Antrodia Salmonea Research Articles

Antrodia salmonea suppresses epithelial-mesenchymal transition/metastasis and Warburg effects by inhibiting Twist and HIF-1α expression in Twist-overexpressing head and neck squamous cell carcinoma cells

Ethnopharmacological relevanceAntrodia salmonea (AS), linked to the genus Taiwanofungus, is a medicinal fungus, and exhibits anti-inflammatory, anti-oxidant, and tumor inhibiting properties. Aim of the studyIn this study, we investigated the metabolic reprogramming and anti-metastasis/epithelial-mesenchymal transition (EMT) effects of AS exposure in Twist-overexpressing head and neck squamous cell carcinoma (HNSCC, OECM-1 and FaDu-Twist) cells. Materials and methodsMTT assay, Western blot, migration/invasion assay, immunofluorescence, glucose uptake assay, lactate assay, oxygen consumption rate (OCR)/Extracellular acidification rate (ECAR) assay, Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS), and qRT-PCR experimental techniques were used to evaluate the therapeutic potential of AS treatment in HNSCC cells. ResultsThis study showed that AS exhibits anti-EMT and anti-metastatic effects as well as metabolic reprogramming in Twist-overexpressing HNSCC cells. AS exposure inhibited Twist and hypoxia-inducible factor-1α (HIF-1α) protein and/or mRNA expression in Twist-overexpressing OECM-1 and FaDu-Twist cells. AS markedly suppressed EMT by enhancing the expression of E-cadherin; while the N-cadherin was suppressed. Furthermore, glucose uptake and lactate accumulation, together with HIF-1α-regulated glycolysis genes were diminished by AS in OECM-1 cells. AS decreased the ECAR, and enhanced the OCR together with basal respiration, ATP production, maximal respiration, and spare respiratory capacity under normoxia and hypoxia (CoCl2) in OECM-1 cells. There was a marked reduction in the level of glycolytic intermediate's; while TCA cycle metabolites were increased by AS treatment in OECM-1 cells. ConclusionsWe concluded that AS treatment suppresses EMT/metastasis and Warburg effects through Twist and HIF-1α inhibition in Twist-overexpressing HNSCC cells.

In vitro and in vivo anti-tumor activity of Antrodia salmonea against twist-overexpressing HNSCC cells: Induction of ROS-mediated autophagic and apoptotic cell death.

Head and neck squamous cell carcinoma (HNSCC) is a relatively common malignancy, characterized by lethal morbidity. Herein, we attempted to investigate the autophagy/apoptosis activities of the submerged fermented broths of Antrodia salmonea (AS) in HNSCC Twist-overexpressing (OECM-1 and FaDu-Twist) cells. AS (0-150μg/mL) effectively reduced cell viability, colony formation, and downregulated Twist expression in OECM-1 and FaDu-Twist cells compared to FaDu cells. AS- induced apoptosis was mainly associated with activation of caspase-3, PARP cleavage, increased expression of VDAC-1 and disproportionation of Bax/Bcl-2. Annexin V/PI staining suggested late apoptosis induction by AS treatment. AS exhibits enhanced autophagy process mediated via LC3-I/II accumulation, increased acidic vesicular organelles (AVOs) formation and p62/SQSTM1 expression feeding into the apoptotic program. However, pre-treatment with autophagy blockers 3-MA and CQ significantly diminished AS-induced cell death. Additionally, suppression of AS-induced ROS release by treatment with antioxidant N-acetylcysteine (NAC) resulted in reduction of apoptotic and autophagic cell death. In vivo studies strengthened the above observations and showed that AS effectively reduced the tumor volume and tumor weight in OECM-1-xenografted nude mice. This study discovered that Antrodia salmonea exhibits a novel anti-cancer mechanism which could be harnessed as a new potent drug for HNSCC treatment.

The in vitro and in vivo anticancer activities of Antrodia salmonea through inhibition of metastasis and induction of ROS-mediated apoptotic and autophagic cell death in human glioblastoma cells

Background:Antrodia salmonea (AS) exhibits anticancer activities against various cancers. ObjectiveThis study investigated the anticancer activities of AS on human glioblastoma (GBM8401 and U87MG) cells both in vitro and in vivo and explained the underlying molecular mechanism. MethodsMTT, colony formation, migration/invasion assay, immunoblotting, immunofluorescence, TUNEL, Annexin V/PI staining, AO staining, GFP-LC3 transfection, TEM, qPCR, siLC3, DCFH2-DA assay, and xenografted-nude mice were used to assess the potential of AS therapy. ResultsAS treatment retarded growth and suppressed colony formation in glioblastoma cells. AS attenuates EMT by suppressing invasion and migration, increasing E-cadherin expression, decreasing Twist, Snail, and N-cadherin expression, and inhibiting Wnt/β-catenin pathways in GBM8401 and U87MG cells. Furthermore, AS induced apoptosis by activating caspase-3, cleaving PARP, and dysregulating Bax and Bcl-2 in both cell lines. TUNEL assay and Annexin V/PI staining indicated AS-mediated late apoptosis. Interestingly, AS induced autophagic cell death by LC3-II accumulation, AVO formation, autophagosome GFP-LC3 puncta, p62/SQSTM1 expression, and ATG4B inhibition in GBM8401 and U87MG cells. TEM data revealed that AS favored autophagosome and autolysosome formation. The autophagy inhibitors 3-MA/CQ and LC3 knockdown suppressed AS-induced apoptosis in glioblastoma cells, indicating that the inhibition of autophagy decreased AS-induced apoptosis. Notably, the antioxidant N-acetylcysteine (NAC) inhibited AS-mediated ROS production and AS-induced apoptotic and autophagic cell death. Furthermore, AS induced ROS-mediated inhibition of the PI3K/AKT/mTOR signaling pathway. AS reduced the tumor burden in GBM8401-xenografted nude mice and significantly modulated tumor xenografts by inducing anti-EMT, apoptosis, and autophagy. AS could be a potential antitumor agent in human glioblastoma treatment.

Antrodia salmonea Extracts Regulate p53-AR Signaling and Apoptosis in Human Prostate Cancer LNCaP Cells.

Antrodia salmonea (AS) is a genus of Antrodia, an epiphyte of Cunninghamia konishii in Taiwan. AS has been reported to have potential therapeutic effects on different diseases, including diarrhea, abdominal pain, and hypertension. AS has been reported to have anticancer effects on numerous cancer types, such as ovarian carcinoma and triple-negative breast cancer. Our previous studies demonstrated that antrocins and triterpenoids are possibly bioactive compositions. However, the effects of AS on prostate cancer remain unknown. Therefore, we investigated the role of AS in prostate cancer growth, apoptosis, and cell cycle regulation. The results showed that AS extracts significantly inhibited the proliferation of prostate cancer LNCaP cells in a dose-dependent manner and increased the levels of apoptotic markers (cleaved PARP and cleaved caspase 3/8/9). In addition, the cell cycle-related proteins CDK1, CDK2, CDK4, and their respective specific regulators Cyclin B1, Cyclin A, and Cyclin D were also affected. Besides, AS treatment increased p53 protein levels and slowed its degradation in LNCaP cells. Interestingly, we found that AS treatment reduced both total protein and Ser-81 phosphorylation levels of the androgen receptor (AR). Notably, the increase of nuclear p53 was accompanied by the down-regulation of AR, suggesting a reverse regulation between p53 and AR in LNCaP cells was triggered by AS treatment. These findings suggest that AS extracts trigger the apoptosis of prostate cancer cells through the reverse regulation of p53 and AR and elucidate that AS extracts might be a potential treatment for androgen-dependent prostate cancer in the near future.

Antrodia salmonea extract inhibits cell proliferation through regulating cell cycle arrest and apoptosis in prostate cancer cell lines.

Antrodia salmonea (AS) is a fungus, which belongs to a fungal family of Taiwanofungus salmoneus with the features of anti-oxidant, anti-inflammatory, and anticancer. Recent studies have shown that AS has anti-cancer functions in ovarian and breast cancer. However, the effects of AS on prostate cancer (PCa) proliferation remain unknown. Therefore, we investigated the role of AS in PCa proliferation through apoptosis, and cell cycle regulation in PCa cell lines. Our results showed that Antrodia salmonea extract (ASE) inhibited PCa cells growth with a dose-dependent manner. In addition, ASE decreased the anchorage-independent growth formation ability in PC3 cells. Moreover, ASE-induced cell growth inhibition in PCa cells (DU145, PC3) was correlated to decreased cell cycle-related proteins such as cyclin A/B and cyclin-dependent kinase CDK1/2/4, and increased cell cycle inhibitor proteins p21. Besides, ASE decreased the total protein level of epidermal growth factor receptor and its downstream signaling pathways Akt and Erk in both PCa cells. We found that apoptotic markers such as cleaved-PARP protein levels increased significantly in DU145 cells indicating ASE might induce apoptosis. In conclusion, our results suggest that ASE may have the ability to induce PCa cell death through regulating cell cycle arrest and apoptosis pathways.

Antrodia salmonea induces apoptosis and enhances cytoprotective autophagy in colon cancer cells.

A traditional Chinese medicinal fungus, Antrodia salmonea (AS), with antioxidant properties is familiar in Taiwan but anti-cancer activity of AS in human colon cancer is ambiguous. Hence, we explored the anti-cancer activity of AS in colon cancer cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that AS showed a remarkable effect on cell viability in colon cancer cells; SW620, HCT116, and HT29. Annexin V/propidium iodide (PI) stained cells indicated that AS induced both early/late apoptosis in SW620 cells. Additionally, cells treated with AS induced caspase-3 activation, poly (ADP-ribose) polymerase (PARP) cleavage, mitochondrial dysfunction, and Bcl-2 associated X (Bax)/B-cell lymphoma (Bcl-2) dysregulation. Microtubule- associated protein 1A/1B-light chain 3B (LC3-II) accumulation, sequestosome 1 (p62/SQSTM1) activation, autophagy related 4B cysteine peptidase (ATG4B) inactivation, acidic vesicular organelles (AVOs) formation, and Beclin-1/Bcl-2 dysregulation revealed that AS-induced autophagy. Interestingly, cells pretreated with 3-methyladenine (3-MA) strengthened AS-induced caspase-3/apoptosis. Suppression of apoptosis by z-Val-Ala-Asp fluoromethyl ketone (Z-VAD-FMK) did not however block AS-induced autophagy, suggesting that autophagy was not attenuated by the AS-induced apoptosis. Application of N-acetylcysteine (NAC) prevented AS-induced cell death, caspase-3 activation, LC3-II accumulation, and AVOs formation, indicating that AS-induced apoptosis and autophagy was mediated by reactive oxygen species (ROS). Furthermore, AS-induced cytoprotective autophagy and apoptosis through extracellular signal-regulated kinase (ERK) signaling cascades. Moreover, in vivo data disclosed that AS inhibited colitis-associated tumorigenesis in azoxymethane (AOM)-dextran sodium sulphate (DSS)-treated mice. For the first time, we report the anti-cancer properties of this potentially advantageous mushroom for the treatment of human colon cancer.

Assessment of the thermal and oxidative stability of Antrodia salmonea extract in dripping pills via DSC and TAM air analyses

Lovastatin, also known as monacolin K, is the major active component in red yeast rice. It is the principal active substance in Monascus fermentation products and a statin (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) that inhibits rate-limiting enzymes in the production of cholesterol. Lovastatin is effective in reducing cholesterol levels in humans and is a lipid-lowering drug. Herein, lovastatin was produced from Antrodia salmonea by a liquid-state fermentation process. The A. salmonea extract contained 431 μg g–1 (d.b) lovastatin. A. salmonea extract with high lovastatin content is insoluble, resulting in poor bioavailability, which is problematic. In this study, we utilized a dripping pill delivery system to improve the absorption of lovastatin. Comparisons of the oxidative and thermal stability properties of the lovastatin medicine, A. salmonea extract and dripping pill were conducted by using high-performance liquid chromatography (HPLC) analyses, differential scanning calorimetry (DSC) tests and isothermal microcalorimetry (TAM Air) measurements. We also investigated the thermal degradation of the A. salmonea extract dripping pill under various atmospheric environments to determine the proper storage conditions, including the onset time, total reaction time and heat power of the exothermic reaction, by isothermal storage experimental analyses. The analysis of the oxidative and thermal stability properties suggested that the A. salmonea extract had a higher lovastatin content than the lovastatin medicine, and the results demonstrated the superiority of the dripping pills. Overall, the results can be applied to determine the suitable conditions for the production, storage, heat treatment and food processing of A. salmonea extract dripping pills, and the results will be beneficial to food and pharmaceutical applications.

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 suppresses invasion and metastasis in triple-negative breast cancer cells by reversing EMT through the NF-κB and Wnt/β-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 β-catenin pathway and upregulation of GSK3β expression were also observed while, suppression of metastasis and EMT in TGF-β1-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.

Inhibition of ROS production, autophagy or apoptosis signaling reversed the anticancer properties of Antrodia salmonea in triple-negative breast cancer (MDA-MB-231) cells

We investigated the in vitro and in vivo anticancer properties of Antrodia salmonea (AS), a well-known edible/medicinal mushroom in Taiwan, on human triple-negative breast cancer (MDA-MB-231) cells and xenografted nude mice; and revealed the underlying molecular mechanisms involved in autophagic- and apoptotic-cell death. Treatment of MDA-MB-231 cells with fermented culture broth of AS (0–200 μg/mL) inhibited cell viability/growth. AS-induced autophagy was evidenced via increased LC3-II accumulation, GFP-LC3 puncta and AVOs formation in MDA-MB-231 cells. These events are associated with increased ATG7, decreased p-mTOR, vanished SQSTM1/p62 expressions and dysregulated Beclin-1/Bcl-2 ratio. AS-induced apoptosis/necrosis through increased DNA fragmentation, Annexin-V/PI stained cells and Bax expression. Both mitochondrial (caspase-9/caspase-3/PARP) and death-receptor (caspase-8/FasL/Fas) signaling pathways are involved in execution of apoptosis. Interestingly, blockade of AS-induced ROS production by N-acetylcysteine pretreatment substantially attenuated AS-induced autophagy, mitochondrial dysfunction and autophagic/apoptotic-cell death. Inhibition of apoptosis by Z-VAD-FMK suppressed AS-induced autophagic-death (decreased LC3-II/AVOs). Similarly, inhibition of autophagy by 3-methyladenine/chloroquine diminished AS-induced apoptosis (decreased DNA fragmentation/caspase-3) in MDA-MB-231 cells. Bioluminescence imaging further confirmed that AS inhibited breast tumor growth in living MDA-MB-231-luciferase-injected nude mice. Taken together, AS crucially involved in execution/propagation of autophagic- or apoptotic-death of MDA-MB-231 cells, and decreased tumor growth in xenografted nude mice.

Antrodia salmonea induces G2 cell-cycle arrest in human triple-negative breast cancer (MDA-MB-231) cells and suppresses tumor growth in athymic nude mice

Ethnopharmacological relevanceAntrodia salmonea (AS), is a well-known folk medicinal mushroom in Taiwan, has been reported to exhibit anti-oxidant, anti-angiogenic, and anti-inflammatory effects. Materials and methodsIn the present study, we examined the effects of AS on cell-cycle arrest in vitro in MDA-MB-231 cells and on tumor regression in vivo using an athymic nude mice model. ResultsAS (0–200μg/mL) treatment significantly induced G2 cell-cycle arrest in MDA-MB-231 cells by reducing the levels of cyclin B1, cyclin A, cyclin E, and CDC2 proteins. In addition, N-acetylcysteine (NAC) pretreatment prevented AS induced G2 cell-cycle arrest, indicating that ROS accumulation and subsequent cell cycle arrest might be a major mechanism of AS-induced cytotoxicity. Further, AS treatment decreased COX-2 expression and induced PARP cleavage was significantly reversed by NAC pretreatment in MDA-MB-231 cells. The in vivo study results revealed that AS treatment was effective in terms of delaying the tumor incidence and reducing the tumor growth in MDA-MB-231-xenografted nude mice. TUNEL assay, immunohistochemical staining and Western blotting confirmed that AS significantly modulated the xenografted tumor progression as demonstrated by induction of apoptosis, autophagy, and cell-cycle arrest. ConclusionOur data strongly suggest that Antrodia salmonea could be an anti-cancer agent for human breast cancer.

Quality of Bread Supplemented with Antrodia salmonea-Fermented Grains.

Fermented grains of buckwheat, oat, embryo rice and wheat, which were prepared by solid-state fermentation with Antrodia salmonea, and the mycelium was used to substitute 7% of wheat flour to make bread. No difference in proximate composition, texture profile and contents of non-volatile taste components was observed among bread samples. White bread and bread supplemented with mycelium and fermented grains looked different. Bread supplemented with fermented grains had similar thermal properties, which differed from those of white bread and bread supplemented with mycelium. Bread supplemented with fermented grains contained substantial mass fractions (on dry mass basis) of adenosine (0.92-1.96 µg/g), ergosterol (24.53-30.12 µg/g), ergothioneine (2.16-3.18 µg/g) and γ-aminobutyric acid (2.20-2.45 µg/g). In addition, bread supplemented with mycelium contained lovastatin (0.43 µg/g). White bread and bread supplemented with fermented grains had similar sensory results. Overall, fermented grains could be incorporated into bread to provide beneficial effects.

Antioxidant and Anti-Inflammatory Properties of Solid-State Fermented Products from a Medicinal Mushroom, Taiwanofungus salmoneus (Higher Basidiomycetes) from Taiwan.

Cooked grains of buckwheat (TSFB), oats (TSFO), embryo rice (TSFR), or wheat (TSFW) were inoculated with the medicinal fungus Taiwanofungus salmoneus (=Antrodia salmonea), and the metabolites formed were evaluated for antioxidant activity using the conjugated diene method, reducing power, scavenging ability, and chelating ability. Overall, the effectiveness was generally in the descending order of TSFB ≥ TSFO > TSFR ≥ TSFW. The correlation between effective concentration (EC50) of antioxidant property and antioxidant component content measured was established. However, flavonoid contents correlated well with antioxidant properties assayed (r = 0.707-0.933). Fermented products contained substantial amounts of adenosine (34.7-73.8 µg/g), ergosterol (1070-1212 µg/g), ergothioneine (81.0-119.8 µg/g), γ-aminobutyric acid (100.9-170.2 µg/g), and lovastatin (3.06-5.60 µg/g). In addition, with the addition of the extracts from fermented products, lipopolysaccharide-induced nitric oxide, tumor necrosis factor-α, interleukin-1β, and interleukin-6 production in RAW 264.7 murine macrophage cells could be effectively suppressed. Accordingly, four types of T. salmoneus fermented products could be beneficially used as food-flavoring materials and food ingredients or as nutritional supplements.

Apoptotic Effect of Extract from Medicinal Mushroom from Taiwan Taiwanofungus salmoneus (Higher Basidiomycetes) Mycelium Combined with or without Cisplatin on Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma is a cancer of high mortality; therefore, the effective therapy on this cancer is an imperative issue. Recently, anticancer agent combined with natural products has been demonstrated to increase apoptosis of various cancer cells effectively. Accordingly, we investigated the apoptotic effect and possible mechanism of the ethanol extract from Taiwanofungus salmoneus (=Antrodia salmonea) mycelium (TsE) alone or in combination with cisplatin in SK-Hep-1 cells. In this study, the proliferation of SK-Hep-1 cells could be inhibited at various concentrations of TsE for 24 h whereas TsE combined with cisplatin would inhibit the cell proliferation more notably. Moreover, the DNA damage and the interruption of cell cycle of SK-Hep-1 cells would be effectively raised after incubation with TsE combined with cisplatin for 24 h. The apoptosis of cells was dramatically induced, and the expression of caspases 3, 8, and 9, apoptosis-related protein, were significantly upregulated. Therefore, we proposed that the TsE combined with cisplatin inhibited cell proliferation by elevating sub-G1 phase, inducing DNA damage, activating caspases 3, 8, and 9 activities, and triggering cells apoptosis. These results reveal that TsE could be a potential adjuvant chemotherapeutic agent.

The anti-tumor activity of Antrodia salmonea in human promyelocytic leukemia (HL-60) cells is mediated via the induction of G1 cell-cycle arrest and apoptosis in vitro or in vivo

Ethnopharmacological relevanceThe medicinal mushroom Antrodia salmonea has been used as a traditional Chinese medicine and has demonstrated antioxidant and anti-inflammatory effects. Materials and methodsIn the present study, we examined the anti-tumor activity of the fermented culture broth of Antrodia salmonea (AS) in vitro and in vivo and revealed its underlying molecular mechanism of action. ResultsTreatment of human promyelocytic leukemia (HL-60) cells with AS (50–150μg/mL) significantly reduced cell viability and caused G1 arrest via the inhibition of cell-cycle regulatory proteins, including cyclin D1, CDK4, cyclin E, cyclin A, and phosphorylated retinoblastoma protein (p-Rb). Furthermore, AS treatment induced apoptosis, which was associated with DNA fragmentation, followed by a sequence of events, including intracellular ROS generation; mitochondrial dysfunction; Fas ligand activation; cytochrome c release; caspase-3, -8, -9, and PARP activation; and Bcl-2/Bax dysregulation. The results of the in vitro study suggested that AS-induced apoptosis in HL-60 cells was mediated by both the mitochondrial and death receptor pathways. Furthermore, we found that AS treatment was effective in delaying tumor incidence in HL-60 xenografted nude mice and reducing tumor burden. ConclusionsTo the best of our knowledge, this is the first report confirming the anti-tumor activity of this potentially beneficial mushroom against human promyelocytic leukemia.

Antrodia salmonea in submerged culture exhibits antioxidant activities in vitro and protects human erythrocytes and low-density lipoproteins from oxidative modification

Antrodia salmonea is well known in Taiwan as a beneficial mushroom. In the present study, we investigated the antioxidant activity of whole fermented broth (AS), filtrate (ASF), and mycelia (ASM) of A. salmonea using different antioxidant models. Furthermore, the effect of A. salmonea on AAPH-induced oxidative hemolysis of human erythrocytes and CuSO4-induced oxidative modification of human low-density lipoproteins (LDLs) was examined. We found that the AS, ASF, and ASM possess effective antioxidant activity against various oxidative systems including superoxide anion scavenging, reducing power, metal chelation, and DPPH radical scavenging. Further, AAPH-induced oxidative hemolysis in erythrocytes was prevented by AS, ASF, and ASM. Notably, AS, ASF, and ASM appear to possess powerful antioxidant activities against CuSO4-induced oxidative modification of LDL as assessed by malondialdehyde (MDA) formation, cholesterol degradation, and the relative electrophoretic mobility of oxidized LDL. It is noteworthy that AS had comparatively strong antioxidant ability compared to ASF or ASM, which is well correlated with the content of their total polyphenols. Thus, A. salmonea may exert antioxidant properties and offer protection from atherogenesis.

Antrodia salmonea inhibits TNF-α-induced angiogenesis and atherogenesis in human endothelial cells through the down-regulation of NF-κB and up-regulation of Nrf2 signaling pathways

Ethnopharmacological relevanceAntrodia salmonea (AS) is known as a traditional Chinese medicine, but very few biological activities have been reported. Materials and methodsThe present study was aimed to investigate the anti-angiogenic and anti-atherosclerotic potential of the fermented culture broth of AS against tumor necrosis factor-α (TNF-α)-stimulated human endothelial (EA.hy 926) cells. ResultsThe non-cytotoxic concentrations of AS significantly inhibited TNF-α-induced migration/invasion and capillary-like tube formation in EA.hy 926 cells. Furthermore, AS suppressed TNF-α-induced activity and expression of matrix metalloproteinase-9 (MMP-9), and cell-surface expression of intercellular adhesion molecule-1 (ICAM-1), which was associated with abridged adhesion of U937 leukocytes to endothelial cells. Moreover, AS significantly down-regulated TNF-α-induced nuclear translocation and transcriptional activation of nuclear factor κB (NF-κB) followed by suppression of I-κB degradation and phosphorylation of I-κB kinase-α (IKKα). Notably, the protective effect of AS was directly correlated with the increased expression of hemeoxygenase-1 (HO-1) and γ-glutamylcysteine synthetase (γ-GCLC), which was reasoned by nuclear translocation and transactivation of NF-E2 related factor-2 (Nrf2)/antioxidant response element (ARE). Furthermore, HO-1 knockdown by HO-1-specific shRNA diminished the protective effects of AS on TNF-α-stimulated invasion, tube formation, and U937 adhesion in EA.hy 926 cells. ConclusionsTaken together, these results suggest that Antrodia salmonea may be useful for the prevention of angiogenesis and atherosclerosis.

Contents of lovastatin, γ-aminobutyric acid and ergothioneine in mushroom fruiting bodies and mycelia

Lovastatin, γ-aminobutyric acid (GABA) and ergothioneine were analysed in mushrooms. Among fruiting bodies, Pleurotus ostreatus (Japan) and Agaricus bisporus contained the highest amount of lovastatin (606.5 and 565.4 mg/kg, respectively) whereas among mycelia, Cordyceps sinensis and Antrodia salmonea contained the highest (1365 and 1032 mg/kg, respectively). Among fruiting bodies, Flammulina velutipes and Boletus edulis contained the highest amount of GABA (229.7 and 202.1 mg/kg, respectively) whereas among mycelia, Cordyceps cicadae, C. sinensis and Agaricus blazei contained the highest (254.9, 220.5 and 200.4 mg/kg, respectively). Among fruiting bodies, Pleurotus citrinopileatus, P. ostreatus (Korea), P. ostreatus (Taiwan) and Pleurotus salmoneostramineus contained the highest amount of ergothioneine (2850.7, 1829.4, 1458.4 and 1245.0 mg/kg, respectively) whereas among mycelia, Pleurotus eryngii contained the highest (1514.6 mg/kg). Ergothioneine was detected in all samples. However, the Pleurotus genus contained considerably high amount of ergothioneine. Overall, these results might be related to their beneficial effects.

232 Relevance of WT1 expression, mutations and single nucleotide polymorphisms in juvenile myelomonocytic leukemia

We investigated the in vitro and in vivo anticancer properties of Antrodia salmonea (AS), a well-known edible/medicinal mushroom in Taiwan, on human triple-negative breast cancer (MDA-MB-231) cells and xenografted nude mice; and revealed the underlying molecular mechanisms involved in autophagic- and apoptotic-cell death. Treatment of MDA-MB-231 cells with fermented culture broth of AS (0–200 μg/mL) inhibited cell viability/growth. AS-induced autophagy was evidenced via increased LC3-II accumulation, GFP-LC3 puncta and AVOs formation in MDA-MB-231 cells. These events are associated with increased ATG7, decreased p-mTOR, vanished SQSTM1/p62 expressions and dysregulated Beclin-1/Bcl-2 ratio. AS-induced apoptosis/necrosis through increased DNA fragmentation, Annexin-V/PI stained cells and Bax expression. Both mitochondrial (caspase-9/caspase-3/PARP) and death-receptor (caspase-8/FasL/Fas) signaling pathways are involved in execution of apoptosis. Interestingly, blockade of AS-induced ROS production by N-acetylcysteine pretreatment substantially attenuated AS-induced autophagy, mitochondrial dysfunction and autophagic/apoptotic-cell death. Inhibition of apoptosis by Z-VAD-FMK suppressed AS-induced autophagic-death (decreased LC3-II/AVOs). Similarly, inhibition of autophagy by 3-methyladenine/chloroquine diminished AS-induced apoptosis (decreased DNA fragmentation/caspase-3) in MDA-MB-231 cells. Bioluminescence imaging further confirmed that AS inhibited breast tumor growth in living MDA-MB-231-luciferase-injected nude mice. Taken together, AS crucially involved in execution/propagation of autophagic- or apoptotic-death of MDA-MB-231 cells, and decreased tumor growth in xenografted nude mice.

Phylogenetic analysis of Antrodia species and Antrodia camphorata inferred from internal transcribed spacer region

The species of Antrodia are one of the difficult-to-classify and obscure groups of poroid Aphyllophorales based on morphological appearance. However, it is becoming increasingly important to reliably identify the entire suite of Antrodia camphorata strains and Antrodia species due to the potential pharmaceutical value of their biologically active ingredients. In this study, the internal transcribed spacer (ITS) region of the ribosomal RNA gene (rDNA) was sequenced and phylogenetically analyzed in a number of Antrodia fungal species and strains. ITS amplicons from the Antrodia species tested ranged in size from 543 to 610 bp; the size of the ITS of A. camphorata strains ranged from 592 to 596 bp. The overall sizes of ITS2 and 5.8S ribosomal RNA gene of all A. camphorata strains tested in this study were shown to be 217 and 158 bp, respectively. A phylogenetic analysis of ITS data generated, which included sequences of 11 A. camphorata strains and nine other Antrodia species, showed three clearly distinct groups. Group 1 includes A. camphorata, Antrodia salmonea, and Antrodia carbinca strains. Within Group 2, Antrodia sinuosa and Antrodia xantha were clustered together. Group 3 contained Antrodia albida, A. heteromorpha, A. serialis, and A. malicola. The observed sequence diversity among ITS alleles provided an effective tool for differentiating strains of A. camphorata, A. salmonea, A. xantha, A. sinuosa, or A. serialis. Polymorphisms arising within the ITS1-5.8S-ITS2 region can provide practical markers for establishing a foundation for the further expansion of an ITS sequence database of medically important fungi.