Title Page
Contents
ABBREVIATIONS 16
ABSTRACT 19
CHAPTER Ⅰ. Background and object of research 22
1. CANCER 23
2. METASTASIS 27
3. PROGRAMMED CELL DEATH 29
4. SERTAD1 31
CHAPTER Ⅱ. The inhibitory role of SERTAD1 in anticancer drug-induced apoptosis and anoikis via the activation of autophagy in 2D and 3D culture 37
1. INTRODUCTION 38
2. MATERIALS AND METHODS 50
2.1. Cell lines, cell culture and cell treatment 50
2.2. Trypane blue staining 51
2.3. CCK assay 52
2.4. Western blot analysis 52
2.5. Immunoflorescene (IF) staining for cells 54
2.6. Mitotracker staining 54
2.7. Lysotracker staining 55
2.8. Breast cancer gene-expression miner v4.5 (bc-GenExMiner v4.5) 56
2.9. Cathepsin B activity assay 56
2.10. siRNA transfection 57
2.11. Real-Time polymerase chain reaction (RT-PCR) 57
2.12. GEPIA Dataset Analysis 58
2.13. Co-Immunoprecipitation (Co-IP) 58
2.14. Isolation of mouse embryo fibroblasts (MEF) 59
2.15. Survival rate studies 60
2.16. Xenograft study 60
2.17. Immunoflorescene (IF) staining for tumors 61
2.18. Statistical analysis 62
3. RESULTS 63
3.1. SERTAD1 expression levels of ten breast cell lines under adhesion (2D) and suspension (3D) culture. 63
3.2. Cell growth curve of ten breast cell lines under adhesion (2D) and suspension (3D) culture. 67
3.3. Cell proliferation and SERTAD1 expression levels of four breast cancer cell lines during long term suspension 3D. 70
3.4. Cell viability of four breast cell lines in response to Dox treatment under 2D and 3D culture. 73
3.5. Inhibitory role of SERTAD1 on apoptosis and anoikis in response to Dox treatment under 2D and 3D culture. 75
3.6. SERTAD1 inhibited Dox-induced apoptosis and anoikis under 2D and 3D culture via activating autophagy. 83
3.7. Mitochondrial function in the suppression of SERTAD1 in Dox-induced apoptosis/anoikis. 86
3.8. Lysosomal function involved in the suppression SERTAD1 in Dox-induced apoptosis/anoikis. 94
3.9. Positive effect of SERTAD1 on lysosome activity via CTSB indirectly. 105
3.10. SERTAD1 suppressed sensitivity of breast cancer cells to Dox treatment in vivo 111
4. DICUSSION 119
CHAPTER Ⅲ. The inhibitory role of SERTAD1 in glucose starvation-induced programmed cell death 124
1. INTRODUCTION 125
2. MATERIALS AND METHODS 136
2.1. Cell lines, cell culture and cell treatment 136
2.2. Induction of glucose starvation 136
2.3. Trypane blue staining 137
2.4. CCK assay 137
2.5. Western blot analysis 138
2.6. Measurement of reactive oxygen species (ROS) 139
2.7. Measurement of cellular ATP levels 140
2.8. DAPI staining 140
2.9. Breast cancer gene-expression miner v4.5 (bc-GenExMiner v4.5) 141
2.10. Statistical analysis 141
3. RESULTS 143
3.1. Cell growth curve of six breast cancer cell lines under glucose-starved condition. 143
3.2. SERTAD1 protected MCF7 breast cancer cells against cell death under glucose-starved condition. 146
3.2. SERTAD1 protected MCF7 breast cancer cells against cell death under glucose-starved condition. 150
3.3. The inhibitory role of SERTAD1 on cell death via regulating ROS and ATP production under glucose starvation. 156
3.4. SERTAD1 repressed AMPK activation under glucose starvation. 160
4. DISCUSSION 165
REFERENCES 169
ABSTRACT IN KOREAN 185
Table 1. Classification of cathepsin family. 45
Table 2. Key characteristics and detection methods of cell death. 128
CHAPTER Ⅰ 24
Figure 1. Estimated new cancer cases and deaths by gender in United States, 2021. 24
Figure 2. Hallmarks of cancer. 26
Figure 3. Overview of the metastatic cascade. 28
Figure 4. Three main morphological types of cell death. 30
Figure 5. Genomic structure of SERTAD family. 32
Figure 6. Cellular function of SERTAD1. 34
Figure 7. SERTAD1 mRNA expression overview in human cancer cell lines in the Human Protein Atlas. 35
CHAPTER Ⅱ 40
Figure 8. Acquisition of anoikis resistance. 40
Figure 9. Mediators of anoikis-resistant cells. 41
Figure 10. LMP-inducers and consequences. 47
Figure 11. SERTAD1 expression levels in normal and cancer cell lines under 3D culture. 66
Figure 12. Cell growth of various breast cell lines under 2D and 3D culture. 68
Figure 13. Cell growth and SERTAD1 expression levels of four breast cancer cell lines during long term suspension 3D. 71
Figure 14. Cell viability of four breast cell lines in response to Dox treatment under 2D and 3D culture. 74
Figure 15. Cell viability of four breast cell lines in response to Dox treatment under 2D and 3D culture. 80
Figure 16. SERTAD1 suppressed Dox-induced apoptosis and anoikis via activating autophagy. 84
Figure 17. Mitochondrial function in the suppression of SERTAD1 in Dox-induced apoptosis/anoikis. 92
Figure 18. Lysosomal function in the suppression SERTAD1 in Dox-induced apoptosis/anoikis. 102
Figure 19. Positive effect of SERTAD1 on lysosome activity via CTSB indirectly. 109
Figure 20. SERTAD1 suppressed sensitivity of breast cancer cells to Dox treatment in vivo. 117
Figure 21. Summary model. 123
CHAPTER Ⅲ 127
Figure 22. Cell death classification. 127
Figure 23. Molecular mechanism of necroptosis. 130
Figure 24. Entosis can be induced by multiple mechanisms. 133
Figure 25. Several types of live internalized cell fate. 134
Figure 26. Cell growth of six breast cancer cell lines under normal and glucose-starved culture. 145
Figure 27. Effect of SERTAD1 on cell viability of MCF7 cell lines under glucose-starved culture. 148
Figure 28. Entosis and necrosis/necroptosis were induced under short- and long-term of low glucose condition, respectively. 155
Figure 29. Effect of SERTAD1 on the ROS and ATP production. 158
Figure 30. SERTAD1 suppressed AMPK phosphorylation via PP2A activity under glucose starvation. 163
Figure 31. Summary model. 168