不同檳榔子成份誘導的細胞凋亡與自體吞噬之研究

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Title:	不同檳榔子成份誘導的細胞凋亡與自體吞噬之研究 Study of the apoptosis and autophagy induced by different areca nut ingredients
Authors:	洪温擇 Wen-Zhai Hong
Contributors:	林美惠劉永超嘉南藥理科技大學：生物科技研究所
Keywords:	自體吞噬檳榔鹼 arecoline 30-100K autophagy
Date:	2009
Issue Date:	2009-03-12 11:33:16 (UTC+8)
Abstract:	檳榔嚼塊 (betel quid, BQ) 與檳榔子 (areca nut, AN) 已經被公認為致癌物，能引起各種口腔疾病，包含口腔鱗狀細胞上皮癌 (oral squamous cell carcinoma, OSCC)。我們過去發現，檳榔子中不同的成分能夠引起不同的細胞死亡方式，檳榔子萃取液 (AN extract, ANE) 與檳榔鹼 (arecoline) 可分別誘導細胞進行自體吞噬 (autophagy) 與細胞凋亡 (apoptosis) 。此外，可誘導自體吞噬的活性位於經部分純化後檳榔子萃取液的30-100 kDa部分 (稱之30-100K)，它也能誘導幾種不同的上皮癌細胞進行自體吞噬的死亡，如OECM-1與CE81T/VGH等。本實驗進一步証實以30-100K刺激正常纖維母細胞 (CMT415)、正常胚胎肺部纖維母細胞 (MRC5) 與白血病T細胞(Jurkat T cell) 後，皆可發現類似於OECM-1和CE81T/VGH有細胞脹大及細胞核濃縮等外型的改變。另外，丫啶橙 (Acrdine Orange) 螢光染色的結果也顯示30-100K能刺激CMT415、白血病T細胞及正常口腔角質細胞(NOK) 產生酸性囊泡。此外，藉由西方墨點法顯示30-100K能誘導CMT415與白血病T細胞中LC3-I的裂解，並呈劑量相依性，並且在30-100K刺激白血病T細胞24小時後，LC3-II/LC3-I的比值達到最高。隨後地也發現STO-609 【Calmodulin-dependent kinase kinase-(CaMKK) 的抑制劑】及Compound C 【AMP-activated protein kinase (AMPK) 的抑制劑】均能顯著地抑制30-100K所造成白血病T細胞與舌頭鱗狀上皮癌細胞 (SAS) 的死亡。我們結論30-100K可能透過CaMKK/AMPK途徑在正常與惡性癌細胞中引起自體吞噬。 Betel quid (BQ) and areca nut (AN) are both recognized as carcinogens causing various oral disease including oral squamous cell carcinoma (OSCC). We preciously found that different ingredients of AN may cause distinct pattern of cell death. Extract of AN (ANE) and arecoline induce autophagic and apoptotic cell death, respectively. The autophagy-inducing activity were traced to the partially purified 30-100 kDa fraction of ANE (designated as 30-100K), capable of inducing autophagic cell death in several types of carcinoma cells, such as OECM-1 and CE81T/VGH. In this study, we further demonstrated that 30-100K triggered the same morphological changes (rounding plasma membrane and condensed nucleus) in some other cell types, including normal oral fibroblast, normal embryonal lung fibroblasts, oral keratinocytes, and Jurkat leukemia T cells, as those in OECM-1 and CE81T/VGH before cell death. Acridine orange staining showed that the acidic vacuoles were generated in CMT415, Jurkat T cell and NOK cells after 30-100K treatment. Furthemor, 30-100K induces LC3-I cleavage in CMT415 and Jurkat T cells in a dose-dependent manner as revealed by Western blotting, and the LC-II/LC3-I ratio reached the highest level after 24-h treatment of Jurkat T cells. Afterwards, we also found that the 30-100k-induced cell death of SAS and Jurkat leukemia T cells were significantly inhibited by STO-609 the inhibitor of calmodulin-dependent kinase kinase-CaMKK】 and Compound C 【the inhibitor of AMP-activated protein kinase (AMPK)】, and the 30-100K-induced LC3-I cleavage of Jurkat leukemia T cells was dose-dependently inhibited by the STO609, Compound C, and 3MA. We conclude that 30-100K is able to trigger autophagy in both normal and malignant cells probably through the CaMKK/AMPK pathway.
Relation:	校內外均一年後公開
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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