臺灣博碩士論文加值系統

肺癌是造成世界癌症患者死者的首要原因。臨床上有許多治療肺癌的方法。然而，臨床上肺癌的發現往往屬於中晚期。截至目前為止並沒有有效的治療方法。本實驗主要目的是找出對人類肺腺癌細胞株A549細胞具有明顯細胞毒殺活性的新化合物及研究新化合物在進行癌症細胞毒殺作用時，會如何影響癌細胞不同蛋白質的表現量。本實驗所使用的六個新合成化合物為4-(3,4,5-三甲氧基苯氧基)苯甲酸類衍生物，分別為 DA-H、 DA-Me、 DA-Et、 DA-Pr、DA-Bu 與DA-Pn。經過細胞毒殺活性篩選後，發現DA-Et對於人類肺腺癌細胞株A549細胞具有最強的細胞毒殺效果。DA-Et治療A549細胞株48小時後，其IC50 (抑制50％細胞生長所需藥物濃度)為15 uM。本實驗利用二維蛋白電泳技術分析後，發現DA-Et會造成A549細胞株細胞死亡，與控制組比較後，發現多種蛋白質有不同的表現量。四十個具有顯著性差異的蛋白質被挑選出經鑑定後，找出十個表現量下降的蛋白質點與三十個表現量上升的蛋白質點。有些蛋白質點，如： B2CL2, GRP75, HSP7C, IFT27, KPYM, PDIA6, RAD51及 SIT1等，發現與細胞生長抑制及凋亡有相關性。初步實驗結果顯示，有助於尋找DA-Et造成A549肺腺癌細胞凋亡之生物標誌，此結果應有助於未來肺癌治療作用機轉的研究。

Lung cancer is the leading cause of cancer deaths in worldwide. The current treatment with anticancer drugs can not completely cure the lung cancer in the mid and late stages of clinical trials. In this study, it was purposed to discover new anticancer compounds which have high cytotoxic activity against lung cancer cells and to study the mechanism why the anticancer compounds affect cancer cell death in the protein expression level. Six new synthesized compounds of 4-(3,4,5-trimethoxyphenoxy)benzoic acid derivatives, DA-H, DA-Et, DA-Me, DA-Pr, DA-Bu and DA-Pn, were examined for cytotoxic activity against A549 human lung adenocarcinoma cell line. All of compounds inhibited the cell proliferation of A549 cells by dose and time dependent manner. DA-Et exhibited the highest cytotoxic activity against A549 cells with an IC50 of 15 uM for 48 hr and selectively used for studying the differential protein expression of A549 cells using a proteomic approaches. The result of 2-dimensional electrophoresis (2-DE) showed the differential protein expression profiles of A549 cells with and without DA-Et treatment. Forty differentially expressed proteins containing 10 down-regulated proteins and 30 up-regulated proteins were identified and searched their category classification according to cellular component, molecular function and biological process. Among these proteins, B2CL2, GRP75, HSP7C, IFT27, KPYM, PDIA6, RAD51 and SIT1 were found to be correlated with major signaling pathways in cellular responses. This finding of differentially expressed proteins provided the useful information, in which these proteins may act as the lung cancer biomarkers that used for further studying the mechanism of lung cancer therapy.

ABSTRACT i
CHINESE ABSTRACT ii
ACKNOWLEDGEMENTS iii
TABLE OF CONTENTS iv
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF ABBREVIATIONS ix

CHAPTER 1 INTRODUCTION 1
1.1 Statistics of lung cancer 1
1.2 Types, stages and treatment of lung cancer 2
1.3 Discovery of anticancer drugs 4
1.4 Proteomic study of lung cancer 6
1.5 Objective 9
1.6 Scope of investigation 9

CHAPTER 2 LITERATURE REVIEW 10
2.1 Lung cancer treatment 10
2.2 Proteomic study of lung cancer 14
2.3 Background of diaryl ether 16

CHAPTER 3 MATERIALS AND METHODS 18
3.1 Materials 18
3.2 Equipments 19
3.3 Procedures 19
3.3.1 Cell culture 19
3.3.1.1 Subculture 19
3.3.1.2 Culture/Maintenance 20
3.3.2 Cell treatment 20
3.3.2.1 Cell treatment 20
3.3.2.2 Harvesting of cultured cells 21
3.3.3 Cell cytotoxicity test using MTT assay 21
3.3.4 Cell cycle analysis by flow cytometry 22
3.3.5 Protein extraction 23
3.3.6 Two-dimensional electrophoresis 23
3.3.6.1 First dimensional IEF 23
3.3.6.2 Second dimensional SDS-PAGE 24
3.3.6.3 Gel staining and scanning 25
3.3.7 In-gel digestion for protein identification 26
3.3.8 Mass spectrometry analysis 27
3.3.9 Bioinformatics 29

CHAPTER 4 RESULTS 30
4.1 Formula of six synthesized compounds 30
4.2 Cytotoxicity screening of active compounds 31
4.3 Cell cycle analysis by flow cytometry 36
4.4 2-DE Analysis of A549 cells treated with DA-Et 37
4.5 Protein identification of differentially expressed proteins 42
4.6 Correlation of differentially expressed proteins with cellular
response 51

CHAPTER 5 DISCUSSIONS 53
5.1 Cytotoxicity screening of active compounds 53
5.2 2-DE Analysis of A549 cells treated with DA-Et 54
5.3 Protein identification of differentially expressed proteins 55

CHAPTER 6 CONCLUSIONS 61
6.1 Conclusions 61
6.2 Suggestions for the future work 61

LITERATURE CITED 62

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