2-苯基萘靶向拓扑异构酶抗肿瘤作用机制研究

发布时间：2018-01-23 00:58

本文关键词： CS1 拓扑异构酶有丝分裂灾难细胞凋亡 DNA损伤　出处：《山东大学》2014年硕士论文　论文类型：学位论文

【摘要】：拓扑异构酶是真核生物体内最重要的酶之一,调节DNA拓扑结构,参与DNA复制、转录、染色体重组以及姐妹染色体分离等多个环节。拓扑异构酶已经成为抗肿瘤药物的一个重要靶点,靶向拓扑异构酶的药物在临床上也取得了很好的疗效。但拓扑异构酶抑制剂在临床使用过程中出现了许多毒副作用,除了常见的胃肠道反应,皮肤病,还有许多严重的不良反应如阿霉素引起的严重新心脏毒性、依托泊苷诱发次生肿瘤(白血病)以及多药耐药等。多年来,开发高效低毒的拓扑异构酶抑制剂一直是抗肿瘤药物研究的热点之一。天然三联苯类化合物主要来源于真菌代谢产物,现已发现的三联苯类化合物表现出多种生物活性包括抗肿瘤,抗氧化,抗细菌等。本课题组前期研究发现对三联苯H1-H8抑制人乳腺导管癌MDA-MB-435细胞增殖,引起细胞G2/M期停滞并诱导细胞凋亡。此外,H1-H8以及从链霉菌sp. LZ35中分离得到的对三联苯echosides A-E能不同程度地抑制拓扑异构酶的活性,但是这类化合物在对正常细胞和肿瘤细胞的选择性以及体内抗肿瘤活性较差。基于前期的研究基础,本实验室以对三联苯为基本结构进行改造针对拓扑异构酶设计合成了一系列2-苯基萘,本论文研究这一系列2-苯基萘的抗肿瘤活性及其对拓扑异构酶的抑制活性,并挑选代表化合物CS1深入研究这类化合物的体内外抗肿瘤活性及其抗肿瘤作用机制。我们首先用SRB法检测了48个2-苯基萘对三株肿瘤细胞MDA-MB-231,A549和HeLa细胞的细胞毒性,结果有11个化合物对人乳腺癌MDA-MB-231细胞的IC5o小于5μM。然后我们用DNA松弛实验检测这11个细胞毒性较强的化合物对拓扑异构酶I和拓扑异构酶II的抑制作用,有5个化合物能完全抑制拓扑异构酶II的活性。我们挑选代表化合物1(CS1)深入研究其抗肿瘤作用机制。为研究CS1体外抑制拓扑异构酶的作用方式,我们采用了pBR322DNA松弛实验、kDNA解除链接实验、DNA嵌入实验以及拓扑异构酶介导的DNA断裂检测实验。结果发现CS1是一个DNA非嵌入剂,能够稳定拓扑异构酶-DNA可切割三元复合物,作为一个拓扑异构酶II毒剂抑制拓扑异构酶的活性。为全面评价CS1的体外抗肿瘤作用,我们选取不同组织来源的十一株细胞检测其细胞毒性,结果显示CS1对多种组织来源的肿瘤细胞均表现出一定的细胞毒性,但对正常细胞株HUVEC和HL7702细胞毒性较小。同时我们还检测了CS1对p-糖蛋白过表达的多药耐药细胞株MCF-7/ADR的细胞毒性,结果发现CS1对敏感细胞MCF-7和耐药细胞MCF-7/ADR表现出相同程度的细胞毒性,并且CS1不引起Topo Ⅱα和Topo Ⅱβ蛋白降解,这表明CS1具有潜在抗多药耐药作用且不易引起耐药性。周期分析和DNA Ladder实验显示CS1诱导细胞凋亡,我们进一步用Anneixn V/PI双染色证明CS1诱导细胞凋亡具有一定浓度依赖性。彗星电泳,免疫荧光和免疫印迹等实验显示CS1能够引起DNA断裂,有趣的是CSl并没有激活典型的ATM-Chk1/Chk2应激反应,只选择性磷酸化DNA损伤信号蛋白ATR但对ATM没有影响。Hoechst33342染色显示,CS1诱导细胞产生多核现象,我们进一步用免疫荧光染色发现CS1诱导细胞不正常有丝分裂,形成多个极点,细胞不对称分裂,诱导细胞发生有丝分裂灾难。最后,我们还有检测了CS1在裸鼠体内对MDA-MB-231肿瘤的抑制作用,结果显示CS1给药15天能显著抑制肿瘤的生长,按瘤重计算抑制率达62.3%,按肿瘤体积计算抑制率达43.6%,并且CS1对裸鼠体重没有任何影响。综上所述,以CSl为代表的2-苯基萘类化合物具有很强的抗肿瘤活性但对正常细胞毒性较小,能够抑制恶性肿瘤细胞迁移并具有潜在的抗多药耐药活性,CS1还能抑制接种裸鼠的人乳腺癌MDA-MB-231肿瘤生长。其作用机制可能是CS1通过抑制拓扑异构酶II活性,诱导DNA损伤,引起肿瘤细胞有丝分裂灾难进而发挥抗肿瘤活性。我们的研究结果为CS1的成药性提供了理论依据,其新颖的作用机制还可能为开发新型拓扑异构酶抑制剂提供新的研究思路。
[Abstract]:Topoisomerase enzyme is one of the most important eukaryotic organisms, regulating the DNA topology, involved in DNA replication, transcription, recombination and chromosome separation and other aspects. Topoisomerase has become an important target of anticancer drugs, drug targeting topoisomerase in clinic also achieved good results but there are many topoisomerase inhibitor side effects in clinical use, in addition to the gastrointestinal tract reaction, common skin disease, there are many serious adverse reactions such as severe new cardiac toxicity induced by adriamycin, etoposide induced secondary tumor (leukemia) and multidrug resistance. Over the years, the development of high efficiency and low toxicity have been topoisomerase inhibitors is one of the hot researches on antitumor drugs.
Natural terphenyl compounds mainly derived from fungal metabolites, terphenyl compounds have been found to exhibit a variety of biological activities including antitumor, antioxidant, anti bacteria and so on. Our previous study found that terphenyl H1-H8 inhibition of human breast cancer MDA-MB-435 cell proliferation, induced G2/M cell cycle arrest and apoptosis. In addition, H1-H8 and echosides A-E terphenyl could inhibit the activity of topoisomerase sp. isolated from Streptomyces LZ35, but this kind of compounds in normal and tumor cells and selective antitumor activity in vivo is poor. Based on the previous research, the laboratory in p-terphenyl as basic structure the transformation for topoisomerase designed and synthesized a series of 2- phenyl naphthalene, this thesis studies a series of 2- phenyl naphthalene and antitumor activity of different topology The inhibitory activity of the enzyme and the selection of the representative compound CS1 are used to study the antitumor activity of these compounds in vivo and in vitro and the mechanism of antitumor action.
We first use the SRB method to detect 48 2- phenyl naphthalene on three tumor cell lines MDA-MB-231, A549 and cytotoxicity of HeLa cells, the results of 11 compounds on human breast cancer cell line MDA-MB-231 IC5o is less than 5 mu M. inhibition and then we used DNA relaxation experiments to detect the 11 strong cytotoxic compounds on topoisomerase I and topoisomerase II, 5 compounds can completely inhibit the activity of topoisomerase II. We selected 1 representative compounds (CS1) in-depth study of the mechanisms of its anti-tumor effect. The inhibitive effect of topoisomerase CS1 in vitro for the study, I have used pBR322DNA relaxation experiments, kDNA unlinking experiments, DNA embedding experiment and topoisomerase mediated the DNA fault detection experiments. The results showed that CS1 is a DNA non intercalating agent can stabilize the topoisomerase -DNA complexes can be cut to three yuan, as a topoisomerase II Agents inhibit topoisomerase activity. To evaluate the antitumor effect of CS1 in vitro, we selected eleven cell strains derived from different tissues to detect the cytotoxicity, the results showed that CS1 of tumor cells showed some cytotoxicity, but the normal HUVEC cells and HL7702 cells with low toxicity. At the same time we also test the cytotoxicity of CS1 on p- glycoprotein expression and multidrug resistance cell line MCF-7/ADR, the results showed that CS1 sensitive cells MCF-7 and MCF-7/ADR cells showed the same degree of cytotoxicity, and CS1 did not induce Topo II Topo II alpha and beta protein degradation, which indicates that CS1 has potential anti multidrug resistance effect and is not easy to due to drug resistance. Cycle analysis and DNA Ladder test showed that CS1 induced apoptosis, we further used Anneixn V/PI double staining showed that CS1 induced apoptosis has certain concentration The degree of dependence. Comet assay, immunofluorescence and Western blotting experiments showed that CS1 can cause DNA fracture, what is interesting is that CSl does not activate ATM-Chk1/Chk2 typical stress responses, only selective phosphorylation of DNA damage signaling protein ATR of ATM but did not affect.Hoechst33342 staining showed that CS1 induced nuclear phenomena, we further use immunity immunofluorescence staining showed that CS1 induced abnormal cell mitosis, forming a plurality of poles, asymmetric cell division, apoptosis induced by mitotic catastrophe. Finally, we have examined the inhibitory effect of CS1 on MDA-MB-231 tumor in nude mice, the results showed that CS1 was administered for 15 days could significantly inhibit the growth of tumor, according to tumor weight calculation the inhibition rate reached 62.3%, according to the calculation of tumor volume inhibition rate was 43.6%, and CS1 had no effect on the weight of nude mice.
In summary, 2- phenyl naphthalene compounds represented by CSl has strong antitumor activity but less toxic to normal cells, can inhibit malignant tumor cell migration and anti MDR activity potential of human breast cancer MDA-MB-231 CS1 can inhibit tumor growth in nude mice. Its mechanism may be through inhibition of topoisomerase CS1 the activity of II, induced DNA damage caused by tumor cell mitotic catastrophe and its antitumor activity. Our results provide a theoretical basis for the medicine of CS1, its novel mechanism of action may also provide a new idea for the development of new topoisomerase inhibitors.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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