【作者】 祁铭华；

【导师】 闫兵；

【作者基本信息】 山东大学 ， 药物化学， 2009， 博士

【摘要】 铜是人和动物组织必需的微量元素之一,也是细胞调整蛋白骨架必需的化学元素。多项癌症研究发现,血清中铜的浓度与肿瘤发生,肿瘤负荷,肿瘤恶性进展及肿瘤复发有关。铜在肿瘤发生过程中发挥着积极的作用,肿瘤细胞的特性之一是积聚高浓度铜,有目的的消耗铜是目前研究者认为比较理想的抗肿瘤策略之一。目前一些上市的抗铜试剂比如四硫钼酸盐Tetrathiomolybdate（TM）、卡托普利（Captoril）、锌（Zinc）和曲恩汀（Trientine）等被发现在抗肿瘤治疗中有一定功效,它们的作用主要是降低肿瘤组织中过量的铜,但是它们的最大局限性表现为疗期过长、副作用高、毒性大等。8-羟基喹啉8-hvdroxyquinoline（Ox）和5-氯-7-碘-8-羟基喹啉5-chloro-7-iodo-8-hydroxyquinoline（CQ）是一类常见的金属配合物。有资料证实CQ与Cu（Ⅱ）形成的复合物可能依赖于8位羟基和吡啶环上的N。近几年有研究发现CQ不但可以有效的治疗老年痴呆症还可以抑制8种不同的人体肿瘤细胞系的生长,而且这个抑制作用会随着金属离子Cu²⁺或者Zn²⁺的介入而增强。有研究还表明Cu²⁺存在的条件下,Ox和CQ是一类新型的蛋白酶体抑制剂。但是Ox及其CQ在Cu²⁺存在的条件下对肿瘤细胞的作用机制目前还不清楚。基于以上研究,本论文将着重探讨Ox、CQ在内源性铜的介入下抑制H460肺癌细胞增殖的作用机制。论文将围绕三个部分展开研究:第一部分研究Ox、CQ对H460肺癌细胞生长的抑制作用,重点探讨Ox、CQ对铜的依赖性与其抗肿瘤活性的关系;第二部分通过将Ox、CQ的8-位羟基进行取代得到70个Ox类似物,研究这些类似物的抗肿瘤活性是否依赖于铜;第三部分初步探讨Ox、CQ及其Ox类似物对H460细胞生长抑制作用的机理。论文另一部分的主要工作是运用HPLC/MS/UV检测器创建一种新的分析方法,对50种结构不同的有机物在储存和生物筛选过程中的自校正绝对定量法的可行性应用进行评价。论文的主要工作及其结果如下:第一部分,为了研究Ox及其CQ对H460细胞的活性是否依赖于Cu²⁺,我们比较了三种不同的加药方式对H460细胞的生长抑制作用,测定三种不同加药条件下H460细胞中Cu²⁺的含量,运用紫外光谱滴定法测定Ox-Cu²⁺和CQ-Cu²⁺在体外细胞环境中的结合常数K_b1和K_b2。结果发现这两种有机配体本身具有抑制H460细胞生长的活性,且Ox的活性比CQ的活性强,内源铜的介入可以促进有机配体对H460细胞的生长抑制作用。通过测定细胞中Cu²⁺含量,发现有机配体CQ、Ox能促使细胞中Cu²⁺含量的升高,利用光谱分析测得Ox、CQ能有效的结合Cu²⁺,Ox-Cu²⁺的结合常数K_b1比CQ-Cu²⁺结合常数K_b2大。第二部分,我们研究了70个Ox类似物对H460细胞的毒性,结果发现当Ox及其CQ的8位-OH被其它取代基取代后对H460细胞没有毒性,Ox类似物的抗肿瘤活性依赖于Cu²⁺,内源性Cu²⁺存在时17个Ox类似物表现出抗肿瘤活性。通过比较17个抗肿瘤活性依赖于铜的Ox类似物的IC₅₀,发现类似物1#、5#、6#和115#对癌细胞生长的抑制作用较强,但是比Ox、CQ弱。利用紫外光谱分析这些化合物与Cu²⁺的结合作用,发现Ox类似物与Cu²⁺不结合。第三部分,通过进一步研究Ox、CQ及其17个有活性的化合物对H460细胞的生长抑制作用,发现17个化合物在内源性铜的介入下对H460细胞的生长抑制作用增强。着重研究115#、CQ、Ox对细胞中Cu²⁺含量的影响,发现Ox及其类似物能促使细胞内铜含量的升高,其中Ox把Cu²⁺带入细胞内的量比CQ带入的多;而类似物115#带入Cu²⁺的量较少。另外我们测定了有无内源性铜的条件下,H460细胞中ROS的水平变化,发现加入Ox、CQ、及其有活性的17个化合物都能促使H460细胞中ROS含量的升高,但是通过比较发现ROS含量的升高不是化合物抑制H460细胞增殖的主要原因。第四部分,运用HPLC/MS/UV检测器,对用于生物活性筛选的50个不同结构的化合物进行绝对定量,发现HPLC/MS/UV检测器绝对定量对用于生物活性筛选的药物具有一定的优势:在化合物的生物活性试验中,有利于高通量筛储存已久的化合物,测定其真实浓度,用于前期筛选和IC₅₀的测定;有利于对因储存已久而被分解的药品进行重新收集和再分析。结论:1)Ox、CQ能有效的抑制H460细胞的增殖,在内源性铜的参与下Ox、CQ对H460细胞的生长抑制作用加强,其抑制作用可能与有机配体和铜的结合能力有关:2)Ox类似物当8位羟基被取代后对H460细胞没有毒性,说明Ox及其CQ的8位羟基是羟基喹啉类化合物的抗癌活性位点。Ox类似物的抗肿瘤活性依赖于内源性铜,但是其机制与化合物和铜的结合机制无关。3)Ox、CQ及其Ox类似物对H460细胞的生长抑制作用均依赖于铜,能促进细胞内铜升高。但是这种依赖机制不完全是由于结合作用产生的。4)OX、CQ及其OX类似物可以促使H460细胞中ROS含量升高,但是ROS升高不是化合物抑制H460细胞生长的主要因素。5)运用HPLC/MS/UV检测器对储存已久的药物进行绝对定量是可行的,但是具有一定的局限性。更多还原

【Abstract】 Cu is an essential trace element in human and animal tissues. The copper ion is also an integral part of many metalloenzymes. It has been found the serum copper concentration correlates with tumor incidence and burden, malignant progression and recurrence in a variety of human cancer. Copper ions seem to play on important role in the stimulation of angiogenesis. Therefore, copper became one of the targets in antiangiogenic cancer treatment. In vitro and in vivo studies have confirmed the efficacy of copper-reducing and copper-chelating agents in antiangiogenic treatment. Based on the experimental data coming from in vitro and in vivo studies, it seems reasonable to use copper chelators or copper-reducing agents in antiangiogenic treatment alone or in combination with other therapeutic approaches like surgery or classic chemotherapy, such as tetrathiomolybdate （TM）, Captoril、Zinc and Trientine . However, there are certain limitations of using anti-copper compounds in antitumour treatment. One of them is the long time of drug administration required to produce therapeutic effects. In addition, more work has to be done to explore the exact mechanisms of action of copper-reducing agents and even more important, to define the effects of a long-term copper deficiency.In recently a kind of organic-copper compounds, （8-hydroxyquinoline） Ox and （5-chloro-7-iodo-8-hydroxyquinoline） CQ were found to inhibit the chymotrypsin-like activity of the proteasome in vitro and in human tumor cell culture. Proteasome inhibition and apoptosis were also studied in copper-containing cancer breast cells treated with 8-hydroxyquinoline. CQ is capable of binding copper, forming new complexes that have proteasome-inhibitory to cancer breast cells. Dou concludes the complex between CQ and copper in tumor cellular is a novel proteasome inhibitor. CQ also has the effects on the viability of eight human cancer cell lines. Varia group analyzed the crystal structure of CQ-Cu （II） complex by the way of X ray diffractometry and proved the binding of CQ - Cu （II） is depended on the 8- hydroxyl and N of pyridine. Based on the research referred we presumed the 8- hydroxyl of Ox and CQ is bind to Cu （II） and has antitumor bioactivity to cancer cells. While 8- hydroxyl is substituted it is unable to bind Cu （II） and have no bioactivity. The content of this paper consists four parts. In the first part, the anti-proliferative effects of Ox and CQ on H460 cell line was studied in copper-enrichment or not. Especially the mechanism of anti-proliferative effects of Ox and CQ on H460 was discussed in this paper. In the second part, the 8- hydroxyl of Ox and CQ was substituted by alkane and the library of 8-OHQ analogs was synthesized by combinatory chemistry approach. The library of Ox analogs were screened for their antitumor activity on H460 cell line. In the third part, the mechanism of antitumor activity for Ox, CQ and Ox analogs were discussed. In the last part the feasibility of a self-calibrated LC/MS/UV method to determine the absolute amount of compounds in their storage and screening lifecycle was evaluated.The major research work and results of the paper are as follows:Part 1 the anti-proliferative effects on H460 cell line were compared under three treatments and UV-Vis titration was used to study the binding constant between Ox, CQ and Cu （II）. The copper level of H460 per cell was determined under three treatments. The cytotoxic activity of Ox, CQ was investigated and the Ox was more potent than CQ on H460 cell line. Moreover the cytotoxic activity was more active for the ligands in copper-enrichment than without copper. The chelators were able to increase the level of copper of per cell when the cell line was cultured in copper-enrichment medium and the binding constants took great effect on the copper level of H460 cell line, which also influenced the cell viability. The binding constant of Ox-Cu²⁺ is larger than the binding constant of CQ-Cu²⁺ so Ox is easier to combine with Cu （II） than CQ, which raised the Cu level of cell line.Part 2, the Ox analogs were found inactive on H460 cell line when 8-hydroxyl was substituted by alkine. While six of 8-hydroxyquinoline derivatives and eleven of 8-hydroxyquinoline analogs were active on H460 cell line when mixed with Cu （II）. By contrast to their IC50 values of six 8-hydroxyquinoline derivatives and eleven of 8-hydroxyquinoline analogs compounds 1#, B, C and 115 # were particularly potent on H460 cell line but were less than Ox and CQ. They are unable to combine with Cu （II） by UV-vis titration.Part 3 In contrast the anti-proliferative effects of Ox, CQ and Ox analogs in four different treatments are more potent to inhibit the cell viability when fully mixed with Cu more time and were determined higher level of Cu in per cell. It was found that the intracellular Cu level was connected with binding constants in four different treatments. In contrast the binding constant of Ox-Cu²⁺ is larger so higher level of Cu²⁺ were transferred into cells than CQ. However 115# was capable to transfer Cu²⁺ into the cells even though it didn’t combine with Cu （II）. In addition Ox, CQ and OX analogs at low concentrations in the presence of copper or not generates reactive oxygen species （ROS）. The level of intracellular ROS was not the main mechanism of inhibition of H460 cells proliferation.Part 4 HPLC/MS/UV detector was applied to determine the self-calibrated quantification for the library of 50 pure compounds. There are several immediate applications of this method: 1) quantification of storage compounds at the time of screening to determine the real concentration for primary screening and IC50 measurements; 2) re-analyze compound collection by quantification of compound loss due to decomposition, freeze/thaw, and other effects during storage.Conclusions:1) Ox and CQ are potent to inhibit H460 cells proliferation. The anti-tumor proliferative effect is related with binding constant2) Ox analogs are inactive on H460 cell line but it is highly potent on H460 cell line in the presence of Cu （II）.3) Ox, CQ and Ox analogs generates reactive oxygen species in the presence of Cu （II） nor not. The generation of ROS was not the main mechanism of inhibition of H460 cells proliferation.4) The self-calibrated LC/MS/UV method to determine the absolute amount of compounds in their storage and screening lifecycle is feasible but it has some limitation.更多还原