臺灣博碩士論文加值系統

結腸直腸癌是全球前三大常見癌症，所以迫切需要開發新的治療手段。TC-N19 （以下簡稱N19）是一種新型表皮生長因子受體及間質表皮轉化因子的雙重抑制劑。儘管先前的研究已經證明了N19對於多種癌症的抗癌功效，例如:前列腺癌、非小細胞肺癌、膠質母細胞瘤和胰臟導管腺癌，但其對結腸直腸癌的影響還尚未被探討過。在本研究中，我們使用了美國國家癌症研究所NCI-60的癌細胞篩選，發現結腸直腸癌對N19治療表現出最高的敏感性。隨後，我們在四種結腸直癌細胞系（HCT116、HT29、RKO和DLD-1）中驗證了N19的體外抗癌的能力，它與其他癌細胞類型中的效果一致。N19在結腸直腸癌細胞中抑制了表皮生長因子受體及間質表皮轉化因子蛋白的表達。而細胞可能通過意外細胞死亡或調節細胞死亡而死亡。意外細胞死亡是一個生物學上不受控制的過程，而調節細胞死亡涉及緊密結構的信號級聯和分子定義的效應機制，如:凋亡、壞死性凋亡、自噬依賴性細胞死亡、鐵死亡等。通過使用一系列調節細胞死亡的抑制劑，我們發現N19主要誘導結腸直腸癌細胞中的自噬依賴性細胞死亡和鐵死亡。將進一步進行研究以闡明N19在結腸直腸癌細胞中的抗癌活性的潛在分子機制和角色。

Colorectal cancer (CRC) stands as the third most prevalent cancer worldwide. The development of novel therapeutic interventions is urgently needed. TC-N19 (hereafter designated as N19) is a novel epidermal growth factor receptor (EGFR)/c-MET dual inhibitor. While previous studies have demonstrated N19's anticancer efficacy in several cancers such as prostate cancer, non-small cell lung cancer (NSCLC), glioblastoma (GBM), and pancreatic ductal adenocarcinoma (PDAC), its impact on CRC remains unexplored. In this study, we employed the National Cancer Institute (NCI)-60 cancer cell panel screening and revealed that colon cancer exhibits the highest sensitivity to N19 treatment. Subsequently, we validated the in vitro anticancer potential of N19 across four CRC cell lines (HCT116, HT29, RKO, and DLD-1). Consistent with its effects in other cancer cell types, N19 demonstrated the inhibition of EGFR and c-MET protein expression in CRC cells. Cells may die by either accidental cell death (ACD) or regulated cell death (RCD). ACD is a biologically uncontrolled process, whereas RCD involves tightly structured signaling cascades and molecularly defined effector mechanisms such as apoptosis, necroptosis, autophagy-dependent cell death, ferroptosis, and so on. Employing a spectrum of RCD inhibitors, we found that N19 predominantly induced autophagy-dependent cell death and ferroptosis in CRC cells. Further investigation will be undertaken to elucidate the underlying molecular mechanisms and their roles in the anticancer activity of N19 in CRC cells.

Abstract vi
中文摘要 vii
Abbreviation viii
Introduction 1
1.1 Colorectal cancer (CRC) 1
1.2 The novel EGFR/c-MET dual inhibitor TC-N19 6
1.3 Mechanism of cell death 7
Motivation 16
Materials and Methods 17
2.1 Reagents, Chemicals, and Antibodies 17
2.2 National Cancer Institute (NCI)-60 screening methodology 18
2.3 Cell Culture 18
2.4 Cell Viability Assay 19
2.5 Protein Expression Analysis 19
2.6 Lipid Peroxidation Assay 20
2.7 Cellular Thermal Shift Assay (CETSA) 20
2.8 Band Depletion Assay 21
2.9 RNA Sequencing (RNA-Seq) Analysis 21
2.10 Statistical Analysis 22
Results 23
3.1 N19 exhibits anticancer activity in CRC cells 23
3.2 N19 inhibits the expression of EGFR and c-MET. 28
3.3 N19 partially triggers caspase-dependent cell death in CRC cells 30
3.4 N19 induces autophagy-dependent cell death in CRC cells. 32
3.5 N19 induces ferroptosis in CRC cells 35
3.6 RNA sequencing analysis reveals the potential mechanism for the anticancer activity of N19 38
Discussion 41
Conclusion 43
Reference 44

Figure 1. CRC stage using the TNM system according to AJCC. 4
Figure 2. The carcinogenic process and mechanisms of CRC. 5
Figure 3. Apoptosis regulatory pathway. 9
Figure 4. Autophagy regulatory pathway. 12
Figure 5. Ferroptosis regulatory pathway. 15
Figure 6. NCI-60 cancer cell screening for N19. 26
Figure 7. N19 exhibits anticancer activity in CRC cells. 27
Figure 8. N19 inhibits the expression of EGFR and c-MET. 29
Figure 9. N19 partially triggers caspase-dependent cell death in CRC cells. 31
Figure 10. N19 induces autophagy-dependent cell death in CRC cells. 33
Figure 11. N19 induces autophagy-dependent cell death in DLD-1 cells. 34
Figure 12. N19-induced ferroptosis also induces autophagy-dependent ferroptosis. 36
Figure 13. KEGG pathway enrichment analysis in N19-treated CRC cells. 39
Figure 14. N19 upregulates genes related to cholesterol biosynthesis in CRC cells. 40

Table 1. Quantification of growth inhibitory and lethal effects of N19 in NCI-60 screening. 24
Table 2. A comparison of the IC50 values and treatment duration of N19 in different cancer cell types. 25

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