| 摘要: | 檳榔嚼塊 (betel quid, BQ) 與檳榔子 (areca nut, AN) 已經被公認為致癌物，然而有關檳榔成分對細胞的影響之研究卻不多。過去我們由檳榔子萃取液中發現一種成分可造成細胞漲大死亡，且誘導LC3-II活化，疑似造成細胞自體吞噬情形。而在此次的研究，我們進一步追蹤到此誘導自體吞噬的成分分子量介於30-100 kDa（稱做LLS-2）。LLS-2的處理可依劑量相關性誘導LC3-II的活化，並於24小時達到高峰。此外，LLS-2也誘導了酸性泡的產生，利用acridine orange螢光染劑染色，以及，觀察細胞內自噬泡的形成（穿透式電子顯微鏡），以及細胞漲大且核濃縮，而細胞質也被消化殆盡（穿透式電子顯微鏡與蘇木紫－伊紅染色）。另一方面，我們也發現檳榔素（Arecoline, Are）可以誘導第三型硫胱胺酸蛋白酶(caspase-3)的活化、DNA片段化、染色質濃縮及細胞核微核化等情形。3-MA及NH4Cl均可抑制Are的細胞毒性，然而3-MA抑制LLS-2細胞毒性的能力比NH4Cl好。ANE、Are及 LLS-2皆可誘導ERK1/2 及 S6K-Thr389的磷酸化，但是只有ANE與LLS-2作用24小時後，可抑制mTOR-Ser2448。綜合以上的結果，我們結論Are 誘導細胞凋亡，而LLS-2誘導自體吞噬。LLS-2是一個新發現的在檳榔子成分，具有誘導自體吞噬的特性，且可能經由抑制mTOR路徑來誘導自體吞噬。
Betel quid (BQ) and areca nut (AN) are both recognized as carcinogens, however, the effects of their ingredients on cells are not poorly studied. We have previously found that extract of AN (ANE) induce an autophagy-like cell death as evidenced by swollen cell morphology and LC3-II activation. In this study, we further addressed that the autophagy-inducing activity of ANE is located between 30-100 kDa (designated as LLS-2). Treatment of LLS-2 results in the dose-dependent activation of LC3-II, which peaked at 24 hr. Furthermore, it also induces the emergence of acidic vacuoles (by acridine orange staining), autophagic vacuoles (by transmission electron microscopy), and swollen cells with the condensed nucleus and nearly empty cytoplasm (by transmission electron microscopy and hematoxylin and eosin staining). On the other hand, arecoline (Are) triggers the caspase-3 activation, DNA fragmentation, and condensed and micronucleated nuclei. Both 3-MA and NH4Cl inhibit the cytotoxicity of arecoline, while only 3-MA exhibits more blocking effect on LLS-2-mediated cytotoxicity than that of NH4Cl. ANE, Are and LLS-2 activates the phosphorylation of ERK1/2 and S6K-Thr389, whereas only ANE and LLS-2 inhibit the phosphorylation of mTOR-Ser2448 after 24-hr treatment. Taken together we conclude that Are and LLS-2 induce apoptosis and autophagy, respectively. LLS-2 may represent a brand-new autophagy-inducing AN ingredient, which acts through the inhibition of mTOR pathway. |
