Title Page

Contents

Abstract 17

Chapter 1. General Introduction 22

Chapter 2. Lichen secondary metabolites in Flavocetraria cucullata exhibit anti-cancer effects on human cancer cells through the induction of apoptosis and suppression of tumorigenic potentials 33

2.1. Introduction 34

2.2. Materials and Methods 36

2.2.1. Preparation of lichen extracts 36

2.2.2. High performance liquid chromatography (HPLC) analysis of lichen materials 36

2.2.3. Cell culture 37

2.2.4. MTT assay 38

2.2.5. Fluorescence microscopy of apoptotic morphology 39

2.2.6. Flow cytometry assay for cell cycle 40

2.2.7. Wound-healing assay 40

2.2.8. Invasion assay 41

2.2.9. Clonogenic assay 42

2.2.10. Soft agar colony-formation assay 42

2.2.11. Detection of proteins in cell lysates 43

2.2.12. qRT-PCR analysis 44

2.2.13. In vivo tumorigenicity assay 44

2.2.14. Statistical analysis 45

2.3. Results 46

2.3.1. Cytotoxic effects of Flavocetraria cucullata acetone extract on cancer cells 46

2.3.2. Lethal concentrations of Flavocetraria cucullata and usnic acid induce apoptosis of cancer cells 55

2.3.3. Sub-lethal concentrations of Flavocetraria cucullata and usnic acid inhibit tumorigenicity and motility of cancer cells 63

2.4. Discussion 76

Chapter 3. Lichen secondary metabolites of Sticta weigelii exhibit anti-cancer effects on human colon cancer stem cells through the induction of apoptosis and inhibition of stemness potentials 81

3.1. Introduction 82

3.2. Materials and Methods 86

3.2.1. Preparation of lichen extracts 86

3.2.1.1. High performance liquid chromatography (HPLC) analysis of lichen materials 86

3.2.1.2. Isolation secondary metabolites of Sticta weigelii extract 87

3.2.2. Cell culture 88

3.2.3. Cytotoxicity assay 89

3.2.4. Fluourescence microscopy of apoptotic morphology 90

3.2.5. Flow cytometry assay for cell cycle 90

3.2.6. Soft agar colony formation assay 91

3.2.7. Spheroid formation assay 92

3.2.8. Reporter assay 93

3.2.9. Plasmids and transfections 94

3.2.10. Detection of protein in cell lysate 95

3.2.11. Quantitative real time RT-PCR (qRT-PCR) analysis 96

3.2.12. Statistical analysis 97

3.3. Results 98

3.3.1. The cytotoxic effect of acetone extract of Sticta weigelii on various cell lines 98

3.3.2. Induction of the apoptosis in human colon stem cancer cell line CSC221 at lethal dose of the acetone extract and secondary metabolites of S. weigelii in time dependent manner 109

3.3.3. Iinhibited the self-renewal and proliferation potentials of CSC221 human colon stem cancer cell line at sub-lethal doses of the acetone extract and subcomponents of S. weigelii in a dose dependent manner 112

3.3.4. Suppression of colon stemness factors in CSC221 human colon stem cancer cell line at sub-lethal doses of the acetone extract and subcomponents of S. weigelii in a dose dependent manner 115

3.3.5. Suppression of stemness factors expression in CSC221 human colon stem cancer cell line through reduction of Wnt/β-catenin and Hedgehog pathways in a dose dependent manner by the acetone extract and subcomponents of S. weigelii at sub lethal dose 120

3.3.6. Recovery of the suppression of self-renewal potential by co-overexpression of targets of Wnt/β-catenin and Hedgehog pathway in CSC221 125

3.4. Discussion 129

Chapter 4. Physciosporin of Pseudocyphellaria granulata exhibits anti-cancer effects on human colon cancer stem cells through the induction of apoptosis and inhibition of stemness potentials 135

4.1. Introduction 136

4.2. Materials and Methods 138

4.2.1. Preparation of lichen extracts 138

4.2.2. High performance liquid chromatography (HPLC) analysis of lichen materials 138

4.2.3. Cell culture 139

4.2.4. Cytotoxicity assay 140

4.2.5. Fluorescence microscopy of apoptotic morphology 141

4.2.6. Soft agar colony formation assay 141

4.2.7. Spheroid formation assay 142

4.2.8. Reporter assay 143

4.2.9. Plasmids and transfections 144

4.2.10. Detection of protein in cell lysate 145

4.2.11. Quantitative real time RT-PCR (qRT-PCR) analysis 146

4.2.12. Statistical analysis 147

4.3. Results 148

4.3.1. Cytotoxic effects Physciosporin, major secondary metabolite of Pseudocyphellaria granulata and Pseudocyphellaria faveolata extract on human colon stem cancer cell line CSC221 148

4.3.2. Pseudocyphellaria granulata extract were decided in the induction of apoptosis on human colon stem cancer cell line CSC221 by physciosporin in a dose dependent manner 157

4.3.3. Inhibitory effect of physciosporin on self-renewal and proliferation potentials on CSC221 human colon stem cancer cell line 159

4.3.4. Inhibitory effect of physciosporin on colon stemness factors of the human colon stem cancer CSC221 cell line 162

4.3.5. Suppression of colon stemness potential of CSC221 human colon stem cancer cell line through inhibition of Notch and Hedgehog pathways 167

4.3.6. Recovery of self-renewal potential suppression of CSC221 cell by co-overexpression of targets signals of Notch and Hedgehog pathway 170

4.4. Discussion 175

Chapter 5. Conclussion 179

References 182

Table 1. IC50 values of the acetone extract from the Romania lichens.(이미지참조) 47

Table 2. HPLC profile of Flavocetraria cucullata acetone extract. 53

Table 3. IC50 values of acetone extract of F. cucullata and usnic acid, lichesterinic acid, on various cancer cells.(이미지참조) 54

Table 4. The IC50 values of 26 specimens of Vietnamese lichen.(이미지참조) 101

Table 5. HPLC profile of Sticta weigelii acetone extracts 106

Table 6. The IC50 values of the single compounds of Sticta weigelii specimens.(이미지참조) 107

Table 7. The IC50 values of the mixtures of Sticta weigelii single compounds.(이미지참조) 108

Table 8. The IC50 values of the acetone extract from f Chilean lichen specimens at 60 hrs treatment.(이미지참조) 152

Table 9. HPLC profile of of acetone extracts of Pseudocyphellaria granulata and Pseudocyphellaria faveolata 155

Table 10. The IC50 values of the single compounds of the Pseudocyphellaria granulate and Pseudocyphellaria faveolata at 60 hrs treatment.(이미지참조) 156

Figure 1. Cytotoxic effects of the acetone extract of F. cucullata and its main component, usnic acid, on human cancer cells. 51

Figure 2. Induction of nuclear condensation of human cancer cells by the acetone extract of F. cucullata and its main component, usnic acid at... 58

Figure 3. Induction of Annexin V positivity and accumulation of sub G1 population of human cancer cells by the acetone extract of F.... 61

Figure 4. Activation of apoptosis pathway on human cancer cells by the acetone extract of F. cucullata and usnic acid at lethal concentrations. 62

Figure 5. Inhibition of anchorage-independent growth of A549 and AGS cancer cells by the acetone extract of F. cucullata and usnic acid at... 68

Figure 6. Inhibition of the motility of A549 and AGS cancer cells by the acetone extract of F. cucullata and usnic acid atsub-lethal... 69

Figure 7. Suppression of epithelial-mesenchymal transition (EMT) by the acetone extract of F. cucullata and usnic acid in sub-lethal... 71

Figure 8. Reduction of phosphor-Akt level by the acetone extract of F. cucullata and usnic acid at sub-lethal concentrations. 72

Figure 9. Inhibition of in vivo tumorigenicity of A549 cancer cells pretreated by the acetone extract of F. cucullata and usnic acid in sub-... 73

Figure 10. In vivo tumorigenicity assay using A549 cells pretreated with DMSO, acetone extract of F. cucullata, usnic acid, or lichesterinic acid.... 75

Figure 11. Cytotoxic effects of the acetone extract of Sticta weigelii and its components on human colon cancer stem cells (CSC221) 105

Figure 12. Activation of apoptosis pathway on human conlon cancer stem cells by the acetone extract of Sticta weigelii (VN120166) and its... 111

Figure 13. Inhibition of self-renewal and proliferation potentials of CSC221 cancer cells by the acetone extract of S.weigelii (VN120166) and... 114

Figure 14. Inhibition of stemness protein expression of CSC221 cancer cells by the acetone extract of S weigelii (VN120166) and its components... 118

Figure 15. Inhibition of stemness mRNA expression of CSC221 cancer cells by the acetone extract of S. weigelii (VN120166) and its components... 119

Figure 16. Expression of target signal in Hedgehog and Wnt/β-catenin pathways by the acetone extract of S. weigelii (VN120166) and its... 122

Figure 17. Compensation of the suppression of self-renewal potential in CSC221 cell by over expression of β-catenin together with Gli activator 124

Figure 18. Compensation of the suppression of self-renewal potential and stem factors expression in CSC221 cell by Over expression of β-... 127

Figure 19. Chemical structure of the secondary metabolites of the acetone extract of S. weigelii 131

Figure 20. Cytotoxic effects of the acetone extract of Pseudocyphellaria granulata and its components on human colon cancer stem cells (CSC221) 150

Figure 21. Activation of apoptosis pathway of human conlon cancer stem cells by the acetone extract of Pseudocyphellaria granulata and its... 158

Figure 22. Inhibition of self-renewal and proliferation potentials of CSC221 cancer cells by the acetone extract of Pseudocyphellaria... 161

Figure 23. Inhibition of stemness protein expression of CSC221 cancer cells by the acetone extract of P.granulata and its components at sub-... 165

Figure 24. Inhibition of stemness mRNA expression of CSC221 cancer cells by the acetone extract of Pseudocyphellaria granulata and its... 166

Figure 25. Decrease in expression level of target signals in Hedgehog and Notch pathways by the acetone extract of Pseudocyphellaria... 169

Figure 26. Compensation of suppression of self-renewal potential in CSC221 cell by over expression of δ-EN1 together with Gli activator 171

Figure 27. Compensation of suppression of self-renewal potential and stem factors expression in CSC221 cell by overexpression of δ-EN1... 173