本文關(guān)鍵詞： CS1 拓?fù)洚悩?gòu)酶 有絲分裂災(zāi)難 細(xì)胞凋亡 DNA損傷　出處：《山東大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：拓?fù)洚悩?gòu)酶是真核生物體內(nèi)最重要的酶之一,調(diào)節(jié)DNA拓?fù)浣Y(jié)構(gòu),參與DNA復(fù)制、轉(zhuǎn)錄、染色體重組以及姐妹染色體分離等多個(gè)環(huán)節(jié)。拓?fù)洚悩?gòu)酶已經(jīng)成為抗腫瘤藥物的一個(gè)重要靶點(diǎn),靶向拓?fù)洚悩?gòu)酶的藥物在臨床上也取得了很好的療效。但拓?fù)洚悩?gòu)酶抑制劑在臨床使用過程中出現(xiàn)了許多毒副作用,除了常見的胃腸道反應(yīng),皮膚病,還有許多嚴(yán)重的不良反應(yīng)如阿霉素引起的嚴(yán)重新心臟毒性、依托泊苷誘發(fā)次生腫瘤(白血病)以及多藥耐藥等。多年來,開發(fā)高效低毒的拓?fù)洚悩?gòu)酶抑制劑一直是抗腫瘤藥物研究的熱點(diǎn)之一。 天然三聯(lián)苯類化合物主要來源于真菌代謝產(chǎn)物,現(xiàn)已發(fā)現(xiàn)的三聯(lián)苯類化合物表現(xiàn)出多種生物活性包括抗腫瘤,抗氧化,抗細(xì)菌等。本課題組前期研究發(fā)現(xiàn)對三聯(lián)苯H1-H8抑制人乳腺導(dǎo)管癌MDA-MB-435細(xì)胞增殖,引起細(xì)胞G2/M期停滯并誘導(dǎo)細(xì)胞凋亡。此外,H1-H8以及從鏈霉菌sp. LZ35中分離得到的對三聯(lián)苯echosides A-E能不同程度地抑制拓?fù)洚悩?gòu)酶的活性,但是這類化合物在對正常細(xì)胞和腫瘤細(xì)胞的選擇性以及體內(nèi)抗腫瘤活性較差�；谇捌诘难芯炕A(chǔ),本實(shí)驗(yàn)室以對三聯(lián)苯為基本結(jié)構(gòu)進(jìn)行改造針對拓?fù)洚悩?gòu)酶設(shè)計(jì)合成了一系列2-苯基萘,本論文研究這一系列2-苯基萘的抗腫瘤活性及其對拓?fù)洚悩?gòu)酶的抑制活性,并挑選代表化合物CS1深入研究這類化合物的體內(nèi)外抗腫瘤活性及其抗腫瘤作用機(jī)制。 我們首先用SRB法檢測了48個(gè)2-苯基萘對三株腫瘤細(xì)胞MDA-MB-231,A549和HeLa細(xì)胞的細(xì)胞毒性,結(jié)果有11個(gè)化合物對人乳腺癌MDA-MB-231細(xì)胞的IC5o小于5μM。然后我們用DNA松弛實(shí)驗(yàn)檢測這11個(gè)細(xì)胞毒性較強(qiáng)的化合物對拓?fù)洚悩?gòu)酶I和拓?fù)洚悩?gòu)酶II的抑制作用,有5個(gè)化合物能完全抑制拓?fù)洚悩?gòu)酶II的活性。我們挑選代表化合物1(CS1)深入研究其抗腫瘤作用機(jī)制。為研究CS1體外抑制拓?fù)洚悩?gòu)酶的作用方式,我們采用了pBR322DNA松弛實(shí)驗(yàn)、kDNA解除鏈接實(shí)驗(yàn)、DNA嵌入實(shí)驗(yàn)以及拓?fù)洚悩?gòu)酶介導(dǎo)的DNA斷裂檢測實(shí)驗(yàn)。結(jié)果發(fā)現(xiàn)CS1是一個(gè)DNA非嵌入劑,能夠穩(wěn)定拓?fù)洚悩?gòu)酶-DNA可切割三元復(fù)合物,作為一個(gè)拓?fù)洚悩?gòu)酶II毒劑抑制拓?fù)洚悩?gòu)酶的活性。為全面評價(jià)CS1的體外抗腫瘤作用,我們選取不同組織來源的十一株細(xì)胞檢測其細(xì)胞毒性,結(jié)果顯示CS1對多種組織來源的腫瘤細(xì)胞均表現(xiàn)出一定的細(xì)胞毒性,但對正常細(xì)胞株HUVEC和HL7702細(xì)胞毒性較小。同時(shí)我們還檢測了CS1對p-糖蛋白過表達(dá)的多藥耐藥細(xì)胞株MCF-7/ADR的細(xì)胞毒性,結(jié)果發(fā)現(xiàn)CS1對敏感細(xì)胞MCF-7和耐藥細(xì)胞MCF-7/ADR表現(xiàn)出相同程度的細(xì)胞毒性,并且CS1不引起Topo Ⅱα和Topo Ⅱβ蛋白降解,這表明CS1具有潛在抗多藥耐藥作用且不易引起耐藥性。周期分析和DNA Ladder實(shí)驗(yàn)顯示CS1誘導(dǎo)細(xì)胞凋亡,我們進(jìn)一步用Anneixn V/PI雙染色證明CS1誘導(dǎo)細(xì)胞凋亡具有一定濃度依賴性。彗星電泳,免疫熒光和免疫印跡等實(shí)驗(yàn)顯示CS1能夠引起DNA斷裂,有趣的是CSl并沒有激活典型的ATM-Chk1/Chk2應(yīng)激反應(yīng),只選擇性磷酸化DNA損傷信號蛋白ATR但對ATM沒有影響。Hoechst33342染色顯示,CS1誘導(dǎo)細(xì)胞產(chǎn)生多核現(xiàn)象,我們進(jìn)一步用免疫熒光染色發(fā)現(xiàn)CS1誘導(dǎo)細(xì)胞不正常有絲分裂,形成多個(gè)極點(diǎn),細(xì)胞不對稱分裂,誘導(dǎo)細(xì)胞發(fā)生有絲分裂災(zāi)難。最后,我們還有檢測了CS1在裸鼠體內(nèi)對MDA-MB-231腫瘤的抑制作用,結(jié)果顯示CS1給藥15天能顯著抑制腫瘤的生長,按瘤重計(jì)算抑制率達(dá)62.3%,按腫瘤體積計(jì)算抑制率達(dá)43.6%,并且CS1對裸鼠體重沒有任何影響。 綜上所述,以CSl為代表的2-苯基萘類化合物具有很強(qiáng)的抗腫瘤活性但對正常細(xì)胞毒性較小,能夠抑制惡性腫瘤細(xì)胞遷移并具有潛在的抗多藥耐藥活性,CS1還能抑制接種裸鼠的人乳腺癌MDA-MB-231腫瘤生長。其作用機(jī)制可能是CS1通過抑制拓?fù)洚悩?gòu)酶II活性,誘導(dǎo)DNA損傷,引起腫瘤細(xì)胞有絲分裂災(zāi)難進(jìn)而發(fā)揮抗腫瘤活性。我們的研究結(jié)果為CS1的成藥性提供了理論依據(jù),其新穎的作用機(jī)制還可能為開發(fā)新型拓?fù)洚悩?gòu)酶抑制劑提供新的研究思路。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

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相關(guān)期刊論文 前1條

1 林菁;王希;;黃癸素對拓?fù)洚悩?gòu)酶的影響及聯(lián)合羥喜樹堿的抗腫瘤作用[J];藥學(xué)學(xué)報(bào);2011年04期

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本文編號：1456230