Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/48509
標題: 探討類鐸受體7配體Imiquimod誘發癌細胞進行細胞自噬及細胞凋亡的相互關係及機制
The Study of Mechanism of Toll-Like-Receptor 7 Ligand Imiquimod Induced Autophagy, Apoptosis and Their Relationship in Cancer Cells
作者: 謝政哲
陳怡如
楊淑美
關鍵字: 基礎研究
Toll like receptor 7
基礎醫學類, 生物技術
類鐸受體 7
Imiquimod
細胞凋亡
細胞自噬
imiquimod
apoptosis
autophagy
摘要: Toll-like receptors (TLRs) have been described to become potent targets in infectious disease treatment, vaccine therapy and in neoplastic disease treatment. Imiquimod (IMQ), a TLR 7 ligand, contains antitumor and antiviral activity through stimulation of cell-mediated immunity in vivo. IMQ shows efficacy toward viral wart, actinic keratoses and some skin cancers. Recent studies have shown that IMQ can directly induce apoptosis in melanoma and bladder cancer cell lines in vitro. However, the effects and mechanism of the IMQ-induced responses in cancer cells are not well understood. In this plan, we will use different cancer cell lines as models to investigate the mechanisms of IMQ-induced cell death. Preliminary results indicated that the IMQ treatment can significantly reduce viability and directly induce apoptosis in p53 wild type cancer cell lines (including A549, AGS, BCC and HepG2). However, T24 cells, a p53 mutant cell line, were resistant to IMQ-induced apoptosis. We demonstrated IMQ could directly induce autophagy in all cancer cells we tested. We also found IMQ could modulate master regulators in apoptosis and autophagy pathway: activate AMPK, enhance p53 expression, but down-regulate mTOR activity. Thus, we assume AMPK and p53 may crucial for IMQ caused effects. In the first year of this proposal, we will deeply demonstrate the IMQ-induced apoptosis is belonging to intrinsic or extrinsic pathway, evaluate whether the IMQ induces a canonical autophagy pathway and the flux of IMQ-induced autophagy. Next, we will determine the temporal and spatial differences of IMQ-induced autophagy and apoptosis, and evaluate their relationship. In the second year, we will determine whether AMPK and mTOR pathway are modulated by IMQ, elucidate the role of AMPK and mTOR in IMQ-induced autophagy by pharmacological and genetic inhibition, and determine which pathway (such as LKB1, CaMKKβ or TAK1) mediates IMQ-induced AMPK activation and autophagy. In the last year, we will examine the mechanism of p53 up-regulation by IMQ, the phosphorylation status and subcellular localization of p53 after IMQ treatment. We will evaluate the role of p53 in IMQ-induced apoptosis by comparing the paired isogenic cancer cell lines (HCT116 p53+/+ and p53-/-; A549 and A549 p53 knock-down) and reconstitute IMQ-induced apoptosis by reactivating wild-type p53 in p53 null cell line. Finally, we will evaluate whether the IMQ-induced autophagy is depend on p53 activated DRAM up-regulation or modulated by cytoplasmic p53 inhibition. Here, we look forward to demonstrate the IMQ not only induce apoptosis but also autophagy, and try to find out the mechanism and interaction of apoptosis and autophagy induced by IMQ treatment in cancer cells. We also wish to provide solid evidences to support the advanced application of IMQ in future.
類鐸受體(Toll-like receptors)被認為在感染疾病、疫苗發展和腫瘤的治療上是種具潛力性的標靶。Imiquimod (IMQ),一種類鐸受體7的配體,在in vivo研究中指出可藉由活化細胞性免疫反應(cell-mediated immunity)達到抗腫瘤及抗病毒的效果。在臨床上,IMQ已被證實對於病毒疣(viral wart)、日光角化症(actinic keratoses)和某些皮膚癌具有治療功效。近期的研究顯示在in vitro下, IMQ可以直接誘導黑色素細胞瘤和膀胱癌的細胞株進行細胞凋亡(apoptosis) 。然而對於在癌細胞中IMQ所直接誘導的反應和機制並不瞭解。在這個計畫中,我們將採用不同種類的腫瘤細胞株作為生物模式來研究IMQ誘導腫瘤細胞死亡的機制。我們初步實驗結果顯示,在p53野生型(wild-type)腫瘤細胞株(包括A549、AGS、BCC和HepG2)中,IMQ能夠顯著的減少其生存能力並且直接地誘導細胞凋亡。然而p53突變(mutant)的細胞株T24,對於IMQ誘導的細胞凋亡具有抵抗性。我們證實IMQ能夠在這些細胞株中直接地誘導細胞自噬 (autophagy)的進行。我們也發現IMQ能夠調節在細胞凋亡和自噬中重要的數種調控蛋白(regulator):如活化AMPK、增強p53的表現並降低mTOR的活性。因此我們認為AMPK和p53是讓IMQ產生生物效應的可能關鍵。此計畫的第一年,我們將深入證實IMQ所誘導的細胞凋亡是屬於內源性(intrinsic)或是外源性(extrinsic)途徑,探討IMQ是否能誘導典型細胞自噬途徑及細胞自噬通量的進行。我們將進一步確認IMQ所誘導的細胞自噬和細胞凋亡在時間上和空間上的差異,且評估其相關性。第二年,我們將證實AMPK和mTOR路徑是否受到IMQ所調控,以藥物(pharmacological)和基因(genetic)抑制活性的方法,探討AMPK和mTOR在IMQ誘導的細胞反應中所扮演的角色,並且進一步確定IMQ經由哪條訊息途徑(如LKB1、CaMKKβ或TAK1)促使AMPK活化及細胞自噬的進行。第三年,我們將分析細胞IMQ的處理後,促使p53表現增加的可能機制、p53磷酸化(phosphorylation) 狀態以及其胞內局限位置 (subcellular localization)。我們將藉由比較成對等基因(paired isogenic)腫瘤細胞株(HCT116 p53+/+和HCT116 p53-/-;A549及A549 p53 knock down)在IMQ誘導之細胞凋亡中,探討p53所扮演的角色,並且在p53 null的細胞株中再活化(re-activating)野生型p53來加以還原IMQ誘導之細胞凋亡,作為反證p53如何參與IMQ所誘導的細胞凋亡。最後,我們將釐清IMQ誘導的細胞自噬是否藉著依賴p53激活DRAM的活化或是受到胞質性(cytoplasmic) p53的負向調控。我們期待藉此計劃能深入了解IMQ誘導腫瘤細胞株進行細胞自噬及細胞凋亡的分子機制及差異性,並期待未來將可據此研究模式,探討類鐸受體7配體對其它腫瘤細胞的作用機制及臨床運用的可行性,在其他癌症治療上提供一新穎的策略與做法。
URI: http://hdl.handle.net/11455/48509
其他識別: NSC99-2320-B005-007-MY3
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=2131817&plan_no=NSC99-2320-B005-007-MY3&plan_year=99&projkey=PC9907-2287&target=plan&highStr=*&check=0&pnchDesc=%E6%8E%A2%E8%A8%8E%E9%A1%9E%E9%90%B8%E5%8F%97%E9%AB%947%E9%85%8D%E9%AB%94Imiquimod%E8%AA%98%E7%99%BC%E7%99%8C%E7%B4%B0%E8%83%9E%E9%80%B2%E8%A1%8C%E7%B4%B0%E8%83%9E%E8%87%AA%E5%99%AC%E5%8F%8A%E7%B4%B0%E8%83%9E%E5%87%8B%E4%BA%A1%E7%9A%84%E7%9B%B8%E4%BA%92%E9%97%9C%E4%BF%82%E5%8F%8A%E6%A9%9F%E5%88%B6
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