Research Article:
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
Hsin-Ling Yanga, Hebron C. Changb,†, Shu-Wei Lina,†, K. J. Senthil Kumare, Chun-Huei Liaoa, Hui-Min Wangc, Kai-Yuan Lind, and You-Cheng Hseue*
Running title: Antrodia salmonea inhibit TNF-α-induced angiogenesis
aInstitute of Nutrition, College of Health Care, China Medical University, Taichung-40402,
Taiwan
bInstitute of Biotechnology and Bioinformatics, Asia University, Taichung-413, Taiwan
cDepartment of Fragrance and Cosmetic Science, Kaohsiung Medical University,
Kaohsiung-80708, Taiwan
dDepartment of Medical Research, Chi-Mei Medical Center, Tainan-710, Taiwan
eDepartment of Cosmeceutics, College of Pharmacy, China Medical University,
Taichung-40402, Taiwan.
*Corresponding authors. Tel.: +886 4 22053366 x 5308; fax: +886 4 22078083.
E-mail addresses: ychseu@mail.cmu.edu.tw (Y.C. Hseu)
Abbreviations:
ARE, antioxidant response element; AS, Antrodia salmonea; HO-1, hemeoxygenase-1; ICAM-1, intercellular adhesion molecule-1; IKKα, I-κB kinase-α; I-κB, inhibitor-κB; MMP-9, matrix metalloproteinase-9; NF-κB, nuclear factor-κB; Nrf2, nuclear factor erythroid 2-related factor 2; TNF-α, tumor necrosis factor-α; γ-GCLC, γ-glutamylcysteine synthetase; DMEM, Dulbecco’s Modified Eagle’s medium; FBS, fetal bovine serum; PBS, phosphate buffer saline; GSH, glutathione; FITC, fluorescein isothiocyanate.
ABSTRACT
Ethnopharmacological relevance: Antrodia salmonea (AS) is known as a traditional Chinese
medicine, but very few biological activities have been reported.
Materials and methods: The 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.
Results: The 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 followed by the augmented nuclear translocation and transcriptional activation of NF-E2 related factor-2 (Nrf2)/antioxidant response element (ARE)
activity. 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.
Conclusions: Taken together, these results suggest that Antrodia salmonea may be useful for
the prevention of angiogenesis and atherosclerosis.
Keywords:
1. Introduction
Angiogenesis, the formation of new vessels from preexisting vasculature, is an essential process in a variety of physiological and pathological conditions, including inflammatory diseases, atherosclerosis, diabetic retinopathy, rheumatoid arthritis, cancer, and metastasis . Complex sequential steps are involved in angiogenesis, such as basement membrane degradation by proteases, endothelial cell proliferation and migration/invasion, formation of capillary tubes, and survival of newly formed blood vessels . Angiogenesis critically depends on several conditions such as endothelial cell proliferation, endothelial cells secretion of matrix metalloproteinases (MMP) required to break down surrounding tissue matrix and the endothelial cell movement/migration . Angiogenesis is tightly regulated by an intricate balance between stimulators and inhibitors . Among these, tumor necrosis factor-α (TNF-α), a soluble angiogenic factor produced by many tumors as well as normal cell lines, plays a key role in regulating normal and pathologic angiogenesis . TNF-α contributes to the angiogenic process by stimulating proliferation, migration/invasion, and the formation of new blood vessels by endothelial cells . Because of the critical dependence of human cancer and inflammatory diseases on angiogenesis, therapeutic strategies have been developed targeting various aspects of the angiogenic processes, many with promising results .
Atherosclerosis is a complicated inflammatory process that can lead to vascular endothelial dysfunction. The early pathogenesis of atherosclerosis appears to be activation of the endothelium in response to a variety of relevant stimuli including cytokines, oxidized lipids, or turbulent flow . The vascular endothelium plays a critical role in the preservation of normal vessel wall structure and function. Vascular endothelial cells control vascular permeability, vessel tone, coagulation, fibrinolysis, and inflammatory responses. Localized accumulation of monocytes/macrophages and T lymphocytes in the arterial intima appears to play a key role in early atherogenesis, as well as in plaque rupture in advanced atherosclerotic lesions . This process appears to be mediated by endothelial-leukocyte adhesion molecules expressed on the
surface of the vascular endothelium covering atherosclerotic and inflammatory lesions . These adhesion molecules include intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, and their expression can be transcriptionally induced by inflammatory cytokines such as TNF-α .
NF-κB, a transcription factor that playing a fundamental role in angiogenesis/atherogenesis, and the expression of MMPs and adhesion molecules is directly coupled with up-regulation of NF-κB . In normal physiological condition NF-κB is localized in the cyctoplasm and tethered with its inhibitor protein called I-κB. Upon activated by a variety of external stimuli including TNF-α, I-κB phosphorylated and degraded via proteosomal degradation. This action further leads to release of NF-κB which then translocates to the nucleus and binds to its promoter region (κB binding site), transcribe a number of genes including MMPs and adhesion molecules (MMP-9 and ICAM-1) .
Heme oxygenase, a microsomal enzyme that catalyzes the first and rate-limiting step in the oxidative degradation of cellular heme into equimolar amounts of carbon monoxide (CO), biliverdin, and free iron . HO-1, an inducible isoform of HO, provides a host defense mechanismthat can protect the body against oxidative injury and also contributes to the anti-inflammatory activity of cells and tissues . These beneficial effects of HO-1 are mediated by the metabolic end products produced from enzymatic degradation of free heme. Biliverdin and its metabolite, bilirubin, confer potent cellular antioxidant capacities and CO acts as an important signaling molecule with anti-apoptotic and anti-inflammatory effects . HO-1 expression is primarily regulated on the transcriptional level and transcription factor NF-E2-related factor 2 (Nrf2) plays a central role for inducible expression of HO-1 . Under normal conditions, Nrf2 is sequestered in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1) and degraded by the ubiquitin-dependent 26S proteasome system. Under activation, Nrf2 released from Keap1 inhibition, translocates to the nucleus, heterodimerizes with Maf, and transactivates HO-1 gene promoter containing several antioxidant response elements
(AREs) .
Antrodia salmonea, a newly identified medicinal fungal species, grow on the empty rotten
trunk of Cunninghamia konishii in Taiwan (Chang and Chou, 2004; Wu et al., 2004). The fruiting body of AS has been used in the food remedy of diarrhea, abdominal pain, hypertension, itchy skin, and cancer, and is also used as a detoxicant in Taiwanese folk medicine (Tsai and Liaw, 1985). Various biological activities of AS have been reported, including anti-inflammatory and antioxidant effects. There were several newly compounds were isolated from the basidiomata of AS, whose in vitro studies displayed anti-oxidative and anti-inflammatory activities in activated inflammatory cells . However, the underlying pharmacological mechanisms of this new drug are still a matter of debate. Therefore, the aim of the present study was to investigate the anti-angiogenic and anti-atherosclerotic properties of the fermented culture broth of Antrodia salmonea (AS) in TNF-α-activated human endothelial (EA.hy 926) cells. Results revealed that AS inhibits TNF-α-induced angiogenesis/atherogenesis through the regulation of NF-κB and HO-1/Nrf2 signaling pathways.
2. Materials and methods
2.1 Chemicals
Dulbecco’s Modified Eagle’s medium (DMEM), fetal bovine serum (FBS), M-199 medium, glutamine, and penicillin-streptomycin-neomycin were obtained from GIBCO BRL (Grand Island, NY). Antibody against MMP-9, ICAM-1, Nrf2, and I-κBα were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Antibodies against anti-NF-κB (p65), phos-IKK, and IKK were obtained from Cell Signaling Technology Inc. (Danvers, MA). Antibody against HO-1 and β-actin was purchased from Abcam (Cambridge, MA). Anti-γ-GCLC antibody was obtained from Gene Tex Inc. (San Antonio, TX). All other chemicals were of the highest grade commercially available and supplied either by Merck (Darmstadt, Germany) or Sigma-Aldrich (St. Louis, MO).
2.2. Preparation of the fermented culture broth of Antrodia salmonea from submerged cultures
The Antrodia salmonea hyphae were separated from the fruiting bodies. The whole colony was cut and placed into a flask with 50 ml of sterile water. After homogenization, the fragmented mycelia suspension was then inoculated with a culture medium composed of 2.0% glucose, 0.1% wheat powder, and 0.1% peptone in distilled water. The medium was adjusted to an initial pH of 5.0. Each shaking flask culture (120 rpm) was conducted in 2 L Erlenmeyer flasks (containing 1 L of medium) and incubated at 25°C for 10 days. Thereafter, 3.5 L of the shaking flask cultures were inoculated into a 500 L fermenting tank containing 300 L of culture medium. The resulting mixtures were then cultured at 25°C for 30 days. The fermentation conditions were the same as the seed fermentation but with an aeration rate of 0.075 vvm to obtain a mucilaginous medium containing the mycelia. The experiments were performed using 2~4 different batches of the whole fermented culture of Antrodia salmonea. The dry matter yield of the fermented culture was about 15 g/L. The freeze-dried samples were ground, shaken with distilled water, and then centrifuged at 3000 × g for 5 min, followed by passage through a
0.2 m filter. The aqueous extracts were concentrated in a vacuum and freeze dried to form a powder. The yield of the fermented culture broth (AS) (1 g) was about 0.375 g. To prepare the stock solution, the powder samples of AS, were solubilized with 10 mM sodium phosphate buffer (pH 7.4) containing 0.15 M NaCl (PBS) at 25C. The solution was stored at -20°C before analysis of analysis of its anti-angiogenic and anti-atherosclerotic potential.
2.3. Endothelial cell culture
The human vascular endothelial cell line (EA.hy 926) was grown in DMEM supplemented with 15% FBS, HAT (100 mM sodium hypoxanthine, 0.4 mM aminopterin, and 16 mM thymidine), 1% glutamine, and 1% penicillin-streptomycin-neomycin at 37 C in a 5% CO2 humidified
incubator. In this study, we used the EA.hy 926 cell line because it possessed endothelial characteristics including the formation of tube-like structures . The use of a cell line also allowed us to overcome the difficulty of obtaining larger numbers of uncontaminated primary cells as well as the requirement of expensive growth factors associated with the use of primary endothelial cells. Cultures were harvested and the cell number was determined using a hemocytometer. For all TNF-α-stimulated experiments, the supernatant was removed following AS supplementation for 1 h, the cells were washed with PBS, and the culture media was replaced with new medium containing 10 ng/mL of TNF-α for the indicated time points.
2.4. MTT Assay
The effect of AS on cell viability was monitored by the MTT colorimetric assay. EA.hy 926 cells at a density of (1 × 105 cells/well) were grown to confluence on 12-well cell culture plates,
pre-incubated with AS (25-100 µg/mL) and allowed to proliferate for 24 h. After treatment, the cells were incubated with 400 μL of 0.5 mg/mL MTT in PBS for 2 h. The culture supernatant was removed and re-suspended with 400 μL of isopropanol to dissolve the MTT formazan, and
the absorbance was measured at 570 nm using ELISA micro-plate reader (Bio-Tek Instruments, Winooski, VT). The effect of AS on cell viability was assessed as the percent of viable cells compared with the vehicle-treated control cells, which were arbitrarily assigned a viability of 100%. The assay was performed in triplicate at each concentration.
2.5. In vitro wound-healing assay
To determine the effects of AS on cell migration, an in vitro wound healing assay was performed. Briefly, EA.hy 926 cells at density of (1 × 104 cells/well) were cultured an Ibidi
culture-insert on 1% gelatin-coated 12-well plate and incubated with the indicated concentration of AS (50 and 100 µg/mL) 1 h in 1% FBS-medium. Cells were then incubated with or without TNF-α (10 ng/mL) in fresh medium containing 1% FBS for 24 h. Then the cells were washed twice with PBS, fixed with 100% methanol, and stained with Giemsa Stain solution. The cultures were photographed using optical microscope (200 × magnification) to monitor the migration of cells into the wounded area, and the closure of wounded area was calculated using Image-Pro® Plus software (Media Cybernetics, Inc., Bethesda, MD).
2.6. Endothelial cell invasion assay
Invasion assay was performed using BD Matrigel™ invasion chambers (BD Biosciences, Bedford, MA). For the invasion assay, 10 µL of Matrigel (25 mg/50 mL) was applied to 8-µm polycarbonate membrane filters, and the bottom chamber of the apparatus contained standard medium. Matrigel is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm mouse sarcoma, a tumor rich in extracellular matrix proteins. Briefly, the top chambers were seeded with EA.hy 926 cells (1 × 105 cells/well) in 500 µL serum-free
medium, and the cells were incubated with AS (50 and 100 µg/mL) for 1 h prior to the addition of 10 ng/mL TNF-α. Cells were placed in the bottom chambers (750 µL), which were filled with serum-free medium. Cells were allowed to migrate for 12 h at 37 °C. After the incubation
period, non-migrated cells on the top surface of the membrane were removed with a cotton swab. The migrated cells on the bottom side of the membrane were fixed in cold 100% methanol for 8 min and washed twice with PBS. The cells were stained with Giemsa stain solution and then de-stained with PBS. Images were obtained using an optical microscope (200 × magnification); invading cells were quantified by manual counting. Percent inhibition of invading cells was quantified and expressed with untreated cells (control) representing 100%.
2.7. Endothelial cell tube formation assay
To determine whether AS affected the angiogenic process, tube formation was evaluated using the BD BioCoat™ angiogenesis system: endothelial cell tube formation assay kit (BD Biosciences, Bedford, MA). In brief, after a treatment with AS (50 and 100 µg/mL), cells were harvested and seeded in a BD Matrigel Matrix coated 96-well plates with EA.hy 926 cells (1 × 105 cells/well) in serum-free medium for 30 min followed by incubating with or without TNF-α
(10 ng/mL) at 37 °C. After 3 h, the capillary networks were photographed using a phase-contrast microscope at 200 × magnification; the number of tubes was quantified from three random fields. The percent inhibition was expressed with untreated cells (control) representing 100%.
2.8. Gelatin zymography assay
The activities of MMP-9 released from cells were measured by gelatin zymography protease assays as described previously . Briefly, EA.hy 926 cells (1 × 105 cells/well) were seeded into
12-well culture dishes and grown in medium with 15% FBS to a nearly confluent monolayer. The cells were resuspended in medium, and then incubated with AS (50 and 100 µg/mL) for 1 h prior to the addition of TNF-α (10 ng/mL). After 24 h, collected media with an appropriate volume (adjusted by vital cell number, 25 µg) were prepared using SDS sample buffer, without boiling or reduction, and were subjected to 1 mg/mL gelatin-8% SDS-PAGE electrophoresis.
After electrophoresis, gels were washed with 2.5% Triton X-100 and then incubated in a reaction buffer (50 mM Tris-base [pH 7.5], 200 mM NaCl, 5 mM CaCl2 and 0.02% Brij 35) at
37 °C for 24 h. Then, the gels were stained with Coomassie brilliant blue R-250. The relative MMP-9 activity was quantified by Matrix Inspector 2.1 software.
2.9. U937 adhesion assay
Human leukemic monocyte lymphoma cell line (U937) were labeled with 10 µg/mL of BCECF-AM for 30 min at 37 °C, washed and resuspended in serum-free media. In other hand, EA.hy 926 cells were pretreated with without various concentrations of AS (25, 50, and 100 g/mL) for 1 h and then stimulated with TNF-α (10 ng/mL) for 4 h. The stimulated EA.hy 926 cells (3 × 104 cells/wells) were cultured in 24 well plate and incubated with reagents prior to
being co-cultured with 1 × 105 cells/mL BCECF-AM-labeled U937 cells for 30 min at 37 °C.
Non-adhering U937 cells were removed by gentle aspiration, and wells were washed with PBS. Cells were lysed using 0.1% Triton X-100 in 0.1 M Tris-HCl, pH 7.4, to evaluate U937 adhesion to EA.hy 926 cells. The fluorescence cells were photographed using fluorescence microscope and the fluorescence intensity was measured using a fluorescence micro-plate reader (Bio-Tek Instruments, Winooski, VT) with excitation at 510 nm and emission at 531 nm.
2.10. Preparation of cell extracts and immunoblot analysis
EA.hy 926 cells (5 × 105 cells/6 cm dish) were incubated with various concentrations of AS
(25-100 µg/mL) in the presence or absence of TNF-α (10 ng/mL) in various time points. After treatment, the cells were detached and washed once in cold PBS and suspended in 100 L lysis buffer (10 mM Tris-HCl [pH 8], 0.32 M sucrose, 1% Triton X-100, 5 mM EDTA, 2 mM DTT, and 1 mM phenylmethyl sulfonyflouride). The suspension was put on ice for 20 min and then
centrifuged at 15,000 g for 20 min at 4 °C. Protein content in total, cytoplasmic, and nuclear fractions were determined using a Bio-Rad protein assay reagent, with bovine serum albumin as the standard as described previously . Protein extracts were reconstituted in sample buffer (0.062 M Tris-HCl [pH 6.8], 2% SDS, 10% glycerol and 5% β-mercaptoethanol), and the mixture was boiled for 5 min. Equal amounts (50 g) of the denatured proteins were loaded into each lane, separated on 8-15% SDS polyacrylamide gel, followed by transfer of the proteins to PVDF membranes overnight. Membranes were blocked with 0.1% Tween-20 in Tris-buffered saline containing 5% non-fat dry milk for 20 min at room temperature, and the membranes were reacted with primary antibodies for 2 h. They were then incubated with a horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibody for 2 h before being developed using the SuperSignal ULTRA chemiluminescence substrate (Pierce Biotechnology Inc., Rockford, IL). Band intensities were quantified by commercially available software (AlphaEase, Genetic Technology Inc. Miami, FL) with the control representing 100% as shown in the histogram data.
2.11. RNA extraction and RT-PCR analysis
EA.hy 926 cells were harvested after pretreatment with the indicated concentration of AS
(25-100 g/mL) for 1 h in the absence or presence of TNF-α (10 ng/mL) for 24 h. Total RNA from cultured cells were prepared using TriZol-Reagent (Invitrogen). 1 µg of total RNA was subjected to RT-PCR using BioRad iCycler PCR instrument (Bio-Rad, Hercules, CA) and SuperScript-III® One-Step RT-PCR platinum taq® Kit (Invitrogen); amplification was achieved
by 30-38 cycles of 94C for 45 s (denaturing), 60-65C for 45 s (annealing), and 72C for 1 min (primer extension). The sequences of primers used were ICAM (forward-5′-AGCAATGTGCAAGAAGATAGCCAA-3′ and reverse -5′-GGTCCCCTGCGTGTTCCACC-3′) and 18S (forward-5′-GTCTGTGATGCCCTTAGATG-3′ and
reverse-5′AGCTTATGACCCGCACTTAC-3′). PCR products were electrophoresed in 1% agarose gel and stained with ethidium bromide (EtBr).
2.12. Luciferase reporter assay for NF-κB and Nrf2 transcriptional activity
To examine promoter activity, we used a dual-luciferase reporter assay system (Promega, Madison, WI). Briefly, EA.hy 926 cells were treated with or without AS (50 and 100 µg/mL) in the presence or absence of TNF-α (10 ng/mL) for 6 h. After AS treatment, cells in 24-well plates at 70%–80% confluence was incubated with serum starved DMEM without antibiotics for 5 h. Cells were then transfected with pcDNA vector or NF-κB or Nrf2 plasmid with β-galactosidase using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Followed by incubation, cells were lysed and luciferase activity was measured using a luminometer (Bio-Tek instruments Inc, Winooski, VA). Luciferase activity was normalized into β-galactosidase activity in the cell lysates and data expressed as the average of three independent experiments.
2.13. Immunofluorescence staining
EA.hy 926 cells at a density of 2 × 104 cells/well were cultured in DMEM medium with 15%
FBS in an eight-well glass Nunc Lab-Tek® chamber and treated with or without AS (50 and
100 μg/mL) in the presence or absence of TNF-α (10 ng/mL) for 1 h. Cells were then fixed in 2% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, washed and blocked with 10% FBS in PBS, and then incubated for 2 h with NF-κB (p65) or anti-Nrf2 primary antibodies in 1.5% FBS. FITC (488 nm) secondary antibody were incubated for another 1 h in 6% bovine serum albumin. 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI) was stained for 5 min. Stained cells were washed with PBS and visualized using a confocal microscope at 630 × magnification.
GSH levels were determined using the method originally described by . Briefly, EA.hy 926 cells were treated with AS (50 μg/mL) for 1 h, washed twice with PBS, and then incubated with monochlorobimane (2 mM) in the dark for 20 min at 37C. After two washes with PBS, the cells were solubilized with 1% SDS and 5 mM Tris-HCl (pH 7.4). Fluorescence was measured by fluorescence micro-plate reader with excitation and emission wavelengths of 380 and 470 nm, respectively. Samples were assayed in triplicate.
2.15. shRNA Transfection
The shRNA was transfected with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. For the transfections, EA.hy 926 cells were grown in DMEM medium containing 15% FBS and plated in 6-well plates to yield a 40−60% confluence at the time of transfection. The next day, the culture medium was replaced with 500 μL of Opti-MEM (Invitrogen), and the cells were transfected using the RNAiMAX transfection reagent (Invitrogen). For each transfection, 5 μL RNAiMAX was mixed with 250 μL of Opti-MEM and incubated for 5 min at room temperature. In a separate tube, shRNA (100 pM for a final concentration of 100 nM in 1 mL of Opti-MEM) was added to 250 μL of Opti-MEM, and the shRNA solution was added to the diluted RNAiMAX reagent. The resulting siRNA/RNAiMAX mixture (500 μL) was incubated for an additional 25 min at room temperature to allow complex formation. Subsequently, the solution was added to the cells in the 6-well plates, giving a final transfection volume of 1 mL. After incubation for 6 h, the transfection medium was replaced with 2 mL of standard growth medium, and the cells were cultured at 37°C. After AS pre-treatment (50 μg/mL) for 1 h and then the cells were subjected to invasion, tube formation, and U937 adhesion assay.
2.16. Statistical analysis
analyzed using an analysis of variance (ANOVA), followed by Dunnett’s test for pair-wise comparison. Statistical significance was defined as p < 0.05 for all tests.
3. Results
In this study, the human endothelial cell lines, EA.hy 926 cells, were used to investigate the anti-angiogenic and anti-atherogenic ability of the fermented culture broth of AS and revealed the molecular mechanism(s) involved in this inhibition.
3.1. Effects of AS on EA.hy 926 cell viability
Prior to the in vitro studies, whether AS had any cytotoxic effects to the cultured human endothelial EA.hy926 cells, the cell viability was examined by MTT colorimetric assay. A result shows that up to the concentration of 100 µg/mL AS was not affected the cell number (Fig. 1). The distinct cellular or morphological changes that are typically associated with apoptosis, such as cell detachment, rounding or chromosomal fragmentation, were barely detected after a 24 h incubation with AS (data not shown). These data suggested that the non-cytotoxic concentrations of AS (i.e., < 100 μg/mL) could be used to evaluate its anti-angiogenic/anti-atherogenic potentials in cultured human endothelial EA.hy 926 cells.
3.2. AS inhibits TNF-a-induced migration and invasion of EA.hy 926 cells
The migration/invasion of endothelial cells through the basement membrane is a crucial step in the establishment of new blood vessels . To determine the effects of AS on endothelial cell migration in vitro, confluent monolayers of EA.hy 926 cells were incubated with or without AS (50 and 100 µg/mL) in the presence or absence of TNF-α (10 ng/mL) for 24 h. As shown in
Fig. 2A, treatment with TNF-α significantly (p < 0.05) induced the migration of EA.hy 926 cells after 24 h (140 ± 3%), whereas the addition of AS significantly (p < 0.05) as well as dose-dependently decreased TNF-α-induced migration of EA.hy 926 cells (Fig. 2A). In addition, we also observed that when compared to control cells, the endogenous migratory potential of EA.hy 926 cells was significantly reduced by AS alone (100 µg/mL) from 100% to 46 ± 3% (Fig. 2A).
Next, the effect of AS on the invasiveness of EA.hy 926 cells was evaluated using the Boyden chamber assay, which allowed us to determine the ability of cells to pass through a layer of extracellular matrix on a Matrigel-coated filter. As shown in Fig. 2B, compared to un-treated control cells, TNF-α significantly (p < 0.05) induced the invasiveness of EA.hy 926 cells (245 ±5%) after 12 h, whereas the addition of AS significantly (p <0.05) as well as dose-dependently decreased the TNF-α-induced invasion of EA.hy 926 cells (Fig. 2B). Moreover, compared to the control cells, the invasive potential of EA.hy 926 cells was significantly reduced by AS alone treatment from 100 % to 32 ± 2% (Fig. 2B).
3.3. AS inhibits TNF-a-induced tube formation by EA.hy 926 cells
Since vascular maturation during angiogenesis is characterized by the formation of tubular structures by capillary endothelial cells . Therefore, we performed a tube formation assay to investigate the effect of exposure to AS for 3 h on TNF-α-induced capillary-like structure formation by EA.hy 926 cells. Following stimulation by TNF-α, EA.hy 926 cells became aligned into cords on the Matrigel, and a tube-like structure was formed (Fig. 2C). Treatment of cells with AS (50 and 100 g/mL) resulted in a significant (p < 0.05) as well as dose-dependent inhibition of TNF-α-induced tube formation by EA.hy 926 cells (Fig. 2C). Moreover, we observed that the control cells also formed tube-like structure, whereas the AS alone (100 µg/mL) treatment significantly inhibited the tube formation from 100% to 46 ±3% (Fig. 2C). These results clearly demonstrate that exposure to AS is effective at controlling the TNF-α-stimulated tube formation by endothelial cells.
3.4. AS suppresses TNF-α-induced MMP-9 activity and expression in EA.hy 926 cells
Since gelatinase and collagenase MMPs are also involved in the angiogenic process , we used gelatin zymography and Western blot assays to determine the TNF-α-induced MMP-9 activity and expression. To examine the effects of AS on MMP-9 activity, EA.hy 926 cells
were treated with or without AS (50 and 100 μg/mL) in the presence or absence of TNF-α for 24 h in serum-free medium. After treatment, the conditioned media was collected and examined for MMP-9 activity using gelatin zymography assay. As shown in Fig. 3A, compared to control, cells exposed to TNF-α markedly increased the MMP-9 activity from 105 to 220 ± 10%. However, cells co-incubated with AS significantly inhibited the TNF-α-induced MMP-9 secretion in a dose-dependent manner (Fig. 3A). In addition, the experimental treatment (AS alone, 100 µg/mL) did not seem to change the amount of detectable MMP-9 secretion in EA.hy 926 cells (Fig. 3A). Next, Western blot analysis was performed to monitor the effect of AS on MMP-9 protein expression in TNF-α-induced EA.hy 926 cells. As shown in Fig. 3B, cells treated with AS (50 and 100 g/mL) for 24 h remarkably reduced the TNF-α-induced MMP-9 protein levels in a dose-dependent manner. Furthermore, the experimental treatment (AS alone, 100 µg/mL) significantly inhibited the amount of detectable MMP-9 protein levels in EA.hy 926 cells (Fig. 3B).
3.5. AS inhibits TNF-α-activated U937 adhesion to endothelial cells
To explore the effects of AS on endothelial cell-leukocyte interactions, we examined adhesion of U937 cells, a monocyte cell line, to TNF-α-activated endothelial cells (EA.hy 926) under static conditions . As shown in Fig. 4A and B, un-stimulated confluent of EA.hy 926 cells exhibited minimal binding to U937 cells; however, U937 adhesion was substantially increased when EA.hy 926 cells were incubated with TNF-α (800 ± 20%). Concurrent incubation of EA.hy 926 cell confluent with various concentrations of AS (25-100 g/mL) dose-dependently inhibited the U937 adhesion to TNF-α-activated EA.hy 926 cells (Fig. 4A and B).
3.6. AS suppresses TNF-α-induced ICAM-1 expression in EA.hy 926 cells
Further we hypothesized that the AS-mediated inhibition of endothelial-leukocyte adhesion may due to the down-regulation of ICAM-1 expression, EA.hy 926 cells were incubated with
or without various concentrations of AS (25-100 µg/mL) in the presence of absence of TNF-α for 24 h, and the ICAM-1 protein and mRNA levels were monitored by Western blotting and RT-PCR analysis, respectively. As shown in Fig. 5A, cells exposed to TNF-α markedly increased the ICAM-1 protein expression from 100% to 390 ± 10%, whereas the concurrent AS treatment significant as well as dose-dependent inhibited the TNF-α-induced ICAM-1 protein expression in EA.hy 926 cells. In addition, a similar effect was also observed in the gene levels that TNF-α-induced ICAM-1 mRNA expression was dose-dependently inhibited by AS (Fig. 5B). These results suggest that the inhibition of TNF-α-activated endothelial-leukocyte adhesion by AS may be due to the down-regulation of ICAM-1 expression in endothelial cells.
3.7. AS attenuates NF-κB activation, I-κBα degradation, and I-κB kinase phosphorylation in TNF-α-activated EA.hy 926 cells
NF-κB activation is a critical event for the TNF-α-induced activation/expression of MMPs and adhesion molecules. Thereby, we examined the effect of AS on TNF-α-induced NF-κB activation in EA.hy 926 cells. NF-κB activation was detected by measuring NF-κB-dependent transcription in EA.hy 926 cells stably transfected with luciferase reporter construct. Cells incubated with TNF-α alone, found remarkable increases of NF-κB activity (10 ± 1.2-fold). However, the TNF-α-induced NF-κB activity was significantly (p < 0.05) as well as dose-dependently reduced to 7 ± 1.1-fold and 4.8 ± 3-fold by 50 and 100 µg/mL of AS, respectively (Fig. 6A). Notably, the AS alone treatment maintained the basal level of NF-κB activity. A similar result was observed in the immunofluorescence assay: in the control cells, NF-κB (p65) was tethered in cytoplasm, and the nuclear NF-κB spontaneously increased after treatment with TNF-α (Fig. 6B). However, AS treatment (50 µg/mL) significantly prevented the TNF-α-induced nuclear translocation of NF-κB in EA.hy 926 cells (Fig. 6B).
Phosphorylation and subsequent proteasomal degradation of I-κBα is a critical step for the export of NF-κB subunits to the nucleus. Thereby, we examined the effect of AS on
TNF-α-induced NF-κB activation in EA.hy 926 cells via detecting I-κBα protein stability by Western blotting. Compare to control, cells exposed to TNF-α, the amount of I-κBα protein level was remarkably reduced, whereas AS treatment prevented the TNF-α-induced I-κBα degradation in a dose-dependent manner (Fig. 6C). Western blot analysis with nuclear fraction also confirms that control cells barely expressed NF-κB (p65) in the nucleus, whereas the increased accumulation of NF-κB (p65) was observed after TNF-α treatment. However, the TNF-α-induced nuclear translocation of NF-κB (p65) was significantly inhibited by AS in a dose-dependent manner (Fig. 6C).
To examine whether the suppression of I-κBα degradation by AS was mediated through inhibition of their up-stream I-κB kinase (IKK) phosphorylation, we investigated TNF-α-induced phosphorylation of IKK proteins in EA.hy 926 cells using immunoblotting. As shown
in Fig. 6D, cells challenged with TNF-α, time-dependently increase the phosphorylation of
IKK, and the phosphorylation was peaked after 15 min, whereas, AS (50 µg/mL) treatment significantly (p < 0.05) suppressed the TNF-α-induced phosphorylation of IKK in a time-dependent manner (Fig. 6D). Results suggest that AS significantly down-regulates TNF-α-induced NF-κB activation followed by the suppression of I-κB degradation and I-κB kinase phosphorylation after challenge with TNF-α.
3.8. AS up-regulates HO-1 and γ-GCLC expression via Nrf2 activation in EA.hy 926 cells
We hypothesized that the protective effects of AS against TNF-α-induced oxidative stress result from the induction of antioxidant genes, such as HO-1 and γ-GCLC, and its transcription factor Nrf2. As expected, we observed that AS significantly increased HO-1 and γ-GCLC expression in a time-dependent manner. The high protein expression of HO-1 and γ-GCLC were observed after 12 and 3 h, respectively (Fig. 7A). Further, we hypothesized that the induction of HO-1 and γ-GCLC by AS is due to the nuclear translocation and transcriptional activation of Nrf2. Therefore, next we monitored the nuclear translocation of Nrf2 by using
immunofluorescence assay. As shown in Fig. 7B, the protein expression and nuclear accumulation of Nrf2 is significantly (p < 0.05) as well as dose-dependently increased by AS treatment.
To further test the hypothesis that AS may promote the transcriptional activation of Nrf2 in EA.hy 926 cells, the promoter activity of Nrf2/antioxidant response element (ARE) was measured using a luciferase reporter assay. Cells treated with AS (50 and 100 µg/mL), indeed displayed a very significant (p < 0.05) increase of ARE-luciferase activity in a dose-dependent manner (Fig. 7C). Compared with un-treated control cells (100%), the ARE promoter activity was increased to 150 ± 23% and 291 ± 37% by 50 and 100 µg/mL of AS, respectively (Fig. 7C). This result confirms that AS treatment not only induces the nuclear translocation of Nrf2 but also promotes ARE-driven transcriptional activity in EA.hy 926 cells.
In the current study, we observed that AS up-regulates γ-GCLC expression in EA.hy 926 cells, which is essential for the de novo synthesis of a rate-limiting enzyme GSH. Therefore, next we examined whether AS treatment could up-regulates the production of GSH. As shown
Fig. 7D, the intracellular GSH level was significantly (p < 0.05) increased by AS (50 µg/mL) after 1 h treatment. The highest level of GSH was observed after 9 h treatment with AS. Taken together, results indicated that AS promotes the induction of HO-1 and γ-GCLC antioxidant genes and GSH production followed by transcriptional activation of Nrf2 in EA.hy 926 cells
3.11. HO-1 knock-down diminishes the protective effect of AS
To demonstrate the importance of HO-1 regulation, we developed an HO-1 knock-down model in EA.hy 926 cells by using shRNA transfection. The effect of knocking down of HO-1 gene was confirmed by Western blot analysis, which showed that the transfection of shHO-1 (100 pM) led to the reduction in the HO-1 protein level (0.3-fold) and that it also decreased the AS (50 µg/mL)-induced HO-1 expression in EA.hy 926 cells from 1.3-fold to 0.6-fold (Fig. 8A). The knockdown of HO-1 was effective even with AS treatment for 6 h. That is, the
transfection with shHO-1 abrogated the protective effect of AS. We further demonstrate that shHO-1 abolishes the inhibitory effects of AS on TNF-α-induced invasion of endothelial cells (Fig. 8B). In addition, the TNF-α-induced tube formation in EA.hy 926 cells were inhibited by AS, whereas the shHO-1 treatment significantly prevented the inhibitory effect of AS (Fig. 8C). Moreover, the TNF-α-induced endothelial-leukocyte adhesion was significantly inhibited by AS, whereas in the shHO-1 treated cells, AS failed to inhibit the TNF-α-induced adhesion activity (Fig. 8D). These results provide positive evidence that the AS-mediated augmentation of HO-1/Nrf2 was involved in the down-regulation of TNF-α-induced angiogenesis/atherogenesis.
4. Discussion
Many medicinal herbs have been shown to be rich sources of phytochemicals with chemoprevention potential for various types of human cancer, inflammatory diseases, and atherosclerosis. --In the present study, we demonstrated that at non-cytotoxic concentrations of submerged culture broth of Antrodia salmonea (AS) markedly inhibited TNF-α-induced angiogenesis/atherogenesis in EA.hy 926 endothelial cells through the down-regulation of NF-κB signaling pathway and up-regulation of HO-1/Nrf2 antioxidant genes. Results support an anti-angiogenic and anti-atherosclerotic activity of Antrodia salmonea that may contribute critically to its cancer, inflammation, and atherosclerosis chemopreventive potential.
We hypothesized that AS could be a promising candidate drug for the treatment of diseases with impaired angiogenesis. Matrix metalloproteinases (MMPs) play a major regulatory role in the degradation and remodeling of the extracellular matrix are clearly implicated in the initiation of angiogenesis and endothelial cell differentiation and spreading during angiogenesis . In particular, the release of MMP-9 from endothelial cells represents an important step in neovascularization, because this major extracellular matrix proteolytic enzyme is secreted when endothelial sprouting takes place, thus enhancing cell migration across the extracellular matrix and tube-like structure formation . Further, MMP-9 is a type IV collagenase that has been shown to be important in tumor migration/invasion in vitro because of their ability to break down basement membrane components, in particular collagen IV . The importance of MMPs in cancer, in particular the contribution of MMP-9 to cancer metastasis and angiogenesis, the anti-angiogenic agents could decrease tumor growth and metastatic dissemination of numerous solid tumor types. Our results demonstrate that AS treatment significantly suppressed the induction of MMP-9 activity in TNF-α-stimulated EA.hy 926 endothelial cells, suggesting that the anti-angiogenic activity of AS is closely related with a decrease in MMP-9 activity. Moreover, the inhibition of migration/invasion and tube formation by AS may also be due to decreased MMP-9 activity.
Leukocyte adhesion to arterial endothelial cells is thought to be an important step in the development of atherosclerosis. Molecular mechanisms mediating the recruitment of monocytes/macrophages and T lymphocytes into atherosclerotic lesions may depend on multiple and complex processes; however, adhesion of monocytes and T lymphocytes to the vascular endothelium is among the earliest and essential processes during atherogenesis, as well as during inflammatory responses . It is well known that adhesion molecules are strong predictors of atherosclerotic lesion development and further onset of cardiovascular events . TNF-α exerts its biological effects mainly by acting on the vascular endothelium, and thereby up-regulates leukocyte recruitment into inflamed tissues . Our present study demonstrates that AS decreased the TNF-α-induced adhesion of U937 leukocytes to the endothelial cells. Endothelial cell adhesion molecules, such as E-selectin, ICAM-1, and VCAM-1 contribute to the development of chronic inflammatory diseases by recruiting leukocytes, especially lymphocytes, into inflammation site . Furthermore, AS significantly diminished TNF-α-induced ICAM-1 expression in both transcriptional and translational levels. Thus, AS could down-regulate VCAM-1 associated with reduced adhesion of leukocytes, which may play a major role in the prevention of atherosclerosis and inflammation.
Recent investigations have shown results that can offer to support this deduction. Notably, Huang et al reported that AS was found to have strong anti-inflammatory effects and the mechanism of its action appears to be by inhibiting the secretion of nitric oxide (NO) and prostaglandin E2 (PGE2) through the suppression of their corresponding mediator inducible
nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-induced murine macrophage cells . However, the effects of AS on the up-stream regulators of inflammatory mediators were poorly investigated. Due to the fact that the production of pro-inflammatory cytokines and mediators are regulated, at least in part, by the transcriptional factor NF-κB, we investigated the role AS play on inhibiting this transcriptional factor. In un-stimulated condition, NF-κB is sequestrated with IκBs such as IκBα, IκBβ, and IκBε in the
cytoplasm, up on stimulation by some physical or chemical stimuli lead to activation of IκB. Once IκB is phosphorylated and degraded, the unbound NF-κB translocates into the nucleus and activates pro-inflammatory genes including genes for cytokines, chemokines, adhesion molecules, and MMPs . A previous study also been demonstrated that TNF-α induces NF-κB activation to stimulate the production of pro-inflammatory cytokines and adhesion molecules in human aortic endothelial cells . In this study, the results demonstrated that AS significantly attenuated TNF-α-induced nuclear translocation and transcriptional activation of NF-κB followed by the suppression of I-κB degradation and I-κB kinase phosphorylation. Taken together, these data confirmed that AS has potent anti-angiogenic and anti-atherogenic effects through the down-regulation of MMP-9 and ICAM-1 via suppressing NF-κB activity.
Mounting evidence suggest that dietary phytochemicals or antioxidants exerts their protective effects not only by scavenging free radicals but, also promoting transcriptional up-regulations of antioxidant genes . It is well demonstrated that HO-1 is a major antioxidant enzyme that play vital role in antioxidant defense against free radicals-induced oxidative destruction in human endothelial cells . Moreover, Nrf2, a basic leucine-zippper (bZIP) transcription factor has been shown to be involved in induction of various antioxidant genes including HO-1 and γ-GCLC . Previous studies have shown that increasing Nrf2 activity in endothelial cells is highly protective during oxidative stress and inflammation . Several investigations have showed that the plant-derived chemical substances enhance Nrf2-dependent ARE activity and induces HO-1 and γ-GCLC expression in variety of cells including endothelial cells . Notably, there is cross-talk between the transcription factors NF-κB and Nrf2 as far as inflammatory genes expression is concerned (Surh and Na, 2008). Over-expression of Nrf2 suppressed pro-inflammatory genes expression in human arotic endothelial cells (Chen et
al., 2006). Unsurprisingly, AS up-regulates the antioxidant genes HO-1 and γ-GCLC through
Nrf2 activation in human endothelial cells. Thus, AS protects endothelial cells from TNF-α-induced oxidative stress mainly by elevating intracellular antioxidant enzymes. The discovery
of RNA interference has revolutionized approaches to decoding specific gene functions and pathways . Notably, shRNA-mediated transcriptional silencing is conserved in mammalian cells and thus provides a means to inhibit specific mammalian gene functions . Our data exhibit that shRNA-mediated knock-down of HO-1 expression leads to enhanced invasion, tube formation, and leukocyte adhesion to endothelial cells after challenge with TNF-α. However, AS barley protects TNF-α-induced invasion, tube formation, and endothelial-leukocyte adhesion in HO-1 knock-down EA.hy 926 cells. These data undoubtedly proved that the protective effect of AS is mainly mediated by HO-1 activation through Nrf2 signaling pathways.
GSH is a well-studied antioxidant enzyme with numerous reported roles in protecting cells from oxidative stress and maintaining the cellular thiol redox status . The cell redox state is particularly important in protein glutathionylation and for the thiol-mediated signal transduction pathways . Nrf2 has been shown to regulates the expression of many thiol-regulating enzymes, including glutathione S-transferase (GST), glutathione peroxidases (GPx), γ-glutamylcysteine synthetase (γ-GCLC), and thioredoxin reductase (TrxRs) . GSH can be act as a non-enzymatic antioxidant by direct interaction of the SH group with ROS, or it can be involved in the enzymatic detoxification reaction for ROS, as a co-factor or co-enzyme . In this study, we also found that AS treatment significantly as well as time-dependently increased GSH secretion in EA.hy 926 cells, which may involved in the protective effect of AS.
Previous studies reported that some bioactive compounds have been identified within extract of the basidiomata of AS and displayed potential with antioxidant and anti-inflammatory effects. A phytochemical investigation reported that four new compounds were isolated from the mycelia of AS: lanosta-8,24-diene-3β,15α,21-triol (1), 24-methylenelanost-8-ene-3β,15α,21-triol , 3-dimethyoxy-5-(2′,5′-dimethoxy-3′m4′- methylenedioxyphenyl-7-methyl-[1,4]-naphthoquinone (3), and 2,3-dimethoxy-6 (2′,5′-dimethoxy-3′,4′-methylenedi-oxyphenyl)-7-methyl-[1,4]-naphthoquinone (4). Among these compounds, compounds 1, 3,
and 4 showed potent anti-oxidative effects in human leukocytes in vitro . Later, Shen et al isolated three new ergostanes, methyl antcinate L (1), antcin M (2), and methyl antcinate K (3), together with nine known compounds. All three new ergostanes (1-3) exhibited anti-oxidative effects by inhibiting NADPH oxidase activity but not through free radical scavenging activity. In addition, the ergostanes (1-3) strongly inhibited nitric oxide production in activated neutrophils and microglia cells . However, the anti-agiogenic and anti-atherogenic effects of these potentially beneficial compounds were not yet being investigated. In the nearing future, we have sought to investigate the anti-angiogenic and anti-atherogenic effect of bioactive compounds isolated from the fermented culture broth of AS.
The results obtained in the present study demonstrate that Antrodia salmonea has potent anti-angiogenic and anti-atherogenic activity in TNF-α-induced human endothelial cells. AS significantly inhibit endothelial cell invasion, tube formation, and leukocyte adhesion after challenge with TNF-α, which may be through the suppression of transcriptional activation of NF-κB. Our present study also reveled that AS induces anti-oxidative genes such as HO-1 and γ-GCLC via Nrf2 signaling pathway that may contribute to its putative beneficial effects in endothelial cells. These finding added some additional information on the biological activities of Antrodia salmonea. However, further in vivo studies may provide new therapeutic insights of this potentially beneficial mushroom.
Conflict of interest
The authors declare that there are no conflicts of interest
Acknowledgements
This work was supported by grants NSC-101-2320-B-039-050-MY3, CMU 100-ASIA-13, and CMU 100-ASIA-14 from the National Science Council, China Medical University, and Asia University, Taiwan.
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Figure legend
Fig. 1. Anti-proliferative activity of the fermented culture broth of Antrodia salmonea (AS) on human endothelial (EA.hy 926) cells. Cells were treated with 25, 50, and 100 g/mL AS and allowed to proliferate for 24 h. Cell numbers were obtained by counting cell suspensions with a hemocytometer. Results are presented as mean SD of three independent assays. AS, Antrodia
salmonea.
Fig. 2. AS inhibits TNF-α-induced migration, invasion, and tube formation in EA.hy 926 cells. (A) Cells were pretreated with 50 and 100 g/mL of AS for 1 h. Subsequently, cells were scratched and then stimulated with or without TNF-α (10 ng/mL) for 24 h. Migration was observed using an optic microscope, at a 200 × magnification, and the closure of area was calculated using commercially available software. (B) Cells were pre-treated with 50 and 100 g/mL AS for 1 h followed by incubation with or without TNF-α (10 ng/mL) for 12 h. Photomicrographs of cells invading under the membrane (for 12 h). The Inhibitory percentage of invading cells was quantified and expressed with untreated cells (control) representing 100%. Invasiveness was determined by counting cells in three microscopic fields per sample. (C) AS inhibits TNF-α-induced tube formation in EA.hy 926 cells. Cells were pretreated with 50 and 100 g/mL AS for 1 h, and then collected and replaced on Matrigel-coated plates at a density of 1×105 cells/well and incubated in the absence or presence of TNF-α (10 ng/mL).
After 3 h, the tube formation was determined using a phase-contrast microscope at 200 × magnification. The capillary networks were photographed, and the number of tubes was quantified from three random fields. One of the typical results from three independent experiments is shown. Results are presented as mean SD of three independent assays; *p <
Fig. 3. AS suppresses/inhibits MMP-9 activity and pexpression in TNF-α-induced EA.hy 926 cells. Cells were pretreated with 50 and 100 g/mL AS for 1 h, and then stimulated with TNF-α (10 ng/mL) for 24 h. (A) An equal amount (50 µg) of conditioned culture media from each sample was subjected to gelatin zymography. The relative density of MMP-9 bands was measured by commercially available uantitative software. (B) Immunoblotting was performed against anti-MMP-9. An equal amount (50 µg) of total lysate from each sample was resolved by 8-15% SDS-PAGE with β-actin as a control. The relative changes in the protein bands were measured by commercially available quantitative software. One of the typical results from three independent experiments is shown. Results are presented as mean SD of three independent assays; *p < 0.05 indicates significant difference in comparison to TNF-α alone vs sample.
Fig. 4. AS inhibits TNF-α-induced U937 cell adhesion in EA.hy 926 cells. Cells were pretreated with 50 and 100 g/mL AS for 1 h, and then stimulated with TNF-α (10 ng/mL) for 4 h. (A) After cells were washed with PBS, BCECF-AM-labeled U937 cells were added to each well and cultures were incubated for an additional hour at 37 °C. The level of U937 cells bound to endothelial cells was photographed by fluorescence microscopy (200 × magnification). One of the typical results from three independent experiments is shown. (B) The fluorescence intensity was quantified and the inhibitory percentage was calculated (control being 100%). Results are presented as mean SD of three independent assays; *p < 0.05
indicates significant difference in comparison to TNF-α alone vs sample.
Fig. 5. AS inhibits TNF-α-induced ICAM-1 expression in EA.hy 926 cells. (A) Cells were harvested after pretreatment with the indicated concentration of AS (25-100 g/mL) for 1 h in the absence or presence of TNF-α (10 ng/mL) for 4 h. An equal amount (50 µg) of total lysate from each sample was resolved by 8-15% SDS-PAGE with β-actin as a control. The relative
changes in the protein bands were measured, the control being 100% as shown just below the gel data. (B) Cells were pretreatment with AS (25-100 g/mL) for 1 h in the presence of absence of TNF-α (10 ng/mL) for 2 h. Total RNA was extracted using TriZol method and subjected to RT-PCR using one-step RT-PCR master mix. The RT-PCR products were separated by 1% agarose gel electrophoresis. GAPDH gene was used as a loading control. Relative changes in mRNA bands were measured, the control being 100% as shown just below the gel data. One of the typical results from three independent experiments is shown. Results are presented as mean SD of three independent assays; * p < 0.05 indicates significant
difference in comparison to TNF-α alone vs sample.
Fig. 6. AS down-regulates NF-κB nuclear translocation through the suppression of IκB degradation in TNF-α-activated EA.hy 926 cells. (A) Immunofluorescence staining shows the changes of nuclear translocation of NF-κB. Cells were grown in chamber slides were exposed to AS (50 µg/mL) in the presence or absence of TNF-α (10 ng/mL) for 1 h, fixed, and permiabilized. Then the cells were incubated with anti-P65 antibody followed by FITC-labeled secondary antibody. Cells were stained with DAPI (1 g/mL) for 5 min and examined by fluorescence microscopy (magnification 200). (B) The SEAP luciferace activity of NF-κB was measured after treatment with AS (50 and 100 µg/mL) in the presence or absence of TNF-α (10 ng/mL) for 6 h. (C) Western blotting was performed to monitor the cytosolic IκBTNF-α and nuclear p65 protein expressions. Cells were treated with indicated concentrations of AS (50 and 100 µg/mL) in the presence or absence of TNF-α (10 ng/mL) for 1 h. (D) The phosphorylation of IKK was determined using Western blot analysis. Cells were treated with AS (50 µg/mL) in the presence or absence of TNF-α (10 ng/mL) for 5-15 min. The total cells lysate was subjected to western blotting. β-actin served as an internal loading control. One of the typical results from three independent experiments is shown. Results are presented as mean SD of three
independent assays; *p < 0.05 indicates significant difference in comparison to TNF-α alone vs
sample.
Fig. 7. AS up-regulates the Nrf2-mediated antioxidant genes in EA.hy 926 cells. (A) Immunofluorescence staining shows the changes of Nrf2 nuclear translocation. Cells were grown in chamber slides were exposed to AS (50 and 100 µg/mL) for 1 h, fixed and permiabilized. Cells were incubated with anti-Nrf2 antibody followed by FITC-labeled secondary antibody. Cells were stained with DAPI (1 g/mL) for 5 min and examined by fluorescence microscopy (magnification 200). (B) The luciferace activity of ARE was measured after treatment with AS 50 and 100 µg/mL for 6 h. (C) Cells were incubated with or without various concentrations of AS (50 µg/mL) for 1-12 h. Total cell lysate was subjected to Western blotting to monitor the HO-1 and γ-GCLC protein using specific antibodies. An equal amount (50 µg) of total lysate from each sample was resolved by 8-15% SDS-PAGE with β-actin as a control. The relative changes in the intensities of the protein bands were measured by densitometry. One of the typical results from three independent experiments is shown. (D) Cells were incubated with AS (50 µg/mL) for 1-9 h. The increasing amount of total GSH was measured using commercially available EIA kit. One of the typical results from three independent experiments is shown. Results are presented as mean SD of three independent assays; *p < 0.05 indicates significant difference in comparison to TNF-α alone vs sample.
Fig. 8. HO-1 shRNA attenuates the protective effect of AS in EA.hy 926 cells. (A) Cells were transfected with a specific shRNA against HO-1 or a non-silencing control. Following transfection 24 h, the cells were incubated with or without AS (50 g/mL) for 6 h. The HO-1 knock-down was evaluated by western blotting. The control and HO-1 shRNA-treated EA.hy 926 cells were pretreated with AS (50 g/mL) for 1h, and the induced by TNF-α for 4-12 h.
The TNF-α-induced invasion (B), tube formation (C), and leukocyte adhesion (D) was measured. One of the typical results from three independent experiments is shown. Results are presented as mean SD of three independent assays; *p < 0.05 indicates significant difference
