【作者】 王海燕；

【导师】 余龙江； 敖明章；

【作者基本信息】 华中科技大学 ， 生物制药工程， 2013， 博士

【摘要】 喜树碱（Camptothecin, CPT）由于高效、广谱的抗肿瘤活性而位居癌症治疗前列，但明显的毒副作用限制了它在临床上的使用，因而开发抗肿瘤活性强、毒副作用小的衍生物成为了国内外研究热点。9-甲氧基喜树碱（9-Methoxycamptothecin, MCPT），一种喜树碱衍生物，体内外试验均证实有明显的抗多种肿瘤活性，但对其有关细胞和分子方面的机制研究极少。本课题首先体外评价了9-甲氧基喜树碱的抗肿瘤细胞活性，并从9-甲氧基喜树碱诱导肿瘤细胞凋亡、影响DNA拓扑异构酶活性及其与DNA分子相互作用等三个方面进行了作用机制的研究。1.评价9-甲氧基喜树碱体外抗肿瘤活性。结果表明，9-甲氧基喜树碱抑制7种肿瘤细胞系，呈剂量依赖性。其中，人卵巢癌细胞A2780和人宫颈癌细胞Hela对9-甲氧基喜树碱最为敏感，其IC50值分别为79±21nM和151±61nM，明显低于阳性对照药物10-羟基喜树碱（Hydroxycamptothecin，HCPT）。选取这两种细胞系开展后续作用机制研究。2.研究了9-甲氧基喜树碱诱导细胞凋亡作用及其机制。结果表明：（1）9-甲氧基喜树碱处理A2780和Hela细胞后，荧光显微镜下观察到诱导的凋亡细胞，流式细胞仪检测到明显的早期和晚期凋亡细胞。9-甲氧基喜树碱诱导A2780细胞的早期凋亡和总凋亡率增加具有时间-剂量依赖性；（2）9-甲氧基喜树碱处理A2780和Hela细胞24h后即表现出强的G2/M期阻滞。1×IC50浓度的9-甲氧基喜树碱处理A2780细胞24h后对G2/M期阻滞最明显，达到81.93%；（3）9-甲氧基喜树碱能使A2780和Hela细胞中产生活性氧（ROS）明显增多。A2780细胞中ROS产生量最高达到对照组的5.22倍，Hela细胞中最高为2.65倍；（4）9-甲氧基喜树碱能不同程度激活caspase-3，-8和-9。在A2780细胞中对caspase-9酶激活程度最强，是对照组细胞的15倍。Hela细胞中caspase-3的酶活性被显著提高，约为对照组细胞的8倍；（5）实时定量PCR检测到9-甲氧基喜树碱调节了17个内、外源性细胞凋亡通路及细胞周期相关基因。其中对细胞周期调控基因p21，p27和cyclinE上调较显著；且明显上调参与外源性细胞凋亡通路的TNFα、Fas和FasL；同时还调节了主要参与内源性细胞凋亡通路的bcl-2家族有关基因表达水平；（6）Westernblot检测到TNFα, Fas, P53和P27的蛋白表达水平上调，与其基因水平调节一致。综上所述，9-甲氧基喜树碱可通过调节细胞周期调控蛋白使细胞周期阻滞于G2/M期，并增加ROS的产生、调节bcl-2家族相关基因激活caspase-9，从而启动内源性细胞凋亡通路；同时激活了caspase-8启动以TNFα和Fas/FasL为主的外源性细胞凋亡通路。以上两条通路共同激活caspase-3，最终导致A2780和Hela细胞凋亡。3.研究了9-甲氧基喜树碱对DNA拓扑异构酶Ⅰ和Ⅱ活性的影响。结果显示，100μM的9-甲氧基喜树碱表现出对拓扑异构酶Ⅰ明显的抑制作用，与阳性对照药物喜树碱一样，可使超螺旋DNA产生明显的缺刻型DNA条带。不同浓度的9-甲氧基喜树碱均未表现出对拓扑异构酶Ⅱ的抑制作用（与阳性对照药物依托泊苷相比）。值得注意的是，100μM的9-甲氧基喜树碱能单独作用于超螺旋DNA并产生明显的缺刻型DNA产物，表明9-甲氧基喜树碱在没有拓扑异构酶Ⅰ存在的情况下也能作用于DNA。提示，9-甲氧基喜树碱的这种直接结合DNA的能力很有可能是其抗肿瘤活性强的一种重要机制。4.采用光谱法研究了9-甲氧基喜树碱与DNA分子相互作用。结果表明，（1）紫外-可见吸收光谱显示，随着不断滴加CT-DNA，9-甲氧基喜树碱最大吸收峰出现了增色效应，谱带未发生偏移，表明其与DNA的作用属于外部键合模式（沟槽作用和/或静电作用）。9-甲氧基喜树碱处理CT-DNA前后体系的解链温度（melting temperature,Tm）值升高不明显，也支持外部键合模式；（2）荧光光谱显示，随着CT-DNA浓度不断增加，9-甲氧基喜树碱荧光强度不断增强，没有发生明显光谱偏移现象，支持外部键合模式；（3）9-甲氧基喜树碱与CT-DNA相互作用的圆二色谱显示，9-甲氧基喜树碱加入后，体系的正峰有小的提升，而其负峰有较明显的回落并伴有轻微的吸收峰红移现象。由于负峰变化程度大于正峰，表明两者结合以沟槽模式为主。综上可知，9-甲氧基喜树碱与CT-DNA的结合方式以沟槽模式为主。综合以上研究结果表明，9-甲氧基喜树碱具有明显的抗多种肿瘤细胞活性，其作用机制主要是通过与拓扑异构酶Ⅰ-DNA形成三元复合物“路障”阻止DNA复制、以沟槽方式结合DNA并对其进行剪切、诱导肿瘤细胞凋亡而起到抗肿瘤作用。更多还原

【Abstract】 Camptothecin（CPT）was a prominent drug due to its high efficient and broad-spectrumanti-tumor activity in cancer treatment, but its clinical application was limited by its obviousside effects. Therefore, an increasing camptothecinoids (CPTs) were developed.9-Methoxycamptothecin (MCPT), one of CPTs, shows strong antitumor activities againstdifferent cancer cells. However, the cellular and molecular mechanisms of MCPT have notbeen well understood. MTT assay was adopted to examine the inhibitory effect of MCPT ondifferent cell lines firstly, then, the mechanisms of MCPT including apoptosis induced, theeffect of MCPT on topoisomeraseⅠand Ⅱ, the interaction with CT-DNA were studied.1. MTT assay was employed to evaluate the anti-tumour activity of MCPT in vitro. Theresults showed that MCPT could inhibit the growth of seven cancer cell lines in adose-dependent manner. Among those cancer cell lines, A2780and Hela cell lines weremore sensitive to MCPT than other cell lines, with the IC50values being79±21nM and151±61nM respectively, lower than that of hydroxycamptothecin（HCPT, the positive control）evidently. So, both A2780and HeLa cell lines were selected for further analysis.2. Apoptosis induced by MCPT and its mechanisms of action were studied. The resultswere listed as follows.(1) Cells including early and late apoptosis cells induced by MCPT inboth A2780and HeLa cells were detected both through fluorescence microscope and flowcytometry. MCPT induced early and total apoptosis of A2780cells in a dose-time dependentmanner.(2) Both A2780and HeLa cells were arrested at the G2/M phase after MCPTtreatment for24h. MCPT at1×IC50caused an accumulation in the G2/M phase with themaximum percentage of81.93%for A2780.(3) MCPT increased significantly ROSgeneration in both A2780and HeLa cells. After treatment with MCPT, the the maximumintracellular level of ROS reached5.22-fold of control cells in A2780cells and2.65-fold inHeLa cells.(4) MCPT could activate caspase-3,-8and-9in different degrees, with thestrongest activation of caspase-9in A2780cells, about15folds relative to control, andcaspase-3in HeLa cells about8folds.(5) MCPT regulated expression of seventeen genesinvolved in extrinsic and intrinsic apoptotic pathway and regulating cell-cycle in A2780and HeLa cells. Among those genes, MCPT up-regulated significantly cell-cycle regulatorygenes-p21，p27and cyclinE, and increased apparently TNFα,Fas and FasL involved inextrinsic apoptotic pathway, meanwhile regulated bcl-2family genes-the key participant inintrinsic apoptotic pathway.(6) Western blot verified that changes of TNFα, Fas, P53andP27protein level were consistent with their gene expression changes. In short, MCPTinduced apoptosis in A2780and HeLa cells through arresting cell cycle at G2/M viaregulating cell-cycle proteins, elevating the ROS level and regulating genes of the bcl-2family, activating caspase-9and thus initiating the intrinsic apoptotic pathway, activatingcaspase-8and initiating the extrinsic apoptotic pathways with the key TNFα and Fas/FasLpathway.Both the intrinsic and extrinsic pathways activated caspase-3and resulted inapoptosis in both cells in the end.3. The effects of MCPT on the catalytic activity of topoisomerase I and Ⅱwere studied.The results showed that100μM MCPT exhibits obvious topoisomerase I poison effects aswell as CPT (the positive control) at the same concentration based on generating nickedforms from supercoiled circular DNA. Gradual increasing concentration of MCPT didn’texhibit topoisomeraseⅡpoison effects based on the lack of generated nicked forms or linearforms from supercoiled circular DNA. It is interesting that MCPT at100μM alone cancleavethe supercoiled DNA. It seems that MCPT can directly bind to DNA without topoisomerase.The ability of MCPT’s direct binding to DNA may be its important mechanism of stronganticancer activity.4. The interaction of MCPT with DNA was studied via spectroscopies.The results wereas follows:(1) UV-vis absorption spectroscopy showed that upon addition CT-DNA toMCPT of fixed concentration, there was an increase in the absorption intensity of MCPTindicative of hyperchromism with no band shift. Moreover, no significant difference in Tmvalues between the MCPT-CT-DNA complexes and CT-DNA alone, indicating MCPTbinding to the CT-DNA with external bonding mode.(2) Fluorescence spectroscopy showedthat upon addition of CT-DNA, the notable emission intensity enhancement without bandshift is observed, indicating that MCPT binding to CT-DNA via external bonding mode notclassical intercalative interaction.(3) Circular dichroism (CD) absorption spectra showedthat MCPT induces a significant decrease in intensity of negative absorption of CT-DNA with slight red-shift in the wavelength, along with a slight enhancement on positiveabsorption intensity. Therefore, groove binding between MCPT with CT-DNA should exist.To sum up, MCPT binds primarily to the CT-DNA with groove bonding mode.In conclusion, MCPT showed strong antitumor activities against different cancer cellsthrough blocking DNA duplicate via Ternary compounds along with TopoisomeraseⅠ-DNA,binding to DNA with groove bonding mode and shearing DNA and inducing cancer cellsapoptosis.更多还原