椴木栽培牛樟芝子實體中三萜類活性成分 Antcin K藉由誘導人類肝癌細胞粒線體及內質網壓力調節之細胞凋亡達到抑癌效果

Abstract

根據中華民國101年行政院衛生署十大癌症死因統計，肝癌佔癌症主要死因第二位，更高居全世界排名第四位，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，本研究欲探討由椴木栽培之牛樟芝子實體中純化出含量最多之麥角甾烷型三萜類─antcin K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，antcin K能有效抑制Hep 3B細胞存活率及促使Hep 3B細胞膜之磷脂醯絲胺酸外翻、染色質濃縮、DNA損傷，但不會明顯地增加細胞中自噬體生成或導致細胞膨脹而破損，故antcin K誘導Hep 3B細胞死亡之主要模式為細胞凋亡，而不是自體吞噬或細胞壞死。深入探討其分子機制後發現，antcin K首先可促進Hep 3B細胞中ROS生成與降低ATP水平而導致內質網壓力產生，內質網壓力伴隨著不完全摺疊反應相關蛋白CHOP表現量上升，進而抑制抗凋亡蛋白Bcl-xL表現量，導致內質網與粒線體膜上之Bax、Bak通道被活化，造成鈣離子由內質網中釋出並部分進入粒線體，最後改變粒線體膜通透性，而釋出促凋亡因子，包括HtrA2/Omi、AIF、Endo-G及cytochrome c，其中cytochrome c進一步活化caspase-9、caspase-3，並切割下游蛋白PARP，最終導致細胞凋亡。綜合以上結果顯示，antcin K可藉由誘導人類肝癌細胞粒腺體及內質網壓力調節之細胞凋亡達到抑癌效果，具有輔助抗癌治療之潛力。

Liver cancer is the second leading cause of cancer deaths in Taiwan as per the 2012 statistics, and ranks the fourth in cancer related mortality in the world. Hence there is a need to focus more on hepato protective foods 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, include hepatoprotection, anti-inflammation, anti-HBV activity, anticancer activity, etc. The active constituents include polysaccharides, triterpenoids, steroids, etc., and among these, m whose triterpenoids are the most prominent because of their potent anticancer effects. In this study, the anti-liver cancer activity and molecular mechanisms of antcin K, the most abundant ergostsne triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea was investigated using the human hepatoma Hep 3B cells. The results show that antcin K effectively reduced Hep 3B cell viability within 48 hours. In addition, antcin K induced phosphatidylserine exposure, chromatin condensation, DNA damage, but not significantly increasing autophagosome content or causing cell expansion and cell lysis. So the principal mode of Hep 3B cell death induced by antcin K was apoptosis, rather than autophagy or necrosis. In depth investigation for the molecular mechanisms, revealed that antcin K firstly promoted ROS generation and ATP depletion, lead to ER stress, followed by CHOP expression, down-regulated Bcl-xL and activated Bax/Bak channel to promote intra-cellular calcium ion release from the endoplasmic reticulum then uptake into the mitochondria, resulting in mitochondrial membrane permeability changes. After losing of mitochondrial membrane potential, the apoptosis related factors were released, including HtrA2/Omi, AIF, Endo-G and cytochrome c, the cytochrome c activated the caspase-9, caspase-3, and cut downstream protein PARP, ultimately leading to cell apoptosis. These results suggested that antcin K induced endoplasmic reticulum stress and mitochondria-mediated apoptosis in human hepatoma cells. Coupled with these findings, antcin K has the potential to be complementary agent in liver cancer therapy.