Antcins from Antrodia cinnamomea and Antrodia salmonea Inhibit Angiotensin-Converting Enzyme 2 (ACE2) in Epithelial Cells: Can Be Potential Candidates for the Development of SARS-CoV-2 Prophylactic Agents.

Antcins are newly identified steroid-like compounds from Taiwan's endemic medicinal mushrooms Antrodia cinnamomea and Antrodia salmonea. Scientific studies of the past two decades confirmed that antcins have various pharmacological activities, including potent anti-oxidant and anti-inflammatory effects. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the coronavirus disease-2019 (COVID-19) pandemic and is characterized as a significant threat to global public health. It was recently identified that SARS-CoV-2 required angiotensin converting enzyme 2 (ACE2), a receptor which supports host cell entry and disease onset. Here, we report a novel function of antcins, in which antcins exhibit inhibitory effects on ACE2. Compared to the untreated control group, treatment with various antcins (antcin-A, antcin-B, antcin-C, antcin-H, antcin-I, and antcin-M) significantly inhibited ACE2 activity in cultured human epithelial cells. Indeed, among the investigated antcins, antcin-A, antcin-B, antcin-C, and antcin-I showed a pronounceable inhibition against ACE2. These findings suggest that antcins could be novel anti-ACE2 agents to prevent SARS-CoV-2 host cell entry and the following disease onset.

Geranium and Lemon Essential Oils and Their Active Compounds Downregulate Angiotensin-Converting Enzyme 2 (ACE2), a SARS-CoV-2 Spike Receptor-Binding Domain, in Epithelial Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as coronavirus disease-2019 (COVID-19), is a pandemic disease that has been declared as modern history's gravest health emergency worldwide. Until now, no precise treatment modality has been developed. The angiotensin-converting enzyme 2 (ACE2) receptor, a host cell receptor, has been found to play a crucial role in virus cell entry; therefore, ACE2 blockers can be a potential target for anti-viral intervention. In this study, we evaluated the ACE2 inhibitory effects of 10 essential oils. Among them, geranium and lemon oils displayed significant ACE2 inhibitory effects in epithelial cells. In addition, immunoblotting and qPCR analysis also confirmed that geranium and lemon oils possess potent ACE2 inhibitory effects. Furthermore, the gas chromatography-mass spectrometry (GC-MS) analysis displayed 22 compounds in geranium oil and 9 compounds in lemon oil. Citronellol, geraniol, and neryl acetate were the major compounds of geranium oil and limonene that represented major compound of lemon oil. Next, we found that treatment with citronellol and limonene significantly downregulated ACE2 expression in epithelial cells. The results suggest that geranium and lemon essential oils and their derivative compounds are valuable natural anti-viral agents that may contribute to the prevention of the invasion of SARS-CoV-2/COVID-19 into the human body.

A mechanistic and empirical review of antcins, a new class of phytosterols of formosan fungi origin.

Antcins are unique phytosterols isolated from A. cinnamomea and A. salmomea, which are the endemic fungus of Taiwan. A. cinnamomea has long been highly valued medicinal mushroom in Taiwan and traditionally used as a folk remedy for various human illness. Recent scientific explorations claimed that the pharmacological activities of A. cinnamomea and A. salmomonea are gone beyond their original usage. The therapeutic efficacy of these medicinal mushrooms was attributed to their high content of unique bioactive secondary metabolites, including terpenoids, benzenoids, ubiquinol derivatives, polysaccharides, lignans, nucleic acids, steroids, and maleic/succinic acid derivatives. Antcins is a group of steroids in Antrodia spp. with ergostane skeleton received much attention in Taiwan's academic circle due to their broad-spectrum of biological activities. At present, twelve antcins, i.e. antcin A, B, C, D, E, F, G, H, I, K, M, and N along with twelve derivatives/epimers (25R/S-antcin A, B, C, H, I and K) and seven analogs (methyl antcinate A, B, G, H, K, L and N) were identified. Several studies have demonstrated that antcins possessed anti-cancer, anti-inflammation, anti-oxidant, anti-diabetic, anti-aging, immunomodulation, hepatoprotection, and hypolipedimic activities. The main goal of this review is to define the chemistry, isolation, advances in production, and biological activities of antcins and their derivatives/analogs. Special attention has been given to a detail view of their biological activities in vitro and in vivo and their pharmacological potentials.

MicroRNA-708 activation by glucocorticoid receptor agonists regulate breast cancer tumorigenesis and metastasis via downregulation of NF-κB signaling.

Therapeutic administration of glucocorticoids (GCs) is frequently used as add-on chemotherapy for palliative purposes during breast cancer treatment. Recent studies have shown that GC treatment induces microRNA-708 in ovarian cancer cells, resulting in impaired tumor cell proliferation and metastasis. However, the regulatory functions of GCs on miR-708 and its downstream target genes in human breast cancer cells (BCCs) are poorly understood. In this study, we found that treatment with either the synthetic GC dexamethasone (DEX) or the natural GC mimic, antcin A (ATA) significantly increased miR-708 expression by transactivation of glucocorticoid receptor alpha (GRα) in MCF-7 and MDA-MB-231 human BCCs. Induction of miR-708 by GR agonists resulted in inhibition of cell proliferation, cell-cycle progression, cancer stem cell (CSC)-like phenotype and metastasis of BCCs. In addition, GR agonist treatment or miR-708 mimic transfection remarkably inhibited IKKß expression and suppressed nuclear factor-kappaB (NF-κB) activity and its downstream target genes, including COX-2, cMYC, cyclin D1, Matrix metalloproteinase (MMP)-2, MMP-9, CD24, CD44 and increased p21CIP1 and p27KIP1 that are known to be involved in proliferation, cell-cycle progression, metastasis and CSC marker protein. BCCs xenograft models indicate that treatment with GR agonists significantly reduced tumor growth, weight and volume. Overall, our data strongly suggest that GR agonists induced miR-708 and downstream suppression of NF-κB signaling, which may be applicable as a novel therapeutic intervention in breast cancer treatment.

Activation of Nrf2-mediated anti-oxidant genes by antrodin C prevents hyperglycemia-induced senescence and apoptosis in human endothelial cells.

In the present study, we investigated the effects of antrodin C (ADC), a maleimide derivative isolated from mycelia of Antrodia cinnamomea, on high glucose (HG, 30 mM)-accelerated endothelial dysfunction in vitro. HG-induced cytotoxicity in human umbilical vein endothelial cells (HUVECs) was significantly ameliorated by ADC. In addition, treatment with ADC significantly prevented HG-induced senescence, growth arrest at the G1-S transition phase and apoptosis in HUVECs. Moreover, the increased level of intracellular reactive oxygen species (ROS) under HG condition was significantly ameliorated by ADC. Further analysis revealed that ADC-mediated anti-oxidant effects were due to up-regulation of cellular anti-oxidant genes, such as HO-1 and NQO-1 via promotion of the transcriptional activity of Nrf2, which was further confirmed by the failure of ADC to protect HUVECs from HG-induced dysfunction under HO-1 inhibition or Nrf2 silencing. Furthermore, hyperosmotic glucose (HOG, 60 mM)-induced uncontrolled production of ROS, rapid apoptotic cell death and HUVEC injury were significantly prevented by ADC, whereas these preventive effects were barely observed in HO-1 inhibited or Nrf2 silenced cells. Taken together, these results suggest that ADC may represent a promising intervention in diabetic-associated cardiovascular diseases by activating the Nrf2-dependent cellular anti-oxidant defense system.

A steroid like phytochemical Antcin M is an anti-aging reagent that eliminates hyperglycemia-accelerated premature senescence in dermal fibroblasts by direct activation of Nrf2 and SIRT-1.

The present study revealed the anti-aging properties of antcin M (ANM) and elucidated the molecular mechanism underlying the effects. We found that exposure of human normal dermal fibroblasts (HNDFs) to high-glucose (HG, 30 mM) for 3 days, accelerated G0/G1 phase arrest and senescence. Indeed, co-treatment with ANM (10 µM) significantly attenuated HG-induced growth arrest and promoted cell proliferation. Further molecular analysis revealed that ANM blocked the HG-induced reduction in G1-S transition regulatory proteins such as cyclin D, cyclin E, CDK4, CDK6, CDK2 and protein retinoblastoma (pRb). In addition, treatment with ANM eliminated HG-induced reactive oxygen species (ROS) through the induction of anti-oxidant genes, HO-1 and NQO-1 via transcriptional activation of Nrf2. Moreover, treatment with ANM abolished HG-induced SIPS as evidenced by reduced senescence-associated ß-galactosidase (SA-ß-gal) activity. This effect was further confirmed by reduction in senescence-associated marker proteins including, p21CIP1, p16INK4A, and p53/FoxO1 acetylation. Also, the HG-induced decline in aging-related marker protein SMP30 was rescued by ANM. Furthermore, treatment with ANM increased SIRT-1 expression, and prevented SIRT-1 depletion. This protection was consistent with inhibition of SIRT-1 phosphorylation at Ser47 followed by blocking its upstream kinases, p38 MAPK and JNK/SAPK. Further analysis revealed that ANM partially protected HG-induced senescence in SIRT-1 silenced cells. A similar effect was also observed in Nrf2 silenced cells. However, a complete loss of protection was observed in both Nrf2 and SIRT-1 knockdown cells suggesting that both induction of Nrf2-mediated anti-oxidant defense and SIRT-1-mediated deacetylation activity contribute to the anti-aging properties of ANM in vitro. Result of in vivo studies shows that ANM-treated C. elegens exhibits an increased survival rate during HG-induced oxidative stress insult. Furthermore, ANM significantly extended the life span of C. elegans. Taken together, our results suggest the potential application of ANM in age-related diseases or as a preventive reagent against aging process.

Antcin C from Antrodia cinnamomea Protects Liver Cells Against Free Radical-Induced Oxidative Stress and Apoptosis In Vitro and In Vivo through Nrf2-Dependent Mechanism.

In this study, we investigated the cytoprotective effects of antcin C, a steroid-like compound isolated from Antrodia cinnamaomea against AAPH-induced oxidative stress and apoptosis in human hepatic HepG2 cells. Pretreatment with antcin C significantly protects hepatic cells from AAPH-induced cell death through the inhibition of ROS generation. Furthermore, AAPH-induced lipid peroxidation, ALT/AST secretion and GSH depletion was significantly inhibited by antcin C. The antioxidant potential of antcin C was correlated with induction of antioxidant genes including, HO-1, NQO-1, γ -GCLC, and SOD via transcriptional activation of Nrf2. The Nrf2 activation by antcin C is mediated by JNK1/2 and PI3K activation, whereas pharmacologic inhibition of JNK1/2 and PI3K abolished antcin C-induced Nrf2 activity. In addition, AAPH-induced apoptosis was significantly inhibited by antcin C through the down-regulation of pro-apoptotic factors including, Bax, cytochrome c, capase 9, -4, -12, -3, and PARP. In vivo studies also show that antcin C significantly protected mice liver from AAPH-induced hepatic injury as evidenced by reduction in hepatic enzymes in circulation. Further, immunocytochemistry analyses showed that antcin C significantly increased HO-1 and Nrf2 expression in mice liver tissues. These results strongly suggest that antcin C could protect liver cells from oxidative stress and cell death via Nrf2/ARE activation.

Hepatoprotective effect of lucidone against alcohol-induced oxidative stress in human hepatic HepG2 cells through the up-regulation of HO-1/Nrf-2 antioxidant genes.

Lucidone was previously reported to exhibit anti-inflammatory activity in vitro and in vivo. In the present study, we characterized the mechanisms underlying the hepatoprotective effect of lucidone against alcohol-induced oxidative stress in vitro. Human hepatoma (HepG2) cells were pretreated with lucidone (1-10µg/mL) and then hepatotoxicity was stimulated by the addition ethanol (100mM). With response to ethanol-challenge, increased amount of alanine aminotransferase (ALT) and aspirate aminotransferase (AST) release were observed, whereas lucidone pretreatment significantly inhibited the leakage of AST and ALT in HepG2 cells without appreciable cytotoxic effects. We also found that lucidone pretreatment significantly decreased ethanol-induced nitric oxide (NO), tumor necrosis factor-α (TNF-α), malondialdehyde (MDA), reactive oxygen species (ROS) and glutathione (GSH) depletion in HepG2 cells. Furthermore, Western blot and quantitative-PCR analyses showed that ethanol-exposure apparently down-regulated endogenous anti-oxidant hemoxygenase-1 (HO-1) expression, whereas pretreatment with lucidone significantly up-regulates HO-1 expression followed by the transcriptional activation of NF-E2 related factor-2 (Nrf-2). Interestingly, the profound up-regulation of HO-1 and Nrf-2 were observed in only ethanol-challenged cells, which evidenced that lucidone-induced induction of HO-/Nrf-2 were specific with oxidative stress. Thus, we concluded that lucidone-mediated up-regulation of phase-II enzymes and HO-1 via Nrf-2 signaling pathway may provide a pivotal mechanism for its hepatoprotective action.