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系統識別號 U0026-2108201315503700
論文名稱(中文) 腫瘤抑制子WWOX在MEK/ERK複合物形成中調控癌細胞凋亡所扮演的角色
論文名稱(英文) Role of tumor suppressor WWOX in MEK/ERK complex formation in cancer cell apoptosis
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 101
學期 2
出版年 102
研究生(中文) 盧貞伃
研究生(英文) Chen-Yu Lu
學號 T16981091
學位類別 碩士
語文別 英文
論文頁數 80頁
口試委員 指導教授-張南山
口試委員-楊倍昌
口試委員-呂增宏
口試委員-許漢銘
口試委員-徐麗君
中文關鍵字 有絲分裂原肌活蛋白激酶激酶1 (MEK1)  含雙色胺酸功能區氧化還原酶 (WWOX/WOX1)  CI-4AS-1  (±)-Blebbistatin  鋅手指樣蛋白 (Zfra) 
英文關鍵字 MEK/WWOX complex  CI-4AS-1  (±)-Blebbistatin  Zfra 
學科別分類
中文摘要 Mitogen-activated protein kinase (MAPK) pathway是一重要的訊息傳導路徑,在許多癌細胞中可發現大量的Extracellular signal-regulated kinase (ERK)活化,使癌細胞不斷增生,故為一重要的藥物標的。我們實驗室發現了一個調控血癌T細胞凋亡的重要開關,MEK/WWOX複合物,我們認為化學藥物無法引起癌細胞凋亡的可能原因為:無法解開MEK/WWOX複合物或是其他複合物,亦或是癌細胞的WWOX有缺失。我們利用急性淋巴母細胞白血病細胞MOLT-4測試23種化學藥物,藉由共軛焦螢光顯微鏡觀察,發現有五種藥物可以解開MEK/WWOX複合物。我們選了其中兩種藥物CI-4AS-1及 (±)-Blebbistatin,進行後續的實驗。利用流式細胞儀分析細胞週期,發現這兩種藥物可以引起MOLT-4細胞、人類大腸癌細胞HCT116及黑色素細胞瘤B16F10不同程度的細胞凋亡。藉由共同免疫沉澱發現,CI-4AS-1或 (±)-Blebbistatin可隨投藥時間進程,解開MEK/WWOX或ERK/WWOX複合物,而且和細胞死亡有關。我們利用免疫健全的BALB/c小鼠,預先注射三個禮拜CI-4AS-1、 (±)-Blebbistatin及同時加入或不加入鋅手指樣蛋白Zfra4-10,休息一個星期後,於小鼠兩側背上進行皮下注射黑色素細胞瘤B16F10,觀察發現以上治療策略皆有效抑制黑色素細胞瘤B16F10生長,經過60天後犧牲實驗小鼠,將其胰臟細胞分離 (primary culture),一部分細胞經由尾靜脈注射,轉移到免疫缺陷小鼠NOD-SCID,一部分細胞藉由流式細胞分選儀分析,發現以上治療策略成功抑制腫瘤生長,很可能是透過誘發一種新型的非T、非B記憶性免疫細胞,我們命名為 Z cells。我們將繼續探究並研擬可行的治療策略。
英文摘要 We have identified a MEK/WWOX (mitogen-activated protein kinase kinase 1/WW domain-containing oxidoreductase) complex as a master switch for apoptosis in T cell leukemia. Failure of chemical drugs-induced cancer apoptosis may due to their inability to dissociate the MEK/WWOX complex, other complex or loss of WWOX. We tested 23 chemicals in MOLT-4 T cells for inducing the MEK/WWOX dissociation. Confocal microscopy analysis revealed that 5 drugs could reduce the co-localization of endogenous WWOX and MEK1. We chose two chemical drugs for further investigation. One is CI-4AS-1, a steroidal androgen receptor agonist (AR agonist), and the other one (±)-Blebbistatin. Cell cycle analysis showed that these drugs induced various extent of apoptosis in MOLT-4 T cells, human colon cancer HCT116 cells and malignant melanoma B16F10. In MOLT-4 T cells, co-immunoprecipitation data revealed that CI-4AS-1 and (±)-Blebbistatin reduced the MEK/WWOX or ERK/WWOX complex binding in a time-dependent manner, which correlates with cell death. Immune competent BALB/c mice were pre-injected with CI-4AS-1 or (±)-Blebbistatin with or without zinc finger-like Zfra4-10 peptide for 3 consecutive weeks, and then inoculated with malignant melanoma B16F10 cells. We determined that these indicated treatments blocked the growth of B16F10 cells may via activation of a new non-T and non-B memory spleen cells, designated called “Z cells”. More efforts are being underway to elucidate the underlying death mechanisms, as well as to search for feasible drugs for therapeutic purposes.
論文目次 Table of contents
中文摘要 I
English abstract II
致謝 III
Index of figures VI
Abbreviation VIII
Introduction 1
Goals of this study 1
WW domain-containing oxidoreductase (WWOX) 2
Chemical drugs: CI-4AS-1 and (±)-Blebbistatin 9
Zinc finger-like protein that regulates apoptosis (Zfra) 10
Adoptive cell transfer (ACT) immunotherapy 12
Materials and Methods 14
Cell lines 14
Chemicals and antibodies 14
Immunofluorescence 15
Förster (fluorescence) resonance energy transfer (FRET) 15
Cell cycle analysis 16
Co-immunoprecipitation and immunoblotting 16
Zfra peptide, CI-4AS-1, (±)-Blebbistatin preparation and in vivo test 17
Wwox Knockout Mice generation 19
Spleen cells isolation and surface marker analysis 19
Results 21
Chemical drugs induce dissociation of the MEK/WWOX complex 21
CI-4AS-1 or (±)-Blebbistatin induces apoptosis and/or G2/M arrest in cancer cells 21
CI-4AS-1 or (±)-Blebbistatin dissociates the MEK/WWOX or ERK/WWOX complex in MOLT-4 T cells, which is associated with cell death 24
Dominant-negative WWOX suppresses CI-4AS-1- or (±)-Blebbistatin-induced apoptosis and/or G2/M arrest in cancer cells 25
Anticancer efficacy of chemical drugs and “Z cells” activation in vivo 27
Z cell characterization and activation in vivo and in vitro 30
Discussion 32
References 36
Figures 41

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