Molecular and cellular signal pathway for programmed cell death ad anti-angiogenic effects by butein
- Alternative Title
- Butein이 세포사멸과 세포주기조절 및 혈관생성에 미치는 기작 규명
- Abstract
- 이번 연구에서는 옻나무유래 물질인 butein이 인체암세포의 성장과 세포주기 조절 및 혈관생성에 미치는 영향과 그 기전에 대하여 조사하였다.
Butein의 처리는 Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)이 매개하는 암세포의 사멸에 강력한 상승효과를 유도하였다. 이러한 결과는 Sp1 전사 인자의 활성화를 통한 Death Receptor5 (DR5)의 발현 증가에 의한 것으로 밝혀졌다. 또한 butein의 처리는 암세포에서 항시적으로 발현이 높은 것으로 보고되어있는 전사인자인 NF-kB의 활성을 저해하여 암세포의 이동과 침윤에 관계하는 MMP-9의 활성을 억제하였고 또한, 혈관생성인자인 VEGF의 발현을 억제시켜 항혈관생성 효과를 보였다. 뿐만 아니라 butein의 처리는 대부분의 체세포에서는 발현이 되지 않지만 암세포의 불멸에 핵심적인 역할을 수행하고 있는 단백질인 telomerase의 활성과 발현을 저해하는 것으로 조사되었다. 마지막으로 butein은 세포 내에서 ROS 생성을 유도하여 Chk1 과 Chk2의 활성을 통한 G₂/M기의 arrest를 유도하였다.
이상에서의 연구결과를 토대로 butein는 암세포주를 이용한 실험에서 효과적인 항암효과를 나타냄을 확인할 수 있었으며, 암 예방 및 암 치료제의 개발에 있어 후보물질로서의 가능성을 가지고 있는 것으로 사료된다.
- Issued Date
- 2009
- Awarded Date
- 2009. 2
- Type
- Dissertation
- Alternative Author(s)
- Moon, Dong Oh
- Affiliation
- 제주대학교
- Department
- 대학원 해양생명과학과
- Table Of Contents
- INTRODUCTION 1
CHAPTERⅠ
1. Introduction 3
2. Materials and methods 7
2.1. Antibodies and reagents 7
2.2. Cell line and cell growth assay 8
2.3. Detection of apoptosis 8
2.4. DNA fragmentation assay 9
2.5. Western blotting assay 9
2.6. Measurement of ROS 10
2.7. In vitro caspase activity assay 10
2.8. RT-PCR analysis 10
2.9. Flow cytometric analysis of death receptors 11
2.10. Plasmids, transfections, and luciferase assays 12
2.11. Small interfering RNA (siRNA) 13
2.12. Electrophoretic mobility shift assay (EMSA) 13
2.13. Chromatin immunoprecipitation (ChIP) assay 14
2.14. Statistical analysis 14
3. Results 15
3.1. Butein sensitizes TRAIL-induced apoptosis regardless of cell type specificity through activation of caspase 15
3.2. Treatment with a combination of butein and TRAIL activates extrinsic and intrinsic pathways 19
3.3. Butein up-regulates synergistic effects with TRAIL through DR5 expression, but not DR4 23
3.4. Butein activates DR5 transcription through the activation of Sp1 in the DR5 promoter regions 28
3.5. Transactivation of the DR5 promoter requires activation of MAPK by butein 33
3.6. Butein inhibits TRAIL-mediated NF-κB activation 37
4. Discussion 41
CHAPTER Ⅱ.
1. Introduction 46
2. Materials and methods 49
2.1. Antibodies and reagents 49
2.2. Cell line and cell growth assay 49
2.3. Detection of apoptosis 50
2.4. Western Blot Analysis 50
2.5. RT-PCR analysis 51
2.6. Flow cytometric analysis of VEGF 51
2.7. Gelatin Substrate Gel Zymography 51
2.8. Plasmids, Transfections, and Luciferase Gene Assays 52
2.9. Electrophoretic mobility shift assay (EMSA) 53
2.10. ELISA for VEGF and MMP-9 53
2.11. Migration assay 54
2.12. Invasion assay 54
2.13. Matrigel in vitro HUVECs tube formation assay 55
2.14. In Vivo angiogenesis 55
3. Results 57
3.1. Butein inhibits cell proliferation and induces apoptosis in Prostate cancer
cells 57
3.2. Butein decreased NF-κB activitysis 59
3.3. Butein reduced VEGF gene expression 62
3.4. Butein decreased MMP-9 gene transcription and its activity 64
3.5. Effect on VEGF and MMP-9 by butein is mediated through the NF-κB pathway 68
3.6. Butein decreased pancreatic cancer cell migration and invasion 70
4. Discussion 73
CHAPTER Ⅲ.
1. Introduction 76
2. Materials and methods 78
2.1. Reagents 78
2.2. Cell line and culture 78
2.3. Cell viability 78
2.4. Flow cytometric analysis 79
2.5. RNA extraction and RT-PCR 79
2.6. Western blot analysis 79
2.7. Telomerase activity assay 80
2.8. Electrophoretic mobility shift assays (EMSAs) 80
2.9. Immunoprecipitation 80
2.10. Chromatin immunoprecipitation (ChIP) assay 81
2.11. In vitro caspase activity assay 81
2.12. DNA fragmentation assay 81
2.13. Statistical analysis 82
3. Results 83
3.1. Butein inhibits cell proliferation of leukemia cells 83
3.2. Butein upregulates proapoptotic Bax, truncated Bid, caspase activity and subsequent cleavage of PARP 86
3.3. Butein inhibits gene expression of hTERT and telomerase activity 89
3.4. Butein downregulates c-Myc-dependent hTERT expression 91
3.5. Butein inactivates Akt and prevents phosphorylation of hTERT, thereby inhibiting nuclear translocation in THP-1 cells 93
4. Discussion 95
CHAPTER Ⅳ.
1. Introduction 98
2. Materials and methods 101
2.1. Reagents and antibodies 101
2.2. Cell culture and viability assay 101
2.3. Flow cytometric analysis 102
2.4. Western blot analysis 102
2.5. ROS measurement 103
2.9. Statistical analysis 103
3. Results 104
3.1. Butein inhibits viability and proliferation of breast cancer cells through G2/M phase arrest 104
3.2. Butein controls the protein expression levels that regulate G2/M transition 109
3.3. The role of JNK activation on butein-induced cell cycle arrest 112
3.4. Involvement of ROS in butein-induced apoptosis 115
4. Discussion 117
ABSTRACT IN KOREAN 120
REFFERENCES 121
- Degree
- Doctor
- Publisher
- 제주대학교
- Citation
- 문동오. (2009). Molecular and cellular signal pathway for programmed cell death ad anti-angiogenic effects by butein
- Appears in Collections:
- General Graduate School > Marine Life Sciences
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