金屬基質蛋白酶(MMPs)在細胞外基質(ECM)的降解中扮演著關鍵角色，金屬基質蛋白酶的上調可促進癌細胞的遷移和侵襲，而表皮生長因子(EGF)引起的表皮生長因子受器(EGFR)活化已被證實參與金屬基質蛋白酶的上調，過去研究亦證實EGFR的訊息傳遞路徑的異常是乳癌的惡性表徵，因此抑制EGF/ EGFR的活化路徑可作為乳癌治療的策略。沒食子酸(Gallic acid)，是一種酚類單寧酸，廣泛分布於自然植物及果實中，過去的研究指出沒食子酸可下調癌細胞中金屬基質蛋白酶MMP-2和MMP-9的表現的活性。因此本論文之研究目標旨在探討乳癌MCF-7細胞中，沒食子酸對表皮生長因子誘導之金屬基質蛋白酶表現的效應，以及其對表皮生長因子的訊息傳遞路徑之調控機制。實驗結果顯示，EGF能夠上調MMP-9表現，提升MCF-7細胞的遷徙與侵襲能力，而此作用係透過活化EGFR及Src，並在EGFR及Src協同調控下，活化Akt/NFκB以及ERK/c-Jun訊息傳遞路徑。沒食子酸透過抑制EGFR與Src間的相互調控，達到抑制下游Akt/NFκB及ERK/c-Jun活化作用，此外沒食子酸的衍生物在MCF-7細胞中亦具有抑制EGF上調MMP-9表現以及細胞的遷徙與侵襲的能力。綜合上述結果顯示，沒食子酸及其衍生物透過抑制表皮生長因子活化EGFR/Src/Akt/ERK的訊息傳遞軸徑，下調MMP-9的表現，進而抑制乳癌MCF-7細胞的遷徙與侵襲。

Matrix metalloproteinases (MMPs) play a key role in the degradation of extracellular matrix, and up-regulation of MMP expression promotes invasion and migration of cancer cells. EGF-elicited EGFR activation has been proven to be involved in up-regulating MMP expression. Aberrant EGFR-mediated signaling pathway causes the malignancy of breast cancer. Thus, suppression of EGF/EGFR-mediated signaling should be a promising strategy in improving breast cancer therapy. Gallic acid is a polyhydroxylphenolic compound naturally occurring in various plants and fruits. Previous studies showed that gallic acid could down-regulate MMP-2 and MMP-9 expression in cancer cells. Thus, the aim of the present study is to explore whether gallic acid regulates EGF-induced MMP expression and EGF/EGFR- mediated signaling pathway in breast cancer MCF-7 cells. EGF treatment promoted MCF-7 cell invasion and migration, and up-regulated MMP-9 mRNA and protein levels via activation of EGFR and Src. Activated EGFR and Src synergistically induced phosphorylation of Akt/NFκB and ERK/c-Jun, leading to MMP-9 up-regulation. Gallic acid abolished the synergy effect of EGFR and Src. Consequently, gallic acid induced Akt/NFκB and ERK/c-Jun inactivation. Meanwhile, gallic acid derivatives including syringic acid, 3, 4, 5-trimethoxybenzoic acid and methyl gallate also showed inhibitory effect on EGF-induced MMP-9 expression and migration/invasion in MCF-7 cells. Taken together, our data reveal that gallic acid and its derivatives suppress EGFR/Src/Akt/ERK axis-mediated MMP-9 expression, leading to inhibit EGF-induced invasion and migration of human breast cancer MCF-7 cells.

論文審定書 i
論文公開授權書ii
致謝 iii
中文摘要 iv
Abstract v
目錄 vi
圖次 viii
前言 1
乳癌 (Breast cancer) 1
表皮生長因子 (Epidermal Growth Factor) 2
基質金屬蛋白酶 (Martrix metalloproteinases ; MMPs) 3
沒食子酸 (Gallic acid) 4
實驗目的 5
縮寫表 6
實驗材料 7
細胞株 7
試劑與分析套組 7
組織培養材料與化學藥品名稱 7
抗體 8
質體 8
實驗方法 9
細胞培養 9
解凍細胞 9
藥物處理細胞 9
細胞存活率測試(Relative survival rate) 9
細胞蛋白質的萃取(Protein Extraction) 10
聚丙烯醯胺膠體電泳法(SDS-PAGE) 10
西方點墨法(Western Blot) 10
DNA轉殖(DNA transfection) 11
逆轉錄聚合酶鏈鎖反應(reverse transcription-PCR, RT-PCR) 11
細胞傷口癒合分析 (Wound healing assay) 12
細胞移動及侵犯試驗 (Transwell migration and invasion assays) 12
明膠酶譜電泳分析(Gelatin zymography) 13
統計分析 13
結果 14
沒食子酸對於細胞存活率的影響 14
沒食子酸對於細胞分泌MMP-2、MMP-9沒有顯著的影響 14
沒食子酸可抑制表皮生長因子誘導MCF-7細胞MMP-9表現 14
沒食子酸可抑制表皮生長因子誘導的細胞遷徙與細胞侵襲 15
沒食子酸可抑制表皮生長因子誘導的ERK、Akt、NFB、AP-1及Src的活化 15
表皮生長因子誘導MMP-9表現需倚賴Src的活化 16
表皮生長因子透過活化ERK、Akt誘導MMP-9表現上升 17
ERK、Akt過度表達可回復沒食子酸所抑制之表皮生長因子誘導的MMP-9表現 17
Src參與調控表皮生長因子誘導的MMP-9表現 17
沒食子酸衍生物可抑制表皮生長因子誘導的MMP-9表現增加、細胞遷徙與侵襲 18
討論 19
附圖 22
參考文獻 35

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