Title Page
Contents
ABBREVIATIONS 14
ABSTRACT IN KOREAN 16
I. INTRODUCTION 20
II. Investigation of the Metabolic Changes of Raji B Lymphoma Cells Undergoing Apoptosis Induced by Zinc Ions 29
1. Introduction 29
2. Material and Method 32
2.1. Materials 32
2.2. Cell culture 33
2.3. Metabolite extraction 34
2.4. Metabolomic analysis 34
2.5. Kinase arrays 36
2.6. Data preprocessing and statistical analysis for the identification of metabolites 37
2.7. Quantitative analysis of choline in the cells 38
2.8. Quantitative analysis of arachidonic acid in the cells 39
2.9. Cell viability assay 40
2.10. Western blot analysis 40
2.11. Annexin-V/propidium iodide staining assay 41
3. Results 42
3.1. Identification of the metabolic changes by metabolic profiling after Zn ion treatment of Raji cells 42
3.2. Protein phosphorylation analysis 57
3.3. Ingenuity pathway analysis (IPA) and metabolic pathway analysis by KEGG 60
3.4. Choline metabolism in cancer altered by Zn ions 64
3.5. Cell survival with fatty acid treatment 68
4. Discussion 73
III. Metabolomics investigation of the metabolic mechanism for apoptosis by anaplastic lymphoma kinase (ALK) inhibition in non-small cell lung cancer cell lines 78
1. Introduction 78
2. Material and Method 82
2.1. Materials 82
2.2. Cell culture 83
2.3. Metabolite extraction 83
2.4. Preparation of QC samples 84
2.5. Metabolomic analysis 84
2.6. Lipidomics analysis 86
2.7. Data pre-processing and statistical analysis for identification of metabolites 88
2.8. Quantitative analysis of 5-methylthioadenosine (5-MTA) 89
2.9. Quantitative analysis of polyamine metabolites in the cells 90
2.10. Measuring folate in cell pellet and media 91
2.11. Cell viability assay 92
2.12. Western blot analysis 93
3. Result 94
3.1.1. Comparison of metabolic profiling between normal lung cells (L132) and ALK dysregulated lung cancer cells (H3122) by UPLC-Orbitrap-MS/MS. 94
3.1.2. Comparison of metabolic change between L132 and H3122 cells 97
3.2.1. Confirmation of ALK expression in H3122 cells 100
3.2.2. Metabolic profiling of ALK-TKIs treated H3122 cells 103
3.2.3. Metabolic profiling of ALK-TKIs treated H3122 cells 107
3.3.1. Confirmation of the ALK-TKIs decrease 5-MTA and MTAP. 117
3.3.2. ALK-TKIs reduced polyamine pathway and increased folate by accumulation 121
4. Discussion 127
4.1. ALK dysregulated lung cancer cells showed different metabolism compared to normal cell 128
4.2. ALK-TKIs altered the metabolism of H3122 cells 130
IV. Investigation of long-term metabolic alteration after stroke in tMCAO (transient middle cerebral artery occlusion) mouse model using metabolomics approach 134
1. Introduction 134
2. Material and Method 137
2.1. Materials 137
2.2. Animal experiment 138
2.3. Transient middle cerebral artery occlusion (tMCAO) model 138
2.4. Cresyl violet staining 140
2.5. Untargeted metabolomics analysis 140
2.6. Data preprocessing and statistical analysis for identification of metabolites 141
3. Result 143
3.1. Infarct volume after tMCAO surgery in mouse model 143
3.2. Multivariate statistics 146
3.3. Alterations of plasma metabolome by tMCAO 151
4. Discussion 162
V. CONCLUSION 168
REFERENCE 171
ABSTRACT 185
Table 1. Significantly changed metabolites after Zn ion treatment. 49
Table 2. The information of identified metabolites in metabolomics study. 53
Table 3. Top altered diseases and functional pathways compared to 12... 62
Table 4. Significantly changed metabolites after ALK-TKIs treatment. 110
Table 5. Significantly changed metabolites after tMCAO surgery. 153
Figure 1. The'-OMICS' cascade. 20
Figure 2. Metabolomics workflow in biomarker and drug discovery. 22
Figure 3. Research of xenobiotics-induced metabolism alterations in... 25
Figure 4. Apoptotic cell population evaluated by flow cytometry... 43
Figure 5. Principal Component Analysis (PCA) plot of quality control... 44
Figure 6. Multivariate statistical analysis based on the time-dependent... 45
Figure 7. Heat map and cluster analysis of the metabolites... 48
Figure 8. The top 10 highest and lowest changes in the... 59
Figure 9. Top enriched diseases and functions by IPA analysis. 63
Figure 10. Proposed choline metabolic pathway after Zn ion treatment. 66
Figure 11. Choline concentration in Zn ion-treated Raji cells. 67
Figure 12. Cell viability test of Raji cells treated with Metabolites. 69
Figure 13. Apoptotic cell population evaluated by flow cytometry... 70
Figure 14. Western blot assays were performed to confirm that AA... 72
Figure 15. Principal Component Analysis (PCA) plot of quality control... 95
Figure 16. PCA model score plots show the different clusters between... 96
Figure 17. Heat map and cluster analysis of the metabolites... 98
Figure 18. Different metabolic pathways were represented according to the characteristics of the L132 and... 99
Figure 19. Western blot assays to confirm that ALK-TKIs reduced... 101
Figure 20. Cell viability tested by ALK-TKIs in H3122 cells. 102
Figure 21. PCA plots built on H3122 cells metabolomics data show QC clustering. 105
Figure 22. Score plots of PCA models showing the different clusters... 106
Figure 23. Heat map and cluster analysis of metabolites discriminated... 112
Figure 24. The ratio of Propionyl carnitine (C3) to carnitine (C0)... 113
Figure 25. Proposed metabolic pathway and relative contents after ALK-TKIs treatment in H3122 cells. 114
Figure 26. IPA pathway analysis summary. 116
Figure 27. 5-MTA concentration in H3122 cells. 118
Figure 28. Cell viability test after the coadministration with 5-MTA... 119
Figure 29. Expression of MTAP after ALK-TKI treatment in H3122... 120
Figure 30. Putrescine, spermidine, and spermine concentration... 123
Figure 31. Changed SMS and SDS protein levels and the cell viability... 124
Figure 32. Measuring folate in H3122 cells after ALK-TKIs treatment. 125
Figure 33. Folate, 5-MTA and polyamine pathways altered by ALK-TKIs treatments in... 126
Figure 34. Measurement of brain damage following tMCAO procedure. 144
Figure 35. Measurement of body weight and NSS following tMCAO procedure. 145
Figure 36. Principal Component Analysis (PCA) plot of quality control... 147
Figure 37. Multivariate statistical analysis based on the time-... 150
Figure 38. Heat map and cluster analysis of metabolites discriminated... 152
Figure 39. Proposed metabolic pathway and relative contents after... 159
Figure 40. Ratio of selected metabolite levels in sham to tMCAO,... 161