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    Please use this identifier to cite or link to this item: http://ir.cnu.edu.tw/handle/310902800/24842

    標題: 檳榔萃取液30-100K與檳榔素所誘導的死亡訊息傳遞之比較
    Comparison of Ane 30-100K and Arecoline-Triggered Signal Transduction
    作者: 林美惠
    貢獻者: 生物科技系(所)
    日期: 2010
    上傳時間: 2011-12-22 09:46:47 (UTC+8)
    摘要: 我們過去證實經部分純化後的檳榔子萃取液(areca nut extract, ANE),其30-100 kDa 的部分(ANE 30-100K)與檳榔素(arecoline)可分別誘導口腔上皮癌細胞(OECM-1)進行自體吞噬(autophagy)與凋亡(apoptosis)。最近我們更進一步發現ANE 30-100K 可同樣在正常口腔纖維母細胞、周邊血淋巴球與Jurkat T 細胞造成自體吞噬的死亡。在試圖以化合物區分細胞對ANE 30-100K 與檳榔素不同的反應後,我們發現兩種自體吞噬的抑制劑(3-MA 與NH4Cl)與自由基的清除劑(NAC)均對ANE 30-100K 與檳榔素的細胞毒性具有保護作用,顯示細胞對這兩種檳榔成份的敏感度並無法由這三種化合物加以區分。有趣的是在分析對AMPK(由AMP 活化之蛋白激酶,已知其具有調節自體吞噬與凋亡功能)之調節作用後,得知ANE 30-100K 與檳榔素分別增強與減弱了AMPK 在第172 個胺基酸位置上的羥丁胺酸(AMPK-Thr172)之磷酸化。此外,AMPK 的抑制劑(compound C)與calmodulin-dependent kinase kinase 的抑制劑(STO-609)降低了所誘導的自體吞噬,並且若將AMPK 以shRNA 抑制之後,也會使 OECM-1 細胞對ANE 30-100K 的耐受力增加,而對檳榔素的耐受力則不變。相對地, AMPK 的活化劑(aminoimidazole-4-carboxamide riboside, AICAR)則可依劑量相依性地減弱由檳榔子所誘導的細胞毒性、AMPK-Thr172 的去磷酸化與caspase-3 的活化。這些結果顯示負責引發自體吞噬的ANE 30-100K 接受器可能廣泛地表現於不同類型的細胞中,並且ANE 30-100K 與檳榔素可能透過對AMPK 相反的調控作用而造成不同類型的死亡方式。未來一年我們預計找到更多被ANE 30-100K 與檳榔素不同調控的分子(特別是位於已知的AMPK 途徑上)。我們預計在未來一年繼續以類似的方法,找出在已知的AMPK/MEK/ERK/mTOR/Beclin-1 路徑上,會受到ANE 30-100K 與檳榔素不同調節作用並參與隨後所誘導的死亡方式之分子。
    We previously demonstrated the induction of autophagy and apoptosis by a partially purified 30-100 kDa fraction of areca nut (AN) extract (ANE 30-100K) and arecoline (the major alkaloid of AN) in oral epidermoid carcinoma Meng-1 (OECM-1) cells, respectively. Recently we have further found that ANE 30-100K can induce the autophagic cell death in normal oral fibroblasts (CMT-415), peripheral blood lymphocytes (PBLs), and Jurkat T cells. After trying to discriminate different cellular responses to the cytotoxic impacts brought byANE 30-100K and arecoline with chemicals, we found that two autophagy inhibitors, 3-methyladenine (3-MA) and NH4Cl, as well as the reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), provided a common protection effect against the cytotoxicity of ANE 30-100K and arecoline, revealing that the sensitivity of cells toward these two AN components can not be distinguished by these three chemicals. Interestingly, after analyzing the regulatory effects on AMP-activated protein kinase (AMPK), known to regulate both apoptosis and autophagy, we found that ANE 30-100K and arecoline respectively enhanced and attenuated the phosphorylation of AMPK-Thr172. Furthermore, the inhibitors of AMPK (compound C) and calmodulin-dependent kinase kinase  (STO-609) crippled ANE 30-100K-induced autophagy, and specific knockdown of AMPK by shRNA also resulted in stronger resistance to ANE 30-100K, but not to arecoline. In contrast, the activator of AMPK, aminoimidazole-4-carboxamide riboside (AICAR), dose-dependently attenuated arecoline-mediated cytotoxicity, decrease of AMPK-Thr172 phosphorylation, and caspase-3 activation. These results suggest that cellular receptor of ANE 30-100K responsible for autophagy initiation may be widely expressed in different cell types, and ANE 30-100K and arecoline may induce different death patterns through the contrary regulation on AMPK.We plane to address more molecules located on AMPK/MEK/ERK/mTOR/Beclin-1 pathway, differentially regulated byANE 30-100K and arecoline, and responsible for induced death pattern with similar strategies in the coming year.
    關聯: 計畫編號:NSC99-2314-B041-002
    Appears in Collections:[生物科技系(所)] 科技部計畫

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