在很多癌症組織或細胞中發現組蛋白乙醯酶(histone acetylases；HATs)
和組蛋白去乙醯酶(histone deacetylases；HDACs)處於不平衡的狀態。
組蛋白去乙醯酶在腫瘤的形成和血管新生扮演關鍵的角色。近年來，
組蛋白去乙醯酶抑制劑快速的開發，它會反轉癌症中不正常表觀遺傳
的改變，對於抗癌是ㄧ個非常有前景的發展。Psammaplin A (PsA)是
ㄧ個天然的溴化酪胺酸衍生物，1987 年首次從Psammaplysilla sponge
這種海綿中萃取出來。過去的文獻指出PsA 會抑制組蛋白去乙醯酶和
DNA 甲基轉移酶，如同腫瘤抑制基因的表觀遺傳修飾，因此為一種
新興的組蛋白去乙醯酶抑制劑。由於還未在人類子宮頸癌細胞上研究
過，其分子機轉也不太清楚，因此我們主要想探討PsA 對人類子宮頸
癌細胞的抗癌效力。結果顯示，PsA 會誘發p21 表現量使細胞週期休
止在G1 期而抑制人類子宮頸癌細胞HeLa 的增殖，並且誘發凋亡性
的細胞死亡。並且PsA 會抑制Mdm2 蛋白的表現量，並且增加它的
泛素化程度，因而降低它與p53 結合的能力，使p53 蛋白泛素化的程
度降低，而影響p53 蛋白的穩定性，延長p53 蛋白的半衰期，而使得
p53 蛋白表現量增加，因而影響下游基因的表現。我們的microarray
資料顯示，處理PsA 會明顯的改變人類子宮頸癌細胞中基因的表現，
有顯著差異的約有266 個基因。並由生物學統整分析這些基因的生物
功能及參與調控轉錄的網路。未來我們將進一步探討p53 蛋白的穩定
性對於PsA 調控細胞生長和基因表現的重要性，並且進一步研究它的
生物相關性。

An imbalance between histone deacetylases (HDAC) and histone
acetyltransferases (HAT) activities has been reported in cancer tissues or cells. HDAC is one of key players in tumorigenesis and angiogenesis. In recent years, inhibition of HDAC is a rapidly growing and very promising target for the development of anticancer drug, and it intended to reverse aberrant epigenetic changes associated with cancer. Psammaplin A (PsA), first identified in the Psammaplysilla sponge in 1987, is a natural bromotyrosine derivative. Recently, it was reported that PsA inhibits both HDAC and DNA methyltransferase (DNMT), as epigenetic modifiers of
tumor suppressor gene, and it has been suggested to be a promising novel HDAC inhibitor. However, it has not been tested in cervical cancer cells, and the accurate mechanism of PsA as a HDAC inhibitor is poorly understood. This study investigated the antitumor effect of PsA on human cervical cancer cells. We found that PsA significantly inhibited the
proliferation of HeLa cells in a dose-dependent manner. Additionally, it induced G1 phase arrest and apoptosis, and it can significantly up-regulate the mRNA and protein expression of p21 by altering p53 stability. We further discovered that PsA inhibited the expression of Mdm2 and decreased the interaction of p53 and Mdm2 by regulating
Mdm2 ubiquitination. Our microarray data showed that PsA treatment of HeLa cells significantly altered the gene expression profile, with about 266 genes undergoing upregulation and downregulation. Bioinformatic analysis revealed that these putative targets belong to diverse functional groups and transcriptional networks. In the future we will confirm the importance of p53 stability in PsA-mediated cell growth and gene expression control, and further investigate its biological relevance.

目錄
指導教授推薦書
口試委員會審定書
長庚大學授權書………………………………………………………...iii
致謝……………………………………………………………………...iv
中文摘要………………………………………………………………...vi
英文摘要……………………………………………………………….viii
目錄……………………………………………………………………….x
第一章 簡介……………………………………………………………1
1.1 組蛋白的修飾作用......................................................................1
1.1.1 組蛋白去乙醯酶.................................................................1
1.1.2 組蛋白去乙醯酶抑制劑.....................................................2
1.1.3 組蛋白去乙醯酶抑制劑結構分類.....................................3
1.2 Psammaplin A...............................................................................3
1.3 p53.................................................................................................5
1.3.1 調控p53 的穩定性.............................................................6
1.3.2 Mdm2 和p53 的降解..........................................................7
1.3.3 Mdm2 的功能......................................................................8
第二章 實驗材料與方法......................................................................10
xi
2.1 藥物來源....................................................................................10
2.2 細胞培養....................................................................................10
2.3 細胞增殖....................................................................................11
2.4 西方墨點法................................................................................11
2.5 細胞RNA抽取...........................................................................13
2.6 反轉錄-聚合酶連鎖反應..........................................................14
2.7 定量PCR....................................................................................14
2.8 細胞轉染....................................................................................15
2.9 Luciferease報導基因分析..........................................................15
2.10 細胞週期及細胞凋亡之分析..................................................16
2.11 微陣列晶片..............................................................................16
第三章 實驗結果與討論......................................................................18
3.1 Psammaplin A抑制人類子宮頸癌細胞的增殖.........................18
3.2 Psammaplin A增加細胞週期G1期的分佈，進而誘發細胞
亡.................................................................................................18
3.3 Psammaplin A 誘發人類子宮頸癌細胞中p21 和p53 的表
量..............................................................................................19
3.4 Psammaplin A經由p53 independent誘發人類骨髓瘤細胞中p21
的表現量.....................................................................................21
xii
3.5 Psammaplin A影響人類子宮頸癌細胞中p53蛋白的半衰期...21
3.6 Psammaplin A 降低p53 蛋白的泛素化....................................22
3.7 Psammaplin抑制Mdm2泛素黏合酶的活性..............................23
3.8 藉由microarray來分析Psammaplin A抑制細胞增殖所扮演的
生物角色.....................................................................................25
第四章 結論.............................................................................................27
第五章 參考文獻.....................................................................................30
第六章 圖示.............................................................................................35
第七章 表格.............................................................................................47
第八章 附錄.............................................................................................67
xiii
圖表目錄
圖一 HeLa細胞處理PsA後，抑制細胞生長狀況和引發凋亡的情
形...............................................................................................................35
圖二 PsA 誘發人類子宮頸癌細胞HeLa 內p53 和p21 mRNA 和蛋白
質表現以及p21 的轉錄性........................................................................38
圖三 PsA 誘發人類骨髓瘤細胞株Saos-2 內p21 mRNA 和蛋白質表
現...............................................................................................................40
圖四 PsA 影響p53 蛋白的穩定性..........................................................41
圖五 PsA 影響p53 的蛋白泛素化..........................................................42
圖六 PsA 抑制Mdm2蛋白的泛素化......................................................43
圖七 Microarray 經電腦分析擷取在兩組間有特別明顯表現差異的基
因，將這266 個基因利用PANTHER 分析其生物能...........................45
圖八 Microarray 中可能被p53 所調控的58 個基因，利用PANTHER
分析其生物功能.......................................................................................46
表一 PsA 改變HeLa 細胞的基因表現...................................................47
表二 Gene Go-轉錄調節分析.................................................................57
表三 p53 轉錄因子所調控的基因..........................................................59

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