표제지
초록
목차
Part Ⅰ. Antiproliferative Activity of Piceamycin by Regulating Alpha-actinin-4 in Gemcitabine-resistant Pancreatic Cancer Cells Piceamycin의 젬시타빈 저항성 췌장암 세포에서 Alpha-actinin-4 조절을 통한 세포 생장 저해 효과 17
1. Introduction 18
2. Materials and Methods 21
2.1. Kaplan-Meier Plotter analysis 21
2.2. Cell culture and reagents 21
2.3. Cell proliferation assay 21
2.4. RNA-seq analysis 22
2.5. Protein-Protein Interaction (PPI) network analysis 24
2.6. Western blot analysis 24
2.7. Cell cycle analysis 25
2.8. Caspase 3/7 activity assay 26
2.9. Colony formation assay 26
2.10. Annexin V-fluorescein isothiocyanate and PI double staining 26
2.11. Wound healing assay (cell migration assay) 27
2.12. Transwell cell invasion assay 27
2.13. Organoid culture medium and 3D culture viability assay 28
2.14. Data analysis 28
3. Results 29
3.1. The clinical significance of ACTN4 expression in patients with pancreatic cancer 29
3.2. Antiproliferative activity of piceamycin (PCM) in pancreatic cancer cells 31
3.3. The effects of piceamycin on gemcitabine-resistant pancreatic cancer cells 34
3.4. The effects of Piceamycin on the ACTN4 expression in gemcitabine-resistant AsPC-1 cells 36
3.5. Piceamycin suppressed actin polymerization signaling pathway in gemcitabine-resistant AsPC-1 cells. 41
3.6. The combination of gemcitabine and Piceamycin is effective in the gemcitabine-resistant AsPC-1 cells. 47
3.7. The effects of Piceamycin on the expressions of focal adhesion biomarkers in gemcitabine-resistant AsPC-1 cells 49
3.8. The effects of Piceamycin on the expressions of epithelial-mesenchymal transition biomarkers in gemcitabine-resistant AsPC-1 cells 53
3.9. The effect of piceamycin on the cell cycle regulation in gemcitabine-resistant AsPC-1 cells 57
3.10. The effect of Piceamycin on apoptosis in gemcitabine-resistant AsPC-1 cells 62
3.11. Antiproliferation activity of piceamycin in patient-derived pancreatic cancer organoids 67
4. Discussion 71
Part Ⅱ. Antiproliferative Activity of Krukovine by Regulating of Transmembrane protein 139 (TMEM139) in Oxaliplatin-resistant Pancreatic Cancer Cells 옥살리플라틴 내성 췌장암 세포에서 막횡단단백질 139 조절을 통한 Krukovine의 성장 억제 효능 연구 75
1. Introduction 76
2. Materials and Methods 78
2.1. Cell cultures and reagents 78
2.2. MTT assay (cell viability assay) 78
2.3. RNA preparation, library preparation, and RNA-seq 78
2.4. Kaplan-Meier Plotter analysis 80
2.5. Protein-Protein Interaction (PPI) network analysis 80
2.6. Western blot analysis 81
2.7. Wound healing assay (cell migration assay) 81
2.8. Transwell cell invasion assay 82
2.9. Organoid medium 82
2.10. Organoid viability assay 82
2.11. Mutation profile of PDPCOs 83
2.12. Data analysis 83
3. Results 85
3.1. Krukovine shows an antiproliferative effect toward KRAS-mutated pancreatic cancer cells. 85
3.2. Antiproliferative activity of KV in oxaliplatin-resistant pancreatic cancer cells 88
3.3. RNA levels of KV-treated pancreatic cancer cell show major metabolic pathway. 90
3.4. The clinical significance of TMEM139 expression in patients with pancreatic cancer 93
3.5. The effects of KV on the TMEM139 expression in oxaliplatin-resistant AsPC-1 cells 95
3.6. The effects of KV on the TMEM139-associated signaling pathway in Oxaliplatin-resistant AsPC-1 cells. 98
3.7. KV inhibits the PI3K-Akt-mTOR pathway in oxaliplatin-resistant AsPC-1 cells. 101
3.8. KV inhibits migration and invasion in oxaliplatin-resistant AsPC-1 cells. 104
3.9. KV showed an antiproliferative effect toward KRAS-mutated patient-derived pancreatic cancer organoids. 108
3.10. KV enhanced the anticancer effects of oxaliplatin in KRAS-mutated patient-derived pancreatic cancer organoids. 113
4. Discussion 117
References 119
Abstract 128
Table 1. Antiproliferative activities of piceamycin against human pancreatic cancer cell lines. 33
Table 2. Drug resistant profiles of AsPC-1 cells with resistance to gemcitabine. 35
Table 3. The effect of drug combination on gemcitabine-resistant AsPC-1 cells. 48
Table 4. TMEM family mRNA expression (fold change) and p-value in oxaliplatin-resistant AsPC-1 cells treated KV. 96
Figure 1. The Kaplan-Meier survival curve according to the ACTN4 expression level. 30
Figure 2. The chemical structure of piceamycin. 32
Figure 3. Heatmap of one-way hierarchical clustering using z-score. 37
Figure 4. Top 20 terms of GO (Gene Ontology) functional analysis. 38
Figure 5. Effect of PCM on ACTN4 mRNA expression. 39
Figure 6. Effect of PCM on ACTN4 protein expression in gemcitabine-resistant AsPC-1 cells. 40
Figure 7. Protein-Protein Interaction (PPI) for ACTN4 from the search tool (STRING) database. 42
Figure 8. Effect of PCM on mRNA expression of actin polymerization related genes. 43
Figure 9. Normalized value (mRNA level) of ACTB and ACTG1. 44
Figure 10. Modified KEGG pathway of the genes related to actin polymerization signaling pathway. 45
Figure 11. Effect of Piceamycin (PCM) on the proteins related to actin polymerization (VCN and ACTB) 46
Figure 12. Heatmap of mRNA expression related to the focal adhesion signaling pathway in gemcitabine-resistant AsPC-1 cells. 50
Figure 13. Modified KEGG pathway of the genes related to focal adhesion signaling pathway from RNA-sequencing data. 51
Figure 14. The protein expressions of focal adhesion-related biomarkers 52
Figure 15. The protein expression levels of epithelial-mesenchymal transition (EMT)-related biomarkers 54
Figure 16. The effect of piceamycin on gemcitabine-resistant AsPC-1 cells migration 55
Figure 17. The effect of Piceamycin (PCM) on gemcitabine-resistant AsPC-1 cell invasion. 56
Figure 18. Piceamycin induced G₀/G₁ cell cycle arrest in gemcitabine-resistant AsPC-1 cells. 58
Figure 19. Heatmap of the genes related to the cell cycle signaling pathway. 59
Figure 20. Normalized value (mRNA level) of CDK2, Cyclin E2, and Cyclin D1 60
Figure 21. The effect of piceamycin on the cell cycle regulatory proteins 61
Figure 22. Induction of apoptosis by piceamycin in gemcitabine-resistant AsPC-1 cells 63
Figure 23. The effect of piceamycin (PCM) on caspase activity in gemcitabine-resistant AsPC-1 cells 64
Figure 24. The effect of piceamycin (PCM) on the expression of apoptotic biomarkers in gemcitabine-resistant AsPC-1 cells. 65
Figure 25. The effect of piceamycin (PCM) on colony formation in gemcitabine-resistant AsPC-1 cells. 66
Figure 26. Drug response (AUC) heatmap based on gemcitabine treatment. 68
Figure 27. Antiproliferative effect of piceamycin on PDPCOs. 69
Figure 28. The level of cell apoptosis by flow cytometry. 70
Figure 29. The structure of Krukovine 86
Figure 30. Cell viability assay of KV on KRAS-mutated pancreatic cancer cell lines. 87
Figure 31. Antiproliferative effect of KV in oxaliplatin-resistant AsPC-1 cells. 89
Figure 32. Heatmap of one-way hierarchical clustering using z-score. 91
Figure 33. Top 19 terms of KEGG pathway. 92
Figure 34. The Kaplan-Meier survival curve according to the TMEM139 expression level. 94
Figure 35. The effect of KV on TMEM139 protein expression in oxaliplatin-resistant AsPC-1 cells. 97
Figure 36. Protein-Protein Interaction (PPI) for TMEM139 from the search tool (STRING) database. 99
Figure 37. Effects of KV on the TMEM139-associated signaling pathway in Oxaliplatin-resistant AsPC-1 cells. 100
Figure 38. Protein-Protein Interaction (PPI) for PI3K-Akt and PI3K-Akt-mTOR signaling pathway from the search tool (STRING) database. 102
Figure 39. Effects of KV on the PI3K-Akt-mTOR signaling pathway in oxaliplatin-resistant AsPC-1 cells. 103
Figure 40. The effect of KV on oxaliplatin-resistant AsPC-1 cells migration. 105
Figure 41. The effect of KV on oxaliplatin-resistant AsPC-1 cell invasion. 106
Figure 42. Top 20 terms of GO (Gene Ontology) functional analysis. 107
Figure 43. Mutation profile of PDPCOs. 109
Figure 44. Multi-drug response heatmap on PDPCOs. 110
Figure 45. Antiproliferative effect of KV on PDPCOs. 111
Figure 46. KV inhibited the growth of the tested PDPCO (SNU-4340-TO) in a dose- dependent manner. 112
Figure 47. Cell viability assay for combination effect of KV with oxaliplatin. 114
Figure 48. Combination effect of KV with oxaliplatin on the growth of multi-drug resistant PDPCO (SNU-4425-TO). 115
Figure 49. Effects of KV on the cleaved-PARP expression in multi-drug resistant organoid (SNU-4425-TO). 116