【作者】 张震；

【导师】 毕彩丰； 范玉华；

【作者基本信息】 中国海洋大学 ， 海洋化学工程与技术， 2013， 博士

【摘要】 自从顺铂应用于临床以来，以金属为基础的抗癌药物引起人们极大关注。近年研究表明某些铜、锌和金等金属配合物能够抑制肿瘤细胞的蛋白酶体活性，从而诱导癌细胞凋亡。泛素-蛋白酶体通路(Ubiquitin proteasome pathway)是真核细胞蛋白质降解的重要途径。它的主要组成部分20S蛋白酶体，是一种高分子量的蛋白酶，其蛋白水解核包含的亚基包括β1、β2和β5，分别与caspase活性、trypsin活性和chymotrypsin (CT)活性相关。特异性抑制蛋白酶体CT活性，可以抑制肿瘤细胞生长，从而诱导肿瘤细胞凋亡。泛素-蛋白酶体系统作为一种新型的分子靶标已被广泛地研究。因此，设计、合成新型金属配合物作为蛋白酶体抑制剂，对抗肿瘤活性药物的研究具有重要的意义。本论文以具有良好生物活性的化合物为配体，合成了一系列具有潜在抗癌活性的过渡金属配合物。考察了所合成配合物对人癌细胞增殖、蛋白酶体抑制和诱导细胞凋亡的能力，深入研究了抗肿瘤作用机制，确定了这些配合物的抗肿瘤作用靶标为蛋白酶体。具体内容如下：(1)利用植物生长激素3-吲哚乙酸、3-吲哚丙酸和3-吲哚丁酸作为配体，合成了二元双核铜配合物和三元铜配合物。探讨了配合物的结构对其转移铜进入癌细胞的影响。研究了配体以及铜配合物抑制人前列腺癌PC3细胞增殖能力，抑制纯化的20S蛋白酶体CT活性和细胞内蛋白酶活性能力以及诱导细胞凋亡的能力；比较了三元铜配合物对人乳腺癌MDA MB231和正常乳腺MCF10A细胞的毒性作用。结果表明：以1，10-邻菲啰啉作为第二配体的三元铜配合物能促进癌细胞吸收铜，从而抑制前列腺癌和乳腺癌细胞的蛋白酶体活性进而诱导癌细胞凋亡，三元铜配合物对正常细胞毒性较小。此类三元铜配合物有望成为临床上治疗前列腺癌和乳腺癌的潜在药物。(2)以3-吲哚丁酸（L1）和3-吲哚丙酸（L2）为配体合成了铜、锌和镉金属配合物。通过MTT法、细胞内蛋白酶体CT活性的测定、免疫蛋白印迹分析，比较了具有相似结构的铜、锌和镉金属配合物对乳腺癌阳性MCF7和阴性MDA MB231细胞增殖和蛋白酶体活性的抑制作用，发现两种镉配合物具有良好的抑制癌细胞增殖和蛋白酶体活性的能力。研究了镉配合物抑制纯化的20S蛋白酶体活性的作用，并通过电子云密度分析初步解释为什么镉配合物可以作为蛋白酶体活性的抑制剂。进一步考察了这两种镉配合物抑制细胞内蛋白酶体CT活性和诱导肿瘤细胞凋亡的能力，结果表明这两种镉配合物可以作为良好的蛋白酶体抑制剂和细胞凋亡的诱导剂，而且对正常细胞MCF10A的毒性相对较小。(3)以第四代头孢菌素头孢吡肟与铜、锌、镍、镉等过渡金属盐混合液作为研究对象，探讨这些混合液抑制阴性乳腺癌MDA MB231细胞增殖的能力。筛选得出头孢吡肟Mn(Ⅱ)混合液具有较强的抑制癌细胞增殖能力。进而研究了头孢吡肟Mn(Ⅱ)混合液抑制纯化的20S蛋白酶体CT活性和细胞内蛋白酶体CT活性以及诱导细胞凋亡能力。结果表明：头孢吡肟Mn(Ⅱ)混合液可以通过抑制乳腺癌MDA MB231细胞的蛋白酶体活性来诱导癌细胞凋亡。通过对乳腺癌细胞MDA MB231和正常细胞MCF10A对比，表明头孢吡肟Mn(Ⅱ)混合液对正常乳腺细胞的毒性较小。(4)合成了3，5-二氨基苯甲酸希夫碱铜、锌等过渡金属配合物。研究了它们抑制细胞增殖能力，筛选出抑制活性较强的配合物为3，5-二氨基苯甲酸缩水杨醛希夫碱镉配合物（LA5）和3，5-二氨基苯甲酸缩邻香草醛希夫碱镉配合物（LC3）。考察了它们对体外纯化20S蛋白酶体抑制活性，结果表明它们对纯化的20S蛋白酶体的抑制作用呈浓度依赖方式。LA5和LC3作用于人乳腺癌细胞MDA MB231的实验表明，二者以浓度及时间依赖方式抑制人乳腺癌细胞MDA MB231内蛋白酶体的CT活性进而诱导癌细胞凋亡。通过研究LC3作用于正常细胞MCF10A，得出配合物LC3对正常细胞的毒性相对较小。(5)探讨了二甲双胍（Metformin）和5-氨基-4-甲酰胺咪唑核糖核苷酸（AICAR）处理前列腺癌细胞后，引起雄激素受体（Androgen receptor, AR）阳性和阴性细胞的不同敏感性以及不同磷酸腺苷活化的蛋白激酶(adenosine monophosphate-activated protein kinase,AMPK)激活效应。结果表明AR在调节前列腺癌细胞对二甲双胍敏感性中起着重要的作用。在前列腺癌细胞中，AMPK激活和AR的存在之间存在很强的相关性，从而导致前列腺癌细胞对二甲双胍的敏感性差异。AR影响AMPK激活剂对AMPK的激活，迅速和长期的激活AMPK可能需要雄激素受体AR的存在。研究表明AR决定前列腺癌细胞对AMPK激活剂的敏感度，长时间的AMPK激活会导致AR蛋白降解。这些发现将有助于在不同的遗传背景和不同的病理阶段阐明前列腺癌中AMPK的作用，最终促进二甲双胍或其他AMPK激活剂在治疗前列腺癌中的临床应用。更多还原

【Abstract】 Metal-based anti-cancer drugs were developed many years ago. In recent years, the study hasshown that a number of the metal-based drugs, including organic copper-, zinc-, and gold-basedcomplexes, are capable of inhibiting the tumor cell proteasome, thereby inducing cancer celldeath. Ubiquitin proteasome pathway is the major proteolytic mechanism which plays a criticalrole in the degradation of the proteins. The20S proteasome, the main component of the UP-S, isa high molecular weight protease complex with a proteolytic core containing subunits includingβ1, β2and β5, which are responsible for its caspase-like, trypsin-like and chymotrypsin-like(CT-like) activities, respectively. It is well established that inhibition of the β5proteasomalsubunit, and therefore its CT-like activity, is primarily associated with apoptosis induction intumor cells. The ubiquitin proteasome pathway has therefore been extensively studied as a novelmolecular target for the development of novel drugs in an attempt to restore protein homeostasisas the ultimate therapeutic strategy.In the current study, several transition metal complexes are synthesised which may havepotential anticancer activities. The abilities to inhibit cell proliferation, chymotrypsin-likeactivity of proteasome and induce apoptosis are investigated in human cancer cells. Themechanism of these complexes to inhibit proteasome and induce apoptosis in cancer cells arestudied, and make sure the antitumor target is proteasome.The details of the contents are as follows,(1) Two types of copper complexes, dinuclear complexes and ternary complexes aresynthesized, to investigate whether a certain structure could easily carry copper into cancer cellsand consequently inhibit tumor proteasome activity and induce apoptosis. The abilities of theligands and copper complexes to inhibit cell proliferation in human prostate cancer PC3cells andchymotrypsin-like activity of purified20S proteasome are investigated firstly. Furthermore, theproteasome-inhibitory and apoptosis–inducing activities of these compounds in the PC3prostatecancer cells are studied. Finally, compare the effects of proteasome inhibition and apoptosisinduction in breast cancer MDA MB231cells and normal MCF10A cells with ternarycomplexes. The new findings suggest that (i) copper binding with1,10-phenanthroline as the third ligand could promote tumor cells to uptake copper, resulting in potent proteasomeinhibition and apoptosis induction in cancer cells, and (ii) breast cancer MDA MB231cells aremore sensitive to the novel candidates ternary complexes than non-tumorigenic cells, suggestingtumor-selective targeting.(2) Novel metal-containing complexes are synthesized by using indole-3-butyric acid (L1)and indole-3-propionic acid (L2) respectively, as ligands. These Cd complexes are potentinhibitors of the proteasome and inducers of apoptosis, effects which appear to be specific totumor cells. Then use proteasome activity, MTT assay and western blot to compare the ability ofthe similar metal complexes containing copper (Cu), zinc (Zn) or Cd to inhibit breast cancer cellproliferation using the estrogen receptor (ER)-positive MCF7and ER-negative MDA MB231breast cancer cell lines. Of the compounds tested, the Cd-containing versions appear to be themost potent inhibitors of cellular proteasome CT-like activity and effective inducers of apoptosisin breast cancer cells, but not in non-tumorigenic breast epithelial MCF10A cells. Additionally,these newly synthesized Cd compounds are superior in potency and cancer selectivity to theDSF-Cd mixture.(3) The ligands (Cefepime) and the mixture of the transition metal with the ligands havebeen prepared. The activity of proteasome inhibition and the apoptosis induction in human breastcancer MDA MB231cells by the mixture are studied. Firstly, compare the ability of thedifferent metal complexes to inhibit breast cancer cell proliferation using the estrogen receptorER-negative MDA MB231breast cancer cell lines. All the results showed that only manganesemixture could inhibit cell proliferation in human breast cancer MDA MB231, and the calpainprotein which plays a critical role in apoptosis involve in manganese mixture induce apoptosis.Finally, compare the effects of the manganese mixture in breast cancer MDA MB231cells withthe effects in non-tumorigenic MCF10A cells. The results also clearly shows that theseimmortalized breast cells remain unharmed and are insensitive to the cytotoxic effects of themanganese mixture.(4)3,5-diaminobenzoic acid Schiff base complexes are synthesized, and their cellproliferation inhibition activity are studied firstly. The results show that LA5and LC3cadmiumcomplexes have anti-proliferation activity in cancer cells. The experiment also show that LC3potently inhibits chymotrypsin-like activity of20S proteasome and induce apoptosis in humanbreast cancer MDA MB231in dose and time dependent manner; finally, compare the effects of LC3in breast cancer MDA MB231cells with the effects in non-tumorigenic MCF10A cells.These immortalized breast cells remain unharmed and are insensitive to the cytotoxic effects ofLC3. These results support the notion that LC3induce proteasome inhibition, followed byapoptosis induction in breast tumor cells.(5) The study on metformin treatment induced greater levels of growth arrest and cell deathin AR positive PCa cells than AR-negative PCa cells. The results clearly shows AR might play arole in mediating differential sensitivity of PCa cells to metformin. Then investigate the differentAMPK activation profiles in AR-positive and AR-negative PCa cells after metformin or AICARtreatment. The results suggest AR influences AMPK activation profile in response to AMPKactivators in PCa cells and the presence of AR is likely required for prompt and prolong AMPKactivation. In summary, the current study demonstrates that AR is involved in determine thesensitivity of prostate cancer cells to AMPK activator and that prolonged AMPK activation leadsto AR degradation. These findings will help elucidate the role of AMPK in prostate cancer withdifferent genetic background and different pathological stages and eventually promote theclinical application of metformin or other AMPK activator in prostate cancer on a personalizedbasis.更多还原