Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20146
標題: 腺苷磷酸活化激酶在Imiquimod與Compound C誘導之細胞自噬所扮演之角色
Role of AMPK in Imiquimod-induced and Compound C-induced Autophagy
作者: 陳育瑜
Chen, Yu-Yu
關鍵字: AMPK
腺苷磷酸活化激酶細胞自噬
autophag
imiquimod
compound c
出版社: 生物醫學研究所
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摘要: Imiquimod,是類鐸受體7 ( Toll-like receptor 7,TLR7 ) 的配合體 ( ligand ),臨床上被用來治療光化角化症或是基底細胞癌 ( basal cell carcinoma,BCC ) 等皮膚腫瘤和病毒疣。之前的文獻指出 imiquimod 可活化巨噬細胞 ( macrophage ) 的 TLR7-MyD88 訊息傳遞而誘導細胞自噬 ( autophagy ) 的進行,而在近期的研究顯示,imiquimod 亦可在 BCC 誘導細胞自噬之進行。細胞自噬作用的其中一個一個最重要的負調控激酶為哺乳動物雷帕霉素標靶蛋白 ( mammalian target of rapamycin, mTOR ),當 mTOR 受到抑制時就會啟動下游訊息而誘發細胞自噬。而腺苷磷酸活化激酶 ( adenosine monophosphate -activated protein kinase, AMPK ) 即為 mTOR 最主要之負調控者之一,AMPK 會去磷酸化下游目標蛋白 raptor,達到抑制 mTOR 之活性。目前為止 Imiquimod 誘發基底細胞癌 ( basal cell carcinoma, BCC ) 細胞自噬的機制尚不明瞭,因此本論文將以 BCC 細胞株 ( cell line ) 作為生物模式,探討 imiquimod 刺激 BCC 進行細胞自噬的分子機制。我們首先證實了 imiquimod 能夠增加 BCC 細胞中 AMPK 之活性,隨著 imiquimod 的刺激時間的增加,使 LC3-II 增加的同時,磷酸化之 AMPK 以及其下游乙醯輔酶 A 羧化酶 ( acetyl-CoA carboxylase,ACC ) 之磷酸化蛋白表現量皆有上升的趨勢;在此同時,imiquimod 亦能夠抑制 mTOR 下游蛋白 p70S6K 之活性。因此我們進一步利用化學抑制劑以及 siRNA 核酸干擾技術與顯性負突變蛋白競爭抑制之不同方法來探討 AMPK 在 imiquimod 引發 BCC 細胞進行細胞自噬所扮演之角色。我們在實驗結果觀察到不論是使用 AMPK 抑制劑或是 siRNA 核酸干擾技術與顯性負突變蛋白競爭抑制方法,皆對於 imiquimod 刺激基底細胞癌細胞株 LC3-I 轉變成 LC3-II 的情況不產生抑制之效果。因此,我們認為增加 AMPK 之活化並不參與 imiquimod 誘導BCC細胞進行之細胞自噬。此外,我們利用流式細胞儀偵測細胞週期以及西方墨點法,也發現到 compound C 不僅具有引發基底細胞癌細胞株進行細胞凋亡以及 G2/M 細胞週期停滯之現象,此外,在 BCC 細胞、人類肝癌細胞株 ( HepG2 和 Hep3B )以及肺癌細胞株 ( A549 ) 中具有單獨使 LC3-I 轉變成 LC3-II、EGFP-LC3 puncta 和自噬囊泡形成以及數量增多的能力。使用 siRNA 核酸干擾技術實驗我們也證實了 compound C 誘導 BCC 細胞進行細胞自噬並不經由 AMPK 訊息傳導路徑。除了細胞自噬,我們也證實了 compound C 具有引發基底細胞癌細胞株進行細胞凋亡以及 G2/M 細胞週期停滯之現象。因此我們認為 compound C 本身即為一個強力的細胞自噬促進劑,且此能力並無細胞特異性。所以當實驗使用 compound C 來當作抑制劑來研究細胞內的反應時,必須特別注意 compound C 本身對於細胞的影響。而我們的實驗結果也指出 compound C 在未來有潛力來當作一個對抗癌症的研究對象。
Imiquimod, a Toll-like receptor 7 ( TLR7 ) ligand, it has shown clinical efficacy toward viral wart, actinic keratose and basal cell carcinoma ( BCC ). Recent studies have shown that imiquimod induce autophagy through TLR7-MyD88 signaling in macrophage cell lines, moreover, imiquimod also induce autophagy in BCC cell line. MTOR ( mammalian target of rapamycin ) is the one of important regulators of autophagy, inhibiting mTOR caused cell autophagy. The adenosine monophosphate ( AMP )-activated protein kinase ( AMPK ) serves as a key energy sensor in cells. AMPK is also an upstream regulator of the mTOR signaling and is able to stimulate the autophagy. However, the mechanism of imiquimod induces autophagy in BCC cells are not well understood. In this study, we used BCC cell line as an experiment model to investigate the role of AMPK in imiquimod-induced autophagy. First, we observed that when imiquimod induced LC3-I to LC3-II conversion in protein level, AMPK and ACC (acetyl-CoA carboxylase, a downstream protein of of AMPK ) phosphorylation were increased in BCC cell line; instead, phosphorylation of p70S6K, which is one of a downstream protein of mTOR, was decreased. Second, we used an AMPK and two upstream kinase of AMPK inhibitors ( compound C, sto-609 and 5-Z-7-oxozeaenol ) , and RNA interference technology, and dominant negative mutant competition to demonstrate whether imiquimod-induced autophagy is going through AMPK signaling pathway. We observed that imiquimod-induced autophagy was not changed by three chemical inhibitors or transfection of AMPK siRNA or dominant negative mutant AMPK competition. In present data indicated that AMPK activation by imiquimod has no benefit to imiquimod-induced autophagy. Furthermore, we also found out that compound C is not only able to cause G2/M cell cycle arrest and induce cell apoptosis by flow cytometry, but also induced the LC3-I to LC3-II conversion in protein level of A549, HepG2 and AGS cell line. Otherwise, we used the EGFP-LC3 stably expressing BCC and AGS cell lines to observe EGFP-LC3 puncta formation with compound C treatment in confocal microscope image. The acridine orange accumulation indicated the increasing of autophagolysosome formation in compound C treated BCC and AGS cell lines. Besides, we also investigated the role of AMPK in compound C-induced autophagy. These results suggested that compound C has potential to be an anticancer agents.
URI: http://hdl.handle.net/11455/20146
其他識別: U0005-2701201115072800
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2701201115072800
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