抗非小细胞肺癌合成小分子及植物提取物的筛选及作用机理研究

发布时间：2018-06-24 11:32

本文选题：非小细胞肺癌 + 合成小分子　；参考：《昆明理工大学》2017年博士论文

【摘要】：癌症是严重威胁人类健康的重大疾病,随着全球工业化和城镇化的加速发展,癌症的发病率呈日益上升趋势,给社会和家庭带来沉重的经济和精神负担。在20种常见癌症中,肺癌已成为危害程度最大的恶性肿瘤之一,其发病率和死亡率均居各癌症之首。根据临床特征及治疗预后的不同,肺癌可分为小细胞肺癌(small cell lung cancer,SCLC)和非小细胞肺癌(non-small cell lung cancer,NSCLC)。NSCLC占肺癌病例的80%～85%,是肺癌的主体,虽然恶性程度较低,局部发展慢,但该种肺癌体内存在微转移的威胁,给临床的诊断与治疗方案的确定带来巨大困难。寻找有效的、毒副作用小的抗癌药物是肺癌治疗的关键。近年来,对生物学与化学进行交叉研究已经成为不可阻挡的趋势,且逐渐形成了一种新兴的学科---化学生物学。利用现有的数量大且化学结构多样的小分子化合物库,针对需要的生物活性进行筛选,并以此为依据进行后续的机制研究,以期获得治疗疾病的前体药物。我们利用购买的Life Chemicals小分子化合物库,在A549细胞系上进行活性筛选,最终获得两种活性较好的小分子化合物rosline、rosbin。Rosline((2Z)-N-[3-(dimethlamino)propyl]-N-(4-fluoro-1,3-benzothizol-2-yl)-3-(4-nitr ophenyl)prop-2-enamide hydrochloride),本研究发现其具有较强的抗A549细胞增殖活性(IC50 为 2.87 ± 0.39μM)。通过 Hoechst 染色,AnnexinV-FITC 和 PI 双染以及细胞周期凋亡峰分析等多种方法分析确定,Rosline具有诱导A549细胞凋亡的作用并引起细胞G2/M期阻滞。RT-PCR结果表明这种阻滞可能与细胞内cyclin B1,cdc2,P21等G2/M周期相关蛋白的变化有关。rosline处理细胞后细胞凋亡的同时伴随着ROS的增加及Ca2+浓度的上升,我们推测rosline诱导细胞凋亡与A549对ROS的敏感性有关。抗氧化剂(N-acetyl-L-cysteine)在抑制A549细胞ROS爆发的同时成功的抑制了rosline引发的A549细胞生长抑制、G2/M期阻滞、MMP的减少、细胞凋亡及caspase3/7、caspase9活性增加等。在进一步关于小分子分子作用机制的研究中,我们发现Rosline处理A549细胞后能够引发胞内PI3K/Akt/mTOR信号通路的变化,明显引起PI3K蛋白及p-Akt蛋白的下调。Rosbin(Thieno[2,3-c]piperidine-3-carboxamide-2-[(3-methoxy-naphtha-1 ene-2-carbonyl)-amino]-6-(benzyl)-,hydrochloride)是一种哌啶并噻吩类小分子衍生物,对A549细胞具有明显的抑制增殖作用,其IC50为2.05 ± 0.31μM;通过在正常细胞上MRC-5上对照,rosbin对MRC-5细胞几乎没有毒性,在最大实验浓度10μM情况下其抑制率仍未达到50%。通过进一步研究表明,rosbin呈剂量依赖性的诱导A549细胞凋亡并能降低线粒体膜电位(MMP);Ca2+浓度上升;增加caspase3/7、caspase9的活性,但是不影响caspase8的活性及A549细胞生长周期。抗氧化剂(N-acetyl-L-cysteine和过氧化氢酶)在抑制A549细胞ROS爆发的同时,成功的阻滞了 MMP的减少,并且抑制了细胞凋亡及caspase3/7、caspase9活性的变化。另外我们发现rosbin处理细胞后rosbin处理细胞后能够引发PI3K/Akt/mTOR及P38/MAPK信号通路的变化,明显引起PI3K蛋白及p-Akt蛋白的下调及P38蛋白的上升通过验证其rosbin诱导P38/MAP K及PI3K/Akt信号通路均与ROS的敏感性相关(P0.001)。天然产物中的中草药具有资源丰富、毒副作用小、长期的临床使用基础、疗效确定等优点。且天然药物具有许多合成药物所不能比拟的优势,如生物相容性较好。2004年4月欧盟《欧洲传统植物药注册程序指令》及2004年6月美国FDA《政策和程序手册:植物药产品审查》包含多种化合物的传统植物药及提取物可作为现代药品,对传统药物的认可度逐步提高。立足于云南省丰富的自然环境资源本研究采取溶剂提取与萃取法建立了包含36种药用植物共252个药用植物提取相,并对其中25种植物的150个提取相采用MTT法进行抑制肺癌A549细胞生长的活性筛选,最终筛选出具有明显抑制A549细胞生长的萃取相25种。其中土荆芥氯仿萃取相和灰灰菜石油醚萃取相抑制A549细胞生长的活性进行了更为深入的探讨。结果显示,这两种萃取相对A549细胞生长的抑制呈显著的剂量效应和时间效应、使A549细胞出现G1期阻滞。Hoechest3342染色、Annxive-FITC及PI双染法研究其对细胞凋亡影响的结果显示,这两种粗提物均能诱导A549细胞出现典型的凋亡形态,包括细胞变圆、皱缩、早期凋亡细胞比例显著增加等。综上所述,无论是合成小分子还是植物提取物均能在不同程度上抑制非小细胞肺癌A549细胞的增殖,在合成小分子方面,我们发现哌啶并噻吩类小分子rosbin及苯并噻唑类小分子rosline的抗癌机理研究,填补了本类化合物在抗癌机理方面的空白。在植物提取物研究中我们发现了灰灰菜石油醚相提取物及土荆芥氯仿相提取物的体外较好的抗肿瘤活性。
[Abstract]:Cancer is a major disease that seriously threatens human health. With the rapid development of industrialization and urbanization in the world, the incidence of cancer is on the rise, bringing heavy economic and spiritual burden to society and family. In the 20 common cancers, lung cancer has become one of the most dangerous malignant tumors, its incidence and mortality are all According to different clinical features and treatment prognosis, lung cancer can be divided into small cell lung cancer (small cell lung cancer, SCLC) and non small cell lung cancer (non-small cell lung cancer, NSCLC).NSCLC accounted for 80% to 85% of lung cancer cases, which is the main body of lung cancer, although the malignant degree is low and local development is slow, but the lung cancer is in the body. The threat of micrometastasis has brought great difficulties to the determination of clinical diagnosis and treatment. To find effective and small side effects is the key to the treatment of lung cancer. In recent years, the cross study of biology and chemistry has become an unstoppable trend, and has gradually formed a new subject - chemical biology. The existing large and chemically diverse small molecular compound library, based on the screening of the biological activity needed, is based on the follow-up mechanism to obtain the precursor drugs for the treatment of disease. We use the purchased Life Chemicals small molecule compound library to screen the activity on the A549 cell line and finally obtain the results. Two small molecular compounds with better activity, rosline, rosbin.Rosline ((2Z) -N-[3- (dimethlamino) propyl]-N- (4-fluoro-1,3-benzothizol-2-yl) -3- (4-nitr ophenyl) prop-2-enamide hydrochloride), have been found to have strong anti proliferative activity (2.87 + 0.39 micron). A variety of methods, such as double staining with PI and analysis of cell cycle apoptotic peak analysis, confirm that Rosline has the effect of inducing apoptosis of A549 cells and causes G2/M phase block of cell.RT-PCR, which indicates that this block may be associated with the changes of cyclin B1, cdc2, P21 and other G2/M cycle related proteins in cells. With the increase of ROS and the increase of Ca2+ concentration, we speculate that rosline induced apoptosis is related to the sensitivity of A549 to ROS. Antioxidants (N-acetyl-L-cysteine) inhibit the rosline induced A549 cell growth inhibition, G2/M phase block, MMP reduction, apoptosis and apoptosis, while inhibiting the outbreak of ROS in A549 cells. In the further study of the mechanism of the action of small molecules, we found that Rosline treatment of A549 cells could lead to changes in the intracellular PI3K/Akt/mTOR signaling pathway, obviously causing the down regulation of the.Rosbin (Thieno[2,3-c] piperidine-3-carboxamide-2-[(3-methoxy-naphtha-1 ene-2-carbonyl) of the PI3K protein and the p-Akt protein (3-methoxy-naphtha-1 ene-2-carbonyl). Mino]-6- (benzyl), hydrochloride), a small molecular derivative of piperidine and thiophene, has a significant inhibitory effect on A549 cells, and its IC50 is 2.05 + 0.31 micron M. Rosbin has almost no toxicity to MRC-5 cells through the control of MRC-5 on normal cells. The inhibition rate is still not reached 50%. through the maximum experimental concentration of 10 micron M. Further studies showed that rosbin could induce apoptosis of A549 cells in a dose-dependent manner and reduce mitochondrial membrane potential (MMP); Ca2+ concentration increased, caspase3/7, caspase9 activity increased, but did not affect the activity of caspase8 and the growth cycle of A549 cells. Antioxidants (N-acetyl-L-cysteine and catalase) inhibited ROS outbreak of A549 cells. At the same time, the decrease of MMP was successfully blocked and the apoptosis and the changes of caspase3/7 and caspase9 activity were inhibited. In addition, we found that after rosbin processing cells of rosbin treated cells, the PI3K/Akt/mTOR and P38/MAPK signaling pathways could be triggered, and the down regulation of PI3K protein and p-Akt protein and the increase of P38 protein were detected. The rosbin induced P38/MAP K and PI3K/Akt signaling pathway are related to the sensitivity of ROS (P0.001). The Chinese herbal medicine in natural products has the advantages of rich resources, small toxic side effects, long-term clinical use basis, and determination of curative effect. And natural drugs have many advantages which can not be compared with many synthetic drugs, such as good biocompatibility in April,.2004. The European traditional plant drug registration procedure directive > and the June 2004 American FDA< policy and procedure manual: plant drug product review > traditional plant drugs and extracts containing a variety of compounds can be used as modern drugs, and the recognition of traditional medicines is gradually improved. Based on the natural environmental resources of Feng Fu in Yunnan Province, this study takes solvent extraction. The extraction and extraction methods were used to establish a total of 252 medicinal plant extracts containing 36 kinds of medicinal plants, and 150 extracts of 25 plants were screened by MTT method to inhibit the growth of lung cancer A549 cells. Finally, 25 extraction phases, which could obviously inhibit the growth of A549 cells, were screened. The activity of A549 cell growth inhibited by the extraction phase was further investigated. The results showed that the inhibition of the two extracts had a significant dose effect and time effect on the inhibition of A549 cell growth, which resulted in.Hoechest3342 staining of G1 phase block in A549 cells. The results of the effect of Annxive-FITC and PI double staining on cell apoptosis showed that this two was the result of the apoptosis. The crude extracts can induce typical apoptosis morphology of A549 cells, including cell circle, shrinkage, and the increase of early apoptotic cells. In summary, both small molecules and plant extracts can inhibit the proliferation of non small cell lung cancer A549 cells in varying degrees. We found piperidine in the synthesis of small molecules. The anticancer mechanism of the small molecule rosbin of thiophene and the small molecule rosline of benzothiazole type has filled the blank of the anticancer mechanism of this kind of compound. In the study of plant extract, we found the good antitumor activity of the extract of the petroleum ether phase and the chloroform extract of the Chinese cabbage in vitro.
【学位授予单位】：昆明理工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

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