Title Page
ABSTRACT
국문 초록
Contents
NOMENCLATURE 13
CHAPTER 1. INTRODUCTION 15
CHAPTER 2. MATERIALS AND METHODS 17
2.1. Reagents and antibodies 17
2.2. Cell culture 17
2.3. Stable HER2 and p95HER2 overexpression in MDA-MB-231 cells 18
2.4. Cell viability assay 18
2.5. Cell cycle analysis and Annexin V/PI assay 18
2.6. Aldefluor-positivity assay and CD44/CD24 staining 19
2.7. HSP90α (C-Terminal) inhibitor screening assay 19
2.8. Western blot analysis 20
2.9. Immunocytochemistry 21
2.10. Cell sorting and cytological centrifugation 21
2.11. Mammosphere formation assay in vitro 22
2.12. In vivo xenograft and mammosphere formation assays 22
2.13. Serum biochemistry profiles for biomarkers of liver and renal injury 23
2.14. Immunohistochemistry and in situ localization of apoptosis (TUNEL) 23
2.15. Statistical analysis 24
CHAPTER 3. RESULTS 25
3.1. NCT-58-dependent apoptosis is mediated by caspase activation in HER2-positive breast cancer cells 25
3.2. NCT-58 targets the C-terminal domain of HSP90 and downregulates the expression of EGFR/HER2/HER3 31
3.3. NCT-58 kills trastuzumab-resistant cells and suppresses HER family members 39
3.4. NCT-58 eradicates HER2-positive BCSCs without triggering the HSR 49
3.5. NCT-58 administration suppresses trastuzumab-resistant tumor growth 59
3.6. Anti-tumor effect of NCT-58 is accompanied by the suppression of BCSC-like characteristics and downregulation of HSF-1/HSP70/HSP90 65
CHAPTER 4. DISCUSSION 70
REFERENCES 74
Figure 1. NCT-58 reduces cell viability and induces apoptosis in HER2-positive breast cancer cells.... 26
Figure 2. NCT-58 reduces cell viability and induces apoptosis in HER2-positive breast cancer cells.... 34
Figure 3. NCT-58 induces apoptosis and inhibits tyrosine kinase activity of HER2 in trastuzumab-resistant cells... 41
Figure 4. NCT-58 targets HER2-positive BCSC-like properties. A-D ALDH1 low (ALDH1-) and high (ALDH1+) BT474 cells were sorted using FACS.... 52
Figure 5. NCT-58 inhibits tumor growth in trastuzumab-resistant JIMT-1 xenografts. A-C Effect of NCT-58 on tumor growth in vivo. JIMT-1 cells (3×10⁶) were injected into the... 60
Figure 6. NCT-58 suppresses BCSC-like properties and downregulates the expression of HSF-1, HSP70 and HSP90 in vivo.... 66
Figure 7. Hypothetical model illustrating multiple actions of NCT-58 on cancer stem-like properties, HER2 signaling and trastuzumab resistance in HER2-positive breast cancer.... 69