臺灣博碩士論文加值系統

非小細胞肺癌是全世界癌症死亡的首要原因，艾瑞莎(gefitinib)是表皮生長因子受體酪氨酸激酶的抑製劑作為非小細胞癌患者的第一線治療藥物，然而，許多患者最終卻對藥物產生抗藥性。因此，有效地開發藥物去治療具抗藥性的患者正迫在眉睫。我們先前發現黃綠青黴素，從真菌物種提煉的毒素之一，可以通過抑制異位表達三磷酸腺苷合成酶的活性進而抑制肺癌細胞生長，但對正常細胞的影響有限。在老鼠模型與CL1-0 (艾瑞莎非抗性肺癌細胞株)證實黃綠青黴素會藉由降低熱休克蛋白中特定絲氨酸(S255)的磷酸化而去抑制絲裂原活化蛋白激酶所參與的訊息傳遞。我們好奇黃綠青黴素在治療具艾瑞莎獲得性抗藥性肺癌細胞的潛在信號傳遞是否不同。在這項研究中，我們發現黃綠青黴素會抑制NCI-H1975(EGFR T790M突變艾瑞莎獲得性抗藥性)細胞生長與群落形成。此外，我們藉由時間性偵測磷酸化蛋白質體學來探討動態分子反應共鑑定出738個磷酸化蛋白，1476條磷酸化胜肽鏈以及1901個磷酸化位置。其中有顯著調控的磷酸化蛋白有174條對應274個磷酸化位置。功能富集分析發現黃綠青黴素會影響染色質的組織，細胞週期和細胞凋亡。有趣的是，我們發現黃綠青黴素藉由磷酸化拓撲異構酶的特定絲氨酸1106位置進而抑制細胞生長，而黃綠青黴素誘發DNA雙股斷裂，進而造成DNA損傷反應(DNA damage response)。DNA受損會促使細胞進行細胞週期停滯在S期去進行修復或是走向細胞凋亡。而所有資料指出黃綠青黴素可能作為治療具艾瑞莎抗藥性的有效藥物。

Non-small cell lung cancer is the leading cause of cancer death worldwide. Gefitinib, epidermal growth factor receptor tyrosine kinase inhibitor, is the first-line treatment of NSCLC, however, many patients eventually become resistant and experience progressive disease. Therefore, development of efficient therapeutic agents to overcome resistance is urgent. We previously found that citreoviridin, one of toxic mycotoxins derived from fungal species, can suppress lung cancer cell growth by inhibiting the activity of ectopic ATP synthase, but has limited effect on normal cells. Citreoviridin suppresses mitogen-activated protein kinase/extracellular signal-regulated kinase signaling by site-specific dephosphorylation of HSP90AB1 on Serine 255 in gefitinib non-resistant lung cancer CL1-0 cells and xenograft model. We are curious whether signaling pathways underlying citreoviridin-treated gefitinib-acquired resistant lung cancer cells are different. In this study, we showed that citreoviridin inhibited cell proliferation and anchorage-dependent growth of gefitinib-acquired resistance NCI-H1975 cells with EGFR T790M mutation. Furthermore, we explored the dynamic molecular response by temporal phosphoproteomic approach. We identified 1476 phosphopeptides corresponding to 738 phosphoproteins and quantified 1901 phosphorylation sites. There were 274 phosphosites corresponding to 174 phosphorylated proteins significantly differential expressed. Functional enrichment analysis demonstrated that citreoviridin treatment affected chromatin organization, cell cycle and apoptosis. Interestingly, we found that citreovirdin suppressed cell proliferation by site-specific phosphorylation of topoisomerase on serine 1106. Citreovirdin induced double strands breaks, and then leaded to DNA damage response. The DNA lesions triggered cells to cell cycle arrest at S phase for repairing or apoptosis for cell death. The results indicated that citreoviridin could potentially be a therapeutic agent against gefitinib-resistant NSCLC.

口試委員會審定書 i
謝辭 ii
中文摘要 iii
Abstract iv
Contents v
List of Figures viii
List of Tables ix
Chapter 1 Introduction 1
1.1 Non-small cell lung cancer 1
1.2 Resistance of EGFR tyrosine kinase inhibitors 2
1.3 Ectopic ATP synthase 2
1.4 ATP synthase inhibitor 3
1.5 Phosphoproteomics 4
1.6 Topoisomerases II α 5
1.7 Motivation 6
Chapter 2 Materials and Methods 8
2.1 Cell culture 8
2.2 Drug treatment 8
2.3 Immunocytochemistry 9
2.4 Proliferation assay 9
2.5 Colony formation assay 10
2.6 Sample preparation (31) 10
2.7 Dimethyl labeling of peptides (31) 11
2.8 Stage tip preparation and Desalting with SDB-XC Stage Tips (31) 12
2.9 Phosphopeptide enrichment with HAMMOC (31) 13
2.10 NanoLC–MS/MS analysis (31) 14
2.11 Data analysis for phosphoproteomes (31) 15
2.12 Functional annotation and clustering analyses 15
2.13 Western blot analysis 16
2.14 Cell cycle analysis 17
2.15 Apoptosis using Annexin V/PI staining by flow cytometry 17
2.16 Reactive oxygen species by flow cytometry 18
Chapter 3 Results 19
3.1 ATP synthase is expressed on the surface of gefitinib-resistant lung cancer NCI-H1975 cells 19
3.2 Ectopic ATP synthase inhibitor citreoviridin suppresses the proliferation of NCI-H1975 cells 20
3.3 Dynamic phosphorylation profiles of citreoviridin-treated NCI-H1975 cells 21
3.4 Functional enrichment map and temporal clustering analysis reveal the functions of differentially regulated phosphoproteins with citreoviridin treatment 23
3.5 Motif analysis reveals the potential kinases of TOP2A at serine 1106 24
3.6 Citreoviridin triggers DNA damage response by double strand break 25
3.7 Citreoviridin induces cell cycle arrest at S phase and causes apoptosis 26
3.8 Inhibition of casein kinase 2 alpha 1 reduces S1106 phosphorylation of topoisomerase IIα and blocks TOP2A-induced DNA damage response in short time. 27
3.9 Citreoviridin induces ROS-dependent DNA damage in 48 hours 28
Chapter 4 Discussion 30
Chapter 5 Conclusion 35
References 36
Figures 41
Tables 58
Appendix. Supplementary Figures 116

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