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標題: | 乳癌細胞株MCF-7引發多重抗藥性之機轉探討 The Mechanisms of Multidrug Resistance Development in the Breast Cancer Cell Line MCF-7 |
作者: | Shang-Hsun Tsou 鄒尚勳 |
指導教授: | 陳燕惠 |
關鍵字: | 癌幹細胞特徵,突變型p53基因,上皮-間葉轉化,P-糖蛋白,艾黴素,細胞凋亡,細胞核因子B,磷脂?肌醇3激?,乳癌, Cancer stem cell-like cells,Mutant p53,Epithelial-mesenchymal transition,P-glycoprotein,Doxorubicin,Apoptosis,NF-,PI3K,Breast cancer, |
出版年 : | 2015 |
學位: | 博士 |
摘要: | 癌細胞產生對於單一或是多種化療藥物的抵抗作用稱為多重抗藥性(MDR)。多重抗藥性的發生是化學治療失敗的重要原因,其細胞機制涉及轉運蛋白表現,增強排除藥物至細胞外的功能、並減少藥物進入細胞的量,或藉由變更藥物標的、抑制細胞凋亡程序、增進細胞DNA受損後修復能力、活化解毒與藥物代謝基因來達成。對於多重抗藥性的研究,過去多是探討野生型與多重抗藥性型癌細胞的各種表現差異,但對於發生過程的機轉仍不清楚。本研究利用人類乳癌細胞株MCF-7野生型 (MCF-7/WT) 作為起始,建立一系列對doxorubicin有抗藥性的細胞株,來探討誘發多重抗藥性過程中有哪些基因參與其中? 而其相互關係如何? 本實驗室將MCF-7/WT培養在含1 nM doxorubicin的培養基中,待細胞生長穩定後,增加藥物濃度,以二的次方的doxorubicin濃度篩選具有抗藥性的細胞株稱為MCF-7/ADR-n,n為1 nM的倍數。總共建立了MCF-7/ADR-1、MCF-7/ADR-2、MCF-7/ADR-4、MCF-7/ADR-8、MCF-7/ADR-16、MCF-7/ADR-32、MCF-7/ADR-64、MCF-7/ADR-128、MCF-7/ADR-256、MCF-7/ADR-512、MCF-7/ADR-1024等11個不同抗藥性程度的細胞株。細胞培養的終點止於MCF-7/ADR-1024,其與抗藥型細胞株MCF-7/ADR具有相同程度的抗藥性及P-glycoprotein (P-gp)表現。研究結果發現MCF-7 / ADR-1024和MCF-7 /ADR具有相近的細胞型態與DNA倍體狀態。基因表現方面,MCF-7/ADR-1024和MCF-7/ADR在細胞修復基因p53、BRCA1/2,細胞凋亡相關基因Bcl-2和上皮-間葉轉換(EMT)的上皮特徵基因E-cadherin的基因表表現量皆下降;而解毒酶GST-π與蛋白激酶PKC-α表達皆上升。上皮特徵基因E-cadherin表現下降以及間葉特徵基因N-cadherin、Slug、Twist,與 Vimentin的表現上升,證實抗藥性發生過程中上皮-間葉特性發生轉化。此外,MCF-7/ADR-1024經PI3K / AKT抑製劑wortmannin處理後,上皮-間葉特徵基因Slug, Twist表現量下降及轉運蛋白P-gp表現量下降。MCF-7 / ADR-1024和MCF-7 /ADR有相似的微球體形成能力及癌幹細胞特徵簇群CD44 high/CD24 low。同時,具有相同突變形式的p53基因,兩者皆缺失密碼子127-133。更進一步證實突變型p53參與了抗藥性發生的過程。本研究將MCF-7/ADR中帶有缺失密碼子127-133的突變型p53基因轉殖進入MCF-7/WT野生型,建立MCF-7/del p53細胞株。結果發現MCF-7/del p53能表現轉運蛋白P-gp,並顯著活化MDR-1啟動子。以NF-κB抑制劑cyclosporine處理,MCF-7/del p53對於doxorubicin的敏感性會增加。此外MCF-7/del p53與MCF-7/ADR細胞型態近似;間葉特徵基因Slug、Vimentin表現上升而上皮特徵基因CDH1表現下降。與MCF-7/WT比較,MCF-7/del p53具有高度的遷徙能力,癌幹細胞特徵簇群表現與微球體形成能力增加。因此具缺失密碼子127-133的突變型p53基因可能是藉由活化NF-κB信號傳導及MDR-1啟動子、並影響上皮-間葉轉化與癌幹細胞特徵表達,來參與多重抗藥性的發生。 結論 多重抗藥性發生過程中,在篩選劑量為1.024 μM doxorubicin下,P-gp才得以表現。P-gp的表現與一些相關基因的表現或是突變有關,這些基因如DNA修復基因,抗細胞凋亡基因、代謝解毒基因、上皮-間葉轉化基因等。因此,本論文提出兩種可能的多重抗藥性的建立機制。模式一、篩選自癌幹細胞特徵簇群; 模式二、突變累積導致抗藥性表現。無論模式為何,皆是在1.024 μM doxorubicin處理下才有的變化。藉由篩選自癌幹細胞特徵族群外,p53突變亦參與抗藥性發生,證實突變的累積亦是造成抗藥性發生的原因。因此,針對癌幹細胞特徵或是突變基因作為藥物設計的標的,在未來癌症治療上將有相當助益。 Cellular mechanisms of multidrug resistance (MDR) are related to ABC transporters, apoptosis, antioxidation, drug metabolism, DNA repair and cell proliferation. It remains unclear whether the process of resistance development is programmable. I aimed to study gene expression profiling circumstances in MCF-7 during MDR development. Eleven MCF-7 sublines with incremental doxorubicin resistance were established as a valued tool to study resistance progression. MDR marker P-gp was overexpressed only in cells termed MCF-7/ADR-1024 under the selection dose approaching 1024 nM. MCF-7/ADR-1024 and authentic MCF-7/ADR shared common features in cell morphology and DNA ploidy status. MCF-7/ADR-1024 and authentic MCF-7/ADR showed downregulation of DNA repair genes BRCA1/2 and wild type p53, apoptosis-related gene Bcl-2 and epithelial-mesenchymal transition (EMT) epithelial marker gene E-cadherin; Whereas, detoxifying enzymes glutathione-S transferase-π and protein kinase C-α were up-regulated. The genes involved in EMT mesenchymal formation were also overexpressed, including N-cadherin, vimentin and the E-cadherin transcription repressors Slug, Twist and ZEB1/2. PI3K/AKT inhibitor wortmannin suppressed expression of Slug, Twist and MDR-1. In addition, MCF-7/ADR-1024 cells exerted CSC-like cell surface marker CD44 high/CD24 low and form mammospheres. Mutant p53 with a deletion at codons 127-133 markedly appeared in MCF-7/ADR-1024 and authentic MCF-7/ADR as well. In order to understand if p53 mutation is related to drug resistance development, a del p53-containing construct was transfected into MCF-7/WT to generate the MCF-7/del p53 which stably expressed del p53. Results showed that MCF-7/del p53 acquired resistance to doxorubicin with increased P-gp efflux function. The MDR-1 promoter was significantly activated by external or integrated del p53 using transient expression assay (p < 0.001). Inhibition of NF-κB by cyclosporine sensitized MCF-7/del p53 cells to doxorubicin toxicity. In addition, morphology of MCF-7/del p53 and MCF-7/adr were alike. EMT was observed in MCF-7/del p53 cells with regard to presence of mesenchymal markers Slug and Vimentin and the decrease in epithelial marker CDH1, as well as significant migration ability (p < 0.001). Furthermore, expression of cancer stem cell-like marker CD44 increased, accompanying with mammosphere formation. Altogether, expression of del p53 renders MCF-7/del p53 to acquire doxorubicin resistance characteristics as MCF-7/adr but less effectively. del p53 may be an important factor for the non-invasive MCF-7 to activate NF-κB signaling and MDR-1 promoter and partly attribute to EMT and stem cell-like properties so as to facilitate drug resistance. Conclusion Resistance marker P-gp arises owing to turn on/off or mutation of the genes involving in DNA repair, apoptosis, detoxifying enzymes, EMT and ABC transporters at a turning point (1.024 μM doxorubicin challenge). Behind this point, no obvious alterations were found in most tested genes. Selection for CSC-like cells under this dose may also importantly attribute to propagation of the population presenting invasive properties and drug resistance. Thereby, two models are suggested in the induction of drug resistance. Model 1: Selection for CSC-like cells and model 2: Mutations for gain-of-resistance. The acclamation of mutations facilitates drug resistance development. del p53 may play a role in drug resistance. Drugs designs for targeting the cancer stem cell characteristics or mutated genes involved in drug resistance will be useful in cancer treatment. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52302 |
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