생몰정보
소속
직위
직업
활동분야
주기
서지
국회도서관 서비스 이용에 대한 안내를 해드립니다.
검색결과 (전체 1건)
원문 아이콘이 없는 경우 국회도서관 방문 시 책자로 이용 가능
목차보기더보기
Title Page
Abstract
Contents
List of Abbreviations 13
Chapter 1. Research Background 15
1. Natural products from marine resources 16
2. Pharmaceuticals from marine-derived fungus 19
3. Diketopiperazines for anticancer reagent 22
4. Apoptosis pathways 26
5. Prostate cancer 30
Chapter 2. Isolation and structure elucidation of bioactive compounds from marine-derived fungus Aspergillus Fumigatus 34
1. Introduction 35
2. Material and methods 37
2.1. Materials and chemicals 37
2.2. Preparation of Aspergillus fumigatus (MFS-150) 38
2.3. Extraction and isolation of secondary metabolites from marine derived A. fumigatus 42
2.4. Cell culture 44
2.5. Cell cytotoxicity assay 44
2.6. Statistical Analysis 45
3. Results 46
3.1. Structure elucidation of secondary metabolites from marine-derived fungus Aspergillus fumigatus 46
3.2. Effect of compound 1-3 from marine-derived fungi Aspergillus fumigatus on cancer cell viability 64
4. Discussion and conclusion 68
Chapter 3. Anti-proliferative and apoptotic effect of Demethoxy-fumitremorgin C from Aspergillus fumigatus, marine fungi on Human Prostate Cancer PC3 cell line 72
1. Introduction 73
2. Material and methods 75
2.1. Materials and chemicals 75
2.2. Prostate cancer PC3 cell culture 75
2.3. Determination of cell proliferation by MTT assay 75
2.4. Hoechst 33342 and propidium iodide (PI) cell staining of PC3 cell line 76
2.5. Flow cytometry analysis for measurement of cell cycle arrest 77
2.6. Annexin V/ propidioum iodide (PI) assay 78
2.7. Determination of mitochondrial membrane potential (MMP) 78
2.8. Assessment of cell viability assay using caspase-3, -8, -9 inhibitor 79
2.9. Western blot analysis 80
2.10. Statistical Analysis 80
3. Results 82
3.1. Anti-proliferative effect of demethoxyfumitremorgin C on PC3 cell line 82
3.2. Observation of DNA damage by demethoxoyfumitremorgin C on PC3 cells using Hoechst 33342 and PI nuclear staining 84
3.3. Cell cycle arrest at G0/G1 phase by demethoxyfumitremorgin C 86
3.4. Induction of apoptosis by demethoxyfumitremorgin C in PC3 cells analyzed by Annexin V-PI staining 88
3.5. Mitochondrial membrane potential (MMP) reduction 90
3.6. Effect of demethoxyfumitremorgin C on oncogenes of p53 signaling pathway and cell cycle related protein levels 92
3.7. Apoptotic Effect of demethoxyfumitremorgin C on PC3 cells via RAS/Bcl-2 family signaling pathway 95
3.8. Effect of demethoxyfumitremorgin C on caspase-3, -8, -9 protein expressions 98
4. Discussion and conclusion 102
Summary 114
References 117
Table 1. Marine pharmaceutical clinical pipeline (2013) 21
Table 2. Diketopiperazines isolated from marine-derived fungi 25
Table 3. ¹H and ¹³C NMR spectral data for cyclotryprostatin A in DMSO-d6.(이미지참조) 49
Table 4. ¹H and ¹³C NMR spectral data for tryprostatin B in DMSO-d6.(이미지참조) 53
Table 5. ¹H and ¹³C NMR spectral data for Demethoxyfumitremorgin C in DMSO-d6.(이미지참조) 59
Figure 1. Natural products from marine sources 17
Figure 2. Bioactivities of new marine natural products. 18
Figure 3. Structures of L-tryptophan, L-proline and diketopeperazines 24
Figure 4. Two kind of apoptosis pathway 29
Figure 5. Purification steps and morphology of the marine-derived fungus, Aspergillus fumigatus. 39
Figure 6. Analysis and blastN report of Aspergillus fumigatus. 40
Figure 7. Extraction procedure of the marine-derived fungus Aspergillus fumigutus. 41
Figure 8. Isolation steps of Compounds 1-3 from the broth extract of marine-derived fungus, Aspergillus fumigatus. 43
Figure 9. Structures of the Compounds 1-3 isolated from marine-derived fungus, Compound 1 (cyclotryprostatin A, 4.3 mg), Compound 2 (tryprostatin B, 3.8 mg),... 47
Figure 10. LREI-MS spectrum of cyclotryprostatin A. 50
Figure 11. ¹H-NMR (a) and ¹³C-NMR (b) spectrums of cyclotryprostatin A in DMSO-d6.(이미지참조) 51
Figure 12. LREI-MS spectrum of tryprostatin B. 54
Figure 13. ¹H-NMR (a) and ¹³C-NMR (b) spectrums of tryprostatin B in DMSO-d6.(이미지참조) 55
Figure 14. DEPT NMR (a) and HMQC NMR (b) spectrums of tryprostatin B in DMSO-d6.(이미지참조) 56
Figure 15. HMBC spectrum of tryprostatin B in DMSO-d6.(이미지참조) 57
Figure 16. LREI-MS spectrum of Demethoxyfumitremorgin C. 60
Figure 17. ¹H-NMR (a) and ¹³C-NMR (b) spectrums of Demethoxyfumitremorgin C in CD₃OD. 61
Figure 18. DEPT NMR (a) and HMQC NMR (b) spectrums of Demethoxyfumitremorgin C in DMSO-d6.(이미지참조) 62
Figure 19. HMBC spectrum of Demethoxyfumitremorgin C in DMSO-d6.(이미지참조) 63
Figure 20. Effects of the isolated compounds 1-3 (C1-C3) from marine-derived fungus, Aspergillus fumigatus on the viability of HeLa and MCF-7 cancer cells. Relatively... 65
Figure 21. Effects of the isolated compounds 1-3 (C1-C3) from marine-derived fungus, Aspergillus fumigatus on the viability of HepG2 and EA.hy926 cells. Relatively... 66
Figure 22. Effects of the isolated compounds 1-3 (C1-C3) from marine-derived fungus, Aspergillus fumigatus on the viability of PC3 cells. Relatively cell viability was... 67
Figure 23. Cell proliferation of demethoxyfumitremorgin C on PC3 prostate cancer cells. Briefly, PC3 cells were cultured at a density of 5 x 10⁴ cells/ml in 96-well plate... 83
Figure 24. Hoechst 33342 staining of PC3 cells treated with demethoxyfumitremorgin C. for nuclei staining, PC3 cells were sub-cultured in 24-well plate and incubated... 85
Figure 25. Cell cycle analysis of PC3 cells treated with different concentrations of demethoxyfumitremorgin C for 72 h. Cells were fixed with 70% of EtOH and resuspended... 87
Figure 26. Flow cytometric analysis of the effect of demethoxyfumitremorgin C in PC3 cells experimented using Annexin V-PI staining assay. The cells appeared with... 89
Figure 27. Flow cytometric analysis of mitochondrial membrane potential. Flow cytometric analysis of demethoxyfumitremorgin C effects on mitochondrial membrane... 91
Figure 28. Effect of demethoxyfumitremorgin C on proteins of cyclin D, E, CDK2, 4, and phospho-cdc2 signaling pathway. Expression of protein levels assayed using... 93
Figure 29. Effect of demethoxyfumitremorgin C on proteins of p-p53 and p-p21 signaling pathway. Expression of protein levels assayed using Western blot analysis following... 94
Figure 30. Effect of demethoxyfumitremorgin C on proteins of Ras, p-Akt, PI3K pathway. Expression of protein levels assayed using Western blot analysis following... 96
Figure 31. Effect of demethoxyfumitremorgin C on proteins of Bax, Bcl-2 and Bcl-xL signaling pathway. Expression of protein levels assayed using Western blot analysis... 97
Figure 32. Effect of demethoxyfumitremorgin C on proteins of caspase-3, -8 and -9 signaling pathway. Expression of protein levels assayed using Western blot analysis... 99
Figure 33. Effect of demethoxyfumitremorgin C on proteins of caspase-3, -8 and -9 signaling pathway. Expression of protein levels assayed using Western blot analysis... 100
Figure 34. Cell proliferation of demethoxyfumitremorgin C on PC3 prostate cancer cells. Briefly, PC3 cells were cultured at a density of 5 x 104 cells/ml in 96-well plate.... 101
Figure 35. The proposed mechanism of anti-proliferation and apoptosis induction in PC3 cells by demethoxyfumitremorgin C. 113
초록보기 더보기
Human prostate cancer is second cause of male death every year in the western countries. Prostate cancer incidence rate is increased in eastern countries such as Japan, Singapore and Korea. Recently, much attention has been paid on chemotherapeutic compounds from natural product as anti-cancer agents. In the present study, marine-derived fungus Aspergillus fumigatus was cultivated and isolated three secondary metabolites (Cyclotryprostatin A (C1), Tryprostatin B (C2), and Demethoxyfumitremorgin (C3)) from marine fungus strain-150 (MFS-150) culture broth extract. The structures of compounds were elucidated via Low resolution electron ionization mass spectrometer (LREIMS), 1D, and 2D nuclear magnetic resonance (NMR) spectra. To investigate the effect of secondary metabolites in inhibition of cell proliferation and induction apoptosis on prostate cancer cells, PC3 cells were treated with different concentrations (25, 50, 100 μM) and various time intervals (24, 48, 72h) of Cyclotryprostatin A (C1), Tryprostatin B (C2), and Demethoxyfumitremorgin (C3). Among them, Demethoxyfumitremorgin (C3) compound has shown anti-proliferative effect on PC3 cells whereas other compounds did not shows significant cytotoxicity. Furthermore, we tested the cell proliferation and apoptosis activity of Demethoxyfumitremorgin C (C3) in PC3 cells and results shows that significant inhibition on cell proliferation in dose and time dependent manner. The level of cell cycle arrest relative protein expression, phospho-cdc2, cyclin A, D, E, CDK 2, and 4 were decreased by Demethoxyfumitremorgin C (C3) in a dose-dependent manner. In addition, Demethoxyfumitremorgin C (C3) regulated the anti- and pro-apoptotic proteins like Bcl-2, Bcl-xL and Bax. Also, relative proteins for apoptosis, cleaved PARP, p-p53, pp21, caspase-3, -8, and -9 were increased by Demethoxyfumitremorgin C (C3). Taken together, these results implicated that Demethoxyfumitremorgin C (C3) has anticancer effect on prostate cancer PC3 cells via suppress cell proliferation through cell cycle arrest at G1 phase and induction of apoptosis via regulating pro- and anti-apoptotic protein expressions in caspase-dependent pathway. Thus, Demethoxyfumitremorgin C (C3) could be used for therapeutic agent for treatment of prostate cancer.
원문구축 및 2018년 이후 자료는 524호에서 직접 열람하십시요.
도서위치안내: / 서가번호:
우편복사 목록담기를 완료하였습니다.
* 표시는 필수사항 입니다.
* 주의: 국회도서관 이용자 모두에게 공유서재로 서비스 됩니다.
저장 되었습니다.
로그인을 하시려면 아이디와 비밀번호를 입력해주세요. 모바일 간편 열람증으로 입실한 경우 회원가입을 해야합니다.
공용 PC이므로 한번 더 로그인 해 주시기 바랍니다.
아이디 또는 비밀번호를 확인해주세요