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標題: | NS240062在人類前列腺癌細胞之凋亡作用研究 Investigation of NS240062-Mediated Apoptotic Effect in Human Prostate Cancer Cells |
作者: | Shu-yin Hsiao 蕭淑尹 |
指導教授: | 顧記華 |
關鍵字: | 前列腺癌細胞,凋亡,凋亡誘導因子,核仁蛋白, NS240062,apoptosis,prostate cancer,AIF,nucleolin, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | 利用sulforhodamine B (SRB) assay大規模的篩選抗癌藥物後,半合成藥物NS240062(5-[4-Acridin-9-ylamino]phenyl]-5-methyl-3-methylenedihydrofuran-2-one)在前列腺細胞癌及許多癌細胞皆展現抑制生長的效果。在荷爾蒙非依賴性前列腺癌PC-3細胞株中,NS240062抑制其生長的IC50值為0.69μg/ml。以流式細胞儀分析丟失少量染色體的亞倍體(subG1)細胞,發現凋亡細胞群的比例與時間和濃度呈現正相關。此外,使用DNA末端轉移酶介導的dUTP DNA末端標記技術(Terminal deoxynucleotidyl transferase- mediated dUTP nick-end labeling assay,TUNEL)對化合物處理過的細胞進行染色,在共軛焦螢光顯微鏡的觀察下也確實偵測到DNA斷裂的螢光亮點。使用西方墨點法進一步檢測胞內細胞凋亡相關的蛋白,結果顯示,Bcl-2家族蛋白(包括Bcl-2、Bcl-xL、Mcl-1、Bax、Bad、Bid、Bad)均沒有明顯的變化,此外,能直接參與細胞凋亡的caspase也沒有活化裂解的跡象。然而,在共軛焦顯微鏡下觀察到凋亡誘導因子(Apoptosis-inducing factor,AIF)在NS240062處理下有逐漸移進細胞核內的趨勢。此外,處理化合物6~12個小時後除了可看到凋亡誘導因子(AIF)移動,也發現核仁蛋白(nucleolin)有斷裂的現象。整體而言,NS240062引起的細胞凋亡的機轉主要是與AIF的分布、nucleolin的斷裂相關,而非經由活化caspase所造成的結果。 After a large scale anticancer screening by sulforhodamine B (SRB) assays, we found that a semi-synthetic compound, numbered NS240062, displayed the anti-proliferative activity in several cancer cell lines including human prostate cancer cell lines. The concentration for the 50% inhibition of cell growth (IC50) was 0.69 μg/ml in PC-3, an androgen-independent prostate cancer cell line. After the detection of cell-cycle progression by FACScan flow cytometric analysis, NS240062 induced time- and concentration-dependent increase in hypodiploid phase (apoptosis). The apoptotic cell death was confirmed by TUNEL reaction assay. The Western immunoblot assay showed that NS240062 did not induce any alteration in the expression of Bcl-2 family protein members, including Bcl-2, Bcl-xL, Mcl-1, Bax, Bad, Bid and Bad. Furthermore, it did not induce any activation of caspases. Intriguingly, NS240062 triggered a time-dependent nuclear translocation of apoptosis-inducing factor (AIF) using immunoblotting of nuclear fraction and confocal immunochemical examination. The onset of AIF translocation was six to twelve hours after the compound exposure. Moreover, the data also demonstrated the occurrence of nucleolin cleavage by NS240062. Collectively, our data suggest that NS240062 induces apoptotic cell death in PC-3 cells through AIF- and nucleolin-involved while caspase-independent pathways. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30416 |
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