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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 李明亭(Ming-Ting Lee) | |
dc.contributor.author | Chun-Yu Lin | en |
dc.contributor.author | 林俊宇 | zh_TW |
dc.date.accessioned | 2021-06-08T05:04:17Z | - |
dc.date.copyright | 2011-02-20 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-02-09 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/23584 | - |
dc.description.abstract | 癌細胞的轉移是造成癌症病患死亡的主要原因之一,而轉移過程則包含著一連串複雜的過程,包括癌細胞原位的入侵,緊接著惡性腫瘤細胞的脫落、於循環系統中的移動,最後再吸附於特定位置上並大量生長,造成癌細胞的轉移。根據目前臨床上的研究,腫瘤細胞的入侵在癌症細胞轉移的過程為一決定性的速率決定步驟,然而其分子機制到目前為止了解仍相當有限,因此需要進一步得探討。
近年來,皮質-間葉細胞型態轉變[epithelial-mesenchymal transisiton (EMT)]被報導在腫瘤細胞轉移的過程中扮演著重要的角色。而在EMT的過程中,有一群zing-finger轉錄因子,包含Snail、Slug、Twist以及matrix metalloproteinases (MMPs)的表現會受到誘導,而這些與癌細胞入侵及移動能力是有關連的。先前實驗室已取得高入侵性的子宮頸上皮皮膚癌子代細胞A431-III sub-line,此細胞具有大量的MMP-9,同時我們也證實,A431-P與A431-III細胞為一可用來探討EMT過程的模式系統,因此為了深入了解EMT在癌細胞中的角色,我們嘗試探討MMP-9與EMT之間的關係。首先我們將MMP-9處理A431母代細胞 (A431-P),發現會造成細胞型態有類似於EMT的轉變,型態的轉變如同A431-III細胞;另外,我們將MMPs抑制劑GM6001同時處理A431-P與III細胞,發現某些EMT標的物質,如Vimentin貨fibronectin的表現量會有所下降。我們也利用MMP-9 siRNA阻斷MMP-9的表現,發現會破壞MMP-9所執行的細胞-細胞間的連接,進而造成細胞入侵,移動能力的下降,連同造成Snail的表現量下降與細胞骨架排列的改變。另外利用Snail siRNA抑制Snail的表現也會造成類似的結果。這些結果闡述著A431-III細胞中MMP-9大量的表現,會直接誘導EMT過程的發生,而Snail蛋白的表現也會同時與MMP-9共同作用,但我們對於兩者間相互調控的機制尚不清楚。 組織轉麩胺酵素(Tissue transglutaminase, TG2)為一廣泛存在於細胞中,催化蛋白質間連結的酵素。近年來被報導與癌細胞的移動、入侵、貼附與轉移有關。實驗室之前的研究證實TG2的大量表現與A431-III細胞貼附、轉移與入侵能力的提升是有關的,而在此篇研究中,我們進一步證實TG2藉由誘導EMT的發生促進A431-III轉移能力的提升。我們利用A431-P大量表現TG2以及A431-III抑制TG2的表現,發現TG2會藉由改變Snail的量,進而造成EMT現象的發生,最終影響MMP-9的釋放以及細胞移動能力。同時,我們的數據亦證實,TG2會藉由NF-kB以及PI3K/Akt的路徑,進而影響Snail的表現,最終影響EMT的現象。我們的實驗結果進一步證實TG2的確為一腫瘤細胞轉移治療的標的,亦期許未來能以抑制TG2的表現,發展出抑制腫瘤轉移相關治療方式。 | zh_TW |
dc.description.abstract | Cancer metastasis is the main cause of human cancer death. The metastatic progression is a complex and multistep process that entails local invasion, followed by dissemination of the malignant cells, and finally re-establishment at distant sites resulting in metastasis formation. Invasion is a critical step in cancer metastasis; however, the underlying molecular mechanism still remains unclear and need to be further elucidated. One of the most fundamental biological processes in tumor metastasis is the process of epithelial-mesenchymal transition (EMT). During EMT, zinc-finger-family of transcription factors such as Snail, Slug, and Twist, and matrix metalloproteinases (MMPs) are up-regulated, and this correlates with increased tumor cell invasion and motility.
We previously obtained a highly invasive A431-III tumor sub-line, which is a rich source of MMP-9 and observed a plausible link between MMP levels and the promotion of EMT. To gain further understanding of EMT, we investigated the contribution of distinct MMPs to the induction of EMT. Exposing A431, cervical carcinoma parental cells, to MMP-9 stimulated a phenotypic alteration and cells became spindle-like as shown for A431-III cells. In the present communication, we document changes in gene expression profiles of A431-P and A431-III cells, including those of genes involved in cell adhesion, cytoskeleton reorganization, polarity, migration and transcription. Treatment of both A431-P and A431-III cells with GM6001, a broad spectrum MMP inhibitor, resulted in the diminution of vimentin and fibronectin, indicating a role for MMP-9 in the induction of EMT. Abrogation of MMP-9-mediated cell-cell contact in both A431-P and A431-III cells using MMP-9 siRNA resulted in decreased cell invasion, motility and altered cytoskeleton arrangement together with a reduction in Snail expression. Knockdown of Snail resulted in similar changes along with diminished MMP-9 expression. We speculate that the overexpression of MMP-9 in A431-III cells might directly induce (or stimulate) EMT and that the transcriptional factor, Snail, could cooperatively engage in this phenomenon. The enzyme, tissue transglutaminase 2, a ubiquitously occurring, Ca2+-dependent enzyme that catalyzes the covalent cross-linking of proteins, is implicated in cell migration, invasion, adhesion and cancer metastasis. Herein we have confirmed that in A431-III cells, TG2 modulated the induction of EMT. In A431-III knockdown and A431-P over-expression study with these cells revealed that TG2 induced a mesenchymal-like phenotype and accentuated cell motility, mediated by changes in the expression of Snail. The findings are also suggestive that TG2 triggers the activation of Akt/PKB pathway and of NF-kB signaling pathway, resulting in the subsequent induction of Snail expression and promotion of EMT in A431-III cells. These results support our contention that TG2 is a promising therapeutic target for reversing drug resistance and inhibiting the early advent of metastasis of tumor cells. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T05:04:17Z (GMT). No. of bitstreams: 1 ntu-100-D94b46008-1.pdf: 11345708 bytes, checksum: e00ba9237dcc55ef1b4274812511b5b0 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | Abstract ..........................................................................................................................1
中文摘要........................................................................................................................3 Introduction...................................................................................................................5 Cancer metastasis and invasion.............................................................................5 The role of Epithelial Mesenchymal transition (EMT) in cancer progression ......7 Matrix metalloproteinases (MMPs) in EMT and cancer metastasis ...................11 Snail in EMT and cancer metastasis....................................................................14 NF-κB signaling in EMT and cancer metastasis .................................................16 PI3K/Akt signaling in EMT and cancer metastasis .............................................20 The role of Tissue transglutaminase (TG2) in cancer biology.............................22 The role of TG2 in EMT.......................................................................................26 Significance and purpose .............................................................................................28 Material and Methods ..................................................................................................30 Materials ..............................................................................................................30 Preparation of cell lysates and nuclear extracts .................................................30 Gene expression microarray analysis..................................................................31 Western blot..........................................................................................................32 Reverse transcriptase-polymerase chain reaction (RT-PCR)..............................32 Transfection of small interference RNA (siRNA) .................................................33 MMP-9, Snail and TG2 genes construction and transfection..............................33 NF-κB reporter luciferase assay..........................................................................35 Immunoprecipitation............................................................................................35 Gelatin zymography.............................................................................................36 Treatments of A431-P and A431-III cells with activated MMP-9........................36 E-cadherin neutralization ....................................................................................36 Immunofluorescence staining ..............................................................................37 In vitro wound-healing migration assay ..............................................................37 Statistical analysis ...............................................................................................38 Result ...........................................................................................................................40 Characterization of the EMT-related markers in A431-P and A431-III cells......40 Potential role of MMP-9 in facilitation of EMT process in A431-P and III sub-line.................................................................................................................41 Potential role of Snail in facilitation of EMT process in A431-P and III sub-line.............................................................................................................................44 Modulation of mesenchymal marker expression in A431-P and A431-III cells by tissue transglutaminase (TG2).............................................................................45 Modulation of Snail by TG2.................................................................................47 ii Association of NF-κB activity with Snail expression and migratory potential in A431-P and III sub-line .......................................................................................47 Effect of TG2-mediated increase of NF-κB signaling on Snail expression and migratory potential ..............................................................................................48 Associaiton of PI3K/Akt-GSK3β axis signaling with Snail expression and migratory potential in A431-P and III sub-line ...................................................51 Effect of TG2-mediated increase of PI3K/Akt signaling on Snail expression and migratory potential ..............................................................................................53 Disscussion ..................................................................................................................75 Supplemental data........................................................................................................90 Appendix......................................................................................................................94 Reference ...................................................................................................................114 | |
dc.language.iso | en | |
dc.title | 轉麩胺酵素2與高入侵性A431子代細胞之皮質-間葉細胞型態轉變關連性之探討 | zh_TW |
dc.title | Involvement of Tissue Transglutaminase 2 in acquisition of mesenchymal-like phenotype in highly invasive A431 sub-line tumor cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 黃火鍊,黃銓珍,張震東,陳宏文,柯逢春,蕭培文 | |
dc.subject.keyword | 轉麩胺酵素,皮質-間葉細胞型態轉型,子宮頸上皮皮膚癌細胞,癌細胞轉移,癌細胞入侵, | zh_TW |
dc.subject.keyword | Transglutaminase,MMP-9,Snail,Epithelial-Mesenchymal Transition,PI3K/Akt,NF-kB,cancer metastasis,cancer invasion, | en |
dc.relation.page | 129 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2011-02-09 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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