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

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

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

Antcin-A Modulates Epithelial-to-Mesenchymal Transition and Inhibits Migratory and Invasive Potentials of Human Breast Cancer Cells via p53-Mediated miR-200c Activation

Antcin-A (ATA) is a steroid-like phytochemical isolated from the fruiting bodies of a precious edible mushroom Antrodia cinnamomea. We previously showed that ATA has strong anti-inflammatory and anti-tumor effects; however, other possible bioactivities of this unique compound remain unexplored. In the present study, we aimed to investigate the modulation of epithelial-to-mesenchymal transition (EMT), anti-migration, and anti-invasive potential of ATA against human breast cancer cells in vitro. Human breast cancer cell lines, MCF-7 and MDA-MB-231, were incubated with ATA for 24 h. Wound healing, trans-well invasion, western blot, q-PCR, F-actin staining, and immunofluorescence assays were performed. We found that treatment with ATA significantly blocked EMT processes, as evidenced by upregulation of epithelial markers (E-cadherin and occludin) and downregulation of mesenchymal markers (N-cadherin and vimentin) via suppression of their transcriptional repressor ZEB1. Next, we found that ATA could induce miR-200c, which is a known player of ZEB1 repression. Further investigations revealed that ATA-mediated induction of miR-200c is associated with transcriptional activation of p53, as confirmed by the fact that ATA failed to induce miR-200c or suppress ZEB1 activity in p53 inhibited cells. Further in vitro wound healing and trans-well invasion assays support that ATA could inhibit migratory and invasive potentials of breast cancer cells, and the effect was likely associated with induced phenotypic modulation. Taken together, the present study suggests that antcin-A could be a lead phyto-agent for the development of anti-metastatic drug for breast cancer treatment

Essential Oils of Alpinia nantoensis Retard Forskolin-Induced Melanogenesis via ERK1/2-Mediated Proteasomal Degradation of MITF

The anti-melanogenic activity of essential oils of Alpinia nantoensis and their bioactive ingredients were investigated in vitro. Treatment with leaf (LEO) and rhizome (REO) essential oils of A. nantoensis, significantly reduced forskolin-induced melanin production followed by down-regulation of tyrosinase (TYR) and tyrosinase related protein-1 (TRP-1) expression at both transcriptional and translational levels. Further studies revealed that down-regulation TYR and TRP-1 were caused by LEO/REO-mediated suppression of Microphthalmia-associated transcription factor (MITF), as evidenced by reduced nuclear translocation of MITF. Also, we found that LEO/REO induce the sustained activation of ERK1/2, which facilitate subsequent proteasomal degradation of MITF, as confirmed by that LEO/REO failed to inhibits MITF activity in ERK1/2 inhibitor treated cells. In addition, a significant increase of ubiquitinated MITF was observed after treatment with LEO and REO. Furthermore, the chemical composition of LEO and REO were characterized by gas chromatography-mass spectrometry (GC-MS) resulted that camphor, camphene, α-pinene, β-pinene, isoborneol and D-limonene were the major compounds in both LEO and REO. Further studies revealed that α-pinene and D-limonene were the active components responsible for the anti-melanogenic properties of LEO and REO. Based on the results, this study provided a strong evidence that LEO and REO could be promising natural sources for the development of novel skin-whitening agents for the cosmetic purposes

Essential Oils of Alpinia nantoensis Retard Forskolin-Induced Melanogenesis via ERK1/2-Mediated Proteasomal Degradation of MITF

The anti-melanogenic activity of essential oils of Alpinia nantoensis and their bioactive ingredients were investigated in vitro. Treatment with leaf (LEO) and rhizome (REO) essential oils of A. nantoensis, significantly reduced forskolin-induced melanin production followed by down-regulation of tyrosinase (TYR) and tyrosinase related protein-1 (TRP-1) expression at both transcriptional and translational levels. Further studies revealed that down-regulation TYR and TRP-1 were caused by LEO/REO-mediated suppression of Microphthalmia-associated transcription factor (MITF), as evidenced by reduced nuclear translocation of MITF. Also, we found that LEO/REO induce the sustained activation of ERK1/2, which facilitate subsequent proteasomal degradation of MITF, as confirmed by that LEO/REO failed to inhibits MITF activity in ERK1/2 inhibitor treated cells. In addition, a significant increase of ubiquitinated MITF was observed after treatment with LEO and REO. Furthermore, the chemical composition of LEO and REO were characterized by gas chromatography-mass spectrometry (GC-MS) resulted that camphor, camphene, α-pinene, β-pinene, isoborneol and D-limonene were the major compounds in both LEO and REO. Further studies revealed that α-pinene and D-limonene were the active components responsible for the anti-melanogenic properties of LEO and REO. Based on the results, this study provided a strong evidence that LEO and REO could be promising natural sources for the development of novel skin-whitening agents for the cosmetic purposes

Schematic representation represents the anti-metastatic potential of ADC.

<p>TGF-β ligand binding with TGF-β receptor II (TGF-βRII) recruit TGF-βRI into a tetrameric receptor complex resulting in transphosphorylation and activation of TGF-βRI, which then phosphorylates Smad2 or Smad3. Phosphorylated Smad2/3 associate with Smad4 and translocate into the nucleus, where they activate transcription of target genes including snail, slug, and Twist. These genes regulate EMT through suppression of epithelial markers and induction of mesenchymal markers. Pretreatment with ADC inhibits TGF-β-induced EMT by inhibiting phosphorylation and transcriptional activation of Smad2/3. In addition, TGF-β activates β-catenin signaling cascade through the suppression of GSK3β, a negative regulator of β-catenin. The free form of β-catenin translocate into the nucleus and transcribe number of migration marker genes including MMP-2, MMP-9, and uPA. Pretreatment with ADC blocks TGF-β1-induced migration and invasion of MCF-7 cells through the suppression of transcriptional activation of β-catenin followed by down-regulation of MMP-2, MMP-9, and uPA. In addition, previous reports demonstrated that MMPs expression also regulated by snail, transcription factor regulates EMT. There is a possible that reduction in snail expression by may influence MMPs activity. Moreover, Twist was reported to be one of the down-stream target of β-catenin. Therefore, we believe that there may be a cross-talk existing between ADC-induced down-regulation of β-catenin and Twist.</p

ADC inhibits TGF-β1-induced breast cancer cell migration and invasion.

<p>(A) Cell migration was determined by wound healing assay, the confluent MCF-7 monolayer was pre-treated with ADC (5–20 μM) for 2 h, cells were scratched by 200 μL pipet tips, and washed to remove the debris followed by addition of freshmedium containing 1% FBS and TGF-β1 (20 ng/mL). Cells were then incubated for 48 h. Photographs were taken at 0 h, and 48 h using inverted microscope with 10 × magnification. TGF-β1-induced cell motility was determined by measuring the area of wound closure as shown by histogram. The closure area at 48 h was compared with 0 h in the same samples. (B) For the invasion assay, the pre-treated cells were seed into the upper chamber of 24-well transwell chamber containing DMEM with 1% FBS. The lower chamber was filled with complete serum media. The cells were allowed to invade for 48 h. Invading cells were then fixed, and stained with Giemsa stain solution and counted in 5 random fields. The average invaded cells in each group was presented by histogram. The data reported as mean ± SD of three independent experiments. ^Θ<i>P</i>< 0.001, significant difference from control and TGF-β1 alone treated group. *<i>P</i>< 0.05, **<i>P</i>< 0.01, and ***<i>P</i>< 0.001 were significantly different from TGF-β1 alone with the ADC treatment groups.</p