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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林榮耀(Jung-Yaw Lin),呂紹俊(Shao-Chun Lu) | |
| dc.contributor.author | Chung-Yu Ku | en |
| dc.contributor.author | 辜琮祐 | zh_TW |
| dc.date.accessioned | 2021-05-14T17:45:52Z | - |
| dc.date.available | 2016-09-25 | |
| dc.date.available | 2021-05-14T17:45:52Z | - |
| dc.date.copyright | 2015-09-25 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-07-03 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/4721 | - |
| dc.description.abstract | 國際上肝細胞癌在癌症發生率中排行第五，在癌症致死率中排行第三。肝細胞癌的生長及進展仰賴於新生血管的形成，而血管內皮生長因子(VEGF)在此過程中扮演非常重要的角色。
肝型脂肪酸結合蛋白（L-FABP）在肝細胞中大量表現，並已知可參與脂質代謝。L-FABP 過度表現已在許多癌症中被發現，但它在肝細胞癌中扮演的角色仍不清楚。本研究中，我們分析了L-FABP 與VEGF 在90 個HCC 患者中的關聯性。我們發現，L-FABP 在肝癌組織中與VEGF-A 呈現正相關性。此外，L-FABP 在異種移植小鼠模式中可顯著促進腫瘤生長及轉移。我們亦討論L-FABP 活性與腫瘤生成的關係：L-FABP 可與細胞膜上脂筏中的VEGFR2 結合，接著活化下游的Akt/mTOR/P70S6K/4EBP1 與Src/FAK/CDC42 路徑，這也使得VEGF-A 表現量增加，並促進血管新生與細胞移行之活性。我們的研究結果證實，L-FABP 可望成為治療肝癌的新目標。 在臨床上，抑制第二型血管內皮生長因子受體(VEGFR2)之活性已被建議作為治療HCC 的重要策略。本研究中，我們發現獼猴桃根部之化合物，科羅索酸（CA），對肝癌細胞表現出顯著的抗癌作用。研究指出， CA 可透過與VEGFR2 上ATP 結合口袋的交互作用，抑制VEGFR2 之活性。 CA 在Huh7 細胞實驗中可抑制性調控VEGFR2/Src/FAK/CDC42 路徑，減少絲狀肌動蛋白（F-actin）之形成，並降低細胞移行能力。在動物實驗中，CA 對腫瘤生長的有效抑制劑量為每隻小鼠給予5 毫克/公斤/天。我們也證實，CA 與蕾莎瓦（Sorafenib）在廣範圍濃度下具有協同效應。 本研究闡明了CA 抗肝癌的細胞分子機制，並建議CA 可作為治療侵襲性肝癌之抗癌藥或佐劑。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-14T17:45:52Z (GMT). No. of bitstreams: 1 ntu-104-D97442007-1.pdf: 24913983 bytes, checksum: e1aa9c96c7d2cadd20eb7e3d683e828b (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | Abbreviations 1
中文摘要 3 Abstract 5 Introduction Hepatocellular carcinoma 7 Vascular endothelial growth factor and HCC 7 Liver fatty acid-binding protein (L-FABP) 8 Lipid rafts, receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases 9 Chinese herbal medicines and Actinidia chinensis 10 Corosolic acid (CA) 11 Materials and methods Part I Antibodies used for western blot analysis and chemical inhibitors 12 Tissue microarray construction and immunohistochemistry 12 Cell culture 13 Creation and culture of L-FABP overexpressed stable clones 13 Western blot analysis and immunoprecipitation 14 Cell migration assay 14 Angiogenesis activity assay 15 Short interference RNA (siRNA) and short hairpin RNA (shRNA) 16 Lipid rafts isolation 17 Confocal microscopy analysis 18 Small GTPase binding assay 18 Construction of human VEGF-A promoter 19 Luciferase reporter assay 19 Animals 20 Cloning of L-FABP mutants 21 Statistical analysis 21 Part II Plant extracts 22 HPLC analysis 22 Reagents 22 Cell culture 23 Cytotoxicity assay 23 Migration assay 24 Immunoprecipitation 24 Western blot analysis 25 Kinase activity assay 25 Rho GTPase activity assay 26 G-actin/F-actin activity assay 26 Confocal microscopy analysis 27 Animal model 27 Immunohistochemistry 29 Synergistic analysis 29 Molecular docking 29 SRB cell growth assay 30 Statistical analysis 30 Results Part I 1. Up-regulation of L-FABP expression in HCC tissues is correlated with VEGF-A overexpression 31 2. L-FABP induces VEGF-A expression and angiogenic potential in immortalized Hus and Huh7 cells 31 3. Association of L-FABP with VEGFR2 in membrane rafts 33 4. L-FABP increases VEGFR2/Src phosphorylation and cell migration by FAK/cdc42 pathway 34 5. L-FABP induced VEGF-A expression by Akt/mTOR/P70S6K/4EBP1 in translation level 35 6. L-FABP promoted tumor growth and metastasis in vivo 36 7. Cholesterol binding and membrane interacting properties are essential for L-FABP induced cell migration and angiogenesis 37 Part II 8. Corosolic acid significantly decreases the migration activity of Huh7 cells 38 9. Corosolic acid inhibits VEGFR2 kinase activity 39 10. Corosolic acid decreases cell motility by inhibiting VEGFR2/Src/FAK/cdc42 activity and actin rearrangement 40 11. Corosolic acid exhibits anti-tumor effect in vivo 41 12. Synergistic effects of corosolic acid and sorafenib on HCC cells 42 13. Corosolic acid interacts with the ATP-binding site of VEGFR2 kinase domain by molecular docking 43 14. Corosolic acid does not exhibit significant inhibitory effects on Huh7 cell 43 Discussion Part I Role of L-FABP in hepatocellular carcinoma 45 Part II Effects of corosolic acid on hepatocellular carcinoma 50 Summary 53 Figures and Figure legend Part I Figure 1. Correlation between the expression levels of L-FABP and VEGF-A 54 Figure 2. L-FABP expression is associated with VEGF-A expression of HCC cells 55 Figure 3. Expression level of VEGF-A is up-regulated in L-FABP stably expressed Hus cells 56 Figure 4. L-FABP promotes in vitro and in vivo angiogenic activity of Hus cells 57 Figure 5. Sequence aliment of L-FABP interacting domains 58 Figure 6. Co- immunoprecipitation of L-FABP and VEGFR2 in Hus/L-FABP cells 59 Figure 7. L-FABP associates with VEGFR2 in apical membrane of Hus/L-FABP cells 60 Figure 8. Localization of L-FABP and signaling molecules in lipid rafts 61 Figure 9. L-FABP increases the phosphorylation level of VEGFR2 in Hus cells 62 Figure 10. L-FABP increases the phosphorylation level of Src and FAK kinases in Hus cells 63 Figure 11. L-FABP promotes cdc42 activity of Hus cells 64 Figure 12. Analysis of migration activity of L-FABP stably expressed Hus cells 65 Figure 13. L-FABP up-regulates migration activity through VEGFR2/ Src pathway 66 Figure 14. L-FABP activates Akt/ mTOR/ P70S6K/ 4EBP1 signaling 67 Figure 15. HIF-1α significantly enriched in the nucleus of L-FABP overexpressed cells 68 Figure 16. Role of HIF-1α in VEGF-A transcriptional activity of L-FABP overexpressed cells 69 Figure 17. Post-transcriptional regulation of VEGF-A in L-FABP stably expressed Hus cells 70 Figure 18. L-FABP promotes tumor growth in vivo 71 Figure 19. L-FABP promotes in vivo metastasis by lung metastasis model 72 Figure 20. Effect of L-FABP mutants in VEGF-A expression 73 Figure 21. Effect of L-FABP mutants in migration activity 74 Figure 22. Cholesterol binding properties are essential for L-FABP induced cell migration and angiogenesis 75 Figure 23. Knockdown of L-FABP in Hus/L-FABP cells reversely decreased VEGF-A expression and migration activity 76 Figure 24. Knockdown of L-FABP in Huh7 cells down-regulates VEGF-A expression and migration activity 77 Figure 25. Reduction of L-FABP and VEGFR2 co-localization on membrane is observed in Huh7 L-FABP stably knockdown cells 79 Figure 26. Aberrant overexpression of L-FABP in HCC tissues (with cirrhosis) is associated with worse outcome 80 Part II Figure 27. Cytotoxicity and migration inhibitory effect of Actinidia chinensis on Huh7 cells 81 Figure 28. HPLC analysis of Actinidia chinensis 82 Figure 29. Migration activity of Huh7 cells is inhibited by corosolic acid without cytotoxicity 83 Figure 30. Corosolic acid reduces phosphorylation level of VEGFR2 84 Figure 31. Corosolic acid reduces VEGFR2 kinase activity 85 Figure 32. CA-induced inhibition of migration activity in Huh7 cells is VEGFR2 dependent 86 Figure 33. Corosolic acid down-regulates VEGFR2 downstream signals 87 Figure 34. Corosolic acid inhibits cdc42 activity 88 Figure 35. Effect of corosolic acid on actin rearrangement 89 Figure 36. Corosolic acid exhibits significant anti-tumor effects on Huh7 cells in vivo 90 Figure 37. Combinatorial effects of corosolic acid and sorafenib on migration activity of Huh7 cells 92 Figure 38. Combinatorial effects of corosolic acid and sorafenib on signaling molecules of Huh7 cells 93 Figure 39. Combinatorial effects of corosolic acid and sorafenib on Huh7 cells by 94 in vivo xenograft model Figure 40. Inhibitory effects of corosolic acid combined with sorafenib on Src and FAK kinases in vivo 95 Figure 41. Corosolic acid interacts with the ATP-binding site of VEGFR2 kinase domain by molecular docking analysis 96 Figure 42. Analysis of relative distance and surface charge distribution between corosolic acid and VEGFR2 ATP binding pocket 97 Figure 43. Corosolic acid inhibits growth of Huh7, HepG2, and Hep3B cells 98 Figure 44. Cytotoxicity and migration-inhibitory effects of corosolic acid on HepG2 cells 99 Figure 45. Cytotoxicity and migration-inhibitory effects of corosolic acid on Hep3B cells 100 Figure 46. Corosolic acid doesn’t exhibit significant inhibitory effect on invasion activity of Huh7 cells 101 Figure 47. Corosolic acid shows no inhibitory effect on NFκB signaling 102 Tables Table 1. Correlation between L-FABP and VEGF-A protein expression in tissue pairs from 90 HCC patients 103 Table 2. Clinical characteristics of the cases included in analyses of L-FABP protein expression evaluated by immunohistochemistry 103 Table 3. Association of L-FABP protein expression with clinical pathologic characteristics in patients with HCC 104 Supplementary data Table 1 105 Figure 1. Knockdown of VEGFR2 in Hus/L-FABP cells decreased the activation of down-stream signaling molecules 107 Figure 2. Prediction of the interaction models of L-FABP and VEGFR2 kinase domain 108 Figure 3. Amino acid substitution of L-FABP in present studies 109 References 110 | |
| dc.language.iso | en | |
| dc.subject | 科羅索酸 | zh_TW |
| dc.subject | 肝細胞癌 | zh_TW |
| dc.subject | 血管新生作用 | zh_TW |
| dc.subject | 第二型血管內皮生長因子受體 | zh_TW |
| dc.subject | 細胞移行 | zh_TW |
| dc.subject | 肝型脂肪酸結合蛋白 | zh_TW |
| dc.subject | 血管內皮生長因子 | zh_TW |
| dc.subject | vascular endothelial growth factor receptor-2 (VEGFR2) | en |
| dc.subject | Hepatocellular carcinoma | en |
| dc.subject | angiogenesis | en |
| dc.subject | liver fatty acid-binding protein | en |
| dc.subject | vascular endothelial growth factor | en |
| dc.subject | corosolic acid | en |
| dc.subject | migration | en |
| dc.title | 肝細胞癌之相關研究：
一、肝型脂肪酸結合蛋白促進肝細胞癌之血管新生及細胞移行 二、科羅索酸針對VEGFR2/Src/FAK路徑抑制肝細胞癌之細胞移行 | zh_TW |
| dc.title | Studies on Hepatocellular Carcinoma (HCC)：
I. Liver Fatty Acid-Binding Protein (L-FABP) Promotes Cellular Angiogenesis and Migration in Hepatocellular Carcinoma II. Corosolic Acid Inhibits Hepatocellular Carcinoma Cell Migration by Targeting the VEGFR2/Src/FAK Pathway | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 方剛(Kang Fang),李德章(Te-Chang Lee),李明學(Ming-Shyue Lee) | |
| dc.subject.keyword | 肝細胞癌,血管新生作用,肝型脂肪酸結合蛋白,血管內皮生長因子,科羅索酸,細胞移行,第二型血管內皮生長因子受體, | zh_TW |
| dc.subject.keyword | Hepatocellular carcinoma,angiogenesis,liver fatty acid-binding protein,vascular endothelial growth factor,corosolic acid,migration,vascular endothelial growth factor receptor-2 (VEGFR2), | en |
| dc.relation.page | 123 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2015-07-03 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
| 顯示於系所單位： | 生物化學暨分子生物學科研究所 | |
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