Title Page
Contents
국문초록 12
ABSTRACT 15
List of Abbreviation 19
Ⅰ. INTRODUCTION 21
A. The significance of anti-cancer treatments 21
B. Orostachys japonicus and flavonoids 23
C. Apoptosis 27
D. Cell cycle arrest 32
E. Anti-metastasis 33
F. Signal transductions in anti-cancer mechanisms 37
1. Mitogen-activated protein kinase (MAPK) cascades 37
2. PI3K-AKT Signaling Pathway 38
Ⅱ. PURPOSE 39
Ⅲ. MATERIALS AND METHODS 40
A. Preparation of the EtOAc fraction from O. japonicus 40
B. Cell Line and Reagents 42
C. Cell culture 43
D. Cell growth inhibition assay 44
E. To assess cell morphology, DAPI staining was performed, and the stained cells were visualized using a confocal microscope. 46
F. Flow cytometry analysis of apoptosis 47
G. Wound-healing assay 48
H. Protein expression analysis using western blotting 49
I. Statistical analysis 50
Ⅳ. RESULTS 51
A. Effect of flavonols, flavonol glucosides, and OJE on cell viability 51
1. Effects of cytotoxicity of astragalin, afzelin, quercitrin, and isoquercitrin on RAW 264.7 macrophage cells 51
2. Effects of cytotoxicity of kaempferol, quercetin, and OJE on RAW 264.7 macrophage cells 53
3. Effects of anti-proliferation on HT-29 colon cancer cells 56
B. Induction of apoptosis 61
1. Nuclear staining with DAPI was performed and the stained nuclei were observed using confocal microscopy. 61
2. Flow cytometry (FACS) analysis 64
3. Effect of OJE on the expression levels of proteins related to the induction of apoptosis 72
C. Inhibition of migration 85
D. Induction of cell cycle arrest 92
E. Upstream signal transduction in anti-cancer activities 97
Ⅴ. DISCUSSION 100
Ⅵ. Conclusion 109
Ⅶ. REFERENCES 112
Fig. 1. Cause of mortality 22
Fig. 2. The appearances of grown Orostachys japonicus 25
Fig. 3. The chemical structure of kaempferol, quercetin, and flavonol glycosides 26
Fig. 4. Mechanisms underlying the apoptotic effects of bioactive compounds derived from O. japonicus 31
Fig. 5. Scheme for extraction and preparation of EtOAc fraction from O. japonicus 41
Fig. 6. Effect of astragalin (A), afzelin (B), quercitrin (C), and isoquercitrin (D) on cell cytotoxicity for 12 h and 24 h in RAW... 52
Fig. 7. Effect of kaempferol (A), quercetin (B), and OJE (C) on cell cytotoxicity for 12 h and 24 h in RAW 264.7 macrophage... 55
Fig. 8. Effect of kaempferol (A) and kaempferol + OJE (B) on cell viability on HT-29 cells 57
Fig. 9. Effect of quercetin (A) and quercetin + OJE (B) on cell viability on HT-29 cells 58
Fig. 10. Effect of gallic acid (A) and gallic acid + OJE (B) on cell viability on HT-29 cells 59
Fig. 11. Effect of kaempferol + quercetin (A) and kaempferol + quercetin + OJE (B) on cell viability in HT-29 cells 60
Fig. 12. Nuclear morphological changes indicative of apoptosis induction were observed in HT-29 cells following treatment... 63
Fig. 13. Flow cytometry analysis of the apoptotic death in HT-29 cells after treatment with kaempferol (A), quercetin (B)... 67
Fig. 14. Effects of kaempferol, quercetin, OJE on viable in HT-29 cells treated with different concentrations of kaempferol... 69
Fig. 15. Effects of kaempferol, quercetin, OJE on early+late apoptosis in HT-29 cells treated with different concentrations of... 71
Fig. 16. Effect of kaempferol treatment on the levels of caspase proteins in HT-29 cells 76
Fig. 17. Effect of kaempferol treatment on the levels of bax proteins in HT-29 cells 77
Fig. 18. Effect of kaempferol treatment on the levels of lamin A cleaved lamin A and in HT-29 cells 78
Fig. 19. Effect of quercetin treatment on the levels of cleaved caspase-9 in HT-29 cells 79
Fig. 20. Effect of quercetin treatment on the levels of cleaved lamin A/C in HT-29 cells 80
Fig. 21. Effect of treatment with kaempferol (100 μM), quercetin (100 μM), K+Q (kaempferol 50 μM+quercetin 50 μM),... 83
Fig. 22. Effect of treatment with kaempferol (100 μM), quercetin (100 μM), K+Q (kaempferol 50 μM+quercetin 50 μM),... 84
Fig. 23. Effect of treatment with kaempferol (100 μM), quercetin (100 μM), K+Q (kaempferol 50 μM+quercetin 50 μM),... 86
Fig. 24. Effect of quercetin treatment on the levels of MMP-9 in HT-29 cells 87
Fig. 25. Effect of treatment with kaempferol (100 μM), quercetin (100 μM), K+Q (kaempferol 50 μM+quercetin 50 μM),... 89
Fig. 26. Effect of treatment with kaempferol (100 μM), quercetin (100 μM), K+Q (kaempferol 50 μM+quercetin 50 μM),... 91
Fig. 27. Effect of quercetin treatment on the levels of cyclin A2 in HT-29 cells 93
Fig. 28. Effect of treatment with kaempferol (100 μM), quercetin (100 μM), K+Q (kaempferol 50 μM+quercetin 50 μM),... 96
Fig. 29. Effect of treatment with kaempferol (100 μM), quercetin (100 μM), K+Q (kaempferol 50 μM+quercetin 50 μM),... 99