The Mechanisms Of Gallic Acid Binding To Human Serum Albumin And The Apoptosis-inducing In Pancreatic Cancer Cells

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.