAE1-NEDD8,and Cullins-NEDD8 were all inhibited by 2803.Then we examined the expression of cyclin p21 regulated by NEDDylation,and the results showed that it accumulated to a large extent.5.The accumulation of p21 can induce cell cycle block.First,from the flow results,2803 of the exact dose-dependent block cell cycle,the percentage of the G2/M period increased from 20%to 60%.The results of clonal formation showed that the cell colony in the experimental group decreased obviously in turn.However,the results of the Western Blot showed that the CDK1,CyclinB1 and CyclinB2 affecting the G2 period did not decrease,while the protein p-Histone H3 associated with the M period was significantly increased,suggesting that 2803inhibited cell proliferation and blocked the cycle during the M period.6.MAPK is an important signaling pathway involved in cell proliferation.It has three main pathways,and the results show that p-c-Jun is not regulated by2803,and both ERK and p38 pathways are inhibited.Among them,the ERK pathway was most inhibited.The upstream protein of the ERK pathway was found to be c-Raf,MEK1/2,and ERK1/2 were inhibited and the initial Ras was not affected.In further testing,it was found that 2803 can directly interact with Ras.This indicates that 2803 interacts with Ras to affect the phosphorylation of Raf by Ras,thereby inhibiting the activation of downstream MEK1/2 and ERK1/2,resulting in inhibition of cell proliferation.To sum up,indole derivative 2803 can be used as a potential microtubule protein inhibitor for targeted anti-tumor research.In addition,it inhibits the NEDDylation process of MGC803 and inhibits the ERK pathway of MAPK through interaction with Ras.The final compound 2803 plays an anti-tumor role by inducing apoptosis of gastric cancer cell MGC803 and inhibiting its proliferation.更多还原