肺癌位居全球癌症死亡之首。而在肺癌中以非小細胞肺癌為大宗，占約85%的所有肺癌病例。Gefitinib (Iressa，艾瑞莎) 為表皮生長因子受體之酪氨酸激酶的抑制劑，治療帶有表皮生長因子受體突變的非小細胞肺癌病患，為第一線用藥，並且在臨床上展現極好的治療成果。然而在艾瑞莎12到18個月治療後，大多數的病患由於抗藥性的產生而導致治癒效果差，因此探討艾瑞莎治療下產生的抗藥性分子機制以及尋找抗藥性生物標誌是極為迫切的。在本研究中，我們培養了人類肺腺癌細胞株PC9和具有艾瑞莎抗藥性的細胞株PC9/Gef來研究這兩株細胞株中抗藥性機制和差異蛋白質表現量的調節。我們使用了離氨酸標定的二維差異電泳結合基質輔助雷射脫附游離飛行式質譜儀 (MALDI-TOF MS) 分析這兩株細胞中的蛋白質表現並鑑定出具有表現量差異的蛋白質。結果顯示，具有抗藥性的細胞株相對於敏感性的細胞株有47個蛋白質具有明顯表現量的差異。而這些有明顯表現差異並具有潛力的蛋白質將由免疫點墨法來驗證。在進一步的實驗中，我們選擇兩個在抗藥性細胞株中有明顯表現差異的蛋白質，分別是PCNT和mPR。分別使用RNA干擾的技術將這兩個蛋白各自進行基因沉默，並藉由這技術來看PCNT和mPR兩個蛋白質在進行完基因沉默後的影響，是否影響抗藥性細胞株的細胞存活能力以及是否影響抗藥性細胞凋亡的能力，並從中觀察這兩個蛋白質在抗藥性機制中所扮演的角色。總結來說，本實驗利用肺腺癌細胞為基礎的蛋白質體學的研究並鑑定到許多有明顯的蛋白質表現量變化，並期望這些蛋白質可以在未來能作為對艾瑞莎有抗藥性的肺腺癌病患的診斷標記以及治療上的標靶物。

Lung cancer is the leading cause of cancer-related mortality worldwide. Non-small cell lung cancer (NSCLC) accounts for nearly 85% of all lung cancer cases. In patients with NSCLC whose tumors harbor epidermal growth factor receptor (EGFR) activating mutation, gefitinib is the first-line treatment. However, most patients ultimately obtain drug resistance after 12–18 months treatment. Hence, it is urgent need to investigate the drug resistant mechanism and biomarker. In this study, we used a pair of lung adenocarcinoma cell lines, PC9, and the gefitinib resistant PC9/Gef as a model system to examine resistant mechanism and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI-TOF/TOF MS) to examine the global protein expression changes between PC9 and gefitinib resistant PC9/Gef. A proteomic study revealed that resistant properties altered the expression of 47 proteins in PC9/Gef cells comparing to PC9 cells. Many potential proteins have been validated by western blotting. Further studies have used RNA interference, cell viability analysis, and analysis of apoptosis against progesterone receptor membrane component 1 (mPR) and Pericentrin (PCNT) proteins, to monitor and evaluate their potency and mechanism in resistance. The proteomic approach allowed us to identify numerous proteins, including PCNT, involved in drug resistance mechanism. Our results provide useful diagnostic markers and therapeutic candidates for the treatment of gefitinib-resistant lung adenocarcinoma.

LIST OF FIGURES AND TABLES
Chapter 1 Introduction
Figure 1.1 Lung cancer. 1
Figure 1.2 Types of lung cancer. 3
Table1.1 The list of the common EGFR mutations in NSCLC patients. 5
Figure 1.3 Overview of current proteomics technologies. 7
Figure 1.4 The concept of 2D-PAGE. 8
Figure 1.5 The chemical structure of N-hydroxy-succinimidyl esters of propyl Cy3, methyl Cy5 and Cy2. Cy3, Cy5 and Cy2 are used for DIGE labeling of lysine residues. 10
Figure 1.6 Comparison of 2D gels with and without internal standard. 11
Figure 1.7 The principle of MALDI-TOF MS. 12

Chapter 2 Masterials and methods
Figure 2.1 The morphology of PC9 cells and PC9/Gef cells. 15
Table 2.1 Distribution of Cy dye labeling in lysine-labeling 2D-DIGE 20
Figure 2.2 The strategy for lysine-labeling two-dimensional gel electrophoresis (2D-DIGE). 21
Table 2.2The voltage program of isoelectric focusing (IEF). 23
Table 2.3The wavelengths of excitation and emission used in 2D-DIGE for detecting Cy dyes andCCB staining. 23
Table 2.4 The list of the primary antibodies used in this project for validation of identified proteins from 2D-DIGE. 27

Chapter 3 Reasults
Figure 3.1 Dose-dependent kinetics of gefitinib-induced loss of cell viability and increased expression and activation of receptor tyrosine kinases and downstream signaling molecules in PC9 and PC9/Gef cells. 30
Figure 3.2 2D-DIGE analysis ofPC9 and PC9/Gef treated with/without gefitinib. 33
Figure 3.3 Peptide mass fingerprint and database searching for the identified protein,Pericentrin. 35
Figure 3.4 Percentage of differentially expressed proteins identified by lysine-labeling 2D-DIGE / MALDI-TOF MS analysis for PC9 cells and PC9/Gef cells. 37
Figure 3.5 Representative immunoblotting analyses and 2D-DIGE images for selected differentially expressed proteins in PC9 cells and PC9/Gef cells. 39
Figure 3.6 Expression profiles for differentially expressed proteins potentially contributing to 41
Figure 3.7 Evaluation of the roles of PCNT on gefitinib resistance in lung adenocarcinomas using siRNA knockdown. 45
Figure 3.8 Effects of PCNT knockdown on resistance-related signaling pathway in lung adenocarcinoma cells. 47
Figure 3.9 Effects of PCNT knockdown on resistance-related cell cycle in lung adenocarcinoma cells. 48
Figure 3.10 Evaluation of the roles of mPR on gefitinib resistance in lung adenocarcinoma using siRNA knockdown. 52
Figure 3.11Effects of mPR knockdown on resistance-related signaling pathway in lung adenocarcinoma cells. 53

Chapter 4 Discussions
Figure 4.1 The hypothetic mechanism of gefitinib drug-resistance in lung adenocarcinoma. 60

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