Anti-angiogenic effect of water extract from the fruiting body of Agrocybe aegerita

LWT - Food Science and Technology 75 (2017) 155e163

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Anti-angiogenic effect of water extract from the fruiting body of Agrocybe aegerita Shaoling Lin a, 1, Lai Tsz Ching b, 1, Kalung Lam b, Peter C.K. Cheung b, * a b

College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, 350002, China School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 January 2016 Received in revised form 1 August 2016 Accepted 20 August 2016 Available online 21 August 2016

The water extract of the mushroom fruiting body of Agrocybe aegerita (AaE) had a total phenolic content of 13.67 ± 0.21 GAE (mM)/mg extract and 5 dominant phenolic acids including gallic acid, protocatechuic acid, chlorogenic acid, ferulic acid and sinapic acid were identified which accounted for 2.5% dry weight of the extract. AaE inhibited the vascular endothelial growth factor (VEGF)-induced proliferation in HUVECs shown by the MTT assay. Down-regulation of intracellular reactive oxygen species (ROS) level and VEGF secretion were also observed in Caco-2 cells treated with AaE. Inhibition of other angiogenic cascades in HUVECs by AaE (at 25 mg/mL) included a decrease in the migration of endothelial cells (ECs) by 46.14 ± 8.53% as evidenced by the wound healing assay and transwell insert assay; as well as a complete blocking of the VEGF-induced formation of capillary network on matrigel matrix. Taken together, these results demonstrated the presence of anti-angiogenic properties of AaE in vitro and among other compounds present in AaE phenolics might contribute to the effect. Therefore, Aa could be considered as potential functional food with antitumor effect modulated by inhibition of angiogenesis. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Agrocybe aegerita Phenolic composition Anti-angiogenesis Endothelial cells

1. Introduction Mushrooms are rich sources of nutritive and bioactive compounds with different biological activities. Modern science is trying to establish medicinal properties of traditionally used mushrooms through modern scientific methods and techniques. As a common edible mushroom, Agrocybe aegerita (Aa, V. Brig.) Singer (Strophariaceae, Agaricomycetideae) which belongs to Phyla Basidiomycota, Class Agaricomycetes and Order Agaricales, is widely cultivated and consumed in the United States and Asia. Many bioactive compounds have been isolated from the fruiting body of Aa including lectins with anti-tumor activity (Zhao, Sun, Tong, & Qi, 2003), agrocybenine with antifungal activity (Zhong & Xiao, 2009) and indole derivatives with free radical scavenging activity nský et al., 1992). Among the variety of bioactive components, (Stra phenolics are considered as important compounds with clinically beneficial activity. It has been reported the antioxidant activity and free-radical scavenging ability of Aa were correlated with its total phenolic content (Lo & Cheung, 2005). However, there are very few

* Corresponding author. E-mail address: [email protected] (P.C.K. Cheung). 1 Co-authors with equal contribution. http://dx.doi.org/10.1016/j.lwt.2016.08.044 0023-6438/© 2016 Elsevier Ltd. All rights reserved.

reports on the specific phenolic compounds found in Aa (Petrovic et al., 2014). Angiogenesis is the physiological process of forming new blood vessel from pre-existing vessels, which has been identified as a hallmark of tumor progression and is a necessary requirement for promoting the tumor growth and metastasis as a continuous supply of nutrients, oxygen and growth factors play a vital role in the development of tumor growth (Carmeliet, 2005). Therefore, the inhibition of angiogenesis is a promising strategy for anti-cancer drug development (Scappaticci, 2002). Resveratrol, a natural compound in red wine and grapes, was found to exert at least part of its anti-cancer effect in fibrosarcoma in mice by inhibiting angiogenesis including the ECs growth and wound healing (Brkenhielm, 2001). Extensive evidences also revealed that cancer cells bearing higher intracellular reactive oxygen species (ROS) level can upregulate the expression of vascular endothelial growth factor (VEGF), which appears to be the most important angiogenic factor to induce blood vessel formation and sustain tumor growth (Kubo, Li, Suzuki, Ohshima, Qin, & Hamano, 2007). Therefore, anti-VEGF strategy is a promising treatment for solid tumor by inhibiting angiogenesis. During the growth of solid malignant tumors, VEGF secreted by the tumor cells also targets on endothelial cells such as

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HUVEC as well as non-endothelial cells including macrophages, fibroblasts, and myofibrils (Boocock et al., 1995; Sunderkotter, Steinbrink, Goebeler, Bhardwaj, & Sorg, 1994). Indeed, VEGF has been found to be a key molecule for the survival of tumor cells during chemotherapy and radiotherapy. Therefore, it is obvious that anti-VEGF strategy also provides an extra beneficial effect for solid tumor treatment. Anti-angiogenic agents derived from natural products have attracted attentions from scientists, as extracts of natural compounds usually contain a mixture of biological components which could exert synergistic actions for anti-angiogenesis (Singh & Agarwal, 2003). Until now, there have been extensive studies on natural compounds and extracts of mushrooms that showed potent anti-angiogenic activity, which highlighted the potential of bioactive components from mushrooms as anti-cancer agents (Fassina et al., 2004; Guimaraes et al., 2016; Muslim et al., 2012). Considering that tumor angiogenesis is a complex process involved with the interplay of tumor cells, endothelial cells, phagocytes and their secretion. Therefore, a model of using both Caco-2 (an epithelial tumor colon cell) and HUVEC (an endothelial cell) cells was chosen in the present study to evaluate the effects of phenolic-rich water extract from the fruiting body of Aa on angiogenesis-related VEGF secretion and angiogenesis-related signaling in tumor cells, respectively.

2. Materials and methods 2.1. Preparation of water extract from Agrocybe aegerita Fresh fruiting body of Aa was washed, frozen and then lyophilized before milled into powders to pass through a hammer mill with a 0.5 mm sieve (MF 10, IKA-WERKE, Germany). The dried mushroom powders (100 g) were then extracted with distilled water (2 L) at room temperature for 3 h with continuous stirring. The extract was centrifuged and the supernatant was lyophilized to obtain a water-soluble extract of Agrocybe aegerita (AaE) (with a yield of 32.28% DW).

2.2. Cell culture A normal monkey kidney epithelial cell line, Vero (CCL-81) from the American Type Culture Collection (ATCC) was cultured in Roswell Park Memorial Institute-1640 (RPMI 1640) medium (Gibco, Cat # 31800-022). An adult human colorectal cancer cell line, Caco2 (HTB-37, ATCC) was cultured in Minimum Essential Medium (MEM) (Gibco, Cat # 41500-034). A human umbilical vein endothelial cell, HUVEC-C (CRL-1730, ATCC) was cultured in Dulbecco's modified Eagle's medium/Ham's Nutrient Mixture F12 (1:1) (DMEM/F12) (Gibco) with 0.1 mg/mL heparin (Sigma), 0.03 mg/mL endothelial cell growth supplement (ECGS) (Sigma), and 1% penicillin-streptomycin. All of these media were supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone).

2.3. Chemical composition analysis The total phenolic content was determined through the folinCiocalteu assay as previously described (Lin, Ching, Chen, & Cheung, 2015) with some modifications. Content of the total carbohydrates and protein content were analyzed colorimetrically using phenol-sulfuric acid (Chen, Xu, Lin, & Cheung, 2014) and bicinchoninic acid assay (Lin, Ching, Chen, & Cheung, 2015), respectively.

2.4. Phenolic acid identification The identification of phenolic compounds in AaE was carried out using the Apex Ultra 7.0 Hybride Qh-FTMS system (Bruker Daltonics Inc., USA) which was equipped with an Apollo II ion source and Dionex Ultimate 3000 2D Nanoflow LC system (Bruker Daltonics Inc., USA). This system was connected to an analytical (2.1
150 mm) C18 column (5 mm) (XBridgeTM, Ireland). The mobile phase was composed of solvent A (0.1% formic acid in milliQ water) and solvent B (HPLC-grade acetonitrile). The elution flow rate was 0.3 mL/min and the temperature of the column was kept at 30  C with Table 1 showing the solvent elution profile. Eleven phenolic compounds were used as the standards to identify the phenolic compounds by comparing the retention time and MS spectrum under the same conditions.

2.5. Limulus amebocyte lysate (LAL) test The endotoxin level in AaE was measured by E-TOXATE™ [Limulus amebocyte lysate (LAL)] test (Sigma, Cat# ET0200) according to manufacturer's instruction. In brief, designated volume of AaE (25 mg/mL and 50 mg/mL), endotoxin-free LAL reagent water and Endotoxin Standard Dilutions (0e400 EU/mL) were added to non-siliconized glass culture tubes. The E-TOXATE Working Solution was then added and the tube was covered with parafilm. Then the tube contents were mixed gently for 10 s and were incubated at 37  C for 1 h. The test tubes were slowly inverted by 180  C to observe the presence of gelatin.

2.6. MTT assay MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay was used to evaluate the cytotoxicity of AaE on growth of normal cell (Vero cell) and HUVEC as well as the antiproliferation of VEGF-induced on HUVEC proliferation. In brief, cells were seeded in a 96-well plate at 1
104 cells/well overnight. After incubation with various concentrations (1.61e800 mg/mL) of AaE solution for 24, 48 and 96 h, 10 mL of 5 mg/mL MTT in phosphate-buffered saline (PBS) was added, and incubation continued for another 4 h at 37  C. After the culture medium was removed, DMSO (100 mL/well) was added. Then, after complete dissolution, absorbance was detected at 570 nm. While for the antiproliferation assay on HUVECs, the cells were seeded in a 0.1% gelatin-coated 96-well microtiter plate (Nunc) at 1
104 cells/well overnight. After starved with DMEM/F12 containing only 0.5% FBS for 24 h, cells were co-treated with various concentrations of AaE and VEGF (20 ng/mL) or treated with fresh medium only (served as control vehicle) or 20 ng/mL VEGF only (served as control) for 24 h. Procedures after sample treatment were the same as those used in determining the viability of Vero.

Table 1 Elution profile used in the FT-ICR MS analysis. Time (min)

1.1% formic acid (%)

100% acetonitrile (%)

0 30 40 50 60 60.01 65

95 70 70 50 50 95 95

5 30 30 50 50 5 5

S. Lin et al. / LWT - Food Science and Technology 75 (2017) 155e163

Cell viability was expressed as % of control vehicle . 
¼ Abs570sample Abs570control vehicle
100%

2.7. Measurement of reactive oxygen species The 20 70 -Dichlorofluorescein diacetate (DCFH-DA, Sigma, St. Louis, MO) assay was used to determine the scavenging activity of ROS in Caco-2 as previously described (Latchoumycandane, Chitra, & Mathur, 2003). Appropriately 2.5
105 cells seeded in a T-25 culture flask were exposed to various concentrations of AaE (6.25e50 mg/mL) for 24 h. The fluorescent signal was measured immediately by a CXP 500 flow cytometer (Beckman Coulter, Miami, FL).

Intracellular ROS scavenging activity was expresses as % of h
control ¼ 1  fluorescent signalsample . i fluorescent signalcontrol
100 %

2.8. VEGF secretion in cancer cells The VEGF secretion in the Caco-2 medium was determined using a Human VEGF ELISA kit (Thermo Scientific, Cat # EHVEGF) following the manufacturer's instructions. After the Caco-2 cells treated with various concentration of AaE, 50 mL culture supernatants was collected for the test. A calibration curve of VEGF standard (0e2000 pg/mL) was prepared at the same time. Subsequently, the amount of human VEGF in the culture supernatant was quantified by interpolating from the sample absorbance value using the VEGF standard curve.

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starved medium with samples (co-treated with various concentrations of AaE (1.61e25 mg/mL) and 20 ng/mL VEGF in starved medium or with starved medium only (served as control vehicle) or 20 ng/mL VEGF only (served as control). Appropriately 1
104 HUVECs (in 100 mL starved medium) were seeded on the upper chamber and the assembled transwell was then incubated in incubator for 6 h. The number of migrated cells was counted under an inverted microscope.

Number of migrated cells was expressed as % of control
vehicle ¼ Number of migrated cellssample .  Number of migrated cellscontrol vehicle
100

2.11. Endothelial tube formation assay The assay was carried out according to previous protocols (Lee et al., 1999) with some modifications. HUVECs (1
104 cells/well) were seeded on a layer of Matrigel (BD Biosciences, Cat # 354234) and exposed to various concentrations of AaE with 20 ng/mL VEGF in starved medium or with starved medium only (served as vehicle control). After incubated for 5 h in 37  C, images of three randomly selected fields were captured using the inverted microscope and the total tubule length, total tubule area and the total number of junctions formed were analyzed and quantified using an automated image analysis tool, MATLAB®-based program (AngioQuant) (The MathWorks, Natick, MA).

Total tubule length was expressed as % of control vehicle . 
¼ Total tubule lengthsample Total tubule lengthcontrol vehicle
100%

2.9. Wound healing assay The assay was determined as previously described (Sato & Rifkin, 1988). In brief, HUVECs (3
105 cells/well) were seeded in a 24-well plate and then the cells were scraped away horizontally using a sterilized P100 pipette tip. The cells were treated with 300 mL various concentrations of AaE (co-treated with 20 ng/mL VEGF) or 20 ng/mL VEGF only (served as vehicle control) for 24 h. Three randomly views along the line were selected and photographed using an inverted microscope (Nikon ECLIPSE TS-100F) at 0 h and at 24 h treatment. Image analysis for determining the extent of migration was performed by Gwydion data analysis software (freeware, GNU General Public License).

Wound size reduction percentage was expressed as % of control h vehicle ¼ wounded area reductionsample . i wounded area reductioncontrol vehicle
100

Total tubule area was expressed as % of control vehicle
 ¼ Total tubule areasample = Total tubule areacontrol vehicle
100% Total number of junctions was expressed as % of control
vehicle ¼ Total number of junctionssample .  Total number of junctionscontrol vehicle
100%

2.12. Statistical analysis All experiments were performed in triplicate. All data were presented as means ± SEM and all statistics were performed using GraphPad Prism 5.0 software. Differences between means were analyzed by one-way ANOVA. Differences were considered to be statistical significant at p < 0.05 using Dunnett's multiple comparison.

2.10. Transwell culture insert assay 3. Results The assay was performed as previously described (Kim et al., 2003). A 24 well plate insert (Polycarbonate (PCTE) membrane with 6.5 mm diameter and 8.0 mm pore size) (SPL Life Science) was used. The lower chamber of the transwell was filled with 600 mL

3.1. The chemical composition and phenolic acid profile of AaE The total carbohydrate and protein contents in AaE were

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36.0 ± 2.0% (w/w) and 17.4 ± 0.8% (w/w), respectively. The extraction yields and total phenolic content (TPC) of AaE were 32.28 ± 1.11% in DW of Aa and 13.67 ± 0.21 GAE (mM)/mg extract, respectively. Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) was used to identify the phenolic acids in AaE in reference to the retention time and MS spectrum of the peaks (deviation between the theoretical mass and observed mass to be <5 ppm) of 11 phenolic acid standards (Fig. 1A). Five phenolic acids (gallic acid, protocatechuic acid, chlorogenic acid, ferulic acid and sinapic acid) were identified in AaE (Fig. 1B) and accounted for 0.31 ± 0.01, 0.39 ± 0.11, 0.47 ± 0.21, 0.44 ± 0.20 and 0.67 ± 0.35% in the total peak area in the chromatography (Table 2), constituting a total of about 2.5% DW of AaE. 3.2. The cytotoxicity evaluation of AaE by Limulus amebocyte lysate (LAL) test and MTT assay on normal cells and HUVECs The presence of any endotoxin in AaE was checked by E-TOXATETM. Lipopolysaccharide (LPS) was used as the reference standard. At a concentration above 0.25 EU/mL, LPS gave a positive result by causing the clotting of lysate leading to the formation of hard gels. AaE could only form soft gel at a concentration of 50 mg/ mL while a clear liquid was found at a concentration of 25 mg/mL. This indicated that the level of endotoxin in AaE was extremely low (<0.25 EU/mL) when compared to the sensitivity limit of the kit and therefore it was not a concern. From the MTT assay results, it was found that AaE substantially inhibited the growth of Vero cells only at very high concentrations and long incubation time (Fig. 2A). When Vero cells were incubated with AaE at a concentration of 800 mg/mL for 72 h, the survival rate dropped to 51.28 ± 1.40%. The MTT assay results also indicated that AaE only had significant inhibitory effect on HUVECs at concentrations above 100 mg/mL (Fig. 2B). Based on the results in Vero cells and HUVECs, a concentration range between 1.61 and 50 mg/mL of

AaE was applied to all subsequent cell culture experiments on Caco2 cells, despite the differences that might exist between the 3 cell lines. 3.3. The effects of AaE on intracellular ROS level in cancer cells The flow cytometry results showed the intracellular ROS scavenging activity (%) in Caco-2 treated with AaE indicated a dosedependent manner measured by based on the determination of the intensity of the fluorescence relative to that of control cells (cells without AaE treatment). Besides, significant difference (p < 0.05) was found in the intracellular ROS scavenging activity between different AaE concentrations with 44.59 ± 3.85% found when the concentration of AaE reached 50 mg/mL (Fig. 3A). 3.4. The effects of AaE on VEGF secretion in cancer cells Since the relatively high ROS level could promote the induction of VEGF in cancer cells (Chua, Hamdy, & Chua, 1998) and the VEGF secretion in cancer cells could stimulate the angiogenesis, with the high ROS scavenging activity in cancer cells, we also detected the secretion level of VEGF on colon cancer cells (Caco-2) by treated with various concentration of AaE. In this study, the highest concentration of VEGF was found in control group which was 435.81 ± 15.54 pg/mL (Fig. 3B), and when treated with AaE at the concentration of 25 mg/mL and above, significant reduction (p < 0.05) in the VEGF secretion was observed (Fig. 3B). 3.5. The effects of AaE on VEGF-induced HUVECs proliferation On the basis of the ROS scavenging activity and inhibition effect of VEGF secretion on cancer cells, the inhibitory effect of AaE on VEGF-induced HUVECs proliferation was examined to determine whether AaE possessed the anti-angiogenic activity in vitro.

Fig. 1. FT-ICR MS chromatogram of phenolic acid profiles in (A) phenolics standard mixture; (B) AaE. 1. Gallic acid; 2. Protocatechuic acid; 3. Folic acid; 4. Chlorogenic acid; 5. Caffeic acid; 6. Syringic acid; 7. Ferulic acid; 8. Sinapic acid; 9. Cinnamic acid; 10. tert-butylhydroxy quinine; 11. Salicylic acid.

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Table 2 Phenolic profile and their % of total peak area identified in AaE. Phenolic acids identified

Retention time (min)

Detected ionization form

m/z (theoretical)

m/z (experimental)

Relative % of total peak area

Gallic acid Protocatechuic acid Chlorogenic acid Ferulic acid Sinapic acid

3.3 5.9 9.4 14.8 16.7

[MþNa2H]þ [MþNa2H]þ [MþH]þ [MþNa2H]þ [MþH]þ

214.99269 198.99777 355.10236 239.02908 225.07575

214.99482 199.00007 355.10236 239.03010 225.07580

0.31 0.39 0.47 0.44 0.67

± ± ± ± ±

0.01 0.11 0.21 0.20 0.35

Fig. 2. Cell viability of Vero cell (A) and HUVECs (B) after treated with AaE. Each value is expressed as mean ± SD (n ¼ 6), statistical significance was assessed by one-way ANOVA (Dunnett's multiple comparison test). *p < 0.05 compared with control group.

Fig. 3. Anti-cancer activity evaluated in colon cancer cells (Caco-2). A. The quantitative analysis of the ROS scavenging activity by flow cytometry after treating cells without or with AaE at various concentrations for 24 h. B. Effect of AaE on VEGF secretion in Caco-2. Each value is expressed as mean ± SD (n ¼ 3), different letters indicate significant difference compared with the control group (p < 0.05, ANOVA, Dunnett's multiple comparisons test), **p < 0.005 and ****p < 0.001 compared with control group.

Treatment of HUVECs with 20 ng/mL VEGF resulted in a significant increase (144.06 ± 4.20%, p < 0.05) in cell proliferation while this increase was significantly decreased by co-incubation together with AaE at various concentrations (1.61e25 mg/mL) for 24 h in a dose-dependent manner (Fig. 4). 3.6. The effects of AaE on VEGF-induced HUVECs migration As ECs migration is critical for the process of angiogenesis, the effect of AaE on HUVECs migration in vitro was found by wound healing and transwell culture insert method. Fig. 5 illustrates the treatment of VEGF-induced HUVECs with different concentrations of AaE (1.61e25 mg/mL) prohibited significantly the increment in migration induced by VEGF to different extents. Significant inhibition (p < 0.005) in HUVECs migration started when AaE applied was as low as 1.61 mg/mL (Fig. 5i). When cells were treated with 25 mg/mL of AaE, the wound size reduction was 46.14 ± 8.53%, which was significantly smaller than that of the control (149.86 ± 4.32%, p < 0.001) (Fig. 5i).

Fig. 4. Effect of AaE on VEGF-induced HUVECs proliferation. Each value is expressed as mean ± SD (n ¼ 6), statistical significance was assessed by one-way ANOVA (Dunnett's multiple comparison test). #p < 0.05 compared with control group in the AaE treatments group while *p < 0.05, **p < 0.005 and ****p < 0.001 compared with VEGFstimulated group. Control vehicle was normalized to 100% for comparison.

Fig. 5. Inhibition of VEGF-induced HUVECs migration by AaE in wound healing assay (aei) and transwell culture insert method (j). (a) wound size at 0 h; (b) wound size at 24 h without VEGF and AaE; (c) with VEGF and without AaE at 24 h; (deh) with VEGF and 1.61, 3.12, 6.25, 12.5 and 25 mg/mL of AaE at 24 h. Length in between two arrows indicated the wound length; (i) Graph showed the wound size reduction in % wound area of AaE (mean ± S.E.M, n ¼ 6) on VEGF-induced HUVECs motility. (j) Graph showed the migrated cells in % of AaE (mean ± S.E.M, n ¼ 3) on control vehicle which was normalized to 100% by transwell culture insert assay. Statistical significance was assessed by one-way ANOVA (Dunnett's multiple comparison). **p < 0.05 and ***p < 0.001 compared with VEGF-stimulated group.

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As an alternative type of 2-D migration assay, the transwell culture insert method was also performed to further demonstrate the inhibitory effect of VEGF-induced HUVECs chemotactic motility by AaE. The presence of VEGF (20 ng/mL) strongly stimulated HUVECs migration as shown in Fig. 5j. Migrated cells increased significantly (p < 0.05) from 100.00 ± 19.49% to 186.70 ± 15.84% after cells were treated with VEGF (20 ng/mL). With the addition of various concentrations of AaE (1.61e25 mg/mL), the VEGF-induced cells migration was suppressed significantly (p < 0.05) in a dosedependent way (Fig. 5j). When the concentration of AaE reached to 12.5 and 25 mg/mL, cells that could migrate from the upper to the lower chamber resumed to almost the same level as in the control vehicle reaching 95.10 ± 9.46% and 96.50 ± 25.54%, respectively. These values were significantly lower (p < 0.001) than that of the control.

The tubule formation pattern of HUVECs treated with low concentrations (1.61 and 3.12 mg/mL) of AaE were similar to those in the control group (Fig. 6bed) whereas those treated with higher concentrations (6.25e25 mg/mL) mostly remained dotted on the Matrigel without extensive network of tubule formation could be seen (Fig. 6eeg). It was also observed that after co-treating HUVECs with VEGF and AaE, the average tubule length and area was decreased due to the decrease in the branching points as shown in Fig. 6h. It showed that AaE significantly reduced (p < 0.005) the VEGF-induced tubule length of tubules, area and branching points formed on Matrigel in a dose-dependent manner. And it also showed the difference among these three parameters including tubule length of tubules, area and branching points showed a significant reduction at the concentration of 6.25 mg/mL and above.

3.7. The effect of AaE on VEGF-induced HUVECs tube formation

4. Discussion

Fig. 6a shows that HUVECs formed a mesh of tubes within 5 h on Matrigel in the control while the tube formation was more extended if cells were incubated with 20 ng/mL VEGF (Fig. 6b). To test whether AaE could decrease the VEGF-induced formation of tubules by HUVECs in matrigel in vitro, cells were co-treated with 20 ng/mL VEGF and various concentrations of AaE (1.61e25 mg/mL).

Agrocybe aegerita (Aa), which belongs to the family of the bark mulch- and wood-colonizing basidiomycete, was found to possess several important bioactivities with antioxidant (Lo & Cheung, 2005), anti-cancer (Yang et al., 2009) and antifungal activities (Zhong & Xiao, 2009). Here, our results also demonstrated AaE with an abundant total phenolic content, possessing antioxidant and

Fig. 6. Inhibition of VEGF-induced HUVECs tubule formation by AaE after 5 h incubation. (a) without VEGF and AaE; (b) with VEGF and without AaE; (ceg) with VEGF and 1.61, 3.12, 6.25, 12.5 and 25 mg/mL of AaE; (h) Graph showed the effect of the total tubule length, total tubule area and total number of junctions of AaE on VEGF-induced HUVECs. Statistical significance was assessed by one-way ANOVA (Dunnett's multiple comparison). **p < 0.05 and ***p < 0.001 compared with VEGF-stimulated group.

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anti-angiogenic properties. Five phenolic acids including chlorogenic acid, ferulic acid, gallic acid, protocatechuic acid and sinapic acid were identified in AaE by FT-ICR MS. It is known that the production of ROS, especially hydrogen peroxide, is significantly higher in a range of tumor cells, including breast carcinoma, colon carcinoma and ovarian carcinoma, than their normal counterparts (Szatrowski & Nathan, 1991). ROS plays an important role in tumor angiogenesis, which marks the pivotal transition from a vascular to vascular tumor growth. Removal of excess ROS could result in anti-angiogenic effect and increase the life expectancy in cancer animals (Sa & Das, 2008; Suckow & Suckow, 2006). Excessive ROS is also important inducer for VEGF expression in cancer cells. It is reported that small amount of hydrogen peroxide (<5 mM) could actually increase VEGF mRNA expression and hence VEGF production in cancer cells (Kubo et al., 2007). Numerous evidences have suggested that over-expression of VEGF in most hematologic malignancies is responsible for increased angiogenesis found in the malignancies (Dong, Han, & Yang, 2007). Therefore, the production of ROS and subsequent VEGF expression in cancer cells were thought to be connected with the triggering of angiogenic process in the development of solid tumor (Monte, Davel, & Sacerdote de Lustig, 1997). The blockage of H2O2 release and inhibition of VEGF secretion from cancer cells were considered to exert anti-angiogenic effects (Ye et al., 2008). For example, EGCG could significantly inhibit angiogenesis through decreasing VEGF production in head and breast carcinoma cells by inhibiting epidermal growth factor receptor-related pathways of signal transduction (Du et al., 2012). Dried longan seed (Euphoria longana Lam.) extract was also found to decrease the expression and secretion of VEGF in colon cancer cells (SW480) and inhibited the angiogenesis of HUVECs (Panyathep, Chewonarin, Taneyhill, Surh, & Vinitketkumnuen, 2013). Furthermore, similar model has been used in several studies which applied both cancer cell lines such as osteosarcoma cancer cell (Yu, Tang, Li, Wang, Wang, & Pan, 2015), prostate cell (Yi et al., 2008) and breast cancer cell (Chang, Ou, Yang, Huang, & Wang, 2016) in conjunction with endothelial cell (HUVEC) to understand the effects of bioactive compounds on the interplay of tumor cells and endothelial cells during the process of angiogenesis. The present study demonstrated that AaE reduced the intracellular ROS level in a concentration-dependent manner as well as significantly suppressed VEGF production in colon cancer cell at a concentration of 25 mg/mL, showing its potential for regulating ROS-induced VEGF secretion in cancer cells. These data together suggested a promising therapeutic application of AaE in cancer treatment. However, the interplay between the cancer cell and ECs during tumor angiogenesis is complex, therefore, the effect of AaE on cancer cell viability may also contribute to the anti-tumor property which need further exploration. It has been well recognized that all successful tumors must undergo neovascularization (angiogenesis) in order to acquire adequate nutrients and oxygen for continuous growth (Folkman, 1971). Endothelial cell proliferation and migration are essential to angiogenesis. After being activated by angiogenic factors secreted from nearby tissues or cells, activated endothelial cells (ECs) produce proteases to degrade the surrounding basement membrane forming dissolved holes in the blood vessels. Then ECs start to proliferate and migrate towards the angiogenic factors through those dissolved holes to create new capillary sprout. Sprout ECs roll up to form new blood vessel tubes followed by recruitment of pericytes, which help to form new basement membrane. The angiogenesis process is finally completed when individual blood vessel tubes fuse with each other to form blood vessel loops and blood flow starts (Folkman, 1971). VEGF is the best characterized angiogenic cytokine and the most important angiogenic factor in sustaining tumor growth (Goto, Goto, Weindel, & Folkman, 1993)

by inducing proliferation, migration and tubule formation of ECs (Belloni et al., 2007; Waltenberger, Claesson-Welsh, Siegbahn, Shibuya, & Heldin, 1994). A range of polyphenolics have been reported to prevent angiogenesis by inhibiting proliferation, migration and tubule formation of ECs. For example, tea catechin, epigallocatechin gallate (EGCG) and resveratrol, were found to suppress ECs proliferation in vitro (Matsubara, Kaneyuki, Miyake, & Mori, 2005). A phenolic acid rich extract from Southern Brazilian autumnal propois containing gallic acid, protocatechui acid and chlorogenic acid also exerted its anti-angiogenic activity via decreasing the cell viability, proliferation and migration, as well as capillary tube formation of endothelial cells (Meneghelli et al., 2013). Besides, inhibition of VEGFR2 phosphorylation was elucidated as the underlying mechanism by which wild roman chamomile extracts (in which the main phenolic compounds were identified as apigenin, apigenin-7-O-glucoside, caffeic acid, chlorogenic acid, luteolin, and luteolin-7-O-glucoside) exerts its antiangiogenic activity (Guimaraes et al., 2016). Recently, it was also reported that extract from the mushroom Pleurotus tuberregium containing chlorogenic acid and syringic acid showed in vitro and in vivo anti-angiogenic activities in HUVEC cells and zebrafish embryos, respectively (Lin et al., 2015). In the present study, our findings demonstrated that VEGF-induced HUVEC proliferation, migration and tube-formation were significantly reduced after AaE treatment within the concentrations range of 1.61e25 mg/ mL. In addition, the anti-cancer properties of phenolics identified in the AaE (gallic acid, chlorogenic acid, ferulic acid) have also been extensively studied for their anti-angiogenic effects in both cancer cells and ECs (Park, Hwang, Park, & Lee, 2015; Yang, Jiang, & Lu, 2015; Yong et al., 2010). For example, chlorogenic acid has been found to be a novel therapeutic agent for treating the lung cancer through the angiogenesis suppression effects via targeting HIF-1a/ AKT pathway (Park et al., 2015); while ferulic acid was found to inhibit EC proliferation, migration and tube formation in response to basic fibroblast growth factor 1 (bFGF1) in vitro as well as suppressed bFGF1-induced angiogenesis and tumor growth in vivo (Yang et al., 2015). Besides, lectin extracted from Aa has also been reported to inhibit the growth of colon cancer cell (SW480) (Zhao et al., 2003). Notably, it has been reported that the role of synergistic compounds in the phenolic-rich extract obtained from dried longan seed contributed to the down-regulation of VEGF expression in SW480 cells and anti-angiogenesis effect in HUVECs (Panyathep et al., 2013). Therefore, among other compounds in AaE phenolics might contribute to the inhibition of tumor growth and angiogenesis in a synergistic way. 5. Conclusion As a phenolic-rich water extract, AaE contained five major phenolic acids including chlorogenic acid, ferulic acid, gallic acid, protocatechuic acid and sinapic acid. At a concentration of 25 mg/ mL, AaE seems to be able to interfere with the multiple steps of angiogenesis during the development of solid tumor, including lowering the intracellular ROS level and the VEGF secretion in cancer cells (Caco-2 cells) and inhibiting the proliferation, migration and the tubule formation in VEGF-induced HUVECs. However, the effective concentration of AaE to exert a similar anti-angiogenic effect in colon and endothelial cells in vivo would be expected to be much higher due to their unknown uptake efficiency. Although individual phenolics can possess anti-angiogenic effect, the combined effects of multiple phenolics on enhancing anti-carcinogenic have been demonstrated (Araujo, Goncalves, & Martel, 2011). It should also be noted that besides the phenolic compounds identified, it is possible that there are other water-soluble compounds

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such as carbohydrates and proteins present in AaE which might contribute to the anti-angiogenic effects. Therefore, the synergistic effects of these phenolics and water-soluble compounds in AaE could not be ruled out. Further studies are needed to isolate pure bioactive compounds from AaE and elucidate in more details the mechanism of their individual as well as synergistic effects. The present study provides some insights for developing AaE as novel anti-angiogenic and anti-tumor agents with low cytotoxicity and adverse events in future cancer treatment. References Araujo, J. R., Goncalves, P., & Martel, F. (2011). Chemopreventive effect of dietary polyphenols in colorectal cancer cell lines. Nutrition Research, 31(2), 77e87. Belloni, D., Scabini, S., Foglieni, C., Veschini, L., Giazzon, A., Colombo, B., et al. (2007). The vasostatin-I fragment of chromogranin A inhibits VEGF-induced endothelial cell proliferation and migration. The FASEB Journal, 21(12), 3052e3062. Boocock, C. A., Charnock-Jones, D. S., Sharkey, A. M., McLaren, J., Barker, P. J., Wright, K. A., et al. (1995). Expression of vascular endothelial growth factor and its receptors flt and KDR in ovarian carcinoma. Journal of the National Cancer Institute, 87(7), 506e516. Brkenhielm, E. (2001). Suppression of angiogenesis, tumor growth, and wound healing by resveratrol, a natural compound in red wine and grapes. The FASEB Journal, 15(10), 1798e1800. Carmeliet, P. (2005). VEGF as a key mediator of angiogenesis in cancer. Oncology, 69(Suppl. 3), 4e10. Chang, C. H., Ou, T. T., Yang, M. Y., Huang, C. C., & Wang, C. J. (2016). Nelumbo nucifera Gaertn leaves extract inhibits the angiogenesis and metastasis of breast cancer cells by downregulation connective tissue growth factor (CTGF) mediated PI3K/AKT/ERK signaling. Journal of Ethnopharmacology, 188, 111e122. Chen, L., Xu, W., Lin, S., & Cheung, P. C. K. (2014). Cell wall structure of mushroom sclerotium (Pleurotus tuber regium): Part 1. Fractionation and characterization of soluble cell wall polysaccharides. Food Hydrocolloids, 36, 189e195. Chua, C. C., Hamdy, R. C., & Chua, B. H. (1998). Upregulation of vascular endothelial growth factor by H2O2 in rat heart endothelial cells. Free Radical Biology and Medicine, 25(8), 891e897. Dong, X., Han, Z. C., & Yang, R. (2007). Angiogenesis and antiangiogenic therapy in hematologic malignancies. Critical Reviews in Oncology/Hematology, 62(2), 105e118. Du, G.-J., Zhang, Z., Wen, X.-D., Yu, C., Calway, T., Yuan, C.-S., et al. (2012). Epigallocatechin Gallate (EGCG) is the most effective cancer chemopreventive polyphenol in green tea. Nutrients, 4(11), 1679e1691. , R., Morini, M., Minghelli, S., Benelli, R., Noonan, D. M., et al. (2004). Fassina, G., Vene Mechanisms of inhibition of tumor angiogenesis and vascular tumor growth by epigallocatechin-3-gallate. Clinical Cancer Research, 10(14), 4865e4873. Folkman, J. (1971). Tumor angiogenesis: Therapeutic implications. New England Journal of Medicine, 285(21), 1182e1186. Goto, F., Goto, K, Weindel, K., & Folkman, J. (1993). Synergistic effects of vascular endothelial growth factor and basic fibroblast growth factor on the proliferation and cord formation of bovine capillary endothelial cells within collagen gels. Laboratory Investigation, 69(5), 508e517. Guimaraes, R., Calhelha, R. C., Froufe, H. J., Abreu, R. M., Carvalho, A. M., Queiroz, M. J., et al. (2016). Wild Roman chamomile extracts and phenolic compounds: Enzymatic assays and molecular modelling studies with VEGFR-2 tyrosine kinase. Food & Function, 7(1), 79e83. Kim, K. S., Hong, Y. K., Joe, Y. A., Lee, Y., Shin, J. Y., Park, H. E., et al. (2003). Antiangiogenic activity of the recombinant kringle domain of urokinase and its specific entry into endothelial cells. Journal of Biological Chemistry, 278(13), 11449e11456. Kubo, M., Li, T. S., Suzuki, R., Ohshima, M., Qin, S. L., & Hamano, K. (2007). Shortterm pretreatment with low-dose hydrogen peroxide enhances the efficacy of bone marrow cells for therapeutic angiogenesis. American Journal of Physiology: Heart and Circulatory Physiology, 292(6), H2582eH2588. Latchoumycandane, C., Chitra, K. C., & Mathur, P. P. (2003). 2,3,7,8Tetrachlorodibenzo-p-dioxin (TCDD) induces oxidative stress in the epididymis and epididymal sperm of adult rats. Archives of Toxicology, 77(5), 280e284. Lee, O. H., Kim, Y. M., Lee, Y. M., Moon, E. J., Lee, D. J., Kim, J. H., et al. (1999). Sphingosine 1-phosphate induces angiogenesis: Its angiogenic action and signaling mechanism in human umbilical vein endothelial cells. Biochemical and Biophysical Research Communications, 264(3), 743e750. Lin, S., Ching, L. T., Chen, J., & Cheung, P. C. (2015). Antioxidant and anti-angiogenic effects of mushroom phenolics-rich fractions. Journal of Functional Foods, 17, 802e815.

163

Lo, K. M., & Cheung, P. C. K. (2005). Antioxidant activity of extracts from the fruiting bodies of Agrocybe aegerita var. alba. Food Chemistry, 89(4), 533e539. Matsubara, K., Kaneyuki, T., Miyake, T., & Mori, M. (2005). Antiangiogenic activity of nasunin, an antioxidant anthocyanin, in eggplant peels. Journal of Agricultural and Food Chemistry, 53(16), 6272e6275. lix, G. L. Q., Somensi, A., Tomazzoli, M., da Silva, Meneghelli, C., Joaquim, L. S. D., Fe et al. (2013). Southern Brazilian autumnal propolis shows anti-angiogenic activity: An in vitro and in vivo study. Microvascular Research, 88(0), 1e11. Monte, M., Davel, L. E., & Sacerdote de Lustig, E. (1997). Hydrogen peroxide is involved in lymphocyte activation mechanisms to induce angiogenesis. European Journal of Cancer, 33(4), 676e682. Muslim, N., Nassar, Z., Aisha, A., Shafaei, A., Idris, N., Majid, A., et al. (2012). Antiangiogenesis and antioxidant activity of ethanol extracts of Pithecellobium jiringa. BMC Complementary and Alternative Medicine, 12(1), 210. Panyathep, A., Chewonarin, T., Taneyhill, K., Surh, Y. J., & Vinitketkumnuen, U. (2013). Effects of dried longan seed (Euphoria longana Lam.) extract on VEGF secretion and expression in colon cancer cells and angiogenesis in human umbilical vein endothelial cells. Journal of Functional Foods, 5(3), 1088e1096. Park, J. J., Hwang, S. J., Park, J. H., & Lee, H. J. (2015). Chlorogenic acid inhibits hypoxia-induced angiogenesis via down-regulation of the HIF-1alpha/AKT pathway. Cellular Oncology (Dordrecht), 38(2), 111e118. Petrovic, J., Glamoclija, J., Stojkovic, D., Nikolic, M., Ciric, A., Fernandes, A., et al. (2014). Bioactive composition, antimicrobial activities and the influence of Agrocybe aegerita (Brig.) Sing on certain quorum-sensing-regulated functions and biofilm formation by Pseudomonas aeruginosa. Food & Function, 5(12), 3296e3303. Sa, G., & Das, T. (2008). Anti cancer effects of curcumin: Cycle of life and death. Cell Division, 3, 14. Sato, Y., & Rifkin, D. B. (1988). Autocrine activities of basic fibroblast growth factor: Regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis. Journal of Cell Biology, 107(3), 1199e1205. Scappaticci, F. A. (2002). Mechanisms and future directions for angiogenesis-based cancer therapies. Journal of Clinical Oncology, 20(18), 3906e3927. Singh, R. P., & Agarwal, R. (2003). Tumor angiogenesis: A potential target in cancer control by phytochemicals. Current Cancer Drug Targets, 3(3), 205e217.  Bude nský, K., Semerd sínský, M., Ubik, K., Stra zieva, M., Otmar, M., Proch azka, Z., et al. (1992). Antifungal antibiotic from the mushroom Agrocybe aegerita (BRIG.) SING. Collection of Czechoslovak Chemical Communications, 57(3), 590e603. Suckow, B. K., & Suckow, M. A. (2006). Lifespan extension by the antioxidant curcumin in Drosophila melanogaster. International Journal of Biomedical Science e IJBS, 2(4), 401e404. Sunderkotter, C., Steinbrink, K., Goebeler, M., Bhardwaj, R., & Sorg, C. (1994). Macrophages and angiogenesis. Journal of Leukocyte Biology, 55(3), 410e422. Szatrowski, T. P., & Nathan, C. F. (1991). Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Research, 51(3), 794e798. Waltenberger, J., Claesson-Welsh, L., Siegbahn, A., Shibuya, M., & Heldin, C. H. (1994). Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. Journal of Biological Chemistry, 269(43), 26988e26995. Yang, G. W., Jiang, J. S., & Lu, W. Q. (2015). Ferulic acid exerts anti-angiogenic and anti-tumor activity by targeting fibroblast growth factor receptor 1-mediated angiogenesis. International Journal of Molecular Sciences, 16(10), 24011e24031. Yang, N., Li, D. F., Feng, L., Xiang, Y., Liu, W., Sun, H., et al. (2009). Structural basis for the tumor cell apoptosis-inducing activity of an antitumor lectin from the edible mushroom Agrocybe aegerita. Journal of Molecular Biology, 387(3), 694e705. Ye, J., Li, Y., Hamasaki, T., Nakamichi, N., Komatsu, T., Kashiwagi, T., et al. (2008). Inhibitory effect of electrolyzed reduced water on tumor angiogenesis. Biological and Pharmaceutical Bulletin, 31(1), 19e26. Yi, T., Yi, Z., Cho, S. G., Luo, J., Pandey, M. K., Aggarwal, B. B., et al. (2008). Gambogic acid inhibits angiogenesis and prostate tumor growth by suppressing vascular endothelial growth factor receptor 2 signaling. Cancer Research, 68(6), 1843e1850. Yong, L., Feng, J., Hao, J., Wu, K., Zheng, X., Cai, Y., et al. (2010). Gallic acid suppresses cell viability, proliferation, invasion and angiogenesis in human glioma cells. European Journal of Pharmacology, 641(2e3), 102e107. Yu, Q. L., Tang, Z. C., Li, J. G., Wang, G. L., Wang, P. J., & Pan, Z. B. (2015). Anticancer and anti-angiogenesis effect of the leaf extract of Sargassum wightii against osteosarcoma cancer cells. Bangladesh Journal of Pharmacology, 10(2), 351. Zhao, C., Sun, H., Tong, X., & Qi, Y. (2003). An antitumour lectin from the edible mushroom Agrocybe aegerita. Biochemical Journal, 374(2), 321e327. Zhong, J. J., & Xiao, J. H. (2009). Secondary metabolites from higher fungi: Discovery, bioactivity, and bioproduction. In J. J. Zhong, F. W. Bai, & W. Zhang (Eds.), Biotechnology in China I (Vol. 113, pp. 79e150).