Antrodia salmonea suppresses invasion and metastasis in triple-negative breast cancer cells by reversing EMT through the NF-κB and Wnt/ß-catenin signaling pathway.

Antrodia salonea (AS), a fungus that is indigenous to Taiwan has been well known for its anti-cancer properties. We investigated the anti-metastatic and anti-epithelial-mesenchymal transition (EMT) properties of AS in TNBC cells. To determine their EMT and metastasis levels, in vitro wound healing, wound invasion, Western blotting, RT-PCR, luciferase activity and immunofluorescence assays were performed, while the in vivo anti-metastatic efficacy of AS was evaluated in BALB/c-nu mice through bioluminescence imaging, HE staining, and immunohistochemical staining. MDA-MB-231 cells, when treated with AS concentrations (25-100 µg/mL) resulted in significant reduction of invasion and migration as well as the downregulation of VEGF, uPAR, uPA and MMP-9 (inhibition of PI3K/AKT/NFκB pathways). AS treatment prevented morphological changes and reversed EMT through the upregulation of E-cadherin and the downregulation of N-cadherin, Slug, Twist, and Vimentin. Inhibition of Smad3 signaling pathway, downregulation of ß-catenin pathway and upregulation of GSK3ß expression were also observed while, suppression of metastasis and EMT in TGF-ß1-stimulated non-tumorigenic MCF-10A cells was observed when treated with AS. Histological analysis confirmed that AS reduced tumor metastasis and upregulated E-cadherin expression in biopsied lung tissues. Our results indicated that AS exhibits anti-EMT and anti-metastatic activity, that could contribute to develop anticancer drugs against TNBC.

Ganoderma tsugae induced ROS-independent apoptosis and cytoprotective autophagy in human chronic myeloid leukemia cells.

The medicinal fungus Ganoderma, known in Chinese as Lingzhi or Reishi, traditionally has various medicinal uses and has been employed in cancer treatment in Asia for centuries. This study used ethanol-extracted Ganoderma tsugae (GT) and examined its antitumor activities on human chronic myeloid leukemia cells as well as its molecular mechanism of action. Treatment with GT (200-400 µg/mL) significantly reduced cell viability and caused G2/M arrest in K562 cells. In addition, GT induced mitochondrial and death receptor mediated apoptosis, correlated with DNA fragmentation, followed by cytochrome c release, caspase-3/8/9 activation, PARP cleavage, Fas activation, Bid cleavage, and Bax/Bcl-2 dysregulation. Cytoprotective autophagy was found to be induced by GT, as was revealed by increased LC3-II accumulation, Beclin-1/Bcl-2 dysregulation, acidic vesicular organelle formation, and p62/SQSTM1 activation. Notably, pretreatment of cells with the autophagy inhibitors 3-MA and CQ enhanced GT-induced apoptosis. Interestingly, reactive oxygen species production in cells was not triggered by GT administration; equally, the antioxidant N-acetylcysteine was found to be incapable of preventing apoptosis and autophagy induced by GT treatment. Finally, this study discovered that cytoprotective autophagy induced by GT was associated with EGFR and PI3K/AKT/mTOR signaling cascade suppression. In summary, GT demonstrated antitumor activity against human chronic myeloid leukemia.

Induction of autophagic cell death in human ovarian carcinoma cells by Antrodia salmonea through increased reactive oxygen species generation.

We reported in our previously executed studies that the fermented culture broth of Antrodia salmonea (AS), a mushroom used in Taiwanese folk medicine induced reactive oxygen species (ROS)-mediated apoptosis in human ovarian carcinoma cells. In this study, we studied the anticancer efficacies of AS (0-240 µg/ml) by examining the key molecular events implicated in cell death associated with autophagy in SKOV-3 and A2780 human ovarian carcinoma cells and clarified the fundamental molecular mechanisms. Treatment of ovarian carcinoma cells with AS-induced autophagic cell death mediated by increased microtubule-associated protein LC3-II, GFP-LC3 puncta, and acidic vesicular organelle (AVO) formation. These events are linked with the activation of p62/SQSTM1, the inhibition of ATG4B, the expression of ATG7, and the dysregulation of Beclin-1/Bcl-2 (i.e., B-cell lymphoma 2). N-acetylcysteine inhibited AS-induced ROS generation, which in turn constricted AS-induced LC3 conversion, AVO formation, and ATG4B inhibition, indicating ROS-mediated autophagy cell death. In addition, the 3-methyladenine (3-MA) or chloroquine (CQ)-induced autophagy inhibition decreased AS-induced apoptosis. Additionally, apoptosis inhibition by Z-VAD-FMK, a pan-caspase inhibitor, substantially suppressed AS-induced autophagy. Furthermore, AS-inhibited HER-2/ neu and PI3K/AKT signaling pathways which were reversed by autophagy inhibitors 3-MA and CQ. Thus, A. salmonea is a potential chemopreventive agent that is capable of activating ROS-mediated autophagic cell death in ovarian carcinoma cells.

Antrodia salmonea-induced oxidative stress abrogates HER-2 signaling cascade and enhanced apoptosis in ovarian carcinoma cells.

Antrodia salmonea is well known in Taiwan as a traditional Chinese medicinal fungus and has demonstrated antioxidant, anti-inflammatory, and anticancer effects. However, the anticancer activity of A. salmonea against human ovarian cancer is still elusive. Therefore, we investigated the antiovarian tumor activity of a fermented culture broth of A. salmonea and exhibits its underlying molecular mechanism. A. salmonea shows a significant effect on cell viability in human ovarian carcinoma (SKOV-3 or A2780) cell lines with an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Increased terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells and annexin V-propidium iodide stained cells indicate that A. salmonea induces late apoptosis in SKOV-3 cells. Notably, treatment with A. salmonea induced the following events: Apoptosis; caspase-3, -8, -9 and poly(ADP-ribose) polymerase activation; first apoptosis signal (Fas) and Fas ligand activation; Bid cleavage; and Bax2-B-cell lymphoma 2 dysregulation. The results show that A. salmonea-induced apoptosis was mediated by both mitochondrial and death receptor pathways. An increase in intracellular reactive oxygen species (ROS) was also observed in A. salmonea-treated cells, whereas the antioxidant N-acetylcysteine (NAC) prevented A. salmonea-induced cell death and DNA fragmentation, indicating that A. salmonea-induced apoptosis was mediated by ROS generation. Interestingly, A. salmonea-induced apoptosis is associated with the suppression of human epidermal growth factor receptor-2 (HER-2/neu) and phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) expression in HER-2/neu overexpressing SKOV-3 cells. NAC significantly prevented A. salmonea-induced HER-2/neu depletion and PI3K/AKT inactivation, indicating that A. salmonea-triggered apoptosis is mediated by ROS-inhibited HER-2/neu signaling cascades. To our knowledge, this is the first report describing the anticancer activity of this potentially beneficial mushroom against human ovarian carcinoma.