椴木栽培牛樟芝子實體之活性成分齒孔酸（三萜類）誘導人類肝癌細胞內質網壓力調節之自體吞噬

Abstract

根據中華民國100年行政院衛生署十大癌症死因統計，肝癌於不分男女之排名中佔癌症主要死因第二位，更高居全世界排名的第四位，所以在臺灣如何維護肝臟健康是值得重視的議題。近年研究發現，臺灣特有之牛樟芝具有護肝、抗發炎、抗B型肝炎病毒、抗癌等生理活性，其主要活性成分為多醣體、苯類、三萜類、固醇類等，又因三萜類通常具有良好的抗癌效果而備受矚目。因此，本研究欲探討椴木栽培之牛樟芝子實體中含量第二多的三萜類──齒孔酸對人類肝癌Hep 3B細胞株之抗癌活性及其分子機制。結果指出，齒孔酸能有效抑制Hep 3B細胞存活率，其處理24小時之半抑制濃度為18.4 μM，相當於8.7 μg/mL。除此之外，齒孔酸會促使Hep 3B細胞中的LC3-Ⅰ轉變為LC3-Ⅱ，並產生大量的自噬體和自噬溶酶體，但不會明顯地增加細胞的亞二倍體比例或導致細胞破損，故齒孔酸誘導Hep 3B細胞死亡之主要模式為自體吞噬，而不是細胞凋亡或細胞壞死。深入探討其分子機制後發現，齒孔酸首先可促進Hep 3B細胞中的ROS生成和降低ATP水平而導致內質網壓力產生，接著促使細胞質中的鈣離子濃度和BiP表現量上升、DAPK的磷酸化下降及Beclin-1、JNK和Bcl-2的磷酸化上升，最終導致自體吞噬。綜合以上結果顯示，齒孔酸的抗肝癌功效相當良好，且機制較為獨特，加上其在椴木栽培之牛樟芝子實體中含量甚高，因此具有量產並輔助抗癌治療的潛力。

Liver cancer is the second leading cause of cancer deaths in Taiwan as per the 2011 statistics, and ranks the fourth in cancer related mortality in the world. Hence to maintain a healthy liver is a big issue in Taiwan. Recent researches have shown that Antrodia cinnamomea, a Taiwan-specific medicinal mushroom, can manipulate biological activities, including hepatoprotection, anti-inflammation, anti-HBV activity, anticancer activity, etc. The active constituents include polysaccharides, benzenoids, triterpenoids, steroids, etc., and among them triterpenoids are the most prominent because of their potent anticancer effects. In this study, the anti-liver cancer activity and molecular mechanisms of eburicoic acid, the second most abundant triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea was investigated using the human hepatoma Hep 3B cells. The results show that eburicoic acid effectively reduced Hep 3B cell viability within 24 hours, and the IC50 was 18.4 μM, which was equivalent to 8.7 μg/mL. Besides, eburicoic acid induced conversion of LC3-Ⅰto LC3-Ⅱ and a large number of autophagosomes/autophagolysosomes formation, but increasing of hypodiploid proportion or cell lysis obviously in Hep 3B cells. So the principal mode of Hep 3B cell death induced by eburicoic acid was autophagy, rather than apoptosis or necrosis. In depth investigation for the molecular mechanisms, revealed that eburicoic acid firstly promoted ROS generation and ATP depletion, leading to ER stress, followed by elevated cytosolic calcium ion concentration and BiP expression, downregulated phosphorylation of DAPK, upregulated phosphorylation of Beclin-1, JNK, and Bcl-2, and finally induced autophagy in Hep 3B cells. These results indicate that eburicoic acid has significant anti-liver cancer effects and more distinctive mechanisms. Coupled with these findings and the high content of eburicoic acid in the fruiting bodies of basswood cultivated Antrodia cinnamomea, eburicoic acid has the potential for mass production and to assist cancer therapy.