| 摘要: | 檳榔子（areca nut, AN），已被公認為致癌物，參與腔鱗狀細胞上皮癌（oral squamous cell carcinoma, OSCC）形成，然而有關AN成分對細胞的作用之研究仍相當有限。已知兩種檳榔子成分，檳榔素（arecoline）與前矢車菊花青素低聚物（procyanidin oligomers），已知可誘導細胞凋亡（apoptosis）因而可合理推論檳榔子對口腔細胞而言是一種促進凋亡的刺激物。但本實驗室卻發現檳榔子萃取液（AN extract, ANE）與其30-100 kDa的部分（ANE 30-100K）透過活潑氧族誘導正常的口腔纖維母細胞、淋巴球、OSCC (OECM-1) 細胞與食道上皮癌（CE81T/VGH）細胞等，進行自體吞噬作用。自體吞噬在被認為可對已經發展出來的癌細胞提供生長優勢，因此值得研究ANE 30-100K所誘導的自體吞噬之機轉。Atg5是屬於在各種刺激下誘發的自體吞噬程式中，較為下游的執行分子。然而最近的研究卻又指出亦有不依賴Atg5的自體吞噬之存在。因此，本研究進行了解，Atg5是否在由ANE 30-100K誘導的自體吞噬中扮演某種角色，我們以慢病毒所媒介的siRNA策略來抑制Atg5的表現，並監測此抑制是否影響OECM-1 與CE81T/VGH細胞，對ANE 30-100K的反應之變化情形。結果顯示，抑制Atg5造成此兩種細胞對ANE 30-100K的細胞毒性抵抗力增加。再者，Atg5抑制亦減低ANE 30-100K所誘導的微管結合蛋白第三型輕鍊-II（microtubule-associated protein light chain 3-II, LC3-II）的堆積，與酸性泡的產生，在與其母株細胞相較之下，在經篩選過Atg5被抑制的純系細胞株中，均有顯著的減弱現象。綜言之，這些數據顯示，在口腔與食道的上皮癌細胞中，可能由ANE 30-100K所誘導的自體吞噬均需要Atg5的參與。
Areca nut (AN), used by about 200 million people worldwide, is regarded as a carcinogen capable of inducing various oral diseases including oral squamous cell carcinoma (OSCC); however, studies of the effects of AN ingredients on cells remain limited. There are two small AN ingredients, arecoline and procyanidin oligomers, known to induce apoptosis, which leads to a reasonable assumption that AN may be an apoptotic stimulant to oral cells. Unexpectedly, we found that AN extract (ANE) and the 30-100 kDa fraction of ANE (ANE 30-100K) induce autophagy in normal oral fibroblasts, lymphocytes, OSCC cells (OECM-1), and esophageal carcinoma cells (CE81T/VGH) through reactive oxygen species. Autophagy has been extensively studied in the past decade and is thought to provide growth advantage for the developed cancer. Thus, it is worthy to investigate the underlying mechanism of ANE 30-100K-induced autophagy. Among numerous reported mediators of autophagy, Atg5 is a relatively down-stream effector in autophagic program initiated by various stresses; however, a recent study shows the existence of the Atg5-independent autophagy. To assess whether Atg5 play a role in ANE 30-100K-induced autophagy, we inhibited its expression by lentivirus-mediated siRNA strategy and monitored the change of the response of OECM-1 and CE81T/VGH toward ANE 30-100K. The results show that expression level of Atg5 protein was successfully inhibited in both cells, which lead to increased resistance against the cytotoxicity of ANE 30-100K. Furthermore, ANE-30-100K-induced microtubule-associated protein light chain 3 (LC3)-II accumulation and acidic vesicle generation were significantly attenuated in Atg5-knocked down clones of OECM-1 and CE81T/VGH as compared to their parental cells. Collectively, these data suggest that Atg5 may be required for ANE 30-100K-induced autophagy in oral and esophageal carcinoma cells. |
