臺灣博碩士論文加值系統

癌細胞轉移是癌症最大的問題，很多癌症患者都是因為在使用抗癌藥物治療過程中出現抗藥性 (drug resistance)進而導致轉移，其中發現大部分患者可以抵抗多種藥物，但不是因為藥物本身的結構或功能因素所產生的抗藥性，這種情況定義為多重抗藥性。而造成多重抗藥性的最主要原因是透過ABC transporter的調節，藉由水解ATP產生能量提供transporter將多種物質運送出細胞外。ABCB1和ABCG2 都是ABC transporter的一種，且過去研究顯示在癌症多重抗藥性發展中它們扮演很重要的角色。為了嘗試克服在癌症患者因ABCB1或ABCG2所造成的多重抗藥性，有許多發明策略已經被考慮及評估。目前，最直接的方法就是直接抑制其功能或是減少與多重抗藥性相關的ABC transporter蛋白表現，重新回復對抗癌藥物的敏感度。在過去二十幾年來，已經投入很多努力在尋找或發展有潛力的ABCB1和ABCG2抑制劑。不幸的是，由於可與ABCB1結合的受質跟ABCG2的受質專一性大幅度重疊，所以要找到一個對單一種ABC transporter具專一性且有很強的交互作用的化學致敏劑，是一種挑戰。因此，我們在尋找新的化學結構，可以被開發並優化成強效具選擇性的ABC transporter抑制劑。在這項研究中我們首次發現niclosamide，一種抗寄生蟲藥物，對於人類肺癌細胞過度表現ABCG2的A549 Bec150細胞我們推斷可能通過抑制ABCG2功能以及降低蛋白質表現使細胞重新回復對傳統藥物的敏感度。此外，透過執行高通量功能性篩選大量的niclosamide衍生物，其中有五個具獨特化學結構的衍生物被確定，在未來可以候選成為ABCG2的抑制劑。

A major problem in cancer is cancer metastasis. Many metastatic cancers are resistant to treatment with a variety of structurally unrelated anti-cancer drugs, a phenomenon that is known as multidrug resistance (MDR). MDR often is mediated by ATP-binding cassette (ABC) transporters that can generate energy from ATP hydrolysis to actively transport a variety of compounds across biological membranes. ABCB1 and ABCG2 are ABC transporters that have been shown to play an important role in the development of MDR in cancers. In an attempt to overcome ABCB1 or ABCG2-mediated MDR in cancer patients, many inventive strategies have been considered and evaluated. Currently, the most direct way to re-sensitize MDR cancer cells is to either inhibit the function directly, or to reduce the protein expression of these MDR-linked ABC transporters. Tremendous effort has been invested in the development of potent modulators of ABCB1 and ABCG2 for the past two decades. Unfortunately, due to a substantial overlapping substrate specificity between ABCB1 and ABCG2, finding a chemical scaffold that interacts strongly and specifically to one ABC drug transporter, has been challenging. Therefore, we are in search of novel chemical structures that can be developed and optimized into potent and selective modulator of ABC drug transporters. In this study, we showed for the first time that niclosamide, an antihelminthic agent, re-sensitizes the chemosensitivity in ABCG2-overexpressing MDR A549 Bec150 human lung cancer cells by directly inhibiting the function of ABCG2, as well as reducing protein expression level of ABCG2. Moreover, by performing high-throughput functional screening of large numbers of niclosamide derivatives, 5 derivatives with distinct chemical structures were identified as candidates to be developed into potent ABCG2 modulators for the future.

指導教授推薦書
口試委員會審定書
授權書 iii
誌謝 iv
中文摘要 v
英文摘要 vii
目錄 ix
圖表目錄 xiii
一、 前言 1
1.1 多重抗藥性 (multidrug resistance, MDR) 2
1.2 ATP Binding Cassette (ABC) transporter 6
1.2.1ABCB1 (P-glycoprotein / MDR1) 7
1.2.2ABCG2 (BCRP/MXR/ABCP) 8
1.3 ABCB1抑制劑 (modulator) 11
1.4 ABCG2 抑制劑 (modulator) 13
1.5 Tyrosine kinase inhibitor (TKI) 14
1.6 耐克螺 (Niclosamide) 15
1.7 研究動機 17
二、 實驗方法 19
2.1 實驗材料 19
2.2 細胞培養 (Cell Culture) 19
2.3 冷凍細胞保存 20
2.4 解凍細胞 21
2.5 細胞存活率 22
2.6 流式細胞儀 (Flow Cytometry) 23
2.7 高通量功能性篩選分析 ( High-throuput Functional Screening Assay) 24
2.8 收集細胞和萃取蛋白質 25
2.9 蛋白質定量 26
2.10 西方墨點法(Western Blot) 26
2.14 ATPase Assay 29
2.15 Photo-Affinity Labeling of ABCG2 with [125I]IAAP 30
三、 結果 32
3.1 各種細胞株ABC transporters蛋白表現情形 32
3.2 利用已知的抗絛蟲藥物niclosamide測試是否會與ABC transporters 產生交互作用，藉由一連串實驗證明 33
3.2.1 Niclosamide在不同ABC transporters 調節螢光染劑運輸的影響 33
3.2.2 藉由[125I] IAAP (iodoarylazidoprazosin) photoaffinity labeling測試niclosamide對ABCB1及ABCG2的影響 35
3.2.3 Niclosamide 對於ABCB1及ABCG2其 ATPase活性的影響 36
3.2.5 Niclosamide在過度表達ABCG2細胞其蛋白質表現及mRNA的影響 40
3.3 建立高通量功能性篩選平台最佳條件 41
3.3.1 測試高通量功能性篩選平台之最佳細胞數目 42
3.3.2 測試高通量功能性篩選平台之最佳培養時間及螢光染劑 (calcein-AM / PhA) 濃度 43
3.3.3 測試高通量功能性篩選平台之最佳positive control 濃度 43
3.3.4 利用高通量功能性篩選平台測試已知可影響ABC transporter功能的化合物 44
3.3.5 利用高通量功能性篩選平台測試niclosamide衍生物對ABC transporter的影響 45
四、 結論 47
參考文獻 (references) 91
附錄 96

















圖表目錄
圖1. 各種細胞株之ABC transporters蛋白表現情形 53
圖2. Niclosamide在不同ABC transporters 調節螢光染劑運輸的影響 55
圖3. 不同ABC transporter處理濃度梯度niclosamide下其細胞內螢光累積的程度 58
圖4. 藉由[125I] IAAP (iodoarylazidoprazosin) photoaffinity labeling測試niclosamide對ABCB1及ABCG2的影響 59
圖5. Niclosamide 對於ABCB1及ABCG2其 ATPase活性的影響 60
圖6. 各種癌細胞對於niclosamide敏感度及重新回復對傳統抗癌藥物敏感度的效果 61
圖7. Niclosamide在過度表達ABCG2細胞其蛋白質表現及mRNA的影響 69
圖8. 測試高通量功能性篩選平台之最佳細胞數目 71
圖9. 測試高通量功能性篩選平台之最佳培養時間及螢光染劑 (calcein-AM / PhA) 濃度 73
圖10. 測試高通量功能性篩選平台之最佳positive control濃度 76
圖11. 利用高通量功能性篩選平台測試已知可影響ABC transporters功能的化合物 77
圖12. 利用高通量功能性篩選平台測試niclosamide衍生物抑制ABC transporters的效果 79
表1. 利用高通量功能性篩選平台測試niclosamide衍生物其抑制ABC transporters的效果 81
表2. 測試 niclosamide衍生物其重新回復對mitoxantrone敏感度的效果在過度表達ABCG2細胞 89

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