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博碩士論文 etd-0118116-095615 詳細資訊
Title page for etd-0118116-095615
論文名稱
Title
探討ABT-751在肝癌細胞株Huh-7經由ROS造成細胞凋亡與自噬作用
Studies on ABT-751-induced apoptosis and autophagy via reactive oxygen species in hepatocellular carcinoma-derived Huh-7 cells
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
56
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2016-01-27
繳交日期
Date of Submission
2016-02-18
關鍵字
Keywords
細胞自噬、細胞凋亡、TP53、ABT-751、ROS
Autophagy, Apoptosis, TP53, ROS, ABT-751
統計
Statistics
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中文摘要
本篇研究目的是探討 ABT-751 對於肝癌細胞株 Huh-7 誘導細胞凋亡與自噬作用之路徑探討。肝癌(Hepatocellular carcinoma)目前在台灣屬於常見的惡性腫瘤且為高居前兩名的高致死率癌症。ABT-751 是一種新穎的抗有絲分裂藥物,是透過抑制微管的合成進而達到產生細胞毒性。然而,目前對於 ABT-751 於肝癌細胞株的研究尚未清楚。在本篇實驗結果顯示在 Huh-7 細胞株中 ABT-751 能夠抑制癌細胞 anchorage-independent growth 的能力,也發現能使粒線體膜電位降低,以及促使細胞產生 reactive oxygen species (ROS) 與造成細胞 DNA damage 現象。ABT-751 能使轉染後具有表現 GFP-LC3 的 Huh-7 產生 LC3 粒泡聚集。利用 Cyto-ID 與西方墨點法分析結果顯示 ABT-751 能使 Huh-7 在於12小時先出現細胞自噬,而分別在24小時與48小時結果是透過促進 TP53 與 DRAM2 蛋白質表現,並透過抑制 PI3K/AKT/mTOR 路徑來誘導細胞自噬。依核質分離結果為 ABT-751 使 TP53 蛋白在細胞核中增加且細胞質減少。利用 Annexin V/PI double staining assay 與西方墨點法分析結果發現 ABT-751 是透過活化 CASP8、CASP9、CASP3、DFFA 及 PARP1 來產生細胞凋亡。當我們加入caspase 抑制劑 ( Z-VAD-FMK) 時能有效的抑制 ABT-751 所誘導的細胞凋亡。在粒線體與細胞核分離實驗中發現 cytochrome c 會釋放到細胞質。最後,我們加入抗氧化 N-acetyl-L-cysteine (NAC) 能有效的抑制 ABT-751 誘導細胞凋亡與自噬作用。總結而言,ABT-751 能對肝癌細胞株 Huh-7 誘導 ROS 產生後,促使細胞經由 PI3K/AKT/mTOR pathway 產生自噬作用以及經由 CASP-dependent pathway 發生細胞凋亡。
Abstract
The objective was to study the regulatory mechanisms of ABT-751 on apoptosis and autophagy in hepatocellular carcinoma-derived Huh-7 cells. Hepatocellular carcinoma (HCC) is a common malignant tumor and the second leading cause of cancer death in Taiwan. ABT-751 is a novel anti-mitotic agent mediated by the inhibition of microtubule synthesis that exerts cytotoxic effects in preclinical studies. However, it is not clear whether the effects of ABT-751 in HCC-derived cells. In this study, the results indicated that ABT-751 in Huh-7 cells inhibited anchorage independent of colony formation ability. ABT-751 also was able to alter mitochondrial membrane potentials, induce the formation of reactive oxygen species (ROS) and cause DNA damage in Huh-7 cells. ABT-751 induced GFP-LC3 puncta formation in Huh-7 cells overexpressed GFP-LC3 under the fluorescent microscope. The Cyto-ID assay and immunoblotting were used to determine ABT-751 induced autophagy in Huh-7 cells, the results indicated ABT-751 can induce autophagy first at 12 h and up-regulation of p53-depend DRAM2 pathway and down-regulation of PI3K/AKT/mTOR pathway for 24 and 48 h. In nuclear/cytosol fractionation data, ABT-751 induced TP53 expression downregulation in cytoplasm followed by the increase in the nuclear. The Annexin V/PI double staining assay and immunoblotting analyzed about ABT-751 induced apoptosis, the results were ABT-751 activated CASP8, CASP9, CASP3, DFFA and PARP1. And a pan-caspase inhibitor, Z-VAD-FMK, also suppressed ABT-751-induced apoptosis. In mitochondrial/cytosol fractionation, ABT-751 treatment was observed cytochrome c release in cytosol under apoptosis. Moreover, antioxidant NAC (N-acetyl-L-cysteine) reduced ABT-751-induced ROS, and then inhibited apoptosis and autophagy in Huh-7 cells. Taken together, those results indicated ABT-751 induced autophagy via PI3K/AKT/mTOR pathway under ROS formation and apoptosis through CASP-dependent pathway in Huh-7 cells.
目次 Table of Contents
Approval page i
Acknowledgements ii
Abstract iii
Chinese iii
English iv
List of figures vii
List of tables viii
Introduction 1
Materials and Methods 8
Results 24
Figures 28
Discussion 38
References 42
Supplementary Data 46
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