臺灣博碩士論文加值系統

乳癌好發於女性，根據2013年統計，女性乳癌發生率占第一名，死亡率為第二名，而乳癌主要的治療方式為手術、放療、化療、賀爾蒙療法，或是針對腫瘤中大量表現的蛋白進行標靶治療，先前研究發現很多癌症的抑癌miR-125b表現下降，且指出ENPEP基因是miR-125b下游抑制的一個目標基因，因此在56 % 乳癌病人當中ENPEP基因表現量是上升的。而胺基肽酶A (aminopeptidase A; APA) 是由ENPEP基因所轉譯出來的一個含有鋅離子結合位的穿膜蛋白，由3個domains組成並以homodimer的形式增加APA的穩定性，這個酵素主要功能是切除蛋白N-terminal上的Asp、Glu，存在於腸刷狀緣、腎表皮細胞、及許多組織血管內皮細胞上。因此我們想要探討APA在乳癌中扮演甚麼角色，首先透過免疫組織化學染色法證明隨著乳癌癌化的程度增加，APA表現量也隨之增加，並且使用之前我們實驗室篩選出來抑制APA活性效果最好的藥物compound A ，來看APA對乳癌的影響。發現compound A 透過促進細胞週期停留在G0/G1期，進而抑制乳癌細胞短期及長期的細胞增生，並且能抑制癌細胞移動能力及EMT (epithelial-mesenchymal transition) 的現象。近年來許多研究指出，癌症幹細胞的存在會促進癌症的復發，而我們透過聚球生成試驗發現compound A能抑制聚球的形成，顯示其能抑制癌症幹細胞的特性。並發現compound A主要是透過抑制ERK訊號傳遞路徑，進而抑制癌細胞的生長、移動、聚球能力及EMT。因此compound A可能當作配合乳癌標靶治療的藥物。但是compound A是否是經由抑制APA活性而達到抑制腫瘤生長的效果，未來還要再進一步研究。

Breast cancer is one of the most common cancers in female. According to 2013 statistics from Cancer Journal for Clinicians, the incidence of female breast cancer accounted for the first and the mortality rate was the second among all cancers. The treatments for breast cancer include surgery, radiotherapy, chemotherapy, hormone therapy and target therapy. Previous studies showed that miR-125b is a tumor suppressor miRNA in many tumor types and ENPEP gene is one of miR-125b target genes. Besides, they found that ENPEP gene was overexpressed in 56% breast cancer patients. APA (aminopeptidase A) is an enzyme encoded by ENPEP that cleaves aspartic or glutamic acidic residues from the N-terminus of a large variety of protein substrates. So, our aim is to study the roles of APA in breast cancer. Our tissue array data demonstrated that more advanced tumor correlated with higher APA expression. Furthermore, we use compound A which was shown to inhibit APA activity in our previous study to investigate the relationship between APA and breast cancer. Here we show that compound A can promote cell cycle arrest in G0/G1 phase and inhibit breast cancer cell proliferation, migration and EMT (Epithelial–mesenchymal transition). In recent years, many studies have indicated that CSC (cancer stem cell) can promote cancer recurrence. In our study, we also show that compound A can inhibit sphere formation. Furthermore, we found that compound A inhibits cancer cell proliferation, migration, sphere formation and EMT mainly through blocking ERK signaling pathway. Thus, compound A may be another therapeutic drug for breast cancer. However, whether compound A inhibits tumor growth through APA activity blockade needs further study.

致謝……………………………………………………………………i
中文摘要………………………………………………………………ii
英文摘要………………………………………………………………iii
目錄……………………………………………………………………iv
第一章 緒論……………………………………………………………1
第一節、乳癌 (Breast cancer)…………………………………………………..1
第二節、癌症的轉移 (Metastasis)……………………………………………..2
第三節、癌症幹細胞（Cancer Stem Cell，CSC）………………….……..….3
第四節、胺基肽酶A (Aminopeptidase A)簡介……………………………..…..4
第五節、化合物A (Compound A)簡介…………………………..……………5
第六節、研究動機與目的………………………………..………………….…6
第二章 材料與方法
第一節、實驗材料………………………………………………………………8
第二節、實驗方法…………………………………………………………..…16
第三章 實驗結果
第一節、隨著乳癌癌化程度增加，APA表現量明顯上升………………….27
第二節、選出實驗使用的細胞株MCF7……………………………………..27
第三節、Compound A可以抑制APA酵素活性…………………………….28
第四節、Compound A可以抑制MCF7及MDA-MB-453乳癌細胞增生…..28
第五節、Compound A不會影響MCF10A正常乳細胞、MDA-MB-231、HCC1937及Hs578T乳癌細胞的增生…………………………….………….29
第六節、Compound A對細胞凋亡影響較小……………………………….30
第七節、Compound A可以抑制乳癌細胞週期，使細胞停在G0/G1期…….30
第八節、Compound A可以抑制乳癌細胞移動及EMT的發生……………31
第九節、Compound A可以抑制ERK訊號傳遞路徑………………………31
第十節、不同乳癌細胞內生性ADORA2A表現量…………………………32
第十一節、Compound A可以抑制乳癌幹細胞生長……………………….32
第四章 實驗討論……………………………………………………....33
第五章 參考文獻……………………………………………………....38
第六章 實驗結果圖表…………………………………………………43
第七章 附錄……………………………………………………………58

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