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研究生:陳懿華
研究生(外文):I-Hua Chen
論文名稱:3,4-Methylenedioxy-β-nitrostyrene抑制三陰性乳癌細胞轉移的作用機轉探討
論文名稱(外文):Studies on the anti-metastatic effects and mechanisms of action of 3,4-methylenedioxy-β-nitrostyrene in triple-negative breast cancer cells
指導教授:吳志中吳志中引用關係
指導教授(外文):Chin-Chung Wu
學位類別:博士
校院名稱:高雄醫學大學
系所名稱:天然藥物研究所博士班
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:129
中文關鍵詞:34-methylenedioxy-β-nitrostyrene (MNS)三陰性乳癌轉移β1整合蛋白蛋白質雙硫鍵轉位&;#37238
外文關鍵詞:34-methylenedioxy-β-nitrostyrene (MNS)triple-negative breast cancermetastasisβ1 integrinprotein disulfide isomerase
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三陰性乳癌是一種臨床上高轉移且易惡化的乳癌類型。已知整合蛋白 (integrin) 所調控的細胞貼附 (adhesion) 及移動 (migration)是促使乳癌轉移的關鍵角色。3,4-Methylenedioxy-β-nitrostyrene (MNS) 是合成的小分子化合物。本研究發現MNS具有抑制三陰性乳癌細胞與不同的細胞外基質貼附的能力。在不影響細胞的存活率之下,透過體外傷口癒合試驗及Boyden chamber實驗也發現MNS明顯地抑制乳癌細胞移動及侵襲 (invasion) 的能力。以共軛焦顯微鏡觀察經MNS處理過的細胞,發現細胞骨架中張&;#63882;纖維 (stress fiber) 的聚合及附著點複合物 (focal adhesion complex) 的形成皆明顯地減少;且西方墨點法測得細胞內訊息傳遞因子FAK及paxillin的磷酸化也受到抑制。由共軛焦顯微鏡及流式細胞儀的實驗結果得知,MNS抑制FAK的磷酸化,是由於減少β1整合蛋白的活化和叢聚現象引起的。當前加或後加不能穿透細胞膜之含雙硫氫(dithiol)抗氧化劑 (2, 3-dimercaptopropane-1-sulfonic acid),能阻斷 MNS的作用發生。這結果指出:MNS可能會與細胞膜蛋白上具有反應性的thiol基團作用,且此牽涉到調節β1整合蛋白的功能以及細胞貼附和移動的能力。細胞膜表面的蛋白質雙硫鍵轉位&;#37238; (protein disulfide isomerase, PDI) 已被證實能影響整合蛋白的活化。本研究發現不論是在完整的癌細胞或純化的重組蛋白試驗中,MNS均能明顯地抑制其活性。總結來說,我們的研究證明MNS經由抑制β1整合蛋白的活化及阻斷FAK的訊息,減少三陰性乳癌細胞轉移的能力。而且此抑制作用至少有部分是透過抑制PDI的活性而來。這些結果說明了MNS可能有潛力發展為一個新型的抗轉移藥物,可望改善三陰性乳癌治療的困境。

Triple negative breast cancer (TNBC) exhibits an aggressive clinical course by high metastatic potential. It is known that integrin-mediated cell adhesion and migration are important for cancer metastasis. In the present study, a synthetic compound, 3, 4-methyenedioxy-β-nitrostyrene (MNS), significantly inhibited adhesion of TNBC cell lines to different extracellular matrix (ECM) components. The antimetastatic capacity of MNS was also observed through reducing TNBC cells migration and invasion without affecting cell viability. Confocal microscopy revealed that MNS disrupted the formation of focal adhesion complex and actin stress fiber networks. Consistent with this finding, MNS inhibited phosphorylation of focal adhesion kinase (FAK) and paxillin as detected by Western blot analysis. In exploring the underlying mechanism, we found that MNS inhibited phosphorylation of FAK as a result of reducing β1 integrin activation and clustering. A cell-impermeable dithiol reagent, 2, 3-dimercaptopropane-1-sulfonic acid abrogated all of MNS’s actions, indicating that MNS may react with thiol groups of cell surface proteins that are involved in regulation of β1 integrin function as well as cell adhesion and migration. Cell surface protein disulfide isomerase (PDI) has been reported to be essential for the affinity modulation of β integrins. We also demonstrated that MNS inhibited PDI activity both in a pure enzyme system and in intact cancer cells. Taken together, our results suggest that MNS inhibits in vitro metastatic properties of TNBC cells through suppression of β1 integrin activation and focal adhesion signaling. Moreover, inhibition of surface PDI may contribute, at least in part, to the actions of MNS. These results suggest that MNS has a potential to be developed as an anticancer agent for treatment of TNBC.

縮寫檢&;#63850;表 3
中文摘要 5
英文摘要 7
第一章 緒論 9
第一節 文獻回顧 10
一 癌症的發生 10
二 癌症的轉移 13
三 三陰性乳癌 20
四 整合蛋白 (Integrin) 24
五 焦點附著激&;#37238; (Focal adhesion kinase) 28
六 蛋白質雙硫鍵轉位&;#37238; (Protein disulfide isomerase) 32
七 化合物MNS (3,4-Methylenedioxy-β-nitrostyrene) 35
第二節 研究動機及目的 37
第二章 實驗材料及方法 48
第一節 實驗材料 49
第二節 實驗方法 52
第三章 實驗結果 63
第四章 討論 91
第五章 結論與展望 98
參考文獻 101
研究成果 126


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