Title Page
Contents
List of Abbreviations 11
Chapter I. Development of oral cancer sentinel lymph node (OCSLN) animal model and identification of key lymphangiogenic factor 12
Abstract 13
Introduction 15
Materials and Methods 18
Mice and cell lines 18
Construction of plasmids 18
Stable transformation 19
Protein extraction, western blot analysis, and Immunoprecipitation 20
Reverse transcription-polymerase chain reaction (RT-PCR) analysis 20
HLMEC cell proliferation assay 21
HLMEC tube formation assay 22
HLMEC migration assay 22
Animals and the oral cancer sentinel lymph node animal model 23
Tissue preparation 23
Immunohistochemistry 23
Statistical analysis 24
Results 25
1. Establishment of oral cancer sentinel lymph node (OCSLN) animal model 25
Discussion 30
References 33
Figure Legends 35
Figures 39
Chapter II. 3-O-acetyloleanolic acid and 6,8-diprenylgenistein for suppressing oral cancer sentinel lymph node (OCSLN) metastasis and analysis of its inhibitory mechanism 50
Abstract 51
Introduction 53
Materials and Methods 56
Cell lines and culture 56
Cell cytotoxicity test 56
HLMEC cell proliferation assay 56
HLMEC tube formation assay 57
HLMEC migration assay 57
Reverse transcription-polymerase chain reaction (RT-PCR) analysis 58
Protein extraction, western blot analysis, and Immunoprecipitation 58
Co-imxnunoprecipitation assay 59
ELISA assay 60
In vivo Matrigel plug assay 60
Animals and an OCSLN animal model 60
Tissue preparation 61
Immunohistochemistry 61
Statistical analysis 62
Results 63
1. Screening of natural compounds for suppressing OCSLN metastasis 63
2. Effects of 3-O-acetyloleanolic acid on VEGF-A-induced Iymphangiogenesis and lymph node metastasis in an OCSLN metastasis 65
3. Effects of 6,8-diprenylgenestein on VEGF-A-induced OCSLN metastasis 69
Discussion 74
References 78
Figure Legends 82
Tables and Figures 89
Abstract (Korean) 110
Table II-1. List of selected 11 natural compounds from 2nd selection procedures[이미지참조] 92
Table II-2. 4th selection procedure: Selection of natural products not reported to...[이미지참조] 94
Figure I-1. Establishment of oral cancer sentinel lymph node (OCSLN) animal model 39
Figure I-2. Detection of sentinel lymph nodes in OCSLN 40
Figure I-3. Investigation of the lymph node metastasis in OCSLN animal model 41
Figure I-4. Identification of key lymphangiogenic factor in CoCl₂-treated... 42
Figure I-5. Establishment of mouse VEGF-A overexpressing SCCVII cell line 43
Figure I-6. Establishment of mouse VEGF-A knockdown SCCVII cell line 44
Figure I-7. Effects of VEGF-A secreted from SCCVII cells on proliferation,... 45
Figure I-8. Effects of VEGF-A on tumor growth in OCSLN animal model 46
Figure I-9. Effects of VEGF-A on sentinel lymph node enlargement and... 47
Figure I-10. Effects of VEGF-A on lymphangiogenesis in OCSLN animal model 48
Figure I-11. Effects of VEGF-A on lymphangiogenesis-related signaling pathway... 49
Figure II-1. The selection procedures of natural compounds for suppressing oral... 89
Figure II-2. 1st selection procedure: Selection of natural products that inhibit...[이미지참조] 90
Figure II-3. 2nd selection procedure: Selection of natural products that are non-...[이미지참조] 91
Figure II-4. 3rd selection procedure: Selection of natural products that inhibit...[이미지참조] 93
Figure II-5. Structure of 3-O-acetyloleanolic acid (3AOA) 95
Figure II-6. Effects of 3AOA on VEGF-A-induced HLMEC proliferation, tube... 96
Figure II-7. Effects of 3AOA on VEGF-A-induced lymphatic vessel formation in... 97
Figure II-8. Effects of 3AOA on the expression of VEGF family proteins in... 98
Figure II-9. Effects of 3AOA on activation of VEGFR-1, VEGFR-2 and... 99
Figure II-10. Effects of 3AOA on tumor growth, sentinel lymph node... 100
Figure II-11. Effects of 3AOA on Iymphangiogenesis in VEGF-A-induced... 101
Figure II-12. Structure of 6,8-diprenylgenistein (6,8-DG) 102
Figure II-13. Effects of 6,8-DG on VEGF-A-induced HLMEC proliferation, tube... 103
Figure II-14. Effects of 6,8-DG on VEGF-A-induced lymphatic vessel formation... 104
Figure II-15. Effects of 6,8-DG on the expression of VEGF family proteins in... 105
Figure II-16. Effects of 6,8-DG on activation of VEGFR-1, VEGFR-2 and... 106
Figure II-17. The mechanism of action of 6,8-DG in oral cancer related lymphangiogenesis 107
Figure II-18. Effects of 6,8-DG on sentinel lymph node enlargement, and lymph... 108
Figure II-19. Effects of 6,8-DG on tumor-induced lymphangiogenesis in a VEGF-... 109