Title Page

Contents

Abstract 11

I. Introduction 21

1. Cancer 21

2. Rheumatoid arthritis 26

3. NF-kB 29

4. Garlic and its components 35

4.1. Effect of garlic on cancer 44

4.2. Effect of garlic on arthritis 63

4.3. Pharmacokinetics 64

4.4. Toxicity and safety 66

4.5. Application 68

II. Materials and Methods 69

1. Preparation of chemicals 69

1.1. Thiacremonone 69

1.2. Docetaxel 70

1.3. Paclitaxel 70

2. Cell culture 72

3. Cell counting 72

4. Cell viability assay 73

5. Nitrite assay 73

6. Western blot analysis 74

7. Electromobility shift assay 76

8. Transfection and assay of luciferase activity 77

9. Detection of apoptosis 78

10. Immunohistochemistry 79

11. Molecular modeling 81

12. Pull-down Assays 82

13. MALDI-TOF Analysis of p50 82

14. Soft agar formation assay 83

15. Xenograft animal model for anti-tumor activity 83

16. Calculation of combination index 85

17. Assay of ear edema in mice 86

18. Carrageenan induced paw edema model 87

19. Mycrobacterium butyricum induced arthritis model 88

20. Data analysis 89

III. RESULTS 90

1. Anti-cancer effect of thiacremonone on human colon cancer cells 90

1.1. Thiacremonone inhibited NF-kB activation in SW620 and HCT116 human colon cancer cells. 90

1.2. Thiacremonone inhibited SW620 and HCT116 human colon cancer cell growth. 95

1.3. Thiacremonone induced apoptotic cell death in SW620 and HCT116 human colon cancer cells. 98

2. NF-kB as a target of thiacremonone 103

2.1. Two stereoisomers of thiacremonone are shown to form two and three hydrogen bonds with p50. 103

2.2. Thiacremonone might directly bind to p50. 105

3. Combination effect of thiacremonoe and docetaxel in human colon cancer cells 108

3.1. Combination treatment of thiacremonone and docetaxel synergically inhibited NF-kB activity in SW620 and HCT116 human colon cancer cells. 108

3.2. Combination treatment of thiacremonone and docetaxel synergically inhibited cell growth of SW620 and HCT116 human colon cancer cells. 113

3.3. Combination treatment of thiacremonone and docetaxel synergically induced apoptotic cell death in SW620 and HCT116 human colon cancer cells. 115

3.4. Combination treatment of thiacremonone and docetaxel synergically inhibited tumor growth in a xenograft model. 121

3.5. Combination treatment of thiacremonone and docetaxel synergically inhibited NF-ΚB activation and controlled the expression of pro-and anti-apoptotic genes in vivo. 129

4. Combination effect of thiacremonoe and paclitaxel in human lung cancer cells 135

4.1. Combination treatment of thiacremonone and paclitaxel synergically inhibited cell growth in NCI-H460 and A549 human lung cancer cells. 135

4.2. Combination treatment of thiacremonone and paclitaxel synergically induced apoptotic cell death in NCI-H460 and A549 human lung cancer cells. 139

4.3. Combination treatment of thiacremonone and paclitaxel synergically inhibited NF-kB activation in NCI-H460 and A549 human lung cancer cells. 144

4.4. Combination treatment of thiacremonone and paclitaxel synergically inhibited colony formation. 147

4.5. Combination treatment of thiacremonone and other chemotherapeutics synergically inhibited human cancer cell growth. 149

5. Anti-arthritis effect of thiacremonone 158

5.1. Thiacremonone inhibited TPA-induced ear edema in mice. 158

5.2. Thiacremonone inhibited carrageenan-induced arthritis. 162

5.3. Thiacremonone inhibited adjuvant-induced arthritis. 166

5.4. Thiacremonone inhibited NF-kB activity in RAW 264.7 cells. 170

5.5. Thiacremonone inhibited LPS-induced NO production as well as expression of iNOS and COX-2 in RAW 264.7 cells. 178

5.6. Thiacremonone-induced inhibition of DNA binding activity of NF-kB and cell growth were reversed by thiol reducing agents. 184

IV. DISCUSSION 187

1. Thiacremonone could be potentially useful in treatment of human colon cancer cells. 187

2. Thiacremonone may exert anticancer effects by interfering with p50. 194

3. Thiacremonone could be potentially useful in combination with docetaxel for the treatment of human colon cancers. 198

4. Thiacremonone could be potentially useful in combination with paclitaxel for the treatment of human lung cancers. 204

5. Thiacremonone could be potentially useful in treatment of arthritis. 210

V. REFERENCES 214

VI. SUMMARY IN KOREAN 256

VIII. LIST OF PUBLICATIONS 259

Table 1. Chemical structure and quantity of some volatile organosulfur compounds present in garlic 43

Table 2. Epidemiological studies showing anticarcinogenic properties of garlic 51

Table 3. In vivo laboratory studies showing anticarcinogenic properties of garlic and its constituents 56

Table 4. In vitro laboratory studies showing anticarcinogenic properties of garlic and its constituents 62

Figure 1. Multistep models of carcinogenesis stages 22

Figure 2. The progression of rheumatic arthritis 28

Figure 3. The NF-kB signaling pathway 32

Figure 4. Structure of thiacremonone, docetaxel and paclitaxel 71

Figure 5. Effect of thiacremonone on NF-kB activation in SW620 and HCT116 colon cancer cells. 92

Figure 6. Competition and super shift assay. 93

Figure 7. Effect of thiacremononeon NF-kB transcriptional activity in SW620 and HCT116 colon cancer cells. 94

Figure 8. Effect of thiacremonone on morphological changes and cell viability of SW620 and HCT116 colon cancer cells. 96

Figure 9. Effect of thiacremonone on morphological changes and cell viability of CCD-112CoN colon normal cells. 97

Figure 10. Effect of thiacremonone on apoptotic cell death of SW620 and HCT116 colon cancer cells. 101

Figure 11. Effect of thiacremonone on the expression of apoptosis regulatory proteins in SW620 and HCT116 colon cancer cells. 102

Figure 12. Modeling study of the binding of thiacremonone to p50 protein. 104

Figure 13. Maldi-tof analysis and pull down assay of the binding of thiacremonone to p50 protein. 107

Figure 14. Effect of the docetaxel on DNA binding activity and cell viability in SW620 and HCT116 human colon cancer cells. 110

Figure 15. Effect of the combination treatment of thiacremonone and docetaxel on NF-ΚB activation in SW620 and HCT116 colon cancer cells. 112

Figure 16. Effect of the combination treatment of thiacremonone and docetaxel on cell viability in SW620 and HCT116 colon cancer cells. 114

Figure 17. Effect of the combination treatment of thiacremonone and docetaxel on apoptotic cell death of SW620 and HCT116 colon cancer cells. 118

Figure 18. Effect of the combination treatment of thiacremonone and docetaxel on the expression of apoptosis regulatory proteins in SW620 and HCT116 colon cancer cells. 120

Figure 19. Scheme of experiments of SW620 xenografts in vivo model. 124

Figure 20. Effect of combination therapy of thiacremonone and docetaxel on the tumor growth in SW620 xenografts in in vivo model. 126

Figure 21. Effect of combination therapy of thiacremonone and docetaxel on tumor cell proliferation and apoptosis in vivo xenograft animal model. 128

Figure 22. Effect of the combination treatment of thiacremonone and docetaxel on NF-kB activation in vivo xenograft animal model. 132

Figure 23. Effect of the combination treatment of thiacremonone and docetaxel on apoptosis regulatory proteins in vivo xenograft animal model. 134

Figure 24. Median-effect analysis of thiacremonone and paclitaxel in combination on NCI-H460 and A549 human lung cancer cell viability. 138

Figure 25. Effect of the combination treatment of thiacremonone and paclitaxel on apoptotic cell death of NCI-H460 and A549 lung cancer cells. 142

Figure 26. Effect of the combination treatment of thiacremonone and paclitaxel on the expression of apoptosis regulatory proteins in NCI-H460 and A549 lung cancer cells. 143

Figure 27. Effect of the combination treatment of thiacremonone and paclitaxel on the translocation of NF-kB into nucleus in NCI-H460 and A549 lung cancer cells. 145

Figure 28. Effect of the combination treatment of thiacremonone and paclitaxel on NF-kB DNA binding activity in NCI-H460 and A549 lung cancer cells. 146

Figure 29. Effect of a combination of thiacremonone with paclitaxel on colony formation of NCI-H460 and A549 lung cancer cells. 148

Figure 30. Effect of the combination treatment of thiacremonone and other chemotherapeutics on cell viability. 152

Figure 31. Effect of the combination treatment of thiacremonone and other chemotherapeutics on cell viability in human colon cancer cells. 153

Figure 32. Effect of the combination treatment of thiacremonone and other chemotherapeutics on cell viability in various human cancer cells. 155

Figure 33. Effect of the combination treatment of thiacremonone and other chemotherapeutics on NF-kB DNA binding activity. 157

Figure 34. Effects of thiacremonone on TPA-induced ear edema in mice. 159

Figure 35. Effects of thiacremonone on TPA-induced ear edema, and expression of iNOS and COX-2 in mice. 160

Figure 36. Effects of thiacremonone on NF-kB activity in TPA-induced mouse ear tissue. 161

Figure 37. Effect of thiacremonone on carrageenan-induced arthritis in rats. 163

Figure 38. Effect of thiacremonone on the expression of inflammatory proteins in carrageenan-induced rat paw tissue. 164

Figure 39. Effect of thiacremonone on NF-kB activity in carrageenan-induced rat paw tissue. 165

Figure 40. Effect of thiacremonone on adjuvant-induced arthritis in rats. 167

Figure 41. Effect of thiacremonone on the expression of inflammatory proteins in adjuvant-induced rat paw tissue. 168

Figure 42. Effect of thiacremonone on NF-kB activity in adjuvant-induced rat paw tissue. 169

Figure 43. Effect of thiacremonone on LPS-induced NF-kB activation in RAW 264.7 cells. 174

Figure 44. Effect of thiacremonone on TNF-α, IL-1α or IFN-γ-induced NF-kB DNA binding activity in RAW 264.7 cells. 176

Figure 45. Effect of thiacremonone on LPS-induced NF-kB activation in THP-1 cells. 177

Figure 46. Effect of thiacremonone on LPS-induced NO generation in RAW 264.7 cells. 180

Figure 47. Effect of thiacremonone on LPS-induced expression of iNOS and COX-2 in RAW 264.7 cells. 182

Figure 48. Effect of thiacremonone on LPS-inducedcell viability in RAW 264.7 cells. 183

Figure 49. Abolition of the inhibitory effect of thiacremonone by DTT and GSH on NF-kB activity. 185

Figure 50. Abolition of the inhibitory effect of thiacremonone by DTT and GSH, on NO generation iNOS transcriptional activity. 186

 NF-κB는 염증성 질병에 관련이 있는 전사인자로서 암 혹은 관절염 조직에 과발현 되어 있다고 보고되고 있다. 마늘에서 추출된 allin, diallyl disulfide, S—allylmercaptocysteine, S-trityl-L-cysteine과 같은 sulfur compound들은 NF-κB의 활성을 억제할 수 있으며, 다양한 질병에 대하여 치료 효능이 있는 것으로 보고되고 있다. 최근, 우리 연구실에서는 한국산 마늘을 고온·고압 처리하여 sulfur compound인 thiacremonone을 추출하였다. 본 연구에서는 이물질을 이용하여 NF-κB의 활성 억제를 통한 암 세포 성장과 관절염 발전 억제에 대해 연구를 수행하였다. 그 결과 docking modeling을 통하여 thiacremonone이 NF-κB p50과 결합함을 증명하였으며 또한 thiacremonone이 NF-κB의 활성 억제를 통하여 사람의 대장암 세포 성장 억제와 세포 사멸을 유도함을 확인하였고 또한, thiacremonone이 현재 항암화학요법제로 사용되고 있는 docetaxel 혹은 paclitaxel과 병용 처리시 in vivo와 in vitro에서 NF-κB의 활성 억제를 통하여 암 세포 성장 억제와 암 세포 사멸을 유의성있게 유도할 뿐만 아니라 다양한 암세포에 대하여 doxorubicin, cisplatin과 병용처리시에도 암세포 성장 억제에 상승효과를 나타냄을 확인하였다. 그외에 thiacremonone이 NF-κB의 활성 억제를 통하여 항염 및 항관절염 효능이 있는지 연구한 결과, thiacremonone이 in vitro에서 NF-κB 활성 억제를 통하여 iNOS와 COX-2 발현 및 NO 생성을 억제하였으며 또한, 만성 혹은 급성 귀 염증과 관절염 동물 모델에서 NF-κB 활성 억제를 통하여 항염 및 항관절염 효능을 나타내었다. 이러한 thiacremonone의 효능은 환원제인 DTT와 GSH를 처리했을 때 억제되는 경향을 보였으며, 이 결과는 cysteine을 많이 함유하는 NF-κB 의 p50 단백질이 thiacremonone의 항염증 및 항관절염 효능을 나타내는 과정에서 중요한 역할을 한다는 것을 알 수 있었다.

결론적으로 본 연구는 마늘에서 추출한 sulfur compound인 thiacremonone이 NF-κB 활성 억제를 통한 암 및 관절염 치료제로서 개발 가능성이 있음을 제시하였다.