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