【作者】 刘作家；

【导师】 牛凤兰；

【作者基本信息】 吉林大学 ， 卫生毒理学， 2013， 博士

【摘要】 药物进入生物体血液系统后大多会与血浆蛋白结合，而血清白蛋白（serumalbumin，SA）是血浆中含量最丰富的载体蛋白，能与许多内源及外源性化合物结合。药物与SA的结合不仅可以调节游离药物的浓度、延长药物起作用的时间，而且最终影响药物的吸收、分布、代谢和排泄等过程。这些特性使得药物与SA的结合能力成为药物设计和评价备选药物分子的重要指标。药物与SA相互作用在药物动力学研究领域的重要意义，使得这一领域的研究一直十分活跃。因此，建立药物-SA结合的体外模型，了解其结合的紧密程度、结合部位、结合力、结合位点数等问题，不仅对于揭示体内的药物动力学具有重要意义，而且对于药物分子设计也具有重要的指导意义。相对于其它生物大分子而言，血清白蛋白来源广泛，尤其是人血清白蛋白（human serum albumin，HSA）分子结构信息确知，使得HSA成为研究药物小分子与SA相互作用的研究对象。HSA三级结构的解析促进了药物-HSA相互作用的研究进入到一个新的阶段，使得人们对于药物-HSA相互作用的预测有了更加坚实的理论基础。尽管对HSA的研究积累了丰富的科学资料，但有关HSA与不同结构化合物的结合情况，包括结合部位、结合方式及竞争作用等，目前尚未完全阐明，故有关HSA的研究仍是被广泛关注的课题。这方面研究的不断深入，对于指导临床合理用药以及新药设计都具有重要意义。胰腺癌（Pancreatic Cancer）是临床上较为常见的消化系统恶性肿瘤，也是国内外医学界的一个重大难题和严峻挑战。在西方国家，胰腺癌的致死率占癌症相关死亡的第八位。2010年美国胰腺癌发生率位列第十位而死亡率位列第四位。根据2012中国肿瘤登记年报公布的数据，胰腺癌在我国的年发病率居恶性肿瘤的第七位。胰腺癌病人的治疗取决于患者在确诊时所处的发病阶段，手术治疗、化疗或联合治疗是当前临床所采用的主要治疗手段。由于胰腺癌具有较高的局部侵袭及远程转移能力，大多数病人在确诊时已处于局部恶化或转移阶段，故排除了手术治疗的可能性。为减少该疾病的致死率，必需对胰腺癌进行有效的治疗。吉西他滨是目前临床治疗恶性胰腺癌的“金标准”，然而很多病人由于先天或在治疗过程中对该药产生了耐药性，限制了吉西他滨的临床应用。近年来，胰腺癌发病机制的阐明为改善胰腺癌的治疗效果提供了新希望。细胞凋亡或程序化细胞死亡是机体在发育过程中基因控制的一个生理过程，通过消除受损或不正常的细胞以保持生物体的体内平衡。生理及调节机制的缺陷能够导致不同的病理状态，包括恶性转化及肿瘤的发生。凋亡逃逸是癌症的特征之一，可导致恶性肿瘤细胞对化疗敏感性的降低。由于细胞凋亡是人胰腺癌发展过程中主要的信号传递通路，所以发展调节凋亡的治疗手段可以激发胰腺癌细胞的凋亡进而达到治疗胰腺癌的目的。细胞凋亡机制的详细理解对于发展更为有效的或靶向的治疗是至关重要的，可以提供新颖的、对正常细胞具有较低毒性的治疗策略。近年来，凋亡诱导剂作为抗癌药物已被广泛接受并应用于试验中。因此，凋亡诱导仍是一个有兴趣的研究领域，也是发展抗癌药物的一个重要方向。没食子酸（3,4,5-三羟基苯甲酸，gallic acid, GA）是广泛存在于菱角中的、具有生物活性的天然多酚类化合物，具有抗炎、抗氧化及抗肿瘤等多种生物活性。研究表明，GA可以选择性诱导癌细胞凋亡，如HL-60RG、HeLa、dRLh-84、 PLC/PRF/5和KB细胞等。迄今为止，GA在治疗胰腺癌中的应用未见报道。为阐明GA对不同癌细胞的不同影响，急需进一步的研究去评价它的生物功能，而对于其分子作用机制的研究有助于胰腺癌的治疗。本论文首先对GA与HSA蛋白的相互作用及其诱导胰腺癌细胞凋亡的机制研究进行了综述。详细介绍了GA的生物活性、血浆蛋白的生物功能及GA与HSA相互作用的生物学意义，以及GA与HSA相互作用的研究方法和研究内容。接着介绍了胰腺癌及其治疗以及凋亡诱导在抗肿瘤治疗中的应用；对细胞凋亡信号传导通路的分子机制和调节机制也进行了详细的综述。其次，本论文采用光谱法和分子模拟技术相结合的技术手段研究了GA与HSA的相互作用。研究结果表明，在生理条件下，GA对HSA的荧光淬灭属静态淬灭；根据Forster能量转移理论求出GA与HSA中色氨酸的距离为2.04nm，表明二者之间的相互作用机制属能量转移；通过同步荧光光谱技术，证明GA的存在使HSA中色氨酸残基所处环境的疏水性增加，表明在HSA与GA的亲和过程中疏水力起着主要作用；分子模拟实验验证了GA与HSA之间可形成疏水力的作用模式。最后，本论文研究了GA诱导人胰腺癌细胞凋亡的机制。实验结果表明：GA对人胰腺癌细胞CFPAC-1和MIAPaCa-2的生长抑制作用呈现时间和剂量的依赖性关系，其48小时的IC₅₀值分别为102.3±2.4μM和135.2±0.6μM。正常人肝细胞HL-7702比胰腺癌细胞MIA PaCa-2对GA的细胞毒作用具有更低的敏感性，表明GA对癌细胞的细胞毒作用具有选择性。进一步的研究证实，GA在诱导癌细胞凋亡过程中激活了caspase-3/9的活性，提高了ROS、Bax、[Ca²⁺]i及胞浆Cyt-c的水平，并降低了细胞线粒体Cyt-c的水平和ΔΨm。结果表明，GA通过激活线粒体介导的凋亡途径诱导MIA PaCa-2细胞凋亡。在GA诱导细胞凋亡过程中存在两个可能的信号传递途径：一个途径是ROS的产生伴随[Ca²⁺]i的增加；另一个途径是ROS的释放和[Ca²⁺]i的提高是由GA分别诱导的，但这两个凋亡事件是凋亡诱导过程中所必需的。本论文验证了GA与HSA的相互作用，首次证实了GA的体外抗胰腺癌活性，为寻找高效低毒的抗胰腺癌新药提供了理论基础和实验依据。更多还原

【Abstract】 The vast majority of drugs after entering into the blood system willbind to plasma proteins, in which serum albumin （SA） is the mostabundant carrier protein that can bind with many endogenous andexogenous compounds. The binding of drugs with SA may regulate theconcentration of free drugs and prolong the action time of drugs，whichtherefore greatly affects the absorption, distribution, metabolism andexcretion of drugs in vivo. On the basis of these properties, the capabilityof drugs binding to SA becomes one of the most important indicatorsassessing drug design and drug candidates. This therefore becomes ahotspot in the field of drug research and promotes the progression of drugresearch. Thus, these questions, such as building the in vitro model ofdrugs binding to SA, understanding the binding degree, binding position,binding affinity and the number of binding sites, will help to reveal thepharmacokinetics and direct drug design.Human serum albumin （HSA） is an important object studying theinteraction between SA and small molecules due to its wide source andconfirmed structure information. The resolving of3-D promotes the studyand the prediction of the interaction between drugs and HSA. Although accumulating abundant materials on HSA, the interactions of HSA withcompounds with various structures including binding position, bindingmodel and competitive effection fail to be clearly clarified. This study ofdrugs binding HSA is of importance to guide clinical pharmacy and drugdesign.Pancreatic cancer （PC） remains an aggressive cancer among digestivesystem tumors and a significant problem and severe challenge in theworld. PC is the8^thleading cause of cancer-related deaths in the westernworld due to its late stage of disease at diagnosis. In the United States, PCwas the10^thmost common new cancer and the4^thmost commoncancer-related death in2010. China Tumor Annual Report2012demonstrated that PC was the7^thmost common new cancer. Treatment ofpatients with PC depends on the stage of the disease at diagnosis and hasmainly relied on surgery, chemotherapy or combined therapeutic methods.Since PC is with high tendency for local invasion and distant metastasis,a wide majority of patients preclude surgery due to a locally advanced ormetastatic stage. Thus treatments are necessary for reducing the mortalityof this disease. Single-agent gemcitabine is considered a gold standard forthe treatment of advanced PC. It was regretful that some patients eitherdeveloped or initially had chemoresistance to gemcitabine. Recently, theunderstanding of the pathogenesis involved in PC provided some newhopes for improving outcomes in PC therapy. Apoptosis, the process of programmed cell death, is a geneticallycontrolled physiological process in the development of tissue homeostasisby eliminating damaged or abnormal cells. Defects in the physiologicaland regulatory mechanisms can result in various pathological states,including malignant transformation and progression of cancer. Evasion ofapoptosis, one of characteristics of cancer, can lead to the loss ofsensitivity for the malignant cells to chemotherapy. Apoptosis is a coresignaling pathway in human PC, which accelerates the development ofapoptosis-modulating therapy to initiate apoptosis in PC cells. Thedetailed understanding of apoptosis is essential for the development ofmore effective or even ‘targeted’ therapy. This might lead to newtherapeutic strategy with less toxicity to normal cells. Recently, a point ofview that apoptotic inducer is a kind of anticancer drugs has been widelyaccepted and experimental applied. Thus apoptosis-induction continues tobe an interested field and important direction for developing therapeuticdrugs for cancers.Gallic acid （3,4,5-triphydroxyl-benzoic acid, GA） as a naturallyoccurring plant phenol is one of the major bioactive compounds isolatedfrom water caltrop. Various pharmacological activities of GA have beenreported, including antiinflammatory, antioxidant, and anticanceractivities. Studies have also demonstrated that GA selectively inducedcancer cells death by apoptosis, such as HL-60RG, HeLa, dRLh-84, PLC/PRF/5and KB cells etc. Thus far, there have been few reports on theeffect of GA on human PC cells. To clarify the discrepancies among thedifferent effects of GA on various cancer cells, further studies are urgentto evaluate its biological functions and roles. Thus studies on themolecular mechanisms of GA action should help to shed light on thetreatment of PC.In this thesis, first, the author reviewed the mechanisms of GA bindingto HSA and the apoptosis-inducing in pancreatic cancer cells. As the basisof drug-protein interaction research, the biological activities of GA, thebiological functions of plasma protein and the significances of theinteraction of GA with HSA were reviewed in more detail.Simultaneously, the assay methods and research contents also werediscussed. Next, the basic knowledge and chemotherapy of PC as well asthe applications of apoptosis-inducing in the field of tumor therapy wereintroduced, respectively. Lastly, the molecular and regulation mechanismsof the apoptosis pathways were summarized detailedly.Secondly, the binding of GA with HSA was investigated by acombined experimental and computational approach in this dissertationfor understanding the action mechanisms between GA and HSA. Thefluorescence properties of HSA and the binding parameters of GAcollectively indicated that the binding was characterized by staticquenching mechanism at one high affinity binding site at physiological conditions. According to the estimated molecular distance （r=2.04nm）between HSA and GA, the binding was related to the fluorescenceresonance energy transfer. As indicated by the synchronous fluorescencespectra, the binding of GA to HSA leaded to the changes in theconformation of HSA. As thermodynamic parameters showed,hydrophobic interactions dominated in the association reaction and thebinding process was entropy driven. Computational mapping of thepossible binding sites of GA revealed the drug bound in the largehydrophobic cavity of subdomain ⅡA. The expected output shouldultimately help one design GA derivatives with altered HSA-bindingproperties.Lastly, the effect of GA on the apoptosis in PC cells was assessed. Theresults indicated that GA inhibited the proliferation of CFPAC-1and MIAPaCa-2cells in a time-and dose-dependent manner, with IC₅₀S of102.3±2.4μM and135.2±0.6μM at48h, respectively. The normalhuman liver HL-7702cells were less sensitive to GA than MIA PaCa-2cells, which means that GA induces selective cell death in MIA PaCa-2cells compared with normal cells. Also, GA activated caspase-3/9andROS, elevated Bax expressions,[Ca²⁺]i and the level of Cyt-c incytoplasm, and reduced mitochondrial membrane potential and the levelof Cyt-c in mitochondria. These results remarkably suggested that GAcould function as a cancer-selective agent by inducing apoptosis in MIA PaCa-2cells via the mitochondria-mediated apoptotic pathways. Theseresults supported the speculations that there were two possible signalingpathways in GA-induced apoptosis. One was that the generation of ROSaccompanied the elevation of [Ca²⁺]i in GA-induced apoptosis. The otherwas that ROS release and [Ca²⁺]i elevation were induced independently,but both were required for apoptosis induction. From this perspective, GAas a cancer-selective agent inducing apoptosis in human PC cells shouldopen up new opportunities for the therapy of patients with pancreaticcancer.In this thesis, the interaction of GA with HSA and the anticancer effectof GA on PC cells were confirmed in vitro. Together with theobservations detailed in this study, these results supported a role for GAas a potent therapeutic agent for PC. Further, the results providedexperimental and theoretical basis for seeking novel drugs withhigh-efficiency and low-toxicity against PC.更多还原