| 摘要: | 檳榔子（Areca nut, AN）中主要的檳榔鹼—檳榔素（Arecoline），已知可誘導細胞凋亡；但我們先前發現檳榔子萃取液（Areca nut extract, ANE）本身與其中介於30~100 kDa的部分（簡稱為ANE 30-100K），卻是誘導OECM-1細胞進行類似自體吞噬的反應，例如LC3-II的堆積以及與細胞凋亡不同的外觀改變。因此我們有興趣探討ANE 30-100K誘導自體吞噬，其中可能牽涉的機制。我們發現在溶小體抑制劑的存在下ANE與ANE 30-100K皆能更進一步地增加口腔纖維母細胞CMT415中LC3-II的堆積，意味著此兩種檳榔子的成分能誘導自體吞噬的流動。在OECM-1細胞中，藉由RNAi的特異性抑制效果發現由ANE 30-100K所誘導的細胞毒性與LC3-II堆積，在Atg5被抑制的純系細胞株明顯地減弱，而在Beclin-1被抑制的細胞株則較不受影響。相反地，Beclin-1被抑制的細胞株卻比Atg5被抑制的細胞株對葡萄糖的缺乏較具耐受性。另一方面，過去已知ANE 30-100K可在Jurkat T細胞中刺激AMPK的磷酸化，而檳榔素則抑制之；然而我們初步的結果顯示ANE 30-100K在OECM-1中僅微幅地增加AMPK的磷酸化，並且在一個AMPK被 RNAi 抑制的OECM-1細胞株中，由ANE 30-100K所誘導的LC3-II堆積與細胞毒性與控制組細胞類似。有趣的是AMPK的活化劑AICAR在Jurkat T細胞中，可恢復由檳榔素所抑制的AMPK磷酸化與細胞活性。這些結果顯示Atg5在由ANE 30-100K誘導的OECM-1細胞自體吞噬中，可能要比Beclin-1與AMPK扮演了更重要的角色，而AMPK則可能是在由檳榔素所誘導的細胞凋亡中不可或缺的。
Abstract
Arecoline, the major alkaloid of areca nut (AN), was known to induce apoptosis; however, we found that AN extract (ANE) itself and its 30-100 kDa fraction (ANE 30-100K) caused autophagy-like responses in oral cancer OECM-1 cells, including microtubule-associated protein 1 light chain 3 (LC3)-II accumulation and morphological changes distinct to those of apoptosis. Therefore, we were interested in assessing whether ANE 30-100K stimulates autophagic flux and the possible involved mechanisms. We found that ANE- and ANE 30-100K-induced LC-II accumulation was further elevated in the presence of lysosomal protease inhibitors in oral fibroblasts CMT415, suggesting autophagic flux induction by these two AN components. After specific inhibition by RNAi strategies in OECM-1cells, ANE 30-100K-mediated cytotoxicity and LC3-II accumulation were significantly attenuated in Atg5 knocked down clones, but not evidently affected in Beclin-1 knocked down clones. In contrast, Beclin-1, but not Atg5, knocked down clones were more resistant to glucose starvation. On the other hand, ANE 30-100K was known to stimulate the phosphorylation of AMP-activated protein kinase (AMPK) in Jurkat T cells, whereas arecoline exerts a contrary effect on this kinase; however, our preliminary results showed that ANE 30-100K only mildly upregulated AMPK phosphorylation in OECM-1, and ANE 30-100K-induced LC3-II accumulation and cytotoxicity in an AMPK RNAi-inhibited OECM-1 clone were similar to control cells. Interestingly, the AMPK activator, AICAR, restored arecoline-inhibited AMPK phosphorylation and cell viability in Jurkat T cells. These results suggest that Atg5 may play a more important role than Beclin-1 and AMPK in the ANE 30-100K-induced autophagy, whereas AMPK may be required for arecoline-induced apoptosis in OECM-1 cells.
Keywords: Areca nut, Arecoline, AMPK, Beclin-1, Atg5, Autophagy |
