Apoptotic effects of a high performance liquid chromatography (HPLC) fraction of Antrodia camphorata mycelia are mediated by down-regulation of the expressions of four tumor-related genes in human non-small cell lung carcinoma A549 cell

Aim of the study: Antrodia camphorata (niu-chang-chih) is a fungus native to Taiwan which is believed to be effective in preventing diseases. Recent reports demonstrate that Antrodia camphorata products induce the apoptosis of various kinds of tumor cells. In this study we determined the inhibitory effects of alcohol extract and individual fractions of alcohol extract on the proliferation of human non-small cell lung carcinoma A549 cell and clarified the mechanism underlying the anti-cancer activities. Materials and methods: Alcohol extracts of Antrodia camphorata mycelia were prepared by the serial extraction with the solvents with increasing polarity and fractionated using HPLC. Cell viability was determined by MTT assay. Apoptosis detection was carried out by subG(1) analysis and annexin V/propidium iodide staining using flow cytometry. The impacts of HPLC fractions on the expression levels of apoptosis- and cancer-related proteins were evaluated by western blotting. Results: Three HPLC fractions, fractions 5-7, had robust inhibition of human A549 cells and among them fraction 6 (Fr-6) possessed the most potent effectiveness. Apoptotic assay showed that Fr-6-induced human A549 cell apoptosis by triggering the mitochondrial pathway and endothelium reticulum (ER) stress. Immunoblotting results demonstrated that Fr-6 possibly activated ER stress by lowering the expression level of calpain 1/2 small subunit and Fr-6-mediated decrease in cell proliferation might attribute to the suppressive effect on the Erk 1/2 pathway, which arose from Fr-6-derived low galectin-1 expression. Furthermore Fr-6 could diminish Rho GDP dissociation inhibitor alpha (RhoGDI-alpha) expression and subsequently activated c-Jun NH2-terminal kinase (JNK) pathway, which is linked to cell apoptosis. Fr-6 also could decrease the production level of eukaryotic translation initiation factor 5A, which is a potential cancer intervention target. Conclusion: These results suggested that the anti-cancer activity of Antrodia camphorata might be due to multiple active metabolites, which work together to induce cell apoptosis via various pathways. (C) 2009 Elsevier Ireland Ltd. All rights reserved.