Dissertations / Theses on the topic 'Human Liver Fatty Acid Binding Protein'

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002.
Includes bibliographical references (leaves 139-166). Also available in electronic version. Access restricted to campus users.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2006.
Title from first page of PDF file (viewed July 21, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 123-155).

Noncovalent interactions are involved in many biological processes in which biomolecules bind specifically and reversibly to a partner. Often, proteins do not have a biological activity without the presence of a partner, a ligand. Biological signals are produced when proteins interact with other proteins, peptides, oligonucleotides, nucleic acids, lipids, metal ions, polysaccharides or small organic molecules. Some key steps in the drug discovery process are based on noncovalent interactions. We have focused our research on the steps involving ligand screening, competitive binding and ‘off-target’ binding. The first paper in this thesis investigated the complicated electrospray ionization process with regards to noncovalent complexes. We have proposed a model that may explain how the equilibrium between a protein and ligand changes during the droplet evaporation/ionization process. The second paper describes an evaluation of an automated chip-based nano-ESI platform for ligand screening. The technique was compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation was obtained between the results obtained with the two methods. As a general conclusion we believe that the automated nano-ESI/MS should have a great potential to serve as a complementary screening method to conventional HTS. Alternatively, it could be used as a first screening method in an early phase of drug development programs when only small amounts of purified targets are available. In the third article, the advantage of using on-line microdialysis as a tool for enhanced resolution and sensitivity during detection of noncovalent interactions and competitive binding studies by ESI-MS was demonstrated. The microdialysis device was improved and a new approach for competitive binding studies was developed. The last article in the thesis reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II.
QC 20100705

Le molecole lipidiche, come acidi grassi, eicosanoidi e acidi biliari (BAs) sono essenziali per la sopravvivenza cellulare, in quanto possono fungere da fonti di energia, da substrati per la formazione di membrane oppure da molecole segnale per la regolazione del metabolismo cellulare. A causa della loro scarsa solubilità e della loro potenziale citotossicità necessitano di chaperon intracellulari che, legandole, aumentano la loro solubilità in solventi acquosi. Le proteine che legano gli acidi grassi, FABPs (Fatty Acid Binding Proteins), appartengono alla famiglia delle iLBPs (intracellular lipid binding proteins), una classe di piccole proteine citoplasmatiche (di circa 14-15KDa) evolutivamente correlate fra loro, probabilmente implicate nel trasporto lipidico trans-cellulare. Le iLBP sono proteine versatili che partecipano ai processi nucleari e al mantenimento dell’omeostasi e del metabolismo lipidico; per questo motivo sono state scelte come bersagli di farmaci contro lo sviluppo di dislipidemie. La FABP di fegato (LFABP), appartenente alla sotto-famiglia II delle FABP, è forse la più peculiare; a differenza degli altri membri può infatti accomodare, nella sua cavità idrofobica, due molecole di acidi grassi a lunga catena, ma anche una grande varietà di molecole idrofobiche come gli esteri dell’acil-CoA, fosfolipidi e acidi biliari. Per le sue particolari caratteristiche e l'alta concentrazione che può raggiungere all'interno degli epatociti (1-5% delle proteine solubili totali), si è ipotizzato che la LFABP umana (HLFABP) sia implicata nello sviluppo dei parassiti malarici, durante la fase epatica della malattia. Questa fase è asintomatica e potrebbe fornire nuove strategie per arrestare l'infezione. UIS3 è una piccola proteina trans-membrana, presumibilmente localizzata nella membrana vacuolare parassitofora (PVM), specificamente espressa negli sporozoiti infettivi ed essenziale per il loro sviluppo nella fase iniziale della malaria all’interno del fegato. Un saggio di tipo yeast two hybrid, basato sulla proteina UIS3 derivante dal parassita malarico dei roditori P. yoelii (Py-UIS3), ha permesso di identificare LFABP come possibile partner di interazione. Inoltre, in un lavoro del 2008 di Ashwani e collaboratori, venne riportata un’interazione diretta fra HLFABP e il dominio solubile di UIS3 di Plasmodium falciparum ( PfUIS3(130-229)). Al fine di ottenere informazioni vincolanti ad un livello atomico di risoluzione, l'interazione tra HLFABP e PfUIS3(130-229) è stata analizzata in dettaglio tramite spettroscopia di Risonanza Magnetica Nucleare(NMR). Inoltre, anche l'associazione con fosfolipidi e acidi grassi è stata analizzata attraverso NMR. I nostri dati, tuttavia, non hanno evidenziato alcuna interazione di PfUIS3(130-229) con HLFABP e/o molecole lipidiche, indicando la necessità di ridefinire il modello attuale di importo di lipidi nei parassiti malarici mediato da LFABP. Nella seconda parte di questo progetto di ricerca è stata analizzata in dettaglio l’interazione fra HLFABP e gli acidi grassi. L’NMR e la spettroscopia di massa (MS) sono state utilizzate per la prima volta insieme per caratterizzare questa associazione. Campioni di HLFABP in complesso con oleato (OA) e palmitato (PA) sono stati preparati in acqua e successivamente analizzati utilizzando la tecnologia ESI-MS (Electron Spray Ionization Mass Spectroscopy) per determinarne la specificità, la stechiometria e l'affinità relativa. I nostri dati sono concordi con la presenza di due siti di legame distinti con una diversa affinità per gli acidi grassi. Sono poi stati allestiti degli esperimenti di competizione, titolando la proteina sia con OA che con PA; i campioni sono stati analizzati tramite ESI-MS ed i dati indicano che OA e PA competono effettivamente per lo stesso sito di associazione all'interno della proteina e che HLFABP ha una maggiore affinità per gli acidi grassi instauri. Successivamente abbiamo sfruttato la potenza delle titolazioni 13C-NMR per indagare sia l'interazione fra HLFABP e acidi grassi marcati in 13C, che lo stato di ionizzazione dei ligandi legati nella tasca idrofobica della proteina. A livello globale, abbiamo sviluppato un metodo adatto per lo studio di altri membri della famiglia FABP, che, nonostante le loro dimensioni molto favorevoli, sono sistemi complessi per essere caratterizzati da una singola tecnica biofisica. Inoltre, questo metodo potrà essere applicato anche per lo studio di HLFABP in complesso con altri ligandi idrofobici. Nell'ultima parte di questo lavoro ci siamo concentrati sull'interazione tra HLFABP e gli acidi biliari, molecole anfipatiche, che facilitano l'assorbimento dei lipidi, del colesterolo e di vitamine liposolubili nell'intestino tenue. Gli acidi biliari subiscono un riciclaggio tra l'intestino ed il fegato, chiamato "Circolazione enteroepatica", che consente il recupero di quasi il 95% di queste preziose molecole. Dal momento che un trasportatore di acidi biliari non è stato ancora stato identificato negli epatociti dei mammiferi, abbiamo esplorato l’associazione tra HLFABP e gli acidi biliari utilizzando una vasta gamma di tecniche biofisiche, coinvolgendo l'NMR, la spettroscopia di fluorescenza e la spettrometria di massa. L'interazione tra HLFABP e l’acido glicocolico (GCA), il sale biliare più abbondante presente nel fegato umano, è stato ampiamente esplorato mediante NMR. L’NMR è una fra le spettroscopie più potenti e versatili per l'analisi molecolare, poiché permette di caratterizzare la struttura delle macromolecole biologiche ed i loro complessi ad un livello atomico di risoluzione. Inoltre, l'NMR fornisce informazioni sulla dinamica proteica su una vasta gamma di scale dei tempi . Le dinamiche possono influenzare la velocità e la via di ripiegamento delle proteine, così come l’aggregazione, la catalisi e l’associazione ad un ligando attraverso l’adattamento indotto o la selezione conformazionale. Così, la determinazione delle dinamiche proteiche in soluzione è importante per comprendere l'intero spettro di funzioni macromolecolari svolte dalle proteine. Esperimenti di titolazione NMR ed esperimenti omonucleari 1H-1H NOESY, eseguiti con diversi schemi di marcatura isotopica, suggeriscono che HLFABP è in grado di legare una sola molecola di GCA. Inoltre, per complementare i dati NMR, è stata eseguita un’analisi computazionale per calcolare la struttura del complesso, utilizzando il programma di docking HADDOCK. Successivamente, per meglio definire il legame, sono stati acquisiti esperimenti di rilassamento 15N sul backbone di HLFABP nella sua forma apo ed in complesso sia con GCA che con OA e sono state ottenute dinamiche residuo-specifiche su una vasta scala di tempi, che va dai ns ai ms. I nostri dati indicano chiaramente chele dinamiche veloci (ps-ns) non vengono influenzate particolarmente dal binding, mentre le dinamiche lente (μs-ms) sono mantenute o accentuate dopo il legame. Infine sono stati eseguiti esperimenti di scambio idrogeno/deuterio e CLEANEX per monitorare le zone più protette della proteina dallo scambio col solvente in presenza ed in assenza dei ligandi. In presenza di GCA è stato poi osservato un aumento della stabilità proteica. La spettroscopia di fluorescenza e l'NMR sono state anche utilizzate per caratterizzare l'interazione fra HLFABP e un pool di acidi biliari con diverse modalità di coniugazione e idrossilazione. I dati NMR mostrano che HLFABP può interagire con un’ampia gamma di acidi biliari in modo complesso, attraverso la formazione di almeno uno stato attivato. In aggiunta, i nostri dati NMR suggeriscono che la struttura della proteina sia preformata per il legame delle diverse molecole idrofobiche. Infatti la rete di legami idrogeno non viene perturbata in modo significativo dall'aggiunta dei vari ligandi. Attraverso la spettroscopia NMR abbiamo poi dimostrato che HLFABP esiste come un insieme di conformeri in scambio veloce fra loro su una scala di tempi NMR. La spettroscopia di fluorescenza è stata impiegata per calcolare l’affinità di HLFABP verso i vari acidi biliari utilizzati nello screening e attraverso un saggio di competizione, utilizzando il DAUDA come composto fluorescente, è stata calcolata un’affinità μM (da 0.6-7.5 μM) in accordo con quella calcolata tramite spettroscopia NMR. L’affinità maggiore è stata ottenuta per quegli acidi biliari con caratteristiche di idrofobicità maggiori. Infine, la presenza di complessi eterotipici, formati da HLFABP in complesso sia con acidi biliari che con acidi grassi, è stata analizzata tramite NMR e ESI-MS.
Lipidic molecules such as fatty acids (FAs), eicosanoids and bile salts (BAs) are essential for cell survival because they serve as metabolic energy sources, substrates for membranes and signaling molecules for metabolic regulation. Due to their low solubility and in some case cytotoxicity they necessitate intracellular chaperons, which bind them, thus increasing their aqueous solubility. Fatty acid binding proteins (FABPs), belong to the Intracellular lipid binding proteins (iLBPs) family, a class of evolutionarily related small (14-15 KDa) cytoplasmic proteins, which have been proposed to be implicated in the transcellular transport of lipophilic ligands. Due to their participation in nuclear processes and lipid metabolism and homeostasis, they have recently been proposed as drug targets against the development of lipid related disorders. Among the other family members, liver fatty acid binding protein (LFABP), belonging to subfamily II of FABPs, is the most unique. Differently from the other FABPs, LFABP is able to accommodate two long chain FAs (LCFAs) molecules, but also a wide range of hydrophobic ligands, such as BAs, eicosanoids, Acyl-CoA esters and phospholipids. Due to its peculiar characteristics and the high concentration that it could reach within the hepatocytes (1-5% of total soluble proteins), human LFABP (HLFABP) has been hypothesized to be implicated in malaria parasites development, during the hepatic stage of the disease. The hepatic stage is asymptomatic and it would provide novel strategies for arresting the infection. UIS3 is a small transmembrane protein, presumably localized to the parasitophorous vacuolar membrane (PVM), specifically expressed in infective sporozoites, and essential for early-stage liver development. A yeast two-hybrid screen based on UIS3 of the rodent malaria parasite P. yoelii (Py-UIS3) identified mouse LFABP as an interacting host protein. In addition, a work of 2008 of Ashwani and collaborators reports a direct interaction between HLFABP and the soluble domain of UIS3 from Plasmodium falciparum (PfUIS3(130-229)). In order to gain binding information at an atomic level of resolution, the interaction between HLFABP and PfUIS3(130-229) was analyzed in detail exploiting Nuclear Mgnetic Resonance (NMR) spectroscopy. Furthermore, the direct binding of phospholipids and FAs to UIS3 was also analyzed by NMR. However, our data did not show any interaction of PfUIS3(130-229) with HLFABP and lipid molecules, calling for a redefinition of the current model of FABP-mediated lipid import by human malaria parasites. In the second part of this research project, we investigated in detail the interaction between HLFABP and fatty acids. For the first time NMR and MS spectroscopy were used in combination to characterize the binding between HLFABP and FAs. Samples of HLFABP in complex with palmitate (PA) or oleate (OA) were prepared in water and analyzed through Electron Spray Ionization mass spectroscopy (ESI-MS) to asses specificity, stoichiometry and relative affinity. Our data are in agreement with the presence of two distinct binding sites with different affinities for FAs. Competition experiments were also performed, titrating the protein with both PA and OA; OA and PA effectively compete for the same binding site within the protein binding pocket. Our results show that HLFABP has an higher affinity for unsaturated FAs. Successively, we exploited the power of 13C NMR titration data to investigate the interaction between HLFABP and 13C FAs and to get information about the ionization state of the bound ligands. Globally, we developed a method suitable for the study of other FABP family members, which, despite their favorable size are really challenging systems to be characterized by only a singular biophysical technique. In addition, this method could be also applied to the study of HLFABP in complex with other hydrophobic ligands. In the last part of this work we focused on the interaction between HLFABP and BAs, amphipathic molecules, which in the small intestine facilitate the absorption of dietary lipids, cholesterol, and fatsoluble vitamins. BAs undergo a recycling pathway between the intestine and the liver, called “enterohepatic circulation”, which allows the recovery of almost the 95% of these precious molecules. Since a BA carrier within the hepatocytes has not been identified yet, we explored the interaction between HLFABP and BAs using a wide range of biophysical techniques, involving NMR, florescence and mass spectroscopy. The interaction between HLFABP and glycocholic acid (GCA), the most abundant bile salt present in human liver, was extensively explored using NMR spectroscopy technique. NMR is one of the most powerful and versatile spectroscopic technique for molecular analysis, since it allows to characterize biological macromolecules and their complexes at an atomic level of resolution. In addition, NMR provides information about protein dynamics on a wide range of time scales. Dynamics can affect the rate and pathway of protein folding, as well as misfolding and aggregation, catalysis and also binding via induced fit or conformational selection. Thus, the determination of protein dynamics in solution is important for realizing the full spectrum of macromolecular functions and for predicting and engineering protein behavior. NMR titration experiments and 1H-1H homonuclear NOESY filtered experiments, performed with different labeling schemes, suggested that HLFABP is able to accommodate only one molecule of GCA. To complement NMR data, a model of the complex was obtained through a computational analysis, using the docking program HADDOCK. To better characterize the binding, 15N backbone relaxation experiments on HLFABP in its apo form and in complex with either GCA or OA were recorded and residue specific dynamics, on a time scale ranging from ps to ms, were obtained. Fast time scale dynamics are not significantly perturbed upon OA/GCA addition, while slow motions are retained or enhanced upon binding. Hydrogen/deuterium exchange and CLEANEX experiments were also performed to get information on solvent accessibility to individual sites and to detect protein dynamics occurring on a much slower time scale. An increase in protein stability upon GCA/OA binding was observed. For the first time NMR and fluorescence spectroscopy were combined on a BA pool, with different pattern of conjugation and hydroxylation. The NMR data show that HLFABP can interact with a wide range of bile salts, through a complex pathway, involving at least one activated state. In addition the hydrogen bond network was not significantly perturbed upon ligand addition, indicating that the scaffold of the protein is preformed to bind such kind of ligands. Through NMR spectroscopy, we demonstrated also that HLFABP exists as an ensemble of conformers in fast exchange on an NMR time scale. Fluorescence spectroscopy was used to calculate the affinity of HLFABP toward the different BAs employed in the study. An affinity in the µM range (spanning form 0.6-7.5µM) was obtained through DAUDA displacement assay, in close agreement with the ones calculated by NMR. The higher affinity was obtained for those BAs displaying high hydrophobic properties. Finally we analyzed both by NMR and mass spectroscopy the existence of an heterotypic complex constituted by HLFABP in complex with both GCA and FAs, which is likely the conformation assumed by the protein in vivo.

Liver fatty acid binding protein (L-FABP) binds and translocates many lipophilic substrates within the cytoplasm including long chain fatty acids. Moreover it was reported that L-FABP possesses antioxidative properties within hepatocytes. However, the mechanism of L-FABP’s antioxidative activity remains to be determined. Peroxisome proliferator activated receptor (PPAR) agonists and antagonists can regulate L-FABP levels. However, it needs to be investigated how PPAR agonists and antagonists regulate L-FABP expression. And whether the altered expression of L-FABP by these agents will affect its antioxidative properties within hepatocytes remains unclear. In this thesis we employed clofibrate (PPARα agonist), MK886 (PPARα antagonist), and GW9662 (PPARγ antagonist) to elucidate the mechanism whereby PPAR regulate L-FABP expression and what effect such expression has on the antioxidant activity of L-FABP in CRL-1548 hepatoma cells. Clofibrate served to upregulate L-FABP expression while MK886 and GW9662 were employed to inhibit L-FABP expression. The principal findings revealed that clofibrate treatment enhanced L-FABP mRNA stability and transcription, which resulted in increased L-FABP levels, while MK866 and GW9662 reduced these levels. We also demonstrated that increases in L-FABP levels were associated with reduced cytosolic reactive oxygen species (ROS), while L-FABP siRNA knockdown resulted in a decrease in L-FABP expression and an associated increase in ROS levels. The antioxidant mechanism of recombinant rat L-FABP in the presence of a hydrophilic (AAPH) and lipophilic (AMVN) free radical generators was also evaluated. Recombinant rat L-FABP was produced in E. coli and its amino acid sequence was confirmed by MALDI QqTOF MS. Antioxidant activity was assayed using the thiobarbituric acid method. Ascorbic acid served as a positive control for the AAPH reaction while α-tocopherol was used as a positive control for the AMVN reaction. The antioxidant activity of recombinant L-FABP was greater when free radicals were generated with AAPH than AMVN. Oxidative modification of L-FABP included up to five methionine oxidative peptides with a total of 80 Da mass shift compared to native L-FABP. These findings suggest that the mechanism of L-FABP’s antioxidant activity involved the reaction of methionine with free radicals. In conclusion, L-FABP expression is regulated by PPAR agonists and antagonists through transcription and mRNA stability. Moreover, methionine residues appear to play an important role in the antioxidative activity of L-FABP.

Liver fatty acid binding protein has been reported to possess antioxidant properties in the liver. The aim of this study was to investigate the effect of this protein in a nonalcoholic fatty liver disease (NAFLD) cell culture model. Rat hepatoma cells were treated with an oleate:palmitate (2:1) mixture for either 1 and 2 days, or further treated with 500 µM clofibrate to induce L-FABP expression. Intracellular lipid accumulation was quantitated by Nile Red. Lipotoxicity was determined using the WST-1 assay. Dichlorofluorescein (DCF) was utilized to assess intracellular reactive oxidative species (ROS) level. Measurement of lipotoxicity showed statistical decreases in cell viability as lipid concentrations increased in a dose-dependent manner. NAFLD cell cultures showed characteristic cellular damage from increased ROS levels in fatty acid treated cells. All groups treated with clofibrate showed statistically increased intracellular L-FABP levels and reduced ROS levels. The results lead to the conclusion that clofibrate induces L-FABP expression and in this manner suppresses hepatocellular ROS generation.

Indiana University-Purdue University Indianapolis (IUPUI)
My thesis comprises of two individual projects which revolve around the importance of post-transcriptional regulation in liver. My first project is studying the integrated miRNA – mRNA network in NAFLD. For fulfillment of the study we conducted a genome-wide study to identify microRNAs (miRs) as well as the miR-mRNA regulatory network associated with hepatic fat and NAFLD. Hepatic fat content (HFC), miR and mRNA expression were assessed in 73 human liver samples. Liver histology of 49 samples was further characterized into normal (n=33) and NAFLD (n=16). Liver miRNome and transcriptome were significantly associated with HFC and utilized to (a) build miR-mRNA association networks in NAFLD and normal livers separately based on the potential miR-mRNA targeting and (b) conduct pathway enrichment analyses. We identified 62 miRs significantly correlated with HFC (p < 0.05 with q < 0.15), with miR-518b and miR-19b being most positively and negatively correlated with HFC, respectively (p < 0.008 for both). Integrated network analysis showed that six miRs (miRs-30b*, 612, 17*, 129-5p, 204 and 20a) controlled ~ 70% of 151 HFC-associated mRNAs (p < 0.001 with q < 0.005). Pathway analyses of these HFC-associated mRNA revealed their key effect (p<0.05) in inflammation pathways and lipid metabolism. Further, significant (p<2.47e-4, Wilcoxon test) reduction in degree of negative associations for HFC-associated miRs with HFC-associated mRNAs was observed in NAFLD as compared to normal livers, strongly suggesting highly dysfunctional miR-mRNA post-transcriptional regulatory network in NAFLD. Our study makes several novel observations which provide clues to better understand the pathogenesis and potential treatment targets of NAFLD. My second project is based on uncovering important players of post-transcriptional regulation (RBPs) and how they are associated with age and gender during healthy liver development. For this study, we performed an association analysis focusing on the expression changes of 1344 RNA Binding proteins (RBPs) as a function of age and gender in human liver. We identify 88 and 45 RBPs to be significantly associated with age and gender respectively. Experimental verification of several of the predicted associations in the mouse model confirmed our findings. Our results suggest that a small fraction of the gender-associated RBPs (~40%) are likely to be up-regulated in males. Altogether, these observations show that several of these RBPs are important developmentally conserved regulators. Further analysis of the protein interaction network of RBPs associated with age and gender based on the centrality measures like degree, betweenness and closeness revealed that several of these RBPs might be prominent players in liver development and impart gender specific alterations in gene expression via the formation of protein complexes. Indeed, both age and gender-associated RBPs in liver were found to show significantly higher clustering coefficients and network centrality measures compared to non-associated RBPs. The compendium of RBPs and this study will help us gain insight into the role of post-transcriptional regulatory molecules in aging and gender specific expression of genes.

碩士
嘉南藥理科技大學
生物科技系暨研究所
95
Acute myocardial infarction ( AMI ) is the result of a sudden occlusion that cause a portion of myocardium cell death, and accounted for the greatest percentages of deaths from heart diseases in Taiwan. In clinical research, human heart-type fatty acid binding protein ( H-FABP ) is a sensitive cardiac marker useful for early diagnosis of AMI. In this study, we establish a self-assembled surface plasmon resonance ( SPR ) optical immunosensor system to detect human H-FABP, and to compare different immobilization methods applied to immobilize the anti-human H-FABP monoclonal antibodies ( MAbs ) on a gold surface including adsorption, Protein A, cystamine, N-succinimidyl-3-(2-pyridyldithio)propionate ( SPDP ) - Protein A, cystamine - glutaraldehyde - Protein A ( CGP ) method. The recent results indicate that the reusability of the sensor chip adopting the cystamine method was found to be better than the other immobilization methods. Ten cycles of measurements could be performed on the same chip regenerated with a 0.1 M glycine-HCl buffer, a 10 M NaOH solution was used for clearing nonspecific binding in mouse serum. A linear relationship between SPR angle shift and the log values of H-FABP concentrations in the range from 0.2 to 100 ng/mL in phosphate buffered saline ( PBS ) and in mouse serum. When used for 10 days, the angle shifts were all > 95% of those on the response at the first day. Correlation coefficient was 0.9986 between this SPR immunosensor system and ELISA for determination of H-FABP in mouse serum samples. This self-assembled SPR optical immunosensor system offers advantages of simplicity of immobilization, low sample requirement, label-free, no pretreatment, high sensitivity, high specificity and high reusability, it will be used for home care and point-of-care in early diagnosis of AMI, and for preventive medicine research.

博士
國立臺灣大學
生物化學暨分子生物學研究所
103
Hepatocellular carcinoma (HCC) is the fifth most commonly occurring cancer and the third most common cause of cancer death worldwide. The progression of HCC relies on the formation of new blood vessels, and VEGF is critical in this process. Liver fatty acid-binding protein (L-FABP) is abundant in hepatocytes and known to be involved in lipid metabolism. Overexpression of L-FABP has been reported in various cancers; however, its role in hepatocellular carcinoma (HCC) remains unclear. In this study, we investigated L-FABP and its association with vascular endothelial growth factors (VEGFs) in 90 HCC patients. We found that L-FABP was highly expressed in their HCC tissues, and its expression level was positively correlated with that of VEGF-A. Additionally, L-FABP significantly promoted tumor growth and metastasis in a xenograft mouse model. We also studied the mechanisms of L-FABP activity in tumorigenesis: L-FABP was found to be associated with VEGFR2 on membrane rafts and subsequently activate the Akt/mTOR/P70S6K/4EBP1 and Src/FAK/cdc42 pathways. This resulted in up-regulation of VEGF-A expression accompanied by an increase in both angiogenic potential and migration activity. Taken together, our results suggest that L-FABP may be a potential target for HCC chemotherapy. Inhibition of VEGFR2 activity has been proposed as an important strategy for the clinical treatment of hepatocellular carcinoma (HCC). In this study, we identified corosolic acid (CA), which exists in the root of Actinidia chinensis (藤梨), as having a significant anti-cancer effect on HCC cells. We found that CA inhibits VEGFR2 kinase activity by directly interacting with the ATP binding pocket. CA down-regulates the VEGFR2/Src/FAK/cdc42 axis, subsequently decreasing F-actin formation and migratory activity of Huh7 cells in vitro. In an in vivo model, CA exhibites an effective dose (5 mg/kg/day) on tumor growth, and we further demonstrate that CA has a synergistic effect with sorafenib within a wide range of concentrations. In conclusion, we elucidate the effects and molecular mechanism for CA on HCC cells and suggest that CA could serve as a therapeutic or adjuvant target for patients with aggressive HCC.