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系統識別號 U0026-0812200915114382
論文名稱(中文) 探討樺木酸 (betulinic acid)抑制轉錄因子Sp1的分子機轉
論文名稱(英文) Mechanism of inhibition of transcription factor specificity protein 1 by betulinic acid
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 97
學期 2
出版年 98
研究生(中文) 黃詩婷
研究生(英文) Shih-Ting Huang
學號 s2696406
學位類別 碩士
語文別 中文
論文頁數 59頁
口試委員 指導教授-張文昌
口試委員-呂增宏
指導教授-洪建中
中文關鍵字 泛素化  類泛素化  轉錄因子  樺木酸 
英文關鍵字 Sp1  caspases  betulinic acid  degradation  rpt6  ubiquitination  SUMOylation 
學科別分類
中文摘要 樺木酸 (betulinic acid)是一種萃取自白樺樹的五環三萜類化合物 (pentacyclic triterpenoid),具有抗腫瘤、抗病毒和抗發炎的活性,其中以抗癌活性的研究最為廣泛。過去的研究指出betulinic acid會誘發癌細胞凋亡 (apoptosis),抑制腫瘤生長和對抗血管新生,且對於正常細胞與癌細胞間具有選擇性的毒殺作用,展現其在癌症治療的潛力,然而,betulinic acid抑制癌症的詳細作用機轉仍有待進一步研究。Sp1 (specificity protein 1)為普遍表現於不同細胞中且調控許多基因表現的轉錄因子,它影響細胞增生、血管新生等相關基因的表現,例如:血管內皮生長因子 (VEGF)、表皮生長因子受體 (EGFR)等,以促進癌細胞的增生和轉移。因此,本研究想要探討在HeLa細胞中betulinic acid對於Sp1的影響及其詳細的作用機轉。實驗結果發現betulinic acid會降低Sp1的表現量,且呈濃度依賴性 (concentration-dependent),同時,betulinic acid會增加Sp1的蛋白質後轉譯修飾SUMOylation以及ubiquitination,使Sp1與蛋白解體次單元 (proteasome subunit) Rpt6間的相互作用提高,促進Sp1進行proteasome-dependent的降解,進而降低Sp1蛋白質的穩定度。我們進一步以過度表現 (overexpression) HA-Sp1-myc和HA-SUMO-1-Sp1-myc的方式,探討betulinic acid對於SUMO-1-Sp1的影響。結果發現,在betulinic acid的處理下,只些微降低HA-Sp1-myc蛋白的表現,但卻能顯著降低HA-SUMO-1-Sp1-myc蛋白的表現。另外,先前的研究顯示當細胞凋亡與caspases活化時,Sp1會被分解產生68 kDa-Sp1的片段。我們觀察到在不同濃度的betulinic acid (10 μM、20 μM、30 μM、40 μM)處理下,相較於處理DMSO的控制組,Sp1皆會被切割成68 kDa-Sp1的片段,且非特異性caspases抑制劑ZVAD-fmk會阻斷betulinic acid所誘導的Sp1切割。綜合上述實驗結果,betulinic acid會增加Sp1的蛋白質後轉譯修飾SUMOylation與ubiquitination,以及促進caspases-dependent的Sp1切割,導致子宮頸癌HeLa細胞中高度表現的Sp1蛋白降低,因此,betulinic acid具有潛力發展成為Sp1-targeting的癌症治療藥物。
英文摘要 Betulinic acid is a pentacyclic triterpene natural product identified as an effective inhibitor of cell proliferation, which induces apoptotic cell death and antiangiogenic responses in various cancer cells. However, the underlying mechanism of these responses is not well understood. Since several tumor-related genes, such as vascular endothelial growth factor and p21WAF1/CIP1, are regulated by specificity protein 1 (Sp1), the primary goal of this research is to investigate the mechanism in which betulinic acid affects the levels of Sp1 in HeLa cancer cells. Using HeLa cervical cancer cells as a model, we found that betulinic acid dose-dependently decreased the levels of Sp1. Besides, betulinic acid increased the ubiquitin levels of Sp1, and then the interaction between Sp1 and the proteasome subunit Rpt6 was enhanced. Previous studies in our laboratory have shown that SUMOylation of Sp1 augments its degradation by increasing the Sp1 proteolytic process. In this study, Western blot analysis revealed that the levels of a high-molecular- weight Sp1 of 125 kDa (SUMO-1-Sp1) are increased by betulinic acid, which facilitates Sp1 ubiquitination and attenuates Sp1 stability. To further examine the possible role of betulinic acid on the SUMOylated Sp1, the HA-SUMO-1-Sp1 was used to mimic the SUMOylated Sp1. We found that the decrease in Sp1 protein within cells expressing SUMO-1-Sp1 was more significant than that in cells expressing wt-Sp1 under betulinic acid treatment. In addition, Sp1 was cleaved into a 68-kDa fragment by caspases during apoptosis. Cleavage did not occur if apoptosis was inhibited by ZVAD-fmk, an inhibitor for caspase 3-like proteases. We next found that betulinic acid induced Sp1 cleavage in a concentration dependent manner, and the betulinic acid-induced Sp1 cleavage could be blocked by ZVAD-fmk, a broad-spectrum inhibitor for caspases. Taken together, the results showed that betulinic acid decreases the levels of Sp1 through both post-translational modification of Sp1, and a caspases-dependent cleavage of Sp1 during betulinic acid-induced apoptosis; hence betulinic acid has the potential for use as a novel class of Sp1-targeting anticancer drugs.
論文目次 中文摘要 I
英文摘要 II
誌謝 III
目錄 IV
圖目錄 V
縮寫檢索表 VII
第一章 緒論 1
第二章 實驗材料 10
第三章 實驗方法 14
第四章 實驗結果 26
第五章 實驗討論 33
參考文獻 39
附圖 45
附表 57
自述 59
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