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General Graduate School
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Interdisciplinary Graduate Program in Advanced Convergence Technology and Science
Bioactive substances derived from subtropical color foods suppress stemness of breast cancer stem cells
- Abstract
- Breast cancer is a major health problem that affects lives worldwide. Breast cancer stem cells(BCSCs) are small subpopulations of cells with capacities for drug resistance, self-renewal,recurrence, metastasis, and differentiation. Herein, powder extracts of beetroot were subjected tosilica gel, gel filtration, thin layer chromatography (TLC), and preparatory high-pressure liquidchromatography (HPLC) for isolation of one compound, based on activity-guided purificationusing tumorsphere formation assays. The purified compound was identified as betavulgarin,using nuclear magnetic resonance spectroscopy and electrospray ionization (ESI) massspectrometry. Betavulgarin suppressed the proliferation, migration, colony formation, andmammosphere formation of breast cancer cells and reduced the size of the CD44+/CD24−subpopulation and the expression of the self-renewal-related genes, C-Myc, Nanog, and Oct4.This compound decreased the total level and phosphorylated nuclear level of signal transducerand activator of transcription 3 (Stat3) and reduced the mRNA and protein levels of sexdetermining region Y (SRY)-box 2 (SOX2), in mammospheres. These data suggest thatbetavulgarin inhibit the Stat3/Sox2 signaling pathway and induces BCSC death, indicatingbetavulgarin might be an anticancer agent against breast cancer cells and BCSCs.
- Issued Date
- 2021
- Awarded Date
- 2021. 8
- Type
- Dissertation
- Alternative Author(s)
- 유인
- Affiliation
- 제주대학교 대학원
- Department
- 대학원 차세대융복합과학기술협동과정
- Advisor
- 이동선
- Table Of Contents
- CHAPTER I 1
1.1 Breast cancer stem cells 2
1.2 Color foods 5
1.3 Breast cancer stem cell and signaling pathway 8
1.4 Breast cancer stem cell and cytokines 11
CHAPTER II 15
2.1 ABSTRACT 16
2.2 INTRODUCTION 16
2.3 MATERIALS AND METHODS 18
2.3.1 Chemical and reagents 18
2.3.2 Plant material 18
2.3.3 Isolation of an inhibitor 18
2.3.4 4 Culture of human breast cancer cells and mammospheres 19
2.3.5 Cell proliferation assay 20
2.3.6 Colony formation assay 20
2.3.7 Assessment of CD44+/CD24- expression 20
2.3.8 Transwell assay 21
2.3.9 Real-time quantitative reverse transcription PCR 21
2.3.8 Immunofluorescence (IF) 22
2.3.10 Western blotting 22
2.3.11 EMSA 23
2.3.12 Statistical analysis 23
2.4 RESULTS 24
2.4.1 Isolation of a BCSC inhibitor from Beta vulgaris 24
2.4.2 Structure analysis of the purified compound 26
2.4.3 Betavulgarin inhibits breast cancer cell growth and mammosphere formation 28
2.4.4 Betavulgarin decreases CD44+/CD24-expressing cancer cell numbers 28
2.4.5 Betavulgarin inhibits the nuclear translocation of Stat3 in BCSCs 28
2.4.6 Betavulgarin inhibits the mRNA levels of BCSC-specific marker genes and mammosphere growth 29
2.5 DISCUSSION 37
2.6 CONCLUSION 39
CHAPTER III 40
3.1 ABSTRACT 41
3.2 INTRODUCTION 41
3.3 MATERIALS AND METHODS 43
3.3.1 Isolation and identification of a breast CSC inhibitor derived from D. carota L. 43
3.3.2 Structure analysis of the purified compound 44
3.3.3 Cell line and culture of mammospheres 45
3.3.4 Cell proliferation 46
3.3.5 Colony formation assay 46
3.3.6 Flow cytometric assay of CD44+/CD24expression 46
3.3.7 Migration 47
2.3.7 Real-time quantitative reverse transcription PCR 47
3.3.8 Western blotting 47
3.3.9 EMSA 48
3.3.10 Quantification of extracellular human IL-6 and IL-8 cytokines using the cytometric bead array (CBA) 48
3.3.11 Statistical analysis 49
3.4 RESULTs 50
3.4.1 Isolation and identification of a breast CSC inhibitor derived from D. carota L. 50
3.4.2 6-Methoxymellein suppresses the growth of breast cancer cells and mammospheres 50
3.4.3 6-Methoxymellein reduces the proportion of CD44+/CD24-expressing breast cancer cells 56
3.4.4 6-Methoxymellein inhibits the protein expression of cancer stem cell-specific markers and inhibits mammosphere growth 56
3.4.5 6-Methoxymellein suppresses the nuclear localization of NF-κB p65 and NF-κB p50 in BCSCs 56
3.4.6 6-Methoxymellein decreases the mRNA and protein levels of secretory IL-6 and IL-8 in mammospheres 57
3.5 DISCUSSION 62
3.6 CONCLUSION 65
CHAPTER IV 66
4.1 ABSTRACT 67
4.2 INTRODUCTION 67
4.3 MATERIALS AND METHODS 70
4.3.1 Chemical and reagents 70
4.3.2 Plant material 70
4.3.3 Isolation and purification of a mammosphere inhibitor from Artemisia princeps 70
4.3.4 Structure analysis of the purified compound 71
4.3.5 Culture of human breast cancer cells and mammospheres 72
4.3.6 Cell proliferation assay 73
4.3.7 CD44+/CD24- expression, aldehyde dehydrogenase (ALDH) activity and apoptosis using a flow cytometric assay 73
4.3.8 Real-time quantitative reverse transcription PCR 74
4.3.9 Western blotting 74
4.3.10 Electrophoretic mobility shift assay (EMSA) 75
4.3.11 Quantitative measurement of human IL-6 and IL-8 using the BDTM CBA human inflammatory cytokines assay kit 76
4.3.12 Stat3 and YAP1 knockdown using small interfering RNA (siRNA) 76
4.3.13 Statistical analysis 76
4.4 RESULTS 77
4.4.1 Isolation of breat cancer stem cells (BCSCs) inhibitor from Artemisia princeps. 77
4.4.2 5-Desmethylsinensetin inhibits mammosphere formation of breast cancer cells 77
4.4.3 5-Desmethylsinensetin decreases CD44+/CD24expressing and ALDH-expressing cancer cells 80
4.4.4 5-Desmethylsinensetin induces apoptosis and inhibits the mRNA levels of cancer stem cell specific marker genes and mammosphere growth 80
4.4.5 5-Desmethylsinensetin decreases the total and nuclear levels of p-Stat3 and Stat3 in BCSCs 81
4.4.6 5-Desmethylsinensetin reduces the secretion and mRNA levels of IL-6 and IL-8 in mammospheres 84
4.4.7 5-Desmethylsinensetin inhibits the nuclear localization of YAP1 84
4.5 DISCUSSION 91
4.6 CONCLUSION 94
REFERENCES 96
SUPPLEMENTARY FIGURES 126
LIST OF TABLE 153
LIST OF PUBLICATION INCLUDED IN THE THESIS 154
Acknowledgment 156
- Degree
- Doctor
- Publisher
- 제주대학교 대학원
- Citation
- Liu, Ren. (2021). Bioactive substances derived from subtropical color foods suppress stemness of breast cancer stem cells
- Appears in Collections:
- Interdisciplinary Programs > Interdisciplinary Graduate Program in Advanced Convergence Technology and Science
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