구강편평상피 암세포와 이종종양 모델에서의 화학요법과 배아줄기세포 유래 조혈모세포, 혈관내피세포의 단백체 분석 및 기능 연구 = Proteomic analysis to identify hematopoietic and endothelial cells derived from embryonic stem cells and chemotherapeutic effect of natural products on oral squamous cancer cells and xenograft tumors

다국어 초록 (Multilingual Abstract)

Recently, biphenolic components derived from the Magnolia family have been studied for anti-cancer, anti-stress, and anti-inflammatory pharmacological effects. However, the pharmacological mechanism of action of 4-O-methylhonokiol (MH) is not clear in oral cancer. The aim of this study was to investigate the role of MH in apoptosis and its molecular mechanism in oral squamous cell carcinoma (OSCC) cell lines, HN22 and HSC4, as well as tumor xenografts. Here, we demonstrated that MH decreased cell growth and induced apoptosis in HN22 and HSC4 cells through the regulation of specificity protein 1 (Sp1). Moreover, MH treatment effectively inhibited tumor growth and Sp1 levels in BALB/c nude mice bearing HN22 cell xenografts. These results indicated that MH inhibited cell growth, colony formation and also induced apoptosis via Sp1 suppression in OSCC cells and xenograft tumors. Thus MH is a potent anti-cancer drug candidate for oral cancer.
In the second subject that OVOL2, a C2H2 zinc finger protein, is a novel binding protein of ER71 which is a critical transcription factor for blood and vessel development. OVOL2 directly interacted with ER71, but not with ETS1 or ETS2, in the nucleus. ER71-mediated activation of Flk1 promoter was further enhanced by OVOL2, although OVOL2 alone failed to activate it. Consistently, co-expression of ER71 and OVOL2 in differentiating embryonic stem cells (ESCs) led to a significant augmentation of FLK1-expressing (FLK1+) cells, endothelial and hematopoietic cells. Such cooperative effects were impaired by the shRNA-mediated inhibition of Ovol2. Collectively, we conclude that ER71 directly interacts with OVOL2 and that such interaction is critical for FLK1+ cell generation and its differentiation into downstream cell lineages.

국문 초록 (Abstract)

최근 목련종 유래의 바이페놀 성분을 갖는 메틸호노키올의 항암, 항 스트레스 그리고 항 염증성에 대한 약물 효과는 연구 되었지만, 구강암에 있어서 메틸호노키올의 약학적인 메커니즘은 아직 명확하게 밝혀지지 않았다. 이 연구의 목적은 인간 구강편평상피 암세포인 HN22와 HSC4 뿐만아니라 이종종양에서의 분자학적 메커니즘과 세포사멸에 관여하는 메틸호노키올의 역할에 대한 연구이며, 여러 가지 실험방법을 이용함으로써, Sp1의 조절을 통해 HN22와 HSC4 세포의 성장을 늦추고 세포사멸을 유도하는 메틸호노키올에 대해 증명하였다. 또한 메틸호노키올을 처리했을 때 효과적으로 HN22 세포의 이종종양과 관련된 BALB/c 누드 마우스에서 Sp1의 수준과, 종양 성장이 억제되었다. 이러한 결과를 통해 메틸호노키올은 이종종양과 인간 구강 편평상피 암세포에서 Sp1 억제를 통한 세포사멸 유도와 세포성장 및 암세포 군집형성을 억제한다는 것을 시사했다. 두 번째 연구에서는 ER71의 신종 결합 단백질인 C2H2 징크핑거 단백질 OVOL2는 혈액과 혈관의 발생을 위해 중요한 전사인자 임을 보고한다. OVOL2는 직접적으로 ER71과 상호작용하지만 세포 내 핵에서는 ETS1 또는 ETS2와는 결합하지 않는다. OVOL2 단독으로는 Flk1 프로모터를 활성화시키지 못하지만, ER71을 통한 Flk1 프로모터의 활성화 조절이 가능하며, ER71은 OVOL2를 통해 Flk1 프로모터 활성화를 더욱 향상 시킨다. 결론적으로, ER71과 OVOL2는 상호작용 하고 있으며, 이러한 상호작용은 FLK1+ 세포 발생과, 이 후 세포 계통별 분화에 중요한 것으로 나타났다.

목차 (Table of Contents)

• Ⅰ. INTRODUCTION 1
• Ⅱ. MATERIALS and METHODS 3
• 1. Materials 3
• 2. Tissue sample preparation 3
• 3. Cell culture 4
• 4. MTS assay 4
• 5. DAPI staining 5
• 6. Propidium iodide staining and Annexin-V/7-AAD-assay 5
• 7. Reverse transcription-polymerase chain reaction 6
• 8. Immunocytochemistry 6
• 9. Pull-down assays 7
• 10. Molecular modeling for MH with Sp1 7
• 11. Western blot analysis 8
• 12. Anchorage-independent cell transformation assay 9
• 13. Sp1 small interfering RNA (siRNA) 9
• 14. Human carcinoma xenograft model 9
• 15. Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling assay 10
• 16. Immunohistochemistry 11
• 17. Data analysis 12
• Ⅲ. RESULTS 14
• 1. MH suppressed the viability of HN22 and HSC4 cells 14
• 2. MH induced the apoptosis and sub-G1 phase cell cycle of HN22 and HSC4 cells 17
• 3. MH regulated Sp1 protein levels in HN22 and HSC4 cells 21
• 4. MH binds with Sp1 and silico model of MH binding with Sp1 24
• 5. MH regulated the expressions of cell cycle arrest- and apoptosis-related molecules in HN22 and HSC4 cells 26
• 6. MH suppress colony formation mediated through Sp1 in HN22 and HSC4 cells 29
• 7. MH inhibits tumor growth and apoptosis by down-regulating the Sp1 protein in a tumor xenograft of HN22 cells in nude mice 32
• Ⅳ. DISCUSSION 36
• Ⅴ. CONCLUSION 40
• Ⅵ. REFERENCES 41
• Ⅶ. 국문초록 49
• Ⅰ. INTRODUCTION 58
• Ⅱ. MATERIALS and METHODS 60
• 1. Plasmid vector construction 60
• 2. ESC culture and flow cytometry 60
• 3. Generation of inducible ES cells 60
• 4. GST-pull down assay 61
• 5. In-solution tryptic digestion 62
• 6. Desalting of tryptic peptide 62
• 7. Mass spectrometer configuration 63
• 8. Data processing and protein identification 63
• 9. In vitro transcription and translation reactions 64
• 10. Peptide competition assay 64
• 11. Co-immunoprecipitation assay 65
• 12. FACS sorting of ER71-VENUS embryonic cells 66
• 13. Hematopoietic replating assay 66
• 14. Luciferase assay 66
• 15. Immunohistochemistry 66
• 16. Immunofluorescence assay 67
• 17. RNA interference 68
• 18. Quantitative RT-PCR 68
• 19. Statistical analysis 69
• Ⅲ. RESULTS and DISCUSSION 72
• Ⅳ. CONCLUSION 97
• Ⅴ. REFERENCES 98
• Ⅵ. 국문초록 105