Previously, we demonstrated that a submerged fermentation culture of Antrodia camphorata (AC) promotes cell-cycle arrest and apoptosis in human
estrogen receptor-positive/negative
breast cancer cells. However, whether AC is effective against HER-2/neu-overexpressing breast
cancers has not been thoroughly elucidated. In the present study, we showed that AC exhibited a significant cytotoxic effect against HER-2/neu-overexpressing MDA-MB-453 and BT-474 cells. Immunoblot analysis demonstrated that HER-2/neu and their
tyrosine phosphorylation were inhibited by AC in a dose-dependent manner. An increase in intracellular
reactive oxygen species (ROS) was observed in AC-treated cells, whereas
antioxidant N-acetylcysteine (NAC) significantly prevented AC induced HER-2/neu depletion and cell death, which directly indicates that AC-induced HER-2/neu depletion and cell death was mediated by ROS generation. Also, AC significantly downregulated the expression of
cyclin D1,
cyclin E, and CDK4 followed by the suppression of PI3K/Akt, and their downstream effectors GSK-3β and β-
catenin. Notably, AC-treatment induced apoptotic cell death, which was associated with sub-G1 accumulation, DNA fragmentation,
mitochondrial dysfunction,
cytochrome c release,
caspase-3/-9 activation, PARP degradation, and Bcl-2/Bax dysregulation. Assays for colony formation also confirmed the growth-inhibitory effects of AC. This is the first report confirming the anticancer activity of this potentially beneficial mushroom against human HER-2/neu-overexpressing breast
cancers.