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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林榮耀 | |
| dc.contributor.author | Bo-Jyun Lin | en |
| dc.contributor.author | 林柏均 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:36:41Z | - |
| dc.date.copyright | 2011-10-05 | |
| dc.date.issued | 2011 | |
| dc.date.submitted | 2011-07-26 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24686 | - |
| dc.description.abstract | Hepatocellular carcinoma (HCC) is the second highest cancer death in Taiwan, and the main risk factors for the development of HCC include infection with hepatitis B virus (HBV), hepatitis C virus (HCV) and chronic alcohol consumption. Therefore, development of effective chemotherapies is crucial for treatment of HCC. In recent years, the use of Chinese Herbal Medicines (CHMs) has been an adjuvant therapy for cancers. In present study, Hep3B, a classic hepatic carcinoma cell line, were used to investigate the anti-tumor effects of four CHMs (Saussurea lappa , Dwu Hwo Jih Sheng Tang , Achyranthes bidentata Bl , Long Daan Shieh Gan Tang ). First, by MTT and migration assay, the comparison of IC50 cell cytotoxicity and IC50 cell migration values indicated that Saussurea lappa (SL) has a higher inhibitory effect on the migration, but low cytotoxicity of Hep3B cells. Therefore, SL was chosen for further study. SL inhibits the migration of Hep3B cells with a medium inhibitory concentration (IC50 cell migration) value thirty-seven times lower than that of proliferation activity (IC50 cell cytotoxicity). In vivo efficacy was determined in a subcutaneous xenograft tumor model, NOD/SCID mice. Daily oral administration with SL at 2.25 g/kg inhibited the growth of tumor xenografts and lung metastases in NOD-SCID mice These results strongly suggest that SL has the inhibitory effect on cancer progression both in vitro and in vivo.
And then, the anti-tumor molecular mechanisms of SL were further investigated. First, SL inhibited the cell migration and proliferation of Hep3B cells by inhibiting C/EBP regulates to the promoter region of VEGFR2, VEGFR3 and TGFßR1. Furthermore, the down-regulation of these receptors blocked the activation of Src, and decreased the translocation of ß-Catenin from cytosol to nuclear. In addition, SL inhibited the transcriptional activity of ß-Catenin leading to the suppression of Slug, fibronectin and N-cadherin expressions. SL also up-regulated E-cadherin which was negative regulated by Slug and Snail. Moreover, the organization of actin cytoskeleton, a key regulator for cell migration, was inhibited by SL. The regulation of actin cytoskeleton was through the modulation of the activity of small GTPase family including Rac1, Cdc42 and Rho A. Our results suggest that SL not only inhibits the migration of Hep3B cells through inhibiting the phosphorylation of Src, but also suppresses the activation of Cdc42, and then decreases the motility of Hep3B cells. In conclusion, present study provides a therapeutic rationale for the inhibitory effects of SL on HCC, and SL may be a potential antitumor therapeutic agent as an alternative medicine for controlling tumor growth and metastasis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T05:36:41Z (GMT). No. of bitstreams: 1 ntu-100-R98442017-1.pdf: 2265186 bytes, checksum: 357f0859f68825fbb7a69218e88445c2 (MD5) Previous issue date: 2011 | en |
| dc.description.tableofcontents | Abbreviations VIII
摘要 X Abstract XII Introduction 1 1 Hepatocellular carcinoma (HCC) 2 2 Chinese herbal medicine (CHM) 3 3 Saussurea lappa 3 4 Vascular endothelial growth factors(VEGFs)/ VEGF receptor (VEGFR) signaling 4 5 Role of ß-catenin in cancer 5 6 Epithelial -mesenchymal transition (EMT) 7 7 Cell adhesion molecules: E-Cadherins and N-cadherin 8 8 Research purpose of present investigation 9 Materials and Methods 10 1 Materials 11 2 Antibodies 12 3 Preparation of CHMs 12 4 Cell culture 12 5 Cell viability analysis 13 6 Wound-healing assay 14 7 Cell migration assay 14 8 Cell invasion assay 15 9 Confocal microscopy 15 10 RNA extraction and reverse transcription 16 10.1 RNAs extraction 16 10.2 Reverse transcription 17 11 Quantitative Real-time PCR (Q-PCR) 18 12 Western blot analysis 19 12.1 Preparation of cell lysates 19 12.2 Quantification of protein concentration 19 12.3 Preparation of sodium-dodecyl-sμlfate-polyacrylamide gels (SDS-PAGE) 20 12.4 Protein sample preparation 21 12.5 Electrophoresis 21 12.6 Semi-dry blotting 22 12.7 Immunoblotting 23 13 Nuclear cytoplasmic fractionation 24 14 GST-PBD pull down assay 24 14.1 Purification of GST-PBD 24 14.2 Cdc42 activity assays 25 15 Luciferase reporter assay 26 16 Tumor xenograft analysis of anti-proliferation and anti-metastasis activity by SL 27 Results 29 1. Cytotoxicity of four CHMs measured with Hep3B cells 30 2. Effects of SL and DHJST on migration of Hep3B cells 31 3. Suppressions of the invasion and the motility ability by SL 31 4. Inhibition of tumor growth in vivo by treating with SL 32 5. SL suppressed tumor metastasis in vivo 32 6. Inhibition the organization of actin cytoskeleton by SL 33 7. Down-regulation of VEGFC, VEGFR2 and VEGFR3 protein by SL 34 8. Suppression the expression of TGFßR1 protein by SL 35 9. SL inhibited the C/EBP-mediated transcription followed by the expression of VEGFR2, VEGFR3, and TGFßR1 36 10. Cadherin switch was suppressed by SL 36 11. Inhibitory effects of SL on the expression of Slug 37 12. Effect of SL on the translocation of ß-catenin and decreasing of nucleus Slug 38 Discussion 40 Figures 46 References 72 List of figures Figure 1. Effects of aqueous extract of four CHMs on cell viability 49 Figure 2. Effects of CHMs on the migration of Hep3B cells 51 Figure 3. Effects of Saussurea lappa on the invasion of Hep3B cells 53 Figure 4. Saussurea lappa inhibited tumor growth in NOD-SCID mice xenograft 55 Figure 5. Saussurea lappa inhibited tumor metastasis in vivo 57 Figure 6. Saussurea lappa interferes with the organization of actin cytoskeleton 58 Figure 7. Inhibitory effects of Saussurea lappa on Cdc42 expression 59 Figure 8. Down-regulation of VEFGC by Saussurea lappa 60 Figure 9. Down-regulation of VEGFR/p-Src by Saussurea lappa 61 Figure 10. Suppression of TGFβR1 by treating with Saussurea lappa for 24 hr 62 Figure 11. Effect of Saussurea lappa on the transcription of genes related to metastasis 63 Figure 12. Saussurea lappa suppressed the promoter activity of C/EBP and TBE 64 Figure 13. Saussurea lappa suppressed the EMT phenotype of Hep3B cells 66 Figure 14. β-catenin and Slug translocation were downregulated by Saussurea lappa 67 Figure 15. Proposal mechanism of SL on the migration and metastasis of HCC cells 68 Table I. Primer sequences used in quantitative real-time RT-PCR 70 Table II. Cytotoxicity and cell migration inhibitory activities of four kinds of CHMs on Hep3B cells 71 | |
| dc.language.iso | en | |
| dc.subject | Slug | zh_TW |
| dc.subject | 肝癌 | zh_TW |
| dc.subject | 第三型血管表皮生長因子接受器 | zh_TW |
| dc.subject | 第二型血管表皮生長因子接受器 | zh_TW |
| dc.subject | 木香 | zh_TW |
| dc.subject | VEGFR2 | en |
| dc.subject | Hepatocellular carcinoma | en |
| dc.subject | Saussurea lappa | en |
| dc.subject | migration | en |
| dc.subject | proliferation | en |
| dc.subject | C/EBP | en |
| dc.subject | VEGFR3 | en |
| dc.title | 木香抑制肝癌細胞之遷徙與增生 | zh_TW |
| dc.title | Saussurea lappa suppresses the migration and proliferation of hepatocellular carcinoma cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 99-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 李德章,李明學,呂紹俊 | |
| dc.subject.keyword | 肝癌,木香,第三型血管表皮生長因子接受器,第二型血管表皮生長因子接受器,Slug, | zh_TW |
| dc.subject.keyword | Hepatocellular carcinoma,Saussurea lappa,migration,proliferation,C/EBP,VEGFR2,VEGFR3, | en |
| dc.relation.page | 76 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2011-07-27 | |
| 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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