新化合物体外抗肿瘤活性及作用机制

发布时间：2018-07-05 04:34

本文选题：微管靶向药物 + 抗肿瘤作用机制　；参考：《中国人民解放军军事医学科学院》2014年硕士论文

【摘要】：肿瘤是当今世界的一大难题,肿瘤发病率、死亡人数持续走高,其治疗仍迫切需要更有效的药物。微管蛋白抑制剂是一类作用于微管蛋白并破坏其动态平衡,从而阻止肿瘤细胞增殖的抗癌药物,在临床多种实体肿瘤治疗中处于重要的地位。新型微管靶向抗肿瘤药物因具有明确的分子靶向性、强效的抗肿瘤以及抑制并破环肿瘤新生血管形成的作用,成为抗肿瘤药物研究的新热点。WX-123-27,WX-127-07, WX-132-18B是由本所通过普筛发现的三个具有极强抗肿瘤活性的新型分子,初步的实验表明该类化合物可能作用于微管蛋白[1]。本课题采用本课题组建立的基于高内涵分析(HCA)的抗肿瘤药物药效及机制研究的技术平台,通过系统、高效的体外实验,确定上述三种新化合物的抗肿瘤活性及作用的分子机制,以便在药物发展早期阶段全面了解这些活性分子的药理学作用特征,为该类化合物的进一步发展奠定基础。在实验研究中以已知的微管靶向药物为阳性对照药物,采用SRB法在HepG2, HeLa, A549, HLF及HUVEC细胞上检测了受试化合物的细胞增殖抑制活性；采用流式细胞术检测周期阻滞作用；通过高内涵细胞毒性、细胞形态、细胞凋亡的多参数分析以及与肿瘤相关的细胞信号通路(包括2个GPCRs,6个核受体、2个生长因子受体和激酶、7个细胞周期及DNA损伤、6个炎性及应激和8个肿瘤非相关的通路)的筛查分析受试化合物的细胞效应特征；最后采用秋水仙碱/荧光长春碱的微管蛋白竞争抑制实验和胰酶消化微管蛋白实验在分子水平确证受试化合物的微管蛋白作用位点。实验结果如下：WX-123-27, WX-127-07, WX-132-18B在HepG2, HeLa, A549, HLF及HUVEC细胞上均具有显著的细胞增殖抑制活性,WX-123-27的IC50分别为6.83±0.15nM,6.72±0.19nM,6.78±0.31nM,5.51±0.11nM,5.37±0.20nM, WX-127-07的IC50分别为4.47±0.05nM,5.18±0.08nM,4.90±0.19nM,4.10±0.16nM,5.04±0.08nM,WX-132-18B的IC50分别为1.03±0.02nM,1.01±0.01nM,1.03±0.06nM,0.86±0.02nM,0.76±0.04nM,作用强度显著强于秋水仙碱(IC50分别为21.17±1.22nM,14.19±0.53nM,43.80±1.64nM,145.89±10.97nM,27.67±1.79nM)和长春新碱(IC50分别为16.51±0.36nM,16.76±0.33nM,27.80±2.75nM,43.80±1.48nM,9.15±0.78nM),在HepG2, HeLa, HLF细胞上作用显著强于紫杉醇(IC50分别为10.68±0.61nM,12.86±0.25nM,102.07±15.17Nm),在A549, HUVEC上作用与紫杉醇相当(IC50分别为4.81±0：61nM,3.04±0.12nM)；高内涵细胞多参数分析显示,与紫杉醇浓度依赖性地诱发A549细胞微管聚合不同,三种受试化合物与长春新碱、秋水仙碱相似,浓度依赖地诱发A549细胞的微管解聚,紫杉醇、长春新碱、秋水仙碱、WX-123-27、WX-127-07、 WX-132-18B使微管含量(以亮度和面积的乘积为指标)改变的EC50分别为75.84nM,293.2nM,105.5nM,25.91nM,17.31nM,9.43nM,即三种受试化合物的微管效应显著强于三种对照药物；与三种阳性对照药物相似,三种受试化合物诱发A549细胞在G2/M期阻滞,正常对照组细胞在Go/Gi, S, G2/M期的分布分别为71.74%,21.95%,6.31%,经紫杉醇、长春新碱、秋水仙碱、WX-123-27、WX-127-07、WX-132-18B作用后,G0/G1期比例明显减少,分别为18.17%,11.63%,19.15%,7.41%,5.62%,3.74%,而G2/M期比例明显增高,分别为62.90%,71.33%,63.61%,80.35%,72.06%,83.80%；同样,与三种阳性对照药物相似,受试化合物使HepG2细胞核膜通透性增加、诱导细胞早凋；在较高的浓度下,诱导轻度的Rad51颗粒的形成(约为阳性药喜树碱的1/4),但未对筛查的其它30种信号通路有影响。在微管蛋白位点的竞争抑制实验中,WX-123-27, WX-127-07, WX-132-18B浓度依赖地抑制秋水仙碱与微管蛋白的结合(IC50分别为1.75±0.54μM,1.28±0.08μM,0.47±0.10μM),仅WX-123-27, WX-127-07在高浓度对荧光长春碱与微管蛋白的结合表现出一定的抑制作用,但显著弱于长春新碱,也不能排除高浓度非特异的抑制作用；微管蛋白经胰酶消化后的SDS蛋白电泳实验显示WX-123-27、WX-127-07、WX-132-18B与微管蛋白作用后的酶解产物与秋水仙碱作用较为相似而不同于长春新碱,进一步证明受试化合物主要作用于微管的秋水仙碱结合位点。综合以上的研究结果可以看出,受试化合物WX-123-27, WX-127-07, WX-132-18B具有较强的体外抗肿瘤细胞增殖活性及抗血管内皮细胞增殖活性,其抗肿瘤作用机制符合微管靶向药物的作用特点,三种受试化合物主要选择性作用于微管秋水仙碱位点,是具有强抗细胞增殖活性的新型微管解聚剂。
[Abstract]:Tumor is a major problem in the world today. The incidence of cancer and the number of deaths continue to rise, and the treatment still needs more effective drugs. Microtubulin inhibitors are an anticancer drug that acts on microtubule protein and destroys its dynamic balance, thus preventing the proliferation of tumor cells. It is important in the treatment of many clinical solid tumors. The new microtubule targeting antitumor drug has become a new hot spot in antitumor drug research because of its specific molecular targeting, strong anti-tumor and inhibition and the formation of neovascularization of tumor ring tumor,.WX-123-27, WX-127-07, WX-132-18B, a new molecule with three extremely strong anti-tumor activities discovered by this institute. Preliminary experiments show that these compounds may act on tubulin [1]..
In this subject, we set up a technical platform based on high intension analysis (HCA) based on high intension analysis (HCA) for anti-tumor drug efficacy and mechanism. Through systematic and efficient in vitro experiments, the molecular mechanism of the antitumor activity and action of the above three new compounds is determined in order to understand the pharmacology of these active molecules at the early stage of drug development. The characteristics of the study lay the foundation for further development of the compounds.
In the experimental study, using the known microtubule targeting drug as the positive control drug, the cell proliferation inhibition activity of the tested compounds was detected on HepG2, HeLa, A549, HLF and HUVEC cells by SRB, and the flow cytometry was used to detect the cycle arrest, and the multi parameter analysis of the high intension cytotoxicity, cell morphology and apoptosis was carried out. And the cell signaling pathways associated with tumor related cells (including 2 GPCRs, 6 nuclear receptors, 2 growth factor receptors and kinases, 7 cell cycle and DNA damage, 6 inflammatory and stress and 8 unrelated pathways) screening and analyzing the cell effects of the tested compounds; the most later microtubulin competing with colchicine / fluorescent Changchun base Competition inhibition experiments and trypsin digestion of tubulin experiments confirmed the tubulin site of the tested compounds at molecular level.
The experimental results are as follows: WX-123-27, WX-127-07 and WX-132-18B have significant inhibitory activity on cell proliferation on HepG2, HeLa, A549, HLF and HUVEC cells. WX-123-27 IC50 is 6.83 + 0.15nM, 6.72 + 0.19nM, 5.51 +, 5.37 +, 5.18, 4.90, 4.10 +, 4.10 +, 4.10 +. 0.16nM, 5.04 + 0.08nM, WX-132-18B IC50 were 1.03 + 0.02nM, 1.01 + 0.01nM, 1.03 + 0.06nM, 0.86 + 0.02nM, 0.76 + 0.04nM, and the action intensity was significantly stronger than colchicine (IC50 respectively 21.17 +, 14.19 + 0.53nM, 43.80 +, 145.89 +, 27.67 +) and vincristine. 75nM, 43.80 + 1.48nM, 9.15 + 0.78nM), the effect on HepG2, HeLa, HLF cells is stronger than Taxol (IC50 is 10.68 + 0.61nM, 12.86 + 0.25nM, 102.07 + 15.17Nm). In A549, the action of the HUVEC is equivalent to Taxol (4.81 + 3.04, 3.04 +), and the high concentration cell multi parameter analysis shows that the concentration dependence of taxol concentration The microtubule polymerization of A549 cells was different. The three tested compounds were similar to vincristine and colchicine, and the concentration dependent induced microtubule depolymerization of A549 cells. Paclitaxel, vincristine, colchicine, WX-123-27, WX-127-07, WX-132-18B changed the EC50 content of microtubules (the product of brightness and area as the index of 75.84nM, 293.2nM, respectively). 105.5nM, 25.91nM, 17.31nM, 9.43nM, that is, the microtubule effect of three kinds of tested compounds is significantly stronger than that of three control drugs; it is similar to three positive control drugs. Three kinds of subjects induce A549 cells to block in the G2/M phase, and the distribution of the normal control group in Go/Gi, S, and G2/M are 71.74%, 21.95%, 6.31%, after paclitaxel, vincristine, autumn. After the action of Narcissus, WX-123-27, WX-127-07 and WX-132-18B, the proportion of G0/G1 phase decreased significantly, which were 18.17%, 11.63%, 19.15%, 7.41%, 5.62%, 3.74%, and the G2/M period increased significantly, respectively, 62.90%, 71.33%, 63.61%, 80.35%, 72.06%, 83.80%; similarly, similar to the three positive control drugs, the tested compound increased the nuclear membrane permeability of HepG2 cells, Induction of cell early withering; at a higher concentration, the induction of mild Rad51 particles (about 1/4 of the positive drug camptothecin) was not affected by the other 30 signaling pathways of screening. In the competition inhibition test of the microtubule loci, the concentration of WX-123-27, WX-127-07, and WX-132-18B inhibited the combination of colchicine and microtubule protein (I C50 was 1.75 + 0.54 mu M, 1.28 + 0.08 mu M, 0.47 + 0.10 M), only WX-123-27, WX-127-07 showed a certain inhibition effect on the combination of fluorescent Changchun alkali and microtubule protein at high concentration, but it was significantly weaker than vincristine, and it could not exclude high concentration and nonspecific inhibition effect. The microtubulin protein electrophoresis after trypsin digestion showed that the electrophoresis of SDS protein electrophoresis showed that the microtubulin was digested by trypsin. The enzymatic hydrolysates of WX-123-27, WX-127-07, WX-132-18B and microtubule were similar to colchicine, but were different from vincristine, which further demonstrated that the tested compounds were mainly used in the colchicine binding site of microtubules.
The above results can be seen that the experimental compounds WX-123-27, WX-127-07, WX-132-18B have strong anti-tumor cell proliferation activity and anti vascular endothelial cell proliferation activity in vitro. The anti-tumor mechanism of the compound is consistent with the role of microtubule targeting drugs. The three kinds of tested compounds are mainly selective in microtubule colchicine. The site is a new microtubule depolymerization agent with strong anti proliferative activity.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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