【作者】 谭君；

【导师】 王伯初；

【作者基本信息】 重庆大学 ， 生物医学工程， 2007， 博士

【摘要】 恶性肿瘤现已成为人类死亡的主要原因之一，寻找抗肿瘤药物已经成为药学研究的重大课题。近年研究表明某些槲皮素金属配合物不但抗氧化活性比槲皮素强，而且其抑制肿瘤细胞生长的作用也比槲皮素强。然而目前槲皮素金属配合物抗肿瘤的作用机制还不清楚。它们能否诱导肿瘤细胞凋亡，其信号转导通路，作用靶点等科学问题还有待探索研究。本课题利用生物化学、细胞生物学、药学、计算化学等多学科交叉手段，主要研究了槲皮素金属配合物（Mn、Co、Ni、Cu、Zn）对肿瘤细胞凋亡的影响，及其与DNA结合和催化DNA断裂作用，探讨了槲皮素金属配合物诱导肿瘤细胞凋亡的作用机制及其与DNA相互作用的机理。得到了如下结论：①采用MTT法、荧光染色法、流式细胞术和免疫细胞化学方法对槲皮素金属配合物抗肿瘤活性和诱导HepG2细胞凋亡进行了研究，初步探讨了槲皮素金属配合物诱导肿瘤细胞凋亡的分子机制。1)结果表明槲皮素金属配合物（Mn、Co、Ni、Cu、Zn）对肝癌细胞HepG2、SMMC-7721和肺腺癌细胞A549的生长均有明显的抑制作用，并呈明显的剂量和时间依赖性。其中槲皮素锰、镍和锌配合物对肿瘤细胞生长的抑制作用明显强于配体槲皮素，它们作用于HepG2细胞48小时的半数抑制浓度IC₅₀分别为20.64、14.44和10.67μmol·L^-1，而槲皮素的IC₅₀为33.69μmol·L^-12)形态学观察结果表明HepG2细胞经槲皮素锰、镍和锌配合物处理后，染色质凝聚，且有明显的凋亡小体出现，表明槲皮素锰、镍和锌配合物能诱导HepG2细胞凋亡。3)细胞周期分析结果表明槲皮素锰、镍和锌配合物对肝癌HepG2细胞生长的抑制作用可能是通过使细胞阻滞于G₀／G₁期，使细胞DNA合成速度降低，细胞周期的循环被抑制而实现的。对G₀/G₁期细胞阻滞可能是配合物诱导HepG2细胞凋亡的一个重要机制。4)免疫细胞化学检测结果表明槲皮素锰、镍和锌配合物诱导HepG2细胞凋亡可能通过下调bcl-2和survivin，上调p53，引发caspase的级联反应，从而激活caspase-3来进行的。下调survivin和bcl-2的表达对槲皮素金属配合物诱导HepG2细胞凋亡既起到了协同作用，又起到了互补作用。②采用紫外光谱法、荧光光谱法和粘度法研究了槲皮素锰、钴、镍、铜和锌配合物与小牛胸腺DNA的结合方式，采用凝胶电泳法、脱水丙二醛分析和T4连接实验较系统地研究了槲皮素锰、钴、镍、铜和锌配合物对质粒pBR322DNA的催化断裂情况。初步探讨了槲皮素金属配合物和DNA的作用方式及其对DNA的催化断裂机理。发现槲皮素金属配合物具有核酸酶类活性。1)槲皮素锰、钴、镍和锌配合物仅以插入方式与DNA结合，而槲皮素铜配合物与DNA的结合方式可能不止一种，除插入方式结合以外还受到静电作用的影响。2)槲皮素锰、钴、镍、铜和锌配合物均能有效断裂DNA，且槲皮素铜配合物断裂DNA的能力最强；对于槲皮素锰、镍、铜和锌配合物，低离子强度促进DNA裂解，高离子强度抑制DNA裂解；离子强度增大能促进槲皮素钴配合物对DNA的断裂，所以槲皮素金属配合物对DNA的断裂作用受离子强度的影响。3)如以超螺旋DNA为底物，槲皮素锰、钴、镍、铜和锌配合物催化断裂DNA的反应符合准一级反应速率定律。其速率常数分别为1.18×10^-4s^-1、0.53×10^-4s^-1、0.76×10^-4s^-1、4.82×10^-4s^-1、1.68×10^-4s^-1。4)DNA断裂机理实验表明槲皮素锰、钴、镍和锌配合物均能以水解途径断裂DNA；槲皮素铜配合物以氧化还原途径断裂DNA，可能与Cu（I）OOH物质有关。③引入量子化学和分子力学方法，对槲皮素金属配合物抗肿瘤作用的机制及其与DNA结合作用的机理进行了理论解释。1)对槲皮素金属配合物结构进行了量子化学计算，通过对其几何构型的讨论，发现槲皮素金属配合物的6个配位氧原子与金属离子形成畸形六配位八面体配合物，其配位结构是合理的；根据NBO电荷分析、前沿分子轨道分析解释了配合物的配位特性，发现羟基氧的配位能力强于羰基氧。2)计算结果表明槲皮素金属配合物的LUMO及附近未占轨道能量比DNA碱基对模型CG／GC的HOMO和NHOMO的能量低，有利于配合物接受DNA碱基对上的电子，增强了配合物与DNA碱基对的π-π相互作用。槲皮素金属配合物抗肿瘤活性及其DNA键合能力与其LUMO的能量和配体所带电荷两方面的因素有关。3)运用分子力学研究了配合物插入DNA的过程，结果表明五种槲皮素金属配合物都可以插入到DNA碱基对之间，倾向于从小沟插入到CG／GC碱基对间。根据槲皮素金属配合物插入DNA碱基对后的势能值，发现配合物与DNA插入能力的理论计算结果与实验结果一致。槲皮素金属配合物与DNA结合作用的大小顺序为zn（Que）₂>Ni（Que）₂>Mn（Que）₂>Co（Que）₂>Cu（Que）₂。提示槲皮素金属配合物有可能抑制富含GC的survivin基因启动子的转录，而抑制survivin的表达，从而促进肿瘤细胞凋亡。本论文对槲皮素金属配合物诱导肿瘤细胞凋亡的分子机制及其与DNA相互作用的机理进行了探索性的研究，为将此类化合物应用于高效、实用化学核酸酶和抗肿瘤药物的开发奠定了基础，为新型抗肿瘤药物的设计提供了新的思路。更多还原

【Abstract】 In recent years malignant tumor has been one of the main causations of death, and antitumor drug screening has been a significant task in pharmaceutics. It was reported that both antioxidant activity and tumor cell proliferation inhibitory effect of some quercetin metal complexes were stronger than those of quercetin alone. However, little attention is paid to antitumor mechanisms of quercetin metal complexe. So in order to elucidating antitumor mechanism of quercetin metal complexes, we focus our study on the effect of quercetin metal complexes（Mn, Co, Ni, Cu, Zn） on tumor cell apoptosis, and their DNA-binding and cleavage activity. Based on these, we discussed the mechanism of cell apoptosis induced by quercetin metal complexes and the interaction mechanism of the complexes with DNA.1) Antitumor activity of quercetin metal complexes and HepG2 cell apoptosis induced by them was studied using MTT assay, fluorescence staining, flow cytometry （FCM） and immunocytochemical method. And the molecular mechanisms of tumor cell apoptosis induced by quercetin metal complexes are discussed preliminarily.i) It is found that quercetin metal complexes have a significant inhibition to growth and proliferation of HepG2, SMMC-7721 and A549 cell lines in a concentration- and time-dependent manner. Furthermore, the proliferation inhibitory effect of quercetin manganese, nickel and zinc complexes on tumor cells is stronger than that of quercetin alone. After tumor cells were treated by quercetin manganese, nickel and zinc complexes for 48h,the half inhibitory concentration (IC₅₀) were 20.64,14.44 and 10.67μmol·L^-1 respectively, while IC₅₀ of quercetin was 33.69μmol·L^-1ii) After treatment with quercetin manganese, nickel and zinc complexes, morphological changes of HepG2 cells apoptosis were observed with fluorescence microscope. Results indicate that they could induce HepG2 cells apoptosis.iii) FCM results suggest that HepG2 cells are arrested at G₀/G₁ after treatment with quercetin manganese, nickel and zinc complexes, which could contribute to tumor cell apoptosis.iv) Evidence is provided that expression levels of bc1-2 and survivin protein are down-regulated and expression level of p53 protein is up-regulated after treatment with quercetin manganese, nickel and zinc complexes, which activates caspase-3 resulting in HepG2 cells apoptosis. Down-regulation of survivin and bcl-2 could exert both synergistic effect and complementation effect in HepG2 cell apoptosis induced by quercetin metal complexes. Therefore HepG2 cells apoptosis induced by quercetin manganese, nickel and zinc complexes is not regulated by single factor, but rather by multiple pathways.2) The interaction of quercetin metal complexes with calf thymus（CT） DNA is investigated by electronic absorption spectroscopy, fluorescence spectroscopy, viscosity measurement. Plasmid pBR322 DNA cleavage is also studied by gel electrophoresis. Furthermore, DNA-binding properties and mechanism of DNA cleavage are discussed preliminarily. We put forward the nuclease-liked activity of quercetin metal complexes for the first time.i) Results infer that quercetin manganese, cobalt, nickel and zinc complexes could interact with DNA through intercalation mode, but DNA-binding of quercetin copper complexes is more than one mode, including intercalation and electrostatic mode.ii) Results suggest that quercetin metal complexes could cleave supercoiled pBR322 DNA to nicked and line form effectively. Among them, DNA cleavage by quercetin copper complex is the most efficient. Moreover, low ionic strength could promote DNA strand breakage induced by quercetin manganese, nickel, copper and zinc complexes, while high ionic strength could prevent the formation of double strand breaks. However, increasing ionic strength could only promote DNA cleavage induced by quercetin cobalt complex. So DNA cleavage induced by quercetin metal complexes depends on ionic strength in the solution.iii) The decrease of supercoiled DNA induced by quercetin metal complexes with time shows the expected exponential nature of the curves, which confirms the process to pseudo-first-order. Their rate constants are 1.18×10^-4s^-1（Mn）, 0.53×10^-4s^-1（Co）, 0.76×10^-4s^-1（Ni）,4.82×10^-4s^-1（Cu）,1.68×10^-4s^-1（Zn）, respectively.iv) Results imply that the process of DNA cleavage by quercetin manganese, cobalt, nickel and zinc complexes occurs via a hydrolytic path, while the process of DNA cleavage by quercetin copper complex occurs via a redox path involved in Cu（Ⅰ）OOH.3) Mechanisms of antitumor activity and interaction of quercetin metal complexes with DNA were elucidated theoretically by quantum chemistry and molecular mechanics methods.i) Based on quantum chemistry calculation of the structure of quercetin metal complexes, it is found that freakish octahedron structure comprises six oxygen atoms and a metal ion, which has good stability. Furthermore, coordinate properties of quercetin metal complexes are discussed through nature population analysis and frontier orbital analysis, which confirms the coordinate action of hydroxyl oxygen is stronger than that of carbonyl oxygen.ii) The calculation results suggest that the energies of LUMO and NLUMO of quercetin metal complexes are lower than the energies of the HOMO and NHOMO for the CG/GC stacking in DNA backbones, which contributes to enhance theπ-πinteraction between complexes and DNA base-pairs. The antitumor activity and DNA-binding properties of quercetin metal complexes could rely heavily on their LUMO energies and total charge of ligand of complexes.iii) The processes of quercetin metal complexes intercalating into DNA are studied by molecular modeling. Results infer that quercetin metal complexes are inclined to intercalate into base stack in CG/GC region from minor groove, which depends on asymmetric charge distribution and steric interaction. Base on calculated potential energy of quercetin metal complexes intercalating into DNA base stack, it is suggested that calculated DNA-binding action decreases in the order Zn（Que）₂>Ni（Que）₂>Mn（Que）₂ >Co（Que）₂>Cu（Que）₂,according with experiment results. It is proposed that quercetin metal complexes may inhibit transcription of survivin promoter with abundant GC,which contributes to down-regulating expression of survivin protein and promote tumor cell apoptosis.In the present paper, molecular mechanisms of tumor cells apoptosis induced by quercetin metal complexes and mechanisms of interaction of the complexes with DNA are discussed, which lay the foundation of development of new efficient chemical nuclease and antitumor drug, and provide a new consideration for novel antitumor drug design.更多还原