Title Page
Contents
ABSTRACT 9
ABBREVIATIONS 14
CHAPTER Ⅰ. Inhibitory role of TRIP-Br1 onco-protein in anti-cancer drug-mediated programmed cell death via the activation of mitophagy 16
1. Introduction 17
1.1. Anti-cancer drug resistance 17
1.2. Programmed cell death (PCD) and mitochondria 18
1.3. Background and importance of TRIP-Br1 in PCD 19
2. MATERIALS AND METHODS 21
2.1. Cell lines, cell culture, and cell treatment 21
2.2. Cell viability analysis 21
2.3. Western blot analysis 22
2.4. Mitophagy assessment 22
2.5. Real-time polymerase chain reaction (RT-PCR) for mtDNA determination 23
2.6. Preparation of mitochondrial DNA fractions 23
2.7. Measurement of cellular ATP 23
2.8. Measurement of mitochondrial membrane potential (MMP) 24
2.9. Measurement of reactive oxygen species (ROS) 24
2.10. Mitochondrial fractionation 24
2.11. Preparation of lysosomal fractionations 25
2.12. Phos-tag SDS-PAGE 25
2.13. Immunofluorescence and confocal imaging 25
2.14. Animal experiment 26
2.15. Statistical analysis 27
3. RESULTS 28
3.1. Mitochondrial distribution/sub-localization of TRIP-Br1 protein in normal breast and cancer cell lines 28
3.2. ROS-triggered TRIP-Br1 translocation into mitochondria and TRIP-Br1-mediated ROS suppression in response to anti-cancer drug treatment. 32
3.3. Mitochondrial translocation of TRIP-Br1 via dephosphorylation by PP2A phosphatase 38
3.4. TRIP-Br1-mediated PCD suppression in cancer cells via induction of autophagy 43
3.5. TRIP-Br1-mediated PCD suppression in cancer cells via induction of mitophagy 48
3.6. Enhanced TRIP-Br1-mediated mitophagy via up-regulation of lysosomal proteins, Cathepsins B and D 56
3.7. Summary model 61
4. DISCUSSION 63
CHAPTER Ⅱ. TRIP-Br1 enhances metastatic recurrence by inducing anoikis resistance and cell cycle arrest in dormant breast cancer cells 65
1. INTRODUCTION 66
1.1. Cancer recurrence and metastasis 66
1.2. Dormancy and Cell cycle 67
1.3. Background and importance of TRIP-Br1 in cell cycle 71
2. MATERIALS AND METHODS 74
2.1. Cell culture and growth analysis 74
2.2. Generation of cell aggregates in suspension culture 75
2.3. Animal studies 75
2.4. Drug and reagents 75
2.5. Cell viability analysis 76
2.6. Western blot analysis 76
2.7. Caspase 3/7 assay 77
2.8. Autophagy detection 78
2.9. Statistical analysis 78
3. RESULTS 79
3.1. Characterization of breast cancer cell lines under SUS condition. 79
3.2. Role of TRIP-Br1 in dormant MCF7 cancer cells under SUS condition. 83
3.3. Upregulated TRIP-Br1 enhance autophagy during dormant state 87
3.4. TRIP-Br1-mediated PCD suppression in response to anti-cancer drug treatment during dormant state. 92
3.5. Inhibitory role of TRIP-Br1 in cell proliferation via negative regulation of cyclin A under SUS condition. 96
3.6. Negative regulation of cyclin A by TRIP-Br1/XIAP complex under SUS condition. 100
3.7. Summary model 103
4. DISCUSSION 104
REFERENCES 107
ABSTRACT IN KOREAN 120
CHAPTER Ⅰ 31
Figure 1. Subcellular distribution of TRIP-Br1 protein in normal breast and cancer cell lines, and mitochondrial localization of TRIP-Br1. 31
Figure 2. Up-regulated TRIP-Br1 protein level in mitochondria in response to anticancer drug treatment and TRIP-Br1-mediated ROS suppression in... 37
Figure 3. Mitochondrial translocation of TRIP-Br1 via PP2A-mediated dephosphorylation upon STS treatment. 41
Figure 4. Suppression of STS-mediated cell death by TRIP-Br1 via induction of autophagy. 47
Figure 5. Suppression of STS-mediated cell death by TRIP-Br1 via induction of mitophagy. 55
Figure 6. Enhanced TRIP-Br1-mediated mitophagy via up-regulation of lysosomal proteases Cathepsins B and D. 60
Figure 7. TRIP-Br1 oncogenic protein suppresses anti-cancer drug-mediated cell death by strongly activating mitophagy in cancer cells. 61
CHAPTER Ⅱ 73
Figure 1. Proposed model of function of TRIP-Br1 in the regulation of cell cycle progression. 73
Figure 2. Homology domains of TRIP-Br (SERTAD) family. 73
Figure 3. Sensitivity of breast cancer cells under SUS condition. 82
Figure 4. TRIP-Br1 provide survival of breast cancer cells under suspended condition. 85
Figure 5. Autophagy activated in Breast cancer cells under suspended culture. 90
Figure 6. Suppression of STS induced anoikis by enhancement of autophagy in suspension culture. 95
Figure 7. Upregulation of TRIP-Br1 suppress cell proliferation by degrading cyclin A in suspension culture. 99
Figure 8. Negative effect of TRIP-Br1/XIAP complex in cell cycle. 101
Figure 9. TRIP-Br1/XIAP degrade Cyclin A through autophagic pathway. 102
Figure 10. TRIP-Br1 oncogenic protein enhances dormant state by negatively regulating cyclin A. 103