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Contents

Abbreviations 11

Introduction 12

Materials and Methods 20

1. Chemicals, cell lines and animals 20

2. HDAC enzyme assay 21

3. Cell proliferation assay 21

4. Cell cycle analysis 22

5. Apoptosis analysis 22

6. Caspase 3/7 assay 23

7. Western immunoblot analysis 23

8. In vivo bioluminescence imaging of tumor growth in a human tumor xenograft model 24

9. RNA extraction 24

10. Reverse transcription-polymerase chain reaction (RT-PCR) 25

11. cDNA microarray and data analysis 25

12. Mouse microsomal stability assay 26

a. Sample preparation 26

b. Data analysis 27

13. Caco-2 permeability assay 28

a. Sample preparation 28

b. Data analysis 29

14. Statistical analysis 30

Results 32

1. Anti-cancer effect of KBH-A42 in colon cancer cells 32

1.1. Enzyme activity of various HDAC isoforms and on the proliferation of human colon cancer cell lines 32

1.2. Effect of KBH-A42 on histone acetylation in SW620 cells 32

1.3. Cell cycle analysis and protein expression in SW620 cells 33

1.4. Induction of apoptosis and activation of caspases in SW620 cells 34

1.5. Effect of KBH-A42 on the growth of SW620 tumor xenografts in nude mice 35

2. Gene expression profiling of KBH-A42 in human leukemia and bladder cell lines 47

2.1. Effect of KBH-A42 on human leukemia and bladder cancer cell lines 47

2.2. Analysis of gene expression changes by KBH-A42 treatment 48

2.3. Effect of KBH-A42 on expressions of apoptosis related genes 49

2.4. Effect of KBH-A42 on human tumor xenograft model. 49

3. KBH-A42 overcomes multi-drug resistance and induces cell death in P-glycoprotein expressing cells 63

3.1. KBH-A42 induces equivalent cell death in both P-gp-positive and P-gp-negative cancer cell lines 63

3.2. Cell cycle analysis and protein expression in both P-gp-positive and P-gp-negative cancer cell lines 64

3.3. Induction of apoptosis and protein expression in both P-gp-positive and P-gp-negative cancer cell lines 65

4. In vitro absorption and metabolism of KBH-A42 and SAHA 80

4.1. Metabolic stability of KBH-A42 and SAHA in mouse liver microsomes 80

4.2. Permeability of KBH-A42 and SAHA across Caco-2 cell monolayers 80

Discussion 86

References 97

Abstract 109

감사의 글 112

List of Tables

Table. 1. Sequences of oligonucleotide primers for 6 over-expressed and 16 under-expressed genes. 31

Table. 2. Effect of KBH-A42 on the activity of various HDAC isoforms. 36

Table. 3. Effect of KBH-A42 and SAHA on the proliferation of colon cell lines. 37

Table. 4. Effect of KBH-A42 and SAHA on the proliferation of various cell lines 51

Table. 5. Genes up-regulated by KBH-A42 (5 μM) treatment in K-562 and UM-UC-3 cells 52

Table. 6. Genes down-regulated by KBH-A42 (5 μM) treatment in K-562 and UM-UC-3 cells 53

Table. 7. Microsomal intrinsic clearance (CLint) of KBH-A42 and SAHA. Data were originally obtained from mouse liver microsomal assay calculated according to the equation described in the Material...(이미지참조) 82

Table. 8. The effect of KBH-A42 and SAHA on the transepithelial electrical resistance (TEER). TEER values of Caco-2 cell monolayer were measured before and after 2 h incubation of KBH-A42 and... 83

List of Figures

Fig. 1. Death receptor and intrinsic apoptotic pathways. 13

Fig. 2. Cyclins, Cdks and the cell cycle. 15

Fig. 3. Chemical structures of SAHA and KBH-A42. 19

Fig. 4. Effect of KBH-A42 on histone acetylation in SW620 cells. (A) Cells were treated with KBH-A42 (5 μM) for the indicated tiae periodr. (B) Cells were incubated with various concentrations of KBH-A42 for 24 h.... 38

Fig. 5. Effect of KBH-A42 on cell cycle progression in SW620 cells. Cells were treated with the indicated concentrations of KBH-A42 for 24 h.... 39

Fig. 6. Effect of KBH-A42 on the expression of cell cycle regulators. Cells were treated with the indicated concentrations of KBH-A42 for 24 h.... 40

Fig. 7. Induction of apoptosis by KBH-A42 in SW620 cells. Cells were treated with the indicated concentrations of KBH-A42 for 24 h.... 41

Fig. 8. Effect of KBH-A42 on caspase 3/7 activation in SW620 cells. Cells were treated with the indicated concentrations of KBH-A42 for 24 h.... 42

Fig. 9. Effect of KBH-A42 on cell growth inhibition by caspase inhibitor. Cells were cultured for 48 h with 0 to 10 μM KBH-A42 with or without Z-VAD-fmk (40 μM) and cell proliferation was... 43

Fig. 10. Effect of KBH-A42 on the expression of apoptosis regulators. Cells were treated with the indicated concentrations of KBH-A42 for 24 h.... 44

Fig. 11. Effect of KBH-A42 on SW620 tumor growth in a human tumor xenograft model. SW620-Luc cells were injected subcutaneously into female BALB/c-nu mice, and KBH-A42 or SAHA was administered as... 45

Fig. 12. Effect of KBH-A42 on the growth of K562 and UM-UC-3 cells. The cells were incubated for 48 h with KBH-A42 at various concentrations indicated and cell proliferation was determined with a... 54

Fig. 13. Effect of KBH-A42 on cell cycle progression in K562 cells. Cells were treated with the indicated concentrations of KBH-A42 for 24 h.... 55

Fig. 14. Effect of KBH-A42 on cell cycle progression in UM-UC-3 cells. Cells were treated with the indicated concentrations of KBH-A42 for 24 h.... 56

Fig. 15. Induction of apoptosis by KBH-A42 in K562 cells. Cells were treated with the indicated concentrations of KBH-A42 for 24 h. Cells were stained with propidium iodide and a FITC-conjugated antibody... 57

Fig. 16. Induction of apoptosis by KBH-A42 in UM-UC-3 cells. Cells were treated with the indicated concentrations of KBH-A42 for 24 h.... 58

Fig. 17. Effect of KBH-A42 on caspase 3/7 activation in K562 and UM-UC-3 cells. Cells were treated with the indicated concentrations of KBH-A42 foF 24 h.... 59

Fig. 18. Confirmation of cDNA microarray results of up-regulated genes by RT-PCR. Total RNA was isoiated from untreated or treated with KBH-A42 (1, 2.5, 5 μM) in K562 and UM-UC-3 cells... 60

Fig. 19. Confirmation of cDNA microarray results of down-regulated genes by RT-PCR. Total RNA was isolated from untreated or treated with KBH-A42 (1, 2.5, 5 μM) In K562 and UM-UC-3 cells... 61

Fig. 20. Effect of KBH-A42 on K562 and UM-UC-3 tumor growth in a human tumor xenograft model. BALB/c-nu mice were injected s.c. with K562 or UM-UC-3 cells.... 62

Fig. 21. KBH-A42 induces equivalent cell death in P-gp-positive and P-gp-negative K562 and MCF7 cells. Cells were cultured for 24 h with 0 to 10 μM KBH-A42 or 1 μM ADR and ceil proliferation was... 67

Fig. 22. Accumulation of calcein which exhibits fluorescence in both P-gp-positive cells. When ekposed to 1 μM Calcein-AM with or without KBH-A42 or Verapamil for 1-h.... 68

Fig. 23. Effect of KBH-A42 on histone acetylation and P-gp expression in P-gp-positive and P-gp-negative cells. Cells were treated with KBH-A42 (5 μM) for the indicated time periods.... 69

Fig. 24. Effect of KBH-A42 on cell cycle progression in P-gp-positive and P-gp-negative K562 and MCF7 cells. Cells were treated with the indicated concentrations of KBH-A42 for 24 h.... 70

Fig. 25. Effect of KBH-A42 on the expression of cell cycle regulators in P-gp-positive and P-gp-negative cells. Cells were treated with KBH-A42 (5 μM) for 24 h.... 71

Fig. 26. Molecular targets of KBH-A42 on cell cycle. Symbols : (▲) induction of protein expression, (×) reduction of protein expression. 72

Fig. 27. Induction of apoptosis by KBH-A42 in P-gp-positive and P-gp-negative K562 and MCF7 cells. Cells were treated with the indicated concentrations of KBH-A42 for 24 h.... 73

Fig. 28. Effect of KBH-A42 on apoptosis-related protein expression in P-gp-positive and P-gp-negative cells. Cells were treated with KBH-A42 (5 μM) for the indicated time periods.... 76

Fig. 29. Effect of KBH-A42 on intrinsic apoptosis-related protein expression in P-gp-positive and P-gp-negative cells. Cells were treated with KBH-A42 (5 μM) for the indicated time periods.... 77

Fig. 30. Effect of KBH-A42 on mitochondria released protein in P-gp-positive and P-gp-negative cells. Cells were treated with KBH-A42 (5 μM) for the indicated time periods.... 78

Fig. 31. Molecular targets of KBH-A42 on apoptosis. (▲) induction of protein expression, (×) reduction of protein expression. 79

Fig. 32. Time course of KBH-A42 depletion in human liver microsomes. KBH-A42 (1 μM) was incubated with 0.5 mg/ml of ICR mouse liver microsomes in the presence of NADPH.... 84

Fig. 33. Permeability of KBH-A42 and SAHA across Caco-2 cell monolayers. Papp values were determined in AP-to-BL direction at 5 μM in HBSS supplemented with 10 mM HEPES (pH 7.4).... 85

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