신규 합성된 Quaternized Amino Glucosamine 유도체의 항암기전 및 기질금속단백질분해효소와 NF-κB의 억제효능에 대한 연구
- Alternative Title
- Anticancer Mechanism of a Novel Quaternized Amino Glucosamine Derivative and Its Inhibitory Effects on Matrix Metalloproteinases and NF-κB
- Abstract
- 최근 연구에서는 몇몇 후보 항암제들의 활성을 결정하는데 분자들의 전하적 성질이 중요한 요소로 작용하는것으로 알려져 있다. 그러나 많은 연구에서 이러한 전하적 성질을 가지는 분자들(molecules)이 목표(target)가 되는 분자들 (molecules)과 작용기전이 서로 일치하지 않는다는 사실도 보고되고 있다. 본 연구에서는 Quaternized Amino (QA)가 분자량이 작은 강력한 전하적 성질을 나타내기 위해 글루코사민(glucosamine)으로 전환하였다. 이들 양이온을 띄는 Quaternized Amino Glucosamine (QAGlc)는 분자량이 적어서 흑색종세포에서 특이적인 독성효과를 나타내며, 이러한 사실은 기존연구에서 아직 밝혀지지 않는 새로운 사실이다. 그러므로 본 연구에서는 새로운 글루코사민(glucosamin) 유도체를 이용하여 흑색종 세포에 미치는 작용을 확인하였다. 즉 이들 분자들(molecules)의 농도와 처리시간에 따른 아포토시스(apoptosis) 현상을 측정하였다. 그 결과 QAGlc는 아포토시스(apoptosis) 신호전달과정에서 중요한 p53 유전자의 전사활성 및 단백질 발현을 증가시켰다. QAGlc는 p53 발현을 활성화시키데 관여하는 아포토시스 (apoptosis) 관련 단백질의 발현을 단계적으로 유도하였다. 뿐만아니라 B16F1 세포에서 QAGlc은 Apaf-1 유전자 활성화을 통해 아포토시스(apoptosis)를 유발한다는 사실을 확인하였다. 또한 QAGlc은 HT1080 fibrosarcoma 세포에서 농도가 증가함에 따라 MMP-2 와 MMP-9 유전자의 전사가 억제된다는 사실을 알 수 있었다. 이들 MMP-2와 MMP-9는 AP-1과 NF-κB 전사인자를 감소시킴으로서 두 유전 자의 발현이 억제됨을 확인하였다. AP-1과 NF-κB 전사인자들은 mitogen-activated protein kinase (MAPKs)를 억제하는데 관여한다. 또한 면역세포들에서 QAGlc이 NF-κB을 강력하게 억제시켰으며, 이러한 사실은 RAW264.7 세포에서 QAGlc이 IKK 신호전달과정에 영향을 줌으로서 NF-κB가 억제된다는 것을 알 수 있었다. 결론적으로 QAGlc는 NF-κB 활성을 억제함으로서 MAPKs과 염증사이토카인 생성을 억제한다는 사실을 확인하였다.
In the present research, cell-specific cytotoxic effect of newly synthesized quaternized amino glucosamine (QAGlc) in melanoma cells was identified. Analysis of apoptosis employing different approaches confirmed that this molecule induces apoptosis in a concentration and time-dependent manner in B16F1, melanoma cells. Treatment of QAGlc increased the promoter activity and protein expression level of p53 proving QAGlc followed a p53-dependent apoptotic pathway. Following treatment, QAGlc induced a cascade of apoptosis-related proteins including p21, PUMA, cytochrome C, caspase-9 and caspase-3. Concurrent activation of Apaf-1 greatly contributed to apoptosis induction effect of QAGlc in melanoma cells. Cell cycle analysis of B16F1 cells in the presence of QAGlc further confirmed that QAGlc was involved in cell cycle arrest at G1 phase. This was favored by decreased protein expressions of cell cycle-related proteins such as cyclin-D, cyclin-E, cdk-2, cdk-4, cdk-6, Bcl-2 and E2F. The potency of QAGlc to affect transcriptional regulation of MMP-2 and MMP-9 enzymes was identified in HT1080 fibrosarcoma cells and it showed a concentration-dependent effect. This inhibition of MMP-2 and MMP-9 was found to work via down-regulation of both AP-1 and NF-κB transcription factors induced by PMA. Further analysis confirmed that functions of these transcription factors were correlated to suppression of mitogen-activated protein kinases (MAPKs). Moreover, it was elucidated that suppression of NF-κB by QAGlc occurs through the inactivation of IKK as investigated in RAW264.7 immune cell study. In addition, indirect NF-κB inactivation by QAGlc was a result of suppression of MAPKs and related inflammatory cytokines.
- Issued Date
- 2007
- Awarded Date
- 2007. 2
- Type
- Dissertation
- Publisher
- 부경대학교 대학원
- Alternative Author(s)
- B. E. P. Mendis
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 화학과
- Advisor
- 김세권
- Table Of Contents
- Part 1 Anticancer Mechanism of Quaternized Amino Glucosamine in B16F1 Melanoma Cells = 1
Introduction = 2
1. Therapeutic potential of cell cycle arrest and checkpoint control in treatment of cancer = 2
2. Therapeutic potential of the induction of apoptosis in treatment of cancer = 3
3. Involvement of p53 in intrinsic pathway of apoptosis = 5
4. Matrix metalloproteinases (MMPs) and NF-κB: Other main targets in anticancer therapy = 8
5. Chemotherapy as a means of treating cancer = 9
6. Drug resistance in melanoma = 10
7. Potential of nutraceuticals to prevent cancer = 12
Materials and Methods = 15
1. Materials = 15
2. Instrumental analysis = 15
3. Preparation of glucosamine (Glc) from chitosan = 16
4. Synthesis and purification of quarternized amino glucosamine (QAGlc) = 16
5. Cell culture = 17
6. Toxicity determination of QAGlc on cancer cells derived from different tissues = 18
7. Determination of apoptosis = 18
7.1. DNA fragmentation assay = 18
7.2. Cell cycle analysis for apoptosis determination- PI method = 19
7.3. Cell cycle analysis for apoptosis determination- Annexin V method = 20
7.4. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay = 20
8. RT-PCR = 21
9. Reporter gene assay = 22
10. Western blot analysis = 23
11. Statistical analysis = 24
Results and Discussion = 25
1. Synthesis of QAGlc = 25
2. Structural confirmation of QAGlc = 27
3. QAGlc exerted differential effects in cancer cell lines derived from different tissues = 34
4. Cytotoxic effects of QAGlc observed in B16F1 cells were due to induction of apoptosis = 43
5. Visualization of apoptotic cells by TUNEL assay = 46
6. Determination of apoptosis by cell cycle analysis = 46
7. QAGlc induces apoptosis via induction of p53 and its target genes p21, PUMA and suppressing MDM2 = 52
8. Other target genes in apoptosis pathway that affected by the treatment of QAGlc = 58
9. QAGlc exerted inhibitory effects on proteins that are necessary for the progression of the cell cycle = 67
10. The proposed special feature of QAGlc-mediated apoptosis in B16F1 melanoma cells = 72
11. Summary = 77
References = 78
Part 2 MMP-2 and MMP-9 Inhibition by Quaternized Amino Glucosamine in HT1080, Fibrosarcoma Cells = 89
Introduction = 90
1. MMPs: Definition, function and regulation = 90
2. Role of MMPs in tumor growth, invasion and metastasis = 92
3. Current and future developments = 97
Materials and Methods = 100
1. Materials = 100
2. Cell culture = 100
3. Cytotoxicity determination of QAGlc in fibrosarcoma cells = 100
4. Gelatin zymography = 101
5. Reporter gene assay for MMP-9, MMP-2, AP-1 and NF-κB = 102
6. Extraction of nuclear and plasma protein = 102
7. Western blot analysis = 103
8. Cell invasion and mobility assay = 104
9. Statistical analysis = 104
Results and Discussion = 105
1. Inhibition of MMP-9 and MMP-2 expression in QAGlc treated HT1080 cells assessed by gelatin zymography = 105
2. Assessment of transcriptional regulation of MMP-9 and MMP-2 following treatment with QAGlc = 109
3. Assessment of transcriptional regulation of NF-κB and AP-1 following treatment with QAGlc = 112
4. Inhibition of invasiveness of HT1080 cells following treatment with QAGlc = 119
5. Inhibition of mitogen-activated protein kinase pathway in HT1080 cells following treatment with QAGlc = 119
6. Summary = 123
References = 124
Part 3. Inhibition of NF-κB Pathway in RAW264.7, Mouse Macrophage Cells by Quaternized Amino Glucosamine = 131
Introduction = 132
1. Background of nuclear factor kappa-B (NF-κB) = 132
2. Involvement of NF-κB in the occurrence of diseases including cancer . = 133
3. Natural compounds as anti-inflammatory remedies = 135
Materials and Methods = 138
1. Materials = 138
2. Cell culture = 138
3. Cytotoxicity determination of RAW264.7 cells following treatment QAGlc = 138
4. Reporter gene assay = 139
5. Extraction of nuclear and plasma protein = 139
6. Western blot analysis = 140
7. Determination IL-1β, IL-6 and TNF-α activity following treatment of QAGlc = 141
8. Determination PGE2 activity following treatment of QAGlc = 141
9. Statistical analysis = 142
Results and Discussion = 143
1. QAGlc inhibits NF-κB promoter activity = 143
2. QAGlc reduces nuclear translocation of NF-κB = 146
3. Effect of QAGlc on activities of inflammatory cytokines = 149
4. Effect of QAGlc on protein expressions of ERK, JNK, and p38 = 150
5. Summary = 157
References = 158
Abstract = 162
- Degree
- Doctor
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신규 합성된 Quaternized Amino Glucosamine 유도체의 항암기전 및 기질금속단백질분해효소와 NF-κB의 억제효능에 대한 연구.pdf
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