Antioxidant and Antiangiogenic Properties of Phenolic Extract from

Article pubs.acs.org/JAFC

Antioxidant and Antiangiogenic Properties of Phenolic Extract from Pleurotus tuber-regium Shaoling Lin,† Tsz ching Lai,† Lei Chen,†,§ Hin fai Kwok,‡ Clara Bik-san Lau,‡ and Peter C. K. Cheung*,† †

School of Life Sciences and ‡Institute of Chinese Medicine and State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China § Institute of Food and Nutraceutical Science, School of Agriculture and Biology School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China ABSTRACT: Pleurotus tuber-regium (Fries) Singer (PTR), both an edible and a medicinal mushroom also known as tiger milk mushroom, has experienced growing popularity and economic importance due to its flavor, nutritive value, and medicinal effects. In this study, the antioxidant and antiangiogenic activities of a 60% ethanol extract (EE) obtained from the sclerotium of PTR were investigated. Typical phenolic compounds including protocatechuic, chlorogenic, syringic, ferulic, and folic acid were identified and quantified in EE by the HPLC-UV-ESI/MS analyses. EE possessed strong antioxidant activity and could dosedependently inhibit vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVEC) migration and tube formation. qPCR results showed that VEGF-induced FGF, ANG-Tie, and MMP gene expression as well as VEGFR were down-regulated at the mRNA level after treated with EE, suggesting that multiple molecular targets related to angiogenesis was involved. Furthermore, EE also inhibited the formation of subintestinal vessel plexus (SIVs) in zebrafish embryos in vivo. All of these suggested that EE of PTR could be the source of potential inhibitors to target angiogenesis. KEYWORDS: phenolic extract, Pleurotus tuber-regium, antioxidant, antiangiogenesis

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INTRODUCTION Pleurotus tuber-regium (PTR) is both an edible and medicinal mushroom with emerging popularity and economic importance from the Basidiomycotina. It is capable of forming a compact mass of hyphae into a dry structure (sclerotium) to survive environmental extremes.1 The sclerotium of this fungus can serve as food in cooking soup with high nutritive value2 as well as for medicinal purposes. It shows great potential in the treatment for many diseases including cancer, smallpox, constipation, stomach pain, fever, and cold.3 The formation of new blood vessels from pre-existing vessels, known as angiogenesis, has been identified as a hallmark of tumor progression and is a necessary requirement for the growth and metastasis of tumors as it supplies nutrients, oxygen, and growth factors to the tumor cells.4 There is extensive evidence suggesting that antiangiogenic therapy represents a novel and promising strategy for controlling cancer progression.5 It has been shown that cancer cells that bear a higher intracellular reactive oxygen species (ROS) level can up-regulate the expression of vascular endothelial growth factor (VEGF), which appears to be the most important angiogenic factor to stimulate blood vessel formation and sustain tumor growth.6−8 Epigallocatechin gallate, an antioxidant in green tea, was found to exert at least part of its anticancer effect in human colon cancer cell (HT29) by inhibiting angiogenesis through blocking the induction of VEGF.9 In the initial stage of cancer development, oxidative stress plays a major role in damaging essential parts of cells.10 Antiangiogenic agents derived from natural products have attracted attention from scientists, as extracts of natural compounds usually contain a mixture of biological components that could exert multistep actions for antiangiogenesis.11 To © 2014 American Chemical Society

date, there have been extensive studies on natural compounds and extracts that showed potent antiangiogenic activity, in conjunction with their antioxidant activities.12,13 Phenolic compounds are commonly found in many plant foods. Research has shown that phenolic compounds exhibit strong antioxidant activities, and therefore they can be used as efficient antioxidants.14 Some common edible mushrooms widely consumed in Asia have currently been found to possess strong antioxidant activities, which are highly correlated with their total phenolic content;15.16 However, phenolics from PTR have not been reported, and little is known about the chemical composition of the total phenolics from this mushroom species. Therefore, this paper aims to evaluate the antioxidant and antiangiogenic properties of a 60% ethanol extract (EE) rich in phenolic profile from the sclerotium of PTR together with the identification of the phenolic components in EE and elucidation of its possible antiangiogenic mechanism.

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MATERIALS AND METHODS

Plant Material, Animals, Chemicals, and Reagents. The sclerotium of PTR was cultivated by the Sanming Mycological Institute in Fujian province, China. Zebrafish embryos used for screening were obtained from natural spawning of wild-type fish bought from local pet stores in Hong Kong. The Tg (fli1a:EGFP)y1 transgenic line was obtained from the Zebrafish International Resource Center (5274, University of Oregon, Eugene, OR, USA). The zebrafish were maintained in flow-through aquaria (36 L) at 28.5 °C on a 14/10 h (light/dark) photoperiod. The Received: Revised: Accepted: Published: 9488

July 8, 2014 August 29, 2014 September 3, 2014 September 3, 2014 dx.doi.org/10.1021/jf5031604 | J. Agric. Food Chem. 2014, 62, 9488−9498

Journal of Agricultural and Food Chemistry

Article

handling of fish was licensed by the Government of the Hong Kong Special Administrative Region and endorsed by the Animal Experimentation Ethics Committee of The Chinese University of Hong Kong. SU5416 (1,3-dihydro-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylene]2H-indol-2-one), a selective vascular endothelial growth factor (VEGF) receptor-2 inhibitor, was from Sigma. Cell Culture. Human umbilical vein endothelial cells, HUVEC-C (CRL-1730, ATCC) at early passages (passages 8−15) were cultured in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F12 (1:1) (DMEM/F12) (Gibco, catalog no. 12400-024) supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA), 0.1 mg/mL heparin (Sigma, catalog no. H3393), 0.03 mg/mL endothelial cell growth supplement (ECGS) (Sigma, catalog no. E2759), and 1% penicillin−streptomycin. Cells were incubated at 37 °C under 5% CO2. Sample Preparation. Samples of sclerotium were lyophilized before they were pulverized into powders to pass through a screen with a 0.5 mm sieve using a cyclotech mill (Tecator, Höganäs, Sweden). The milled powders (400 g) were extracted with 4 L of 60% ethanol three times at room temperature for 24 h, and the extract was centrifuged and filtered through no. 2 filter paper (Whatman International Ltd., Maidstone, Kent, UK), and concentrated in a rotary evaporator (Rotavapor R-200; Buchi, Postfach, Swizerland). Folin−Ciocalteu Assay. The total phenolic content of EE was determined by using the Folin−Ciocalteu method with some modifications.17 One milliliter of EE solution (1 mg/mL) was mixed with Folin−Ciocalteu reagent (1 mL). After 3 min of incubation at room temperature, 1 mL of saturated Na2CO3 (35% aqueous solution) was added to the mixture and adjusted to 10 mL with distilled water. The mixture was kept in the dark for 90 min, followed by the absorbance being read at 725 nm. Gallic acid was used to prepare the standard curve (0.01−0.4 mM). The total phenolic content of EE was expressed as gallic acid equivalents (GAE) as the amount of gallic acid (mg) per gram of extract. DPPH Radical Scavenging Assay. The DPPH radical scavenging activity of EE was assayed according to the method described previously18 with some modifications. Various concentrations (0.05−2 mg/mL) of EE or negative control (60% ethanol) (0.5 mL) were mixed with 1.0 mL of 0.1 mM DPPH radical solution. The mixture was vortex-mixed and left to stand for 30 min in the dark, the reduction of DPPH radical was determined by measuring the absorbance at 520 nm. tert-Butylhydroquinone (TBHQ) (1.5 g/mL) was used as standard. The DPPH radical scavenging activity (%) was calculated by using the following equation:

concentration (mM) of Trolox, having the same activity as 1 mg of sample. Hydrogen Peroxide Scavenging Assay. The scavenging activity of hydrogen peroxide was measured using a commercial kit (PeroXOquant Quantitative Peroxide Assay Kits, Pierce, catalog no. 23280) according to the manufacturer’s instruction. Working reagent was prepared by mixing reagent A (25 mM ammonium ferrous(II) sulfate, 2.5 M H2SO4) with reagent B (100 mM sorbitol, 125 M xylenol orange in water) (1:100, v/v). EE at various concentrations (0.02−2 mg/mL) was mixed with 0.1 mM H2O2 (Wako, catalog no. 080-01206) for 15 min before assay, whereas standard was prepared by diluting 30% H2O2 to various concentrations (0−1000 μM) with water. An aliquot (20 μL) of sample or standard and 200 μL of working reagent were added into a 96-well plate. The plate was then incubated at room temperature for 20 min for the reaction. The absorbance was read at 595 nm. The concentration of H2O2 present in the sample was determined by interpolating from the sample absorbance value using the standard curve. The hydrogen peroxide scavenging activity (%) was calculated by using the following equation: scavenging activity (%) = {1 − ([H 2O2 ]in sample /[H 2O2 ]in control )} × 100 HPLC-UV-ESI/MS Analyses. Thirteen phenolic compound standards including flavonoids (quercetin) and phenolic acids (ellagic acid, sinapic acid, gallic acid, protocatechuic acid, chlorogenic acid, caffeic acid, syringic acid, cinnamic acid, ferulic acid, folic acid, salicylic acid, and coumaric acid) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The standard stock solutions (30.3, 100, and 1000 μM) were made with the appropriate HPLC grade solvents. All standard calibration curves showed high degrees of linearity (R2> 0.99) (data not shown). Sample compounds were identified on the basis of the retention times, UV spectrum, and MS spectrum of standard compounds and were quantified by comparing their peak areas with those of standard curves. The FT-ICR MS analysis was performed using the Apex Ultra 7.0 Hybride Qh-FTMS (Bruker Daltonics Inc., USA) equipped with an Apollo II ion source and Dionex Ultimate 3000 2D Nanoflow LC system (Bruker Daltonics Inc., USA). EE was redissolved in 60% aqueous methanol (HPLC grade) (J. T. Baker, Phillipsburg, NJ, USA) and filtered through a 0.45 μm nylon membrane filter (TITAN, Rockwood, TN, USA). A 3 μL filtrate was loaded on the HPLC and FT-MS system. Separation was achieved on an analytical (2.1 × 150 mm) C18 column (5 μm) (XBridge, Waters, Dublin, Ireland). The system was connected to two solvents used as mobile phases: Solvent A was 0.1% formic acid in Milli-Q water, and solvent B was 100% acetonitrile (Scharlau, catalog no. AC03402500). Table 1 shows the elution profile. The flow rate was 0.3 mL/min, and the temperature of the column was kept at 30 °C.

scavenging activity (%) = [1 − (Abs520nm, sample /Abs520nm, control )] × 100 The IC50 value (mg/mL) refers to the effective concentration to scavenge the DPPH radicals by 50%. ABTS+ Radical Scavenging Assay. The ABTS+ scavenging potency of EE was assayed according to the method described previously19 with some modifications. Five milliliters of 7 mM ABTS was mixed with 88 μL of 140 mM potassium persulfate and kept for 12−16 h at room temperature in the dark to allow the completion of radical generation. The reagent was diluted with 95% ethanol to give an absorbance of 0.70 ± 0.05 at 734 nm. Then 1 mL of ABTS solution was mixed thoroughly with 10 μL of EE solution of different concentrations, and the absorbance was recorded at 734 nm at 6 min after the initial mixing, using the ethanol as blank. Trolox, with a concentration range of 0.5−2 mM, was prepared as a standard. The percentage scavenging effect was calculated as

Table 1. Elution Profile Used in the HPLC and LC-MS Analyses time (min)

% formic acid (%)

100% acetonitrile (%)

0 40 45 65 70 71

95 70 50 30 30 95

5 30 50 70 70 5

MTT Assay. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to evaluate the cytotoxicity of EE on normal cell growth. In brief, cells were seeded in a 0.1% gelatincoated 96-well plate at 2 × 104 cells/well overnight. After incubation with various concentrations of EE solution for 24 h, 20 μL of 5 mg/ mL MTT in phosphate-buffered saline (PBS) was added, and incubation was continued for another 4 h at 37 °C. The culture

scavenging rate (%) = [1 − (Abs734nm, sample /Abs734nm, control )] × 100 The antioxidant activity of sample was expressed as Trolox equivalent antioxidant capacity (TEAC), which represented the 9489

dx.doi.org/10.1021/jf5031604 | J. Agric. Food Chem. 2014, 62, 9488−9498


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Table 2. Primers Used in Real-Time qPCR gene

accession no.

VEGFR

NM_002253.2

5′-GGAACCTCACTATCCGCAGAGT-3′ 5′-CCAAGTTCGTCTTTTCCTGGGC-3′

primer sequence

132

Tie2

NM_000459.3

5′-GTTCTGTCTCCCTGACCCCTAT-3′ 5′-TAAGCTTACAATCTGGCCCGTA-3′

107

Ang-2

NM_001147.2

5′-AGATTTTGGACCAGACCAGTGA-3′ 5′-GGATGATGTGCTTGTCTTCCAT-3′

139

MMP-2

NM_004530.4

5′-AGCGAGTGGATGCCGCCTTTAA-3′ 5′-CATTCCAGGCATCTGCGATGAG-3′

138

bFGF

NM_002006.4

5′-AGCGGCTGTACTGCAAAAACGG-3′ 5′-CCTTTGATAGACACAACTCCTCTC-3′

138

β-actin

NM_001101.3

5′-CACCATTGGCAATGAGCGGTTC-3′ 5′-AGGTCTTTGCGGATGTCCACGT-3′

135

medium was removed, and DMSO (100 μL/well) was added. Then, after complete dissolution, absorbance was detected at 570 nm.

expected size (bp)

scored for migrated cells. The quantification of the invaded cells was made by submerging the chambers in 1% SDS and measuring the absorbance of the solution at 570 nm. The mean of triplicate assays for each experimental condition was calculated, and values were normalized to the untreated cells.

cell viability expressed as % of control vehicle = (Abs570nm, sample /Abs570nm, control vehicle ) × 100%

invasiveness expressed as % of control vehicle

Lactate Dehydrogenase (LDH) Toxicity Assay. The LDH release assay was performed using a cytotoxicity detection kit plus (LDH) (Cayman) according to the manufacturer’s instructions. In brief, HUVECs were seeded in a 96-well plate at a density of 2 × 105 cells/well. After incubation with various concentrations of EE solution for 24 h, cell supernatants were collected and analyzed. The absorbance of formazan formed was read at 490 nm. Wound-Healing Assay. The wound-healing method was performed according to protocols reported previously20 with some modifications. In brief, cells were seeded in a 24-well plate at a density of 3 × 105 cells/well overnight. After two washings with PBS, the cells were then scraped away horizontally in each well using a sterilized P100 pipet tip. After two washings with PBS, the cells were treated with 300 μL of various concentrations of EE (cotreated with 20 ng/ mL VEGF) or 20 ng/mL VEGF only (serving as vehicle control) for 24 h. Three random views along the line were selected and photographed using an inverted microscope (Nikon ECLIPSE TS100F) at 0 h. After 24 h, another set of images were taken by using the same method. Image analysis for determining the extent of migration was performed by ImageJ software (NIH, Bethesda, MD, USA). The percentage of wound size after 24 h was calculated using the following equation (wound size was expressed as % of wound area when it was made):

= (Abs570nm, sample /Abs570nm, control ) × 100 Endothelial Tube Formation Assay. The tube formation assay was performed according to previous protocols21 with some modifications. Cells (1 × 104 cells/well) were seeded on a layer of Matrigel (BD Biosciences, catalog no. 354234) and exposed to various concentrations of EE with 20 ng/mL VEGF in starved medium or with starved medium only (served as vehicle control) . After incubation for 6 h in 37 °C, images of three randomly selected fields were captured using the inverted microscope. Measurement of Reactive Oxygen Species. The 2′,7′dichlorofluorescein diacetate (DCFH-DA, Sigma) assay was used to determine the scavenging activity of ROS in HUVECs following the exposure to EE as previously described.22 After exposure to different concentrations of EE with 20 ng/mL VEGF in starved medium or with starved medium only (serving as vehicle control) for 24 h, endothelial cells (ECs) were then incubated in DCFH-DA (0.5 μg/mL) at 37 °C for 30 min. Cells were washed with PBS three times to remove DCFHDA that had not entered into cells. The fluorescence was measured immediately by a fluorescent plate reader (λex = 485 nm, λem = 535 nm). RT-PCR Study for mRNA Expression. Total RNA was isolated by using an SV Total RNA Isolation System according the manufacturer’s protocol (Z3100, Promega, Madison, WI, USA) followed by reverse transcription (cDNA synthesis). Real-time qPCR was performed on the ABI7500 real-time PCR detection system in a volume of 20 μL containing 10 μL of SYBR Premix Ex TaqII (2×), 0.4 μL of ROX reference Dye II (50×), 2 μL of cDNA, 6 μL of dH2O, and 0.8 μL of each 10 μM primer. The primers of all tested genes are listed in Table 2. The reaction mixture was subjected to denaturation at 95 °C for 2 min followed by 40 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 15 s, and elongation at 72 °C for 35 s. A melt-curve analysis was performed at the end of the reaction consisting of 40 cycles. The relative expression levels of the mRNAs of the target genes were normalized using the β-actin internal standard. Quantitative Endogenous Alkaline Phosphatase Assay on Zebrafish Embryo. During zebrafish development, the stage between 48 and 72 h past fertilization (hpf) has the highest angiogenic activity,23 and a quantitative endogenous alkaline phosphatase (EAP) assay was performed as previouly described.24 In brief, embryos (24

wound area (%) = [wound area (t = 24 h) /wound area (t = 0 h)] × 100 Transwell Culture Insert Assay. Cell invasion properties was carried out in a modified Boyden chamber (Transwell, Corning, NY, USA) with a poly(ethylene terephthalate) filter inserted coated with a Matrigel matrix in 24-well plates containing 8 mm pores. In brief, 1 × 104 cells suspended in 100 μL of starved medium were seeded on the upper chamber followed by filling the lower chamber with 600 μL of medium with or without EE under VEGF-induced migration. After 6 h of incubation, the nonmigrated cells on the upper side of the membrane were carefully removed by cotton swab. The migrated cells on the bottom side of the membrane were fixed with methanol for 20 min and then stained with 0.1% crystal violet for 10 min. Four randomly selected fields under 10× magnification of each well were 9490



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hpf) was arrayed in a 96-well plate (one embryo per well) and incubated with 100 μL of embryo water per well containing various concentrations of EE, whereas only 100 μL of embryo water without samples was set as control. After further incubation at 28.5 °C for 48 h, the medium in each well was removed, and embryos were treated with 70% ethanol (100 μL) at room temperature for 15 min and then with 100% ethanol (100 μL) at −20 °C for 30 min for dehydration purposes. The embryos were washed three times with 1× diethanolamine buffer (Pierce, Rockford, IL, USA). Then, 100 μL of staining solution was added to each well. After staining, 50 μL of the 2 M NaOH was added to stop the reaction. The optical density was determined at the wavelength of 405 nm. SU5416, a known effective inhibitory compound on angiogenesis, was chosen to conduct the parallel experiments.24 Vessel growth was presented as percentage formation of vessels in optical density compared to control: %vessel formation = (OD405nm, sample, day 3 − OD405nm, sample, day 1) /(OD405nm, control, day 3 − OD405nm, control, day 1) × 100 Microscopic Imaging. The embryos from the Tg (fli1a: EGFP) y1 zebrafish line were used to further evaluate the antiangiogenic effect of EE. The enhanced green fluorescent protein-expressing ECs of the subintestinal vessel plexus (SIVs) were observed and recorded at 72 hpf using an Olympus IX71S8F-2 inverted microscope (Olympus, Tokyo, Japan). Statistical Analysis. All experiments were performed at least three times. All data are presented as means ± SEM, and all statistics were performed using GraphPad Prism 5.0 software. Differences between means were determined by one-way analysis of variance (ANOVA) or Student’s t test. Differences were considered to be statistically significant at p < 0.05.

Figure 1. Chromatogram of phenolic compound analysis by HPLC: (A) standard mixture of 13 phenolic compounds (peaks: 1, gallic acid; 2, protocatechuic acid; 3, chlorogenic acid; 4, caffeic acid; 5, syringic acid; 6, coumaric acid; 7, ferulic acid; 8, sinapic acid; 9, ellagic acid; 10, salicylic acid; 11, cinnamic acid; 12, quercetin; 13, folic acid); (B) phenolic prolile of 60% ethanol extract from sclerotium of PTR.

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RESULTS Amount of Total Phenolics in EE and HPLC-UV-ESI/MS Analysis. The total phenolic content (TAC) was measured and expressed in terms of gallic acid equivalents (GAE; mg/g of GAE). The amount of total phenolics in EE from sclerotium of PTR was 420 ± 0.33 μg/mg extract, indicating that EE contained a substantial amount of phenolic compounds. At least five phenolic acids (including protocatechuic, chlorogenic, syringic, ferulic, and folic acid) were positively identified and quantified in EE by HPLC-UV-ESI/MS on the basis of their chromatographic characteristics and absorption spectra with reference to standard compounds (Figure 1) and confirmed by mass analysis. The results are summarized in Table 3. Antioxidant Activity of EE. Three methods were utilized to analyze the antioxidant activity of EE. The results on DPPH free radical scavenging activity of EE are shown in Figure 2A. EE inhibited DPPH radicals in a dose-dependent manner with an IC50 at about 1 mg/mL. Furthermore, EE also had demonstrated both ABTS+ radical scavenging activity (13.54 ± 2.51% at 5 mg/mL) (Figure 2B) and hydrogen peroxide scavenging activity (30.82 ± 4.28% at 5 mg/mL). The above results suggested that EE demonstrated scavenging activity toward DPPH radical, ABTS+ radical, and hydrogen peroxide, which might be related to its high phenolic content. Cytotoxicity Evaluation of EE on HUVECs. MTT results showed that EE did not significantly affect the viability of the HUVECs when they were cultured in normal cell culture medium (DMEM/F12 supplemented with 10% FBS) at a concentration below 200 μg/mL (Figure 3A). The MTT

results were further confirmed by the LDH release assay (Figure 3B). Therefore, a concentration range of EE from 3.125 to 200 μg/mL was chosen for the following cell culture experiments. Effects of EE on VEGF-Induced HUVEC Migration. To find out the effects of EE on HUVEC migration, the scratch wound healing assay was performed. After scratching the monolayer HUVEC cells using a scraping tool, a denuded area was produced with the same width (red arrow shown in Figure 4A,a). Because activated motile ECs could move randomly in the direction of the angiogenic factors across a horizontal surface, the size of the wound could indicate the extent of wound closure and, therefore, the rate of cell migration. Figure 4 illustrates the wound size of the monolayer HUVEC cells in different treatment groups after 24 h. The inhibitory effect on wound healing was increased in a dose-dependent manner after 24 h of treatment of EE. At the highest concentration of 200 μg/mL (Figure 4A,f), the percentage of wound size at 24 h was 40.90 ± 2.59%, which was significantly larger than that of the control (2.88 ± 1.76%, p < 0.001) (Figure 4A,c). It was noted that quantification of the wound healing test was only arbitrary, and there were difficulties to ensure the consistency of identical growth conditions of confluence and the scraped area between control and different treatment groups.25 Therefore, an alternative type of 2-D migration assay, the transwell culture insert method, was also performed to further confirm the inhibitory effect of VEGF-induced HUVECs migration by EE. 9491



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Table 3. Identification of Phenolic Compounds in EE peak

RT

UV spectrum

detected ionization form

observed mass (Da)

identified known phenolics

proportion in dry extract (%)

2 3 5 7 13

5.583 11.484 13.117 20.517 58.468

259.4, 293.7 217, 240.5, 324.6 217, 274.8 217, 234.6, 322.2 239.3

[2M + K]+ [M + Na]+ [3M + K]+ [2M + K]+ [M + H]+

347.00773 377.08874 633.12038 427.0789 442.14923

protocatechuic acid chlorogenic acid syringic acid ferulic acid folic acid

0.11 0.218 0.004 0.016 0.012

Figure 2. Scavenging activity of 60% ethanol extract from sclerotium of PTR (mean ± SD n = 3) on DPPH (A), ABTS+ (B), and H2O2 (C) radicals.

Figure 3. Cell viability (mean ± SD n = 6) after treatment with 60% ethanol extract from sclerotium of PTR by MTT assay (A) and LDH release activity (B) from HUVECs after treatment with 60% ethanol extract from sclerotium of PTR. Statistical significance was assessed by one-way ANOVA (Student’s t test). (∗∗) p < 0.01 compared with the control group.

(Figure 6A), whereas those treated with 200 μg/mL (Figure 6F) mostly remained dotted on the Matrigel without extensive network of tube formation. Effects of EE on Intracellular ROS Level in HUVECs. ROS derived from NADPH oxidase, which is the major source of ROS in ECs,26 could enhance VEGFR2 autophosphorylation by inactivating protein tyrosine phosphatases (PTPs) and subsequently mediates related gene expression or signal pathways activities, such as the MMP family, angiopoetin family, Tie, PI3-kinase/Akt, and ERK1/2, and leads to the angiogenic responses including EC proliferation and migration.27,28 We also detected the ROS levels by the DCFH-DA assay. The results indicated that VEGF (20 ng/mL) induced an 80% increase in ROS formation in untreated HUVECs after 24 h of exposure as compared to the control (Figure 7). Such an increase was alleviated after exposure to EE at a concentration of 3.125−50 μg/mL. However, when the concentration of EE was ≥100 μg/mL, the ROS level was similar to that of the control (Figure 7). This could be due to the possible activation of cell apoptosis, causing an up-regulation of the ROS level.29 EE Suppress Angiogenesis-Related Gene Expression in HUVECs. To investigate how EE could attenuate the progression of angiogenesis, the expression of the genes

As marked by red arrows in Figure 5, the stimulation of VEGF (20 ng/mL) could strongly promote cell migration. In contrast, co-incubation with VEGFR2 inhibitor SU5416 (4 μM) could strongly inhibit HUVEC migration by 15.02 ± 2.23%. Similarly, with the addition of various concentrations of EE, this VEGF-induced cell migration was suppressed significantly in a dose-dependent way (Figure 5C−E). When the concentration of EE reached 25 μg/mL, the number of cells migrating from the upper to the lower chamber was similar to that of the positive control (SU5416, 4 μM) (Figure 5F). These values were significantly lower (p < 0.001) than that of the control, indicating EE could effectively inhibit VEGF-induced HUVEC migration. EE Inhibit VEGF-Induced HUVEC Tube Formation. Under normal culture conditions, ECs cultured on a Matrigel matrix could form tube-like structures within 6 h (Figure 6A), which could be further promoted by VEGF treatment (Figure 6B). However, when treated with EE at various concentrations (6.25−200 μg/mL), this formation of a mesh of tubes on Matrigel could be inhibited dose-dependently (Figure 6C−F). The tube formation pattern of HUVECs treated with 25 μg/ mL (Figure 6E) of EE was similar to that in the control group 9492



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MMP gene expression as well as VEGFR at the mRNA level (Figure 8) in HUVECs treated with EE in a dose-dependent manner. These results indicated that the VEGF−VEGFR signal pathway was inhibited in the presence of EE in HUEVCs, which provides a critical link between the bioactive compounds in EE and VEGF-mediated activation of angiogenesis. Taken together, these results suggested that EE could regulate the angiogenesis-related signal transduction pathway and suppress the expression of angiogenesis regulation genes. EE Inhibits Vessel Formation in Zebrafish Embryos. The above observations were also further examined in a zebrafish model. Quantitative EAP assay was used to measure the formation of blood vessels in the animal. It showed that incubation with 50 μg/mL EE significantly inhibited vessel formation at around 21.84 ± 0.24% in wild-type zebrafish embryos, which was similar to the positive control SU5416 at 4 μM (Figure 9). Further experiments were conducted using transgenic zebrafish embryos [Tg (fli1a: EGFP)y1], which provides higher resolution and more definitive results. The Tg (fli1a:EGFP)y1 zebrafish contains the enhanced green fluorescent proteins (EGFP) cDNA under control of the fli1 promoter, which is expressed in all ECs of the vasculature.30 Therefore, the ECs of the vasculature in the SIVs could be observed more clearly at 72 hpf through a fluorescent microscope on the basis of their EGFP. The microscopic imaging results clearly showed that the control group (Figure 10A,B) treated with 0.1% DMSO had normal vessel formation, the SIVs developed normally as a smooth basket-like structure with approximately 5−6 arcades (Figure 10A,B), whereas in the transgenic zebrafish treated with 100 μg/mL EE, the development of SIVs was clearly inhibited (highlighted in arrows) (Figure 10C,D).

Figure 4. Inhibition of VEGF-induced HUVECs migration by 60% ethanol extract from sclerotium of PTR in wound healing assay: (A,a) microscopic image of wound size at 0 h; (A,b) without VEGF at 24 h; (A,c) with VEGF at 24 h; (A,d−f) with VEGF and 6.25, 25, and 200 μg/mL of 60% ethanol extract from sclerotium of PTR at 24 h (length between arrowheads indicates wound length); (B) graph of healing effect in percent wound area of EE (mean ± SEM n = 6) on VEGFinduced HUVECs motility (statistical significance was assessed by oneway ANOVA (Student’s t test); (∗∗) p < 0.01; (∗∗∗∗) p < 0.001 compared with VEGF-stimulated group).

involved in this process was evaluated. It was found that EE significantly decreased VEGF-induced FGF, ANG-Tie, and

Figure 5. Effect of 60% ethanol extract from sclerotium of PTR on VEGF-induced HUVEC migration measured by transwell culture insert method: (A) microscopic image of HUVEC migration induced with 20 ng/mL VEGF; (B) with 20 ng/mL VEGF and SU5416 (4 μM); (C−E) with 20 ng/ mL VEGF and 6.25, 25, and 100 μg/mL 60% ethanol extract from sclerotium of PTR; (F) graph shows percent invasion of 60% ethanol extract from sclerotium of PTR (mean ± SEM n = 6) on VEGF-induced HUVECs migration. Red arrows represent the migrated cells from the upper chamber to the lower chamber. Statistical significance was assessed by one-way ANOVA (Student’s t test). (∗∗) p < 0.01 and (∗∗∗) p < 0.001 compared with VEGF-stimulated group. 9493



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Figure 6. Inhibition of VEGF-induced HUVEC tubule formation by 60% ethanol extract from sclerotium of PTR after 6 h of incubation: (A) microscopic image of inducement without VEGF; (B) with 20 ng/mL VEGF; (C) with 20 ng/mL VEGF and SU5416 (4 μM); (D−F) with 20 ng/ mL VEGF and 6.25, 25, and 200 μg/mL 60% ethanol extract from sclerotium of PTR.

acids had been identified and quantified. Not surprisingly, these identified phenolic compounds had also been reported in other mushroom species, including protocatechuic acid in Lepista nuda and Ramaria botrytis 37 and chlorogenic acid in Flammulina Velutipes and Pleurotus ostreatus.38 It was also noted that there were other phenolics or nonphenolic compounds present in EE, which could also act as antioxidants and need to be further studied. Several research studies have investigated the effects of polyphenolic compounds on VEGF expression and angiogenesis process. Oak et al.39 reported that the red wine polyphenolic compounds (RWPCs) with their antioxidant properties could significantly inhibit the PDGFAB-induced expression of VEGF, which was associated with the prevention of the ROS formation. In addition, epigallocatechin-3-gallate (EGCG) has also been shown to inhibit the expression of subunits of NADPH oxidase, which plays a pivotal role in producing superoxide in endothelial cells, namely, p47phox induced by angiotensin II (Ang II) in human umbilical vein endothelial cells.40 Meanwhile, EGCG could also decrease VEGF production in head and breast carcinoma cells by inhibiting epidermal growth factor receptor-related pathways of signal transduction.41 Meanwhile, as one of the main compounds identified in EE, folic acid has been shown to inhibit EC proliferation through activating the cSrc/ERK 2/ NF-κB/p53 pathway mediated by folic acid receptor;42 besides, it was also found to inhibit EC migration through inhibiting RhoA activity mediated by activating the folic acid receptor/ cSrc/p190RhoGAP-signaling pathway.43 Considering the strong link between antioxidant and antiangiogenesis reported previously,28,44 the antiangiogenic effects of EE on HUVECs were evaluated. In the present study, our findings demonstrated that VEGF-induced HUVEC migration, tube formation, and intracellular ROS level were significantly reduced after EE treatment. However, in the presence of VEGF, EE seemed to be less effective in its antiangiogenic effect when applied at a high concentration (100 μg/mL) and could even up-regulate the ROS level. This might be explained by the possible triggering of cell apoptosis due to the high concentration of phenolics. Previous studies demonstrated that high concen-

Figure 7. Scavenging activity of 60% ethanol extract from sclerotium of PTR on VEGF-induced ROS production in HUVECs. Serumstarved HUVECs were incubated with 60% ethanol extract from sclerotium of PTR (0−200 μg/mL) in the presence or not of VEGF (20 ng/mL) for 24 h. Statistical significance was assessed by one-way ANOVA (Student’s t test). (∗∗) p < 0.01 and (∗∗∗) p < 0.001 compared with VEGF-stimulated group.

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DISCUSSION PTR has been reported for its high nutritive value and use as a functional food,31 although the present study is the first to report the antioxidant and antiangiogenic activities of extract from the sclerotium of PTR. In this study, the 60% ethanol extract of PTR enriched with phenolic compounds had demonstrated strong antioxidant activity and inhibition of the VEGF-induced HUVECs angiogenesis in vitro and in vivo. Three different antioxidant assays confirmed that EE could significantly scavenge free radicals (DPPH, ABTS+, and hydrogen peroxide), implying its powerful antioxidant activity. Phenolic compounds from plants have been reckoned as the main source of antioxidants, which have beneficial implications for cancer treatments.32,33,34 It has also been suggested that the antioxidant activities of mushrooms are contributed by their phenolic compounds.35,36 Several studies suggested a strong correlation between the total phenolic content in mushrooms and their antioxidant activity.19 In the present study, the HPLC-UV-ESI/MS analysis confirmed the presence of phenolic compounds in EE, of which five known phenolic 9494



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Figure 8. Effect of 60% ethanol extract from sclerotium of PTR on mRNA levels (mean ± SEM n = 3) of related intracellular genes (VEGFR, FGF, ANG2, Tie2, and MMP2) determined by RT-PCR. Total RNAs and cDNAs were prepared from HUVECs that had been treated with increasing concentrations of 60% ethanol extract from sclerotium of PTR for 7 h. Statistical significance was assessed by one-way ANOVA (Student’s t test). (∗∗) p < 0.01 and (∗∗∗) p < 0.001 compared with VEGF-stimulated group.

could significantly decrease VEGF-induced ROS up-regulation, which was presumed to be mediated by its antioxidant activity and contribute to its antiangiogenic activity through ROS scavenging. Another important stimulator of angiogenesis is the fibroblast growth factors (FGF), first identified in 197558 and subsequently demonstrated to play an important role in modulating the angiogenesis process.59 VEGF receptor signals for the effects in ECs include migration, proliferation, cell survival, and expression of downstream genes. MMP2 is also an important enzyme that degrades components of the extracellular matrix to allow the progression of angiogenesis to occur.60 These major molecules participating in angiogenesis including VEGFR, FGF, and MMP2 were thus investigated after the HUVECs treated with EE. Indeed, mRNA expression of these angiogenic biomarkers was down-regulated by EE in a dose-dependent manner (Figure 8), suggesting a stronger effect of EE on migration and tube formation of HUVEC can be reflected in a decreased level of these angiogenic biomarkers. Besides, two other endothelial cell-specific surface receptors (Tie-1 and Tie-2) with tyrosine kinase activity are known so far and are found to hold unique functions in vascular biology as revealed by gene knockout experiments.61 Tie-2 is present on

tration of an apple extract rich in polyphenols increased the formation of ROS in HT-29 cells.45 On the basis of these results, it is possible that the antiangiogenic effect observed in EE might be partly due to its phenolic compounds and is mediated by its antioxidant effect. Angiogenesis is a process regulated by complex enzymatic and signal transduction pathway. Among the known mediators networks, VEGFA-VEGFR and angiopoietin (angpt)-Tie are two most widely studied signaling pathways involved in angiogenesis;46−48 VEGF is the best characterized angiogenic cytokine and the most important angiogenic factor in sustaining tumor growth49 by inducing proliferation, migration, and tubule formation of ECs.50−52 There is much evidence suggesting that overexpression of VEGF in most hematologic malignancies is responsible for increased angiogenesis found in the malignancies.53 Furthermore, VEGF stimulation could increase ROS production via activation of Rac1-dependent NADPH oxidase in HUVECs54,55 and subsequently evoke the redox signaling pathway leading to angiogenic responses such as EC proliferation and migration. Many researchers56,57 have shown that ROS are involved in VEGF-induced VEGFR2 autophosphorylation in ECs. In this research, we found EE from PTR 9495



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vasculature system is highly characteristic in the developing zebrafish embryo. The small molecules added directly to the fish culture media could diffuse into the embryo and induce observable angiogenesis effect.70 Besides, the blood vessel formation through angiogenic sprouting requires the same proteins that are necessary for blood vessel growth in mammals.71 Here, our results clearly demonstrated that EE exposure altered the development of SIVs in zebrafish. More importantly, EE exposure did not induce apoptosis in zebrafish, which was in agreement with the toxicity assay in normal cells. This paper further demonstrated that the zebrafish model is a very promising platform not only for screening antiangiogenic compounds but also for delineating the drug action mechanism when incorporated with the use of powerful genetic tools and other molecular techniques. In conclusion, to our knowledge, this is the first study reporting the profile of phenolic compounds and the antioxidant and antiangiogenic activities of ethanol extract (EE) isolated from the sclerotium of PTR. The antiangiogenic effect of EE was demonstrated by the inhibition of EC migration and tube formation. The mechanism underlying this inhibitory effect might be due to a decrease in both the ROS production and the mRNA expression of angiogenic biomarkers (such as VEGF, FGF, MMP2, Tie2, and Ang2). These antiangiogenic effects of EE were also clearly observed in the zebrafish model. Therefore, our findings strongly suggest that the phenolicsenriched extract obtained from the sclerotium of PTR might be a potential target for further investigation of its antiangiogenic effect in cancer treatment. Mushroom phenolic antioxidants might be potential anticancer agents to target tumor angiogenesis, tumor development, and metastasis.

Figure 9. Antiangiogenic activities of 60% ethanol extract from sclerotium of PTR (mean ± SEM n = 3) in zebrafish through EAP assay. Effects were assessed by measuring the vessel formation of samples as compared to the control using the quantitative EAP assay. Statistical significance was assessed by one-way ANOVA (Student’s t test). (∗∗∗∗) p < 0.001 compared to control.

ECs in human glioblastoma tissues,62 suggesting a connection to tumor angiogenesis. Its activation has been related to EC migration.63 Our results showed that EE inhibited HUVEC migration, which might be due to its down-regulation effect on Tie2 expression (Figure 8). Besides, angiopoietin-2 (Ang2) exhibits broad expression in the remodeling vasculature of human tumors but has very limited expression in normal tissues, making it an attractive candidate target for antiangiogenic cancer therapy.64 Our results also revealed that Ang2 gene expression was decreased after treatment with EE (Figure 8). It has been well recognized that all successful tumors must undergo neovascularization (angiogenesis) to acquire adequate nutrients and oxygen for continuous growth.65 To further ascertain the effect of EE on inhibiting angiogenesis, studies were then conducted in the zebrafish, which has been successfully used previously as the model system for evaluating antiangiogenic agents.66 Many researchers67−69 have shown that embryonic, young, and growing zebrafish are ideal animal models to observe blood vessel formation. The zebrafish

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AUTHOR INFORMATION

Corresponding Author

*(P.C.K.C.) E-mail: [email protected]. Author Contributions

Conceived and designed the experiments: P.C.K.C., S.L., T.L., L.C. Performed the experiments: S.L. Analyzed the data: S.L, P.C.K.C. Contributed reagents/materials/analysis tools: S.L., H.K., C.B.L., P.C.K.C. Wrote the paper: S.L., P.C.K.C. Notes

The authors declare no competing financial interest.

Figure 10. Lateral view of Tg (fli 1a: EGFP) y1 zebrafish embryos at 72 hpf: (A, B) control (live fluorescence microscopy highlights EGFP expressing subintestinal vessel plexus (SIVs), which appears as a smooth basket-like structure with five to six arcades); (C, D) treatment with 100 μg/mL (60% ethanol extract from sclerotium of PTR) (development of SIVs was inhibited after treatment). The arrow represents the regression of the SIVs. Treatments were done in triplicate. Sample size n = 30. The data represented were pooled from the experiments. 9496



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ABBREVIATIONS USED EAP, endogenous alkaline phosphatase assay; ECs, endothelial cell; EE, ethanol extract of sclerotium from Pleurotus tuberregium; GAE, gallic acid equivalents; Hpf, hour past fertilization; HUVEC, human umbilical vein endothelial cells; LDH, lactate dehydrogenase; PTR, Pleurotus tuber-regium; SIVs, subintestinal vessel plexus; TBHQ, tert-butylhydroquinone

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