Eburicoic Acid, an Active Triterpenoid from the Fruiting Bodies of Basswood Cultivated Antrodia cinnamomea, Induces ER Stress-Mediated Autophagy in Human Hepatoma Cells

Yu-Cheng Su；Chun-Ting Liu；Yung-Lin Chu；Rajasekaran Raghu；Yueh-Hsiung Kuo；Lee-Yan Sheen

Liver cancer ； Antrodia cinnamomea ； Eburicoic acid ； Autophagy ； ER stress

Journal of Traditional and Complementary Medicine

2卷4期（2012 / 10 / 01）

312 - 322

英文

Antrodia cinnamomea, a Taiwan-specific medicinal mushroom, can manipulate biological activities, including hepatoprotection, anti-inflammation, anti-hepatitis B virus activity, anticancer activity, etc. 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 IC_(50) was 18.4 μM, which was equivalent to 8.7 μg/mL. Besides, eburicoic acid induced conversion of LC3-I to LC3-II and a large number of autophagosomes/autolysosomes formation. In depth investigation for the molecular mechanisms, revealed that eburicoic acid firstly promoted reactive oxygen species generation and ATP depletion, leading to endoplasmic reticulum 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.

1. Ao, Z.H.,Xu, Z.H.,Lu, Z.M.,Xu, H.Y.,Zhang, X.M.,Dou, W.F.(2009).Niuchangchih (Antrodia camphorata) and its potential in treating liver diseases.J. Ethnopharmacol,121,194-212.
2. Bradford, M.M.(1976).A rapid and sensitive method for The quantitation of microgram quantities of protein ultilizing The principle of protein-dye binding.Anal. Biochem,72,248-254.
3. Chang, T.T.,Chou, W.W.(1995).Antrodia cinnamomea sp. nov. on Cinnamomum kanehirai in Taiwan.Mycol. Res,99,756-758.
4. Chen, J.C.,Lin, W.H.,Chen, C.N.,Sheu, S.J.,Huang, S.J.,Chen, Y.L.(2001).Development of Antrodia camphorata mycelium with submerged culture.Fung. Sci.,16,7-22.
5. Colombo, M.,Donato, M.F.(2005).Prevention of hepatocellular carcinoma.Semin. Liver Dis,25,155-161.
6. Crouch, S.P.,Kozlowski, R.,Slater, K.J.,Fletcher, J.(1993).The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity.J. Immunol. Methods,160,81-88.
7. Decker, T.,Lohmann-Matthes, M.L.(1988).A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity.J. Immunol. Methods,15,61-69.
8. Hussain, S.P.,Schwank, J.,Staib, F.,Wang, X.W.,Harris, C.C.(2007).TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer.Oncogene,26,2166-2176.
9. Kaminskyy, V.,Abdi, A.,Zhivotovsky, B.(2011).A quantitative assay for the monitoring of autophagosome accumulation in different phases of the cell cycle.Autophagy,7,83-90.
10. Kaufman, R.J.(2002).The cellular response to accumulation of unfolded proteins in the endoplasmic reticulum.Faseb J.,16,A891.
11. Kerr, J.F.,Wyllie, A.H.,Currie, A.R.(1972).Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.Br. J. Cancer,26,239-257.
12. Kroemer, G.,Reed, J.C.(2000).Mitochondrial control of cell death.Nat. Med,6,513-519.
13. Krysko, D.V.,Vanden Berghe, T.,D'Herde, K.,Vandenabeele, P.(2008).Apoptosis and necrosis: detection, discrimination and phagocytosis.Methods,44,205-221.
14. Kuo, P.L.,Hsu, Y.L.,Cho, C.Y.,Ng, L.T.,Kuo, Y.H.,Lin, C.C.(2006).Apoptotic effects of Antrodia cinnamomea fruiting bodies extract are mediated through calcium and calpain-dependent pathways in Hep 3B cells.Food Chem. Toxicol.,44,1316-1326.
15. Kuo, Y.H., Lin, B.F., 2011. Compounds from Antrodia camphorata. Official gazette of the United States patent and trademark office patents.
16. Laszczyk, M.N.(2009).Pentacyclic triterpenes of the lupane, oleanane and ursane group as tools in cancer therapy.Planta Med,75,1549-1560.
17. León, F.,Quintana, J.,Rivera, A.,Estévez, F.,Bermejo, J.(2004).Lanostanoid triterpenes from Laetiporus sulphureus and apoptosis induction on HL-60 human myeloid leukemia cells.J. Nat. Prod,67,2008-2011.
18. Mizushima, N.,Levine, B.,Cuervo, A.M.,Klionsky, D.J.(2008).Autophagy fights disease through cellular self-digestion.Nature,451,1069-1075.
19. Mosmann, T.(1983).Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.J. Immunol Methods,65,55-63.
20. Shen, Y.C.,Chen, C.F.,Wang, Y.H.,Chang, T.T.,Chou, C.J.(2003).Evaluation of the immuno-modulating activity of some active principles isolated from the fruiting bodies of Antrodia camphorata.Chinese Pharm. J.,55,313-318.
21. Thuerauf, D.J.,Marcinko, M.,Gude, N.,Rubio, M.,Sussman, M.A.,Glembotski, C.C.(2006).Activation of the unfolded protein response in infarcted mouse heart and hypoxic cultured cardiac myocytes.Circ. Res,99,275-282.
22. van der Vlies, D.,Makkinje, M.,Jansens, A.,Braakman, I.,Verkleij, A.J.,Wirtz, K.W.,Post, J.A.(2003).Oxidation of ER resident proteins upon oxidative stress: effects of altering cellular redox/antioxidant status and implications for protein maturation.Antioxid. Redox Sign,5,381-387.
23. Verfaillie, T.,Salazar, M.,Velasco, G.,Agostinis, P.(2010).Linking ER stress to autophagy: potential implications for cancer therapy.Int. J. Cell Biol,2010,930509.
24. Wallen, C.A.,Higashikubo, R.,Dethlefsen, L.A.(1982).Comparison of two flow cytometric assays for cellular RNA-acridine orange and propidium iodide.Cytometry,3,155-160.
25. Wu, C.C.,Chung, J.G.,Tsai, S.J.,Yang, J.H.,Sheen, L.Y.(2004).Differential effects of allyl sulfides from garlic essential oil on cell cycle regulation in human liver tumor cells.Food Chem. Toxicol.,42,1937-1947.
26. Wu, S.H.,Ryvarden, L.,Chang, T.T.(1997).Antrodia camphorata ("niu-chang-chih"), new combination of a medicinal fungus in Taiwan.Bot. Bull. Acad. Sinica,38,273-275.
27. Yeh, C.T.,Rao, Y.K.,Yao, C.J.,Yeh, C.F.,Li, C.H.,Chuang, S.E.,Luong, J.H.T.,Lai, G.M.,Tzeng, Y.M.(2009).Cytotoxic triterpenes from Antrodia camphorata and their mode of action in HT-29 human colon cancer cells.Cancer Lett,285,73-79.
28. Zachwieja, J.,Zaniew, M.,Bobkowski, W.,Stefaniak, E.,Warzywoda, A.,Ostalska-Nowicka, D.,Dobrowolska-Zachwieja, A.,Lewandowska-Stachowiak, M.,Siwinska, A.(2005).Beneficial in vitro effect of N-acetyl-cysteine on oxidative stress and apoptosis.Pediatr. Nephrol.,20,725-731.
29. Zhou, F.,Yang, Y.,Xing, D.(2011).Bcl-2 and Bcl-xL play important roles in the crosstalk between autophagy and apoptosis.FEBS J.,278,403-413.

1. Yung-Lin Chu,Yueh-Hsiung Kuo,Yu-Cheng Su,Lee-Yan Sheen,Chi-Tang Ho,Chiao-I. Lai(2016).Antcin K, an active triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea, induces mitochondria and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells.Journal of Traditional and Complementary Medicine,6(1),24-32.