PI3K Complex and

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Bioactive Constituents, Metabolites, and Functions

Mulberry polyphenol extract inhibits FAK/Src/PI3K complex and related signaling to regulate the migration in A7r5 cells Meng-Hsun Yu, Tsung-Yuan Yang, Hsieh-Hsun Ho, Hui-Pei Huang, Kuei-Chuan Chan, and Chau-Jong Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00958 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 4, 2018

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Journal of Agricultural and Food Chemistry

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Mulberry polyphenol extract inhibits FAK/Src/PI3K complex and related signaling to regulate

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the migration in A7r5 cells

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Meng-Hsun Yu†, Tsung-Yuan Yang‡,§, Hsieh-Hsun Ho†, Hui-Pei Huang†, , Kuei-Chuan Chan‡,§, ,

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Chau-Jong Wang†,

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‡

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N. Road, Taichung 402, Taiwan

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†

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Sec. 1, Jianguo N. Road, Taichung 402, Taiwan

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§

⊥

＃

⊥,＃

Department of Internal Medicine, Chung-Shan Medical University Hospital, No. 110, Sec. 1, Jianguo

Institute of Biochemistry, Microbiology and Immunology, Chung Shan Medical University, No. 110,

School of Medicine, Institute of Medicine, Chung-Shan Medical University, No. 110, Sec. 1, Jianguo

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N. Road, Taichung 402, Taiwan

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⊥

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Jianguo N. Road, Taichung 402, Taiwan

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＃

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Kuei-Chuan Chan, Department of Internal Medicine, Chung-Shan Medical University Hospital, No.

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110, Sec. 1, Jianguo N. Road, Taichung 402, Taiwan

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Chau-Jong Wang, Chung Shan Medical University, No. 110, Sec. 1, Jianguo N. Road, Taichung 402,

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Taiwan

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Tel: 886-4-24730022 ext. 11670

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E- mail: [email protected], [email protected]

Department of Biochemistry, School of Medicine, Chung Shan Medical University, No. 110, Sec. 1,

These authors are the corresponding authors:

ACS Paragon Plus Environment


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Abstract

Atherosclerosis is characterized by the buildup of plaque inside arteries. Our recent studies

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demonstrated that polyphenolic natural products can reduce oxidative stress, inflammation,

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angiogenesis, hyperlipidemia, and hyperglycemia. A previous study also showed that mulberry water

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extract (MWE) can inhibit atherosclerosis and contains considerable amounts of polyphenols.

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Therefore, in the present study, we investigated whether mulberry polyphenol extract (MPE) containing

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high levels of polyphenolic compounds could affect vascular smooth muscle cell (VSMC; A7r5 cell)

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motility. We found that MPE inhibited expression of FAK, Src, PI3K, Akt, c-Raf, and suppressed

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FAK/Src/PI3K interaction. Further investigations showed that MPE reduced expression of small

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GTPases (RhoA, Cdc42, and Rac1) to affect F-actin cytoskeleton rearrangement, down-regulated

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expression of MMP2 and VEGF mRNA through NFκB signaling, and thereby inhibited A7r5 cell

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migration. Taken together, these findings highlight MPE inhibited migration in VSMC through

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FAK/Src/PI3K signaling pathway.

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Key words: Mulberry; polyphenol; migration; FAK signaling; vascular smooth muscle cell


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Introduction

Atherosclerosis occurring mainly in large and medium-sized arteries has reached epidemic

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proportions in the elderly and remains the leading cause of mortality and morbidity worldwide.1-3

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Atherosclerosis is a complex and progressive pathogenic process involving endothelial cell dysfunction,

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inflammation, vascular smooth muscle cell (VSMC) proliferation/migration, and matrix

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metalloproteinase (MMP) expression alteration.4-7 Proliferation contributes to the phenotypic

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remodeling of VSMCs in culture. Recent studies have provided mechanistic insights such as the

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findings that increased MMP activity can result in degradation of ECM,8 VEGF overexpression can

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facilitate the repair of vascular injury by stimulating MMP,9 and the inhibition of MMP by tissue

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inhibitors of MMP (TIMP) can facilitate VSMC migration and proliferation.10, 11 Focal adhesion kinase

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(FAK) is an 125-kDa protein tyrosine kinase (PTK) with a role in the cell motility.12 As well,

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phosphorylation of FAK in VSMC increases not only in cell migration but also vascular injury.13 On the

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other hand, phosphorylated of FAK can activate Src.14 The interaction of FAK with Src complex plays

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a role in the signaling of integrin and other signaling molecules.15, 16 In addition, integrin-β3 structural

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interaction with ECM may trigger intracellular signals that regulate F-actin cytoskeleton remodeling.17

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Moreover, our previous study demonstrated that the PI3K and Akt phosphorylation signal pathways

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can stimulate VSMC proliferation.18 However, presence of both growth factors and inflammatory

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cytokines indicates that proliferation of VSMCs occurs through the Nuclear factor κB (NF-κB)

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pathway in atherosclerosis.19, 20 A previous study found that the binding of NF-kB to IkB in the



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cytoplasm prevents it from entering the nucleus.21 NF-κB is an important regulator of Rho GTPases

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(RhoA, RhoB, Rac1, and Cdc42)22 and inflammatory responses.23

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Mulberry (M. alba L.) is a common fruit in temperate, subtropical, and tropical areas, and

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contains abundant polyphenols and anthocyanin components.24, 25 According to the study, mulberry

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anthocyanins (MACs) could inhibited the metastasis of B16-F1 cells by suppression of the Ras/PI3K

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signaling pathway.26 Our studies have shown that mulberry water extract (MWE) can prevent

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atherosclerosis in cholesterol-fed rabbits.27 In addition, MWE is rich in polyphenols that have

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antioxidant, anti-inflammatory, anti-aging, anti-obesity, and anti-tumor effects.28-32 Previous studies

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demonstrated that extracts containing polyphenols from natural sources such as N.nucifera leaf

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polyphenol extract (NLPE) and S. nigrum polyphenol extract (SNPE) also improve cardiovascular

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health and have anti-angiogenic effects.33, 34 Therefore, in the present study, our aim was to identify the

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mechanisms underlying the effect of MPE on VSMC migration, which may also be basis for other

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benefits of this extract.


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Materials and Methods

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Preparation of MPE

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Fresh fruits of the mulberry tree (M. alba L.) were harvested in the Dadu District (Taichung City,

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Taiwan) and freeze-dried. Briefly, the dried fruit (100 g) was macerated, mixed with distilled water

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(1.0 L), and centrifuged at 6,000 rpm for 10 min. The aqueous solution was lyophilized (−80°C, 12 h)

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to obtain the MWE (yield about 5.8%).35 For the preparation of MPE, we heated 100 g of dried MWE

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powder in 500 mL of methanol to 50°C for 180 min, filtered and then lyophilized the extract under

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reduced pressure at room temperature. The powder was re-suspended in 500 mL of 50°C distilled water,

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extracted with 180 ml of ethyl acetate three times, redissolved in 300 mL of distilled water, stored at –

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80°C overnight, and lyophilized.

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High performance liquid chromatography (HPLC) Assay

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MPE polyphenols were separated on an RP-18 column (4.60 mm × 150 mm, 5.0 µm, inner diameter)

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using an HPLC system with diode array (DA) detector (Hewlett-Packard, Palo Alto, CA, USA)

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connected to a Vectra 436/33N personal computer. The mobile phase contained solution A (Acetic

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acid/Water = 2:98) and solution B (Acetic acid/Water/Acetonitrile = 0.5:49.5:50), and the elution

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program time was 60 min.36 The flow rate was set at 1 ml/min, and the absorbance at 280 nm was

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monitored by an ultraviolet (UV) detector. The fifteen standard polyphenols used for analysis with their

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retention times (RT) were as follows: gallic acid (GA, 7.85 min), protocatechuic acid (PCA, 15.17 min),

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catechin (23.49 min), epigallocatechin gallate (EGCG, 23.95 min), caffeic acid (26.08 min),



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epicatechin (27.27 min), P-coumaric acid (31.57 min), rutin (33.44 min), ferulic acid (34.00 min),

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gossypin (41.72 min), hesperetin (42.85 min), resveratrol (45.17 min), quercetin (51.83 min),

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naringenin (56.28 min), and hydroxyflavin (57.44 min), respectively.

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Cell culture

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The rat aortic smooth muscle cell line A7r5 was purchased from American Type Culture Collection

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(ATCC, CRL-1444; Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium

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(DMEM; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS; Hyclone, Logan,

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UT, USA), 1 mmol/L sodium pyruvate, 4 mmol/L L-glutamine, 4.5 g/ L glucose, 1.5 g/L sodium

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bicarbonate, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in a humidified atmosphere

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containing 5% CO2.

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Wound healing

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A7r5 cells were placed in a 6-well culture plates (1 × 106 cells /well) for 48 h and grown to 90%

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confluence. The wound healing assay was performed as described in our previous study.27 Cell

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monolayers on the surface of a 6-well plate were wounded by scratching the monolayers with a 200

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micro-pipette tip. Non-adhering cells were removed through washing with phosphate-buffered saline

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(PBS), and the remaining cells were treated with MPE (0.05−1.0 mg/mL). Cells were photographed

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through a 40X phase-contrast objective, and the images of the linear wounds were taken in nine fields

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per well at days 0–4 after wounding. Three independent experiments were performed.

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Boyden chamber assay


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The cell ability was performed Boyden chamber assay in described previously.37 A7r5 cells were

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seeded in 6-well culture plates (5 × 105 cells /well), and treated with various concentrations of MPE.

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After 48h incubation, the cells were trypsinized, and the in vitro migration was tested in a Boyden

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chamber assay. Subsequently, the A7r5 cells were seed on the upper chamber at a density of

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2 × 104 cells in serum-free medium (50µL) with 8 µm pore polycarbonate filters, and the lower

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chamber medium containing 10% FBS. The chamber was incubated for 8 h at 37 °C. The cells

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migrating across to the lower surface of the membrane were fixed with methanol for 30 min, and

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stained with 5% Giemsa solution for 60 min. The average number of cells was carried out in three

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independent experiments by randomly chosen fields.

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Gelatin zymography

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Gelatin zymography was performed as previously described.38 MPE-treated A7r5 cells were plated

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onto 6-well tissue culture plates (5 × 105 cells /well) and then starved in 1 ml of DMEM containing

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0.5% FBS for 24 h. After treatment, the culture medium was collected and centrifuged at 12,000 rpm

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for 5 min at 4°C for cell debris removal. The samples were dialyzed against loading buffer and

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subjected to SDS-PAGE 8% (Bio-Rad, Hercules, CA, USA) on gels containing 0.1% gelatin

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(Sigma-Aldrich, St. Louis, MO, USA). After electrophoresis, the gels were incubated at room

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temperature, washed twice with 2.5% Triton X-100 on a gyrating shaker for 30 min to remove SDS,

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incubated in reaction buffer (40 mmol/L Tris-HCl, 10 mmol/L CaCl2, and 0.01% NaN3) at 37°C for 16

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h, stained with 0.1% Coomassie Brilliant Blue (R-250), and de-stained with 1.0 L of solution



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containing 50 ml of methanol, 75 ml of acetic acid, and 875 ml of distilled water. With MMP-2 and

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MMP-9 used as positive controls, gelatinolytic activity was indicated by the intensity of horizontal

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white bands on a blue background.

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Immunoprecipitation (IP) analysis

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Cell lysates (approximately 0.5 mg of protein) were immunoprecipitated with monoclonal integrin β3

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antibody for 2 h at 4°C (Santa Cruz Biotechnology, Dallas, TX, USA). The resulting immune

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complexes were analyzed by Western blotting (using PI3K, FAK, and Src antibodies) after harvesting

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them by incubation with Protein A/G Plus Sepharose beads (Santa Cruz Biotechnology, Dallas, TX,

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USA) and then by centrifugation at 2,500 rpm.

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Electrophoresis mobility shift assay (EMSA)

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The binding reaction was performed using a LightShift chemiluminescent EMSA kit (Promega,

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Madison, WI, USA). A total of 10 µg of nuclear extract was mixed with the binding reaction buffer

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containing 10 mmol/L Tris, 50 mmol/L KCl, 1 mmol/L dithiothreitol (DTT; Promega Corporation), 5

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mmol/L MgCl2, 2 µg of Poly-dIdC (Sigma-Aldrich, St. Louis, MO, USA), and 2 pmol of

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oligonucleotide probe with or without protein extract for 20 min at room temperature. We used

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as oligonucleotide probe of NF-kβ, Forward (5′-AGTTGAGGGGACTTTCCCAGGC-3′) Reverse

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(5′-GCCTGGGAAAGTCCCCTCAACT-3′). After separating the complexes by electrophoresis on a

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6% non-denaturing acrylamide gel, the bands were transferred to a positively charged nylon membrane

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and then UV cross-linked by using a Stratalinker® UV crosslinker (Stratagene, La Jolla, CA). Gel


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shifts were visualized with streptavidin−horseradish peroxidase (HRP), followed by chemiluminescent

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detection.

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Immunofluorescence labeling

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Approximately 5 × 104 A7r5 cells were seeded onto 12-well plates, treated with 0.1−1.0 mg/mL MPE

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for 48 h, washed, fixed in 4% formaldehyde/PBS for 10 min, and permeabilized with detergent 0.2%

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Triton X-100 (Sigma)/PBS for 5 min. To visualize polymerized F-actin, cells were stained with

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TRITC-phalloidin dye (Sigma) for 30 min at room temperature, washed with PBS, and briefly

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counterstained with 4′, 6-diamidino-2-phenylindole (DAPI [1 mg/mL]; Sigma) to visualize the nuclei

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Fluorescence images were obtained on a confocal laser scanning microscope LSM 510-Meta (Zeiss)

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and assessed using image analysis software.

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Reverse transcription polymerase chain reaction (RT-PCR)

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Total RNA was isolated from culture cell using a RNA Isolation Kit (Ultraspec, Biotecx, Houston, TX)

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according to the manufacturer's instructions and quantified spectrophotometrically. As previously

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described,27 the primers were as follows: MMP, Forward (5′-ACACCCAGTACTCATTCCCTG-3′ )

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Reverse (5′-GTCCTGACCAAGGATATAGCC-3′) ; VEGF, (5′-TGCACCCACGACAGAA

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GGGGA-3′) Reverse (5′-TCACCGCCTTGGCTTGTCACA-3′); GADPH, Forward

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(5′-ACCACAGTCCATGCCATCAC-3′) Reverse (5′- TCCACCACCCTGTTGCTG

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TA-3′). The PCR conditions were 94°C for 1 minute as an initial step, 72°C for 2 minutes, finally 20

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minutes at 72°C, followed by 30 cycles of 1 minute at an annealing temperature. The RT-PCR products



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were analyzed using agarose gel electrophoresis, and the intensity of the bands corresponding to

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MMP-2, VEGF, and GADPH were measured using Fujifilm Image Gauge software (version 3.1,

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Fujifilm Co., Ltd., Tokyo, Japan). Each experiment was repeated three times.

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Western blotting

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The cell lysates from MPE-treated A7r5 cells were placed on ice in 1X RIPA lysis buffer

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(Sigma-Aldrich) containing protease and phosphatase inhibitor (Thermo-Scientific, Waltham, MA,

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USA). Total protein concentration was measured using a Bradford protein assay kit (Bio-Rad). The

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proteins in cell lysates (50 µg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel

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electrophoresis (SDS-PAGE) gels and electro-transferred to a nitrocellulose membrane. The membrane

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was incubated with 0.5% non-fat milk solution for 1 h at room temperature to block nonspecific

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binding; incubated with antibodies specific for TIMP-2 (Santa Cruz Biotechnology, SC-21735, dilution:

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1:1000), VEGF (Santa Cruz Biotechnology, SC-4570, dilution: 1:1000), FAK (Cell Signaling

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Technology, CST#3285, dilution: 1:1000), integrinβ3 (Cell Signaling Technology, CST#4702, dilution:

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1:1000), c-Raf (Cell Signaling Technology, CST#9422, dilution: 1:1000), Src (Cell Signaling

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Technology, CST#2108, dilution: 1:1000), PI3K (Cell Signaling Technology, CST#4255, dilution:

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1:1000), Akt (Cell Signaling Technology, CST#9272, dilution: 1:1000), Cdc42 (Santa Cruz

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Biotechnology, SC-8401, dilution: 1:1000), RhoA (Santa Cruz Biotechnology, SC-179 , dilution:

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1:1000), RhoB (Santa Cruz Biotechnology, SC-180, dilution: 1:1000), Rac1 (Santa Cruz Biotechnology,

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SC-217, dilution: 1:1000), p-FAK (Cell Signaling Technology, CST#3281, dilution: 1:1000), IκB (Cell


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Signaling Technology, CST#9242, dilution: 1:1000), NF-κB (Cell Signaling Technology, CST#6956,

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dilution: 1:1000), p-Akt (Cell Signaling Technology, CST#9275, dilution: 1:1000), and β-actin (Sigma,

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A-5316, dilution: 1:1000) at 4°C overnight; washed with Tris-buffered saline containing 0.1% Tween

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20 (TBST) three times (10 minutes/each time); incubated with horseradish-peroxidase conjugated

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second antibody (Sigma, St. Louis, MO) at room temperature for 1 h; washed with TBST again;

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incubated with ECL Western Blotting Detection Reagents (Millipore, Bedford, MA) to visualize the

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protein bands, and analyzed by densitometry using Alpha Imager Series 2200 software.

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Statistical analysis

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Statistical significance was evaluated using one-way analysis of variance (ANOVA). Results are

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presented as the means ± standard deviation (SD) of three independent experiments and were compared

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between groups by using the Student t-test. P value < 0.05 was considered statistically significant.



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Results

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Polyphenolic compounds of MPE

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Spectrophotometry and HPLC were used to identify and assay the content of polyphenolic compounds

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in MPE. These included gallic acid (2.67%), protocatechuic acid (13.76%), catechin (3.19%),

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epigallocatechin gallate (6.26%), caffeic acid (6.15%), epicatechin (4.69%), p-coumaric acid (2.48%),

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rutin (18.17%), ferulic acid (0.99%), gossypin (1.04%), hesperetin (2.08%), resveratrol (0.92%),

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quercetin (5.97%), naringenin (6.71%), and hydroxyflavin (1.43%) (Table 1).

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MPE effectively inhibited migration of A7r5 cells

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We used the scratch wound healing and Boyden chamber assay to determine whether MPE suppresses

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A7r5 cell migration. In the wound healing assay, substantially fewer A7r5 cells were observed by light

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microscopy (400 x) to migrate into the wound area after treatment with MPE (0.1−1.0 mg/mL) for 0−4

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d than in the absence of MPE (the control) (Figure 1A and B). This result suggested that MPE may

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suppress A7r5 cell migration.

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MPE affects MMP activity and VEGF secretion in A7r5 cells

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To assess the effect of 0.5 mg/mL MPE on the activities of MMPs (MMP2 and MMP9) in A7r5 cells

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over time (0–48 h), we used gelatin zymography. As shown in Figure 2A, the activities of MMP2 and

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MMP9 were time-dependently suppressed in the MPE group relative to the control group. In addition,

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we determined whether the MPE-induced decrease in MMP activity affects TIMP-2 and VEGF protein

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expression. Western blotting revealed that MPE also time-dependently decreased protein expression of


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TIMP2 and VEGF (Figure 2B, C).

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Treatment with MPE reduced FAK complex-related protein expression in A7r5 cells

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FAK/PI3K/Akt signaling pathways, MAPK family proteins, and c-Raf play key roles in cell

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migration.39, 40 Western blotting was used to investigate the effects of different MPE concentrations

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(0.1–1.0 mg/mL) over 0–48 h on A7r5 cell migration. As shown in Figure 3A and 3B, MPE (unlike the

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control) dose- and time- dependently decreased the expression of the FAK, PI3K, Akt, and c-Raf.

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Previous study has shown that the FAK/Src complex mediates cell survival and migration via PI3K.

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Using immunoprecipitation to determine whether MPE (0–1 mg/mL) affects FAK/Src complex and

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PI3K expression, we showed that treatment with MPE downregulated FAK, Src, and PI3K (Figure 3C).

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Taken together, these results suggest that MPE significantly suppresses the expression of

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migration-related proteins.

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MPE suppressed protein expression and the activity of NF-κB

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Previous study has demonstrated that NF-κB and IκB participate in numerous pathologies involving

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mitogenic, pro-inflammatory, and anti-apoptotic factors.40 Because NF-κB and IκB play a role in cell

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migration, we investigated whether MPE affects NF-κB/IκB protein expression in A7r5 cells. As

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expected, MPE inhibited NF-κB expression and increased IκB expression (Figure 4A and 4B).

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Immunoprecipitation with anti-NF-κB and immunoblotting with anti-IκB found that binding of NF-κB

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and IκB was significantly increased in MPE-treated cells relative to control cells (Figure 4C).

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Moreover, we used the EMSA assay to assess NF-κB DNA binding activity in nuclear extracts. The



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results showed that MPE attenuated NF-κB DNA binding activity relative to that in control cells. Using

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RT-PCR to investigate MPE regulation of the expression of MMP-2 mRNA and VEGF mRNA, we

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found that MPE downregulated MMP-2 and VEGF mRNA expression. Collectively, these results

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demonstrate that the dose- and time- dependent effects of MPE on NF-κB signaling are mediated via

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regulation of MMP and VEGF mRNAs expression (Figure 5).

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MPE affected the F-actin cytoskeleton via inhibiting the expression of small GTPase proteins

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Since the small GTPase superfamily including RhoA, Rac1, and Cdc4241 plays a major role in cell

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growth, migration, and cytoskeletal formation,42, 43 we evaluated the potential effect of MPE on small

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GTPase protein expression in A7r5 cells. As shown in Figure 6A, treatment with various concentrations

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0.1–1 mg/mL of MPE reduced levels of RhoA, Rac1, and Cdc42. Over 0–48 h, MPE lowered small

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GTPase protein levels relative to those in control cells (Figure 6B). In addition, previous studies have

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demonstrated small GTPase proteins regulate F-actin cytoskeleton rearrangement.44 To determine

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whether MPE mediates cytoskeletal change and cellular motility, we used immunofluorescence

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microscopy to assess the MPE-induced change in DAPI (nuclei)- and phalloidin (F-actin)-staining in

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A7r5 cells. We found that MPE altered cytoskeletal distribution (Figure 6C). These data support

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findings that MPE causes F-actin cytoskeleton rearrangement through coordinate reduction in the

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expression of small GTPase proteins.


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Discussion

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Atherosclerosis is associated with other lifestyle-related diseases.45 The major established risk

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factors for atherosclerosis include hyperglycemia, hyperlipidemia, smoking, high-calories, obesity, and

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diabetes mellitus.46 According to WHO statistics, the death toll from cardiovascular diseases is higher

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than those from other causes. An estimated 17.7 million people died of cardiovascular disease

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(coronary heart disease and stroke) in 2015, accounting for 31% of global deaths. In addition, efforts to

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improve and prevent atherosclerosis are continuing. Several studies have utilized VSMCs as a model to

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elucidate the atherosclerosis process.47, 48 In the present study, MPE was found to reduce A7r5 cell

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motility. Moreover, the inhibition of cell motility may be attributed to the presence of numerous

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polyphenols as determined by HPLC analysis, such as gallic acid (GA), protocatechic acid (PCA),

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catechin, epigallocatechin gallate (EGCG), caffeic acid (CA), epicatechin, rutin, and so forth (Table 1).

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On the other hand, recent studies show that GA can increase nitric oxide (NO) levels in human

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umbilical vein endothelial cells (HUVECs) and its effect against hypertension could reduce

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cardiovascular disease risk.49, 50 In 2017, Luo KW et al. reported that EGCG inhibited the proliferation

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and migration of bladder cancer cells (SW480).51 CA and rutin has been previously described to

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mitigate accumulation of fatty acid, and inhibit hepatic lipogenesis of HepG2 cell.52, 53 Some previous

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reports have also suggested the epicatechin could reduce inflammation, oxidative stress, and inhibit

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NF-kB and JNK pathway. In the present study, MPE dose- and time-dependently reduced the migration

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of A7r5 cells.



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Increased activity of MMP is typically associated with migration.54 To better understand the

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molecular mechanism of migration, we used gelatin zymography and the Western blotting assay to

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assess MMP activity in MPE-treated A7r5 cells. The results showed the MPE not only decreased MMP

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(MMP2 and MMP9) activity, but also time-dependently suppressed TIMP2 and VEGF expression. The

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FAK and PI3K/Akt signaling pathways regulate melanoma cell migration55 as well as embryonic cell

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differentiation.56

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High levels of FAK induce metastasis and have been associated with poor prognosis in colon

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cancer. In addition, PI3K/Akt pathway is implicated in the development of cancers and in the

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promotion of corneal endothelial cell proliferation.57 Interestingly, our data showed that MPE

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significantly reduced phosphorylation of FAK and activation of PI3K/Akt, which inhibits A7r5 cell

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migration. Moreover, we used immunoprecipitation to detect the interaction of PI3K with FAK, which

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may explain the MPE-induced reduction in cell migration.

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The NFκB activates angiogenesis, cell migration, and invasion by augmenting VEGF and MMP

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mRNA expression in colon cancer cells.58 Similarly, increased synthesis of VEGF mRNA and the

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bioactivity of MMP9 raises the possibility of greater intrinsic angiogenesis and inflammation.59 Our

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findings are consistent with these studies in that they show that MPE not only reduces the activity and

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protein expression of NFκB, but also the mRNA expression of MMP2 and VEGF. Because of the

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importance of the small GTPase pathway in epithelial cell migration, we examined their effect on

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regulators of the F-actin cytoskeleton. Our experiments showed that MPE dose- and time-dependently


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inhibited small GTPases, resulting in cytoskeletal change.

Our 2014 report showed treatment with MWE inhibits atherosclerosis in vivo and reduces

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migration and proliferation in vitro. HPLC found large amounts of polyphenolic constituents in MWE.

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Therefore, we hypothesized that the polyphenols of MWE are important components affecting

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atherosclerosis, cell migration, and cell proliferation.27 In conclusion, we demonstrated that MPE

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improves A7r5 cell motility. Furthermore, we showed that the very rich complement of MPE

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polyphenols regulates migration via three mechanisms targeting: (i) FAK/Src/PI3K signaling; (ii) the

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secretion of VEGF and MMP regulated by inhibiting NFκB; and (iii) GTPase regulation to improve the

292

differential cytoskeleton remodeling as shown in Figure 7. Future experiments will explore in more

293

detail the kinds of polyphenols acting on the small GTPase or PI3K pathway to inhibit VSMC

294

migration and assess their value in treating cardiovascular disease.



295

Acknowledgements

296

This study was supported by a grant from Chung Shan Medical University Hospital, Taichung, Taiwan

297

(CSH-2016-C-008).

298 299

Conflict of interest

300

The authors declare that they have no competing financial interests.


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Figure legends

462

Figure 1. Migration of A7r5 cells treated with MPE. The migration potential of cultured A7r5 cells

463

treated with MPE (A) at various concentrations of for 0–96 h were assessed by the wound healing

464

assay or (B) for 48 h were assessed by the Boyden chamber assay. The data are the means ± SD from

465

three experiments with four samples per group. (*p＜0.05,**p＜0.001, compared with control)

466 467

Figure 2. MPE treatment decreased MMP activity, increased TIMP2 protein expression, and

468

decreased VEGF expression in A7r5 cells. A7r5 cells were treated with indicated concentration of

469

MPE for 0-48 h (A) Gelatin zymography was used for measuring MMP2 and MMP9 activity. (M

470

denoted the marker) (B) Western blotting was used for measuring TIMP2 and VEGF expression.

471

β-actin and albumin were used as loading controls. Values are the means ± SD from three independent

472

experiments for each group. (*p＜0.05,**p＜0.001, compared with control)

473 474

Figure 3. MPE inhibited the expression of FAK/integrin-β3, PI3K/Akt, and c-Raf proteins in A7r5

475

cells. A7r5 cells were treated with MPE (A) at various concentrations for 48 h or (B) 0.5 mg/ml for

476

0~48 h. The levels of FAK, PI3K, Akt, integrin-β3, and c-Raf protein expression were analyzed by

477

Western blotting with their specific antibodies. (C) The immunoprecipitated (IP) proteins were

478

separated via SDS-PAGE and their levels were quantitated by immunoblotting with anti-FAK, Src, and

479

PI3K antibodies. IB, immunoblotting; IP, immunoprecipitation. β-actin and albumin were used as


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480

loading controls. Each value is the mean ± SD from three experiments per group. (*p＜0.05,**p＜

481

0.001, compared with control)

482 483

Figure 4. MPE inhibited the activation of NF-κB. A7r5 cells were treated with MPE (A) at various

484

concentrations for 48 h or (B) at 0.5 mg/ml for 0~48 h. The protein expression levels of NFκB and IκB

485

were analyzed with Western blotting by using their specific antibodies. (C) Cell lysates were

486

immunoprecipitated (IP) with anti-NFκB antibody and then immunoblotted (IB) with anti-IκB antibody

487

as indicated. β-actin was used as a loading control. Each value is the mean ± SD from three

488

experiments for each group. (*p＜0.05, compared with control)

489 490

Figure 5. MPE inhibited NF-κB DNA binding, MMP-2 and VEGF mRNA expression. Nuclear

491

extracts of A7r5 cells treated with various concentrations of MPE for 12 h were assayed by (A)

492

Western blotting to determine relative levels of NF-κB expression, with C23 used as a negative/

493

loading control or (B) EMSA to determine DNA binding activity of NF-κB. (S; starvation, P; unlabeled

494

probe, and C; control. (C) Nuclear extracts of A7r5 cells treated with various concentrations of MPE

495

for 24 h were assayed by RT–PCR to determine MMP-2 and VEGF mRNA expression. Data are the

496

means ± SD of at least three independent experiments.

497 498

Figure 6. MPE inhibited the expression of small GTPase proteins and cytoskeletal F-actin



499

patterns with phalloidin–TRITC. A7r5 cells treated with MPE (A) at various concentrations for 48 h

500

or (B) 0.5 mg/ml for 0~48 h. The expression levels of small GTPase proteins in lysates were analyzed

501

with Western blotting using specific antibodies to Cdc42, RhoA, and Rac1. β-actin was used as a

502

loading control. (C) A7r5 cells seeded onto 12-well plates were stained with phalloidin-TRITC (red) to

503

detect F-actin, and with DAPI (blue) to detect nucleic acid. Data are the means ± SD from three

504

experiments for each group. (*p＜0.05,**p＜0.001, compared with control)

505 506

Figure 7. Schematic of the proposed mechanism underlying MPE-induced inhibition of A7r5 cell

507

migration.


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Table 1

Table 1. Polyphenolic compounds of MPE.a

a

Free polyphenols in the mulberry polyphenol extract (MPE; 10 mg/ml) were analyzed by HPLC. The amount of each polyphenol is expressed as

percentage of the polyphenolic compounds in MPE, quantified relative to standards, and represents the average of three independent experiments. Abbreviations: Gallic acid, GA; Protocatechuic acid, PCA; Catechin, CA; Epigallocatechin gallate, EGCG; Caffeic acid, CaA; Epicatechin, EC; P-coumaric acid, pCA; Rutin, R; Ferulic acid, FA; Gossypin, GN; Hesperetin, HP; Resveratrol, RSV; Quercetin, Q; Naringenin, NG; Hydroxyflavin, FlOH.



Fig. 1 (A)

600 control 0.05 (mg/ml) 0.1 0.2 0.5 1

Number of cells

500 400 300 200 100 0

0

1

2

3

4

Time (day)

Number of cells

(B)

MPE (mg/ml)


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Fig. 2 (A)

(B)

(C)



Fig. 3 (A)

(B)

(C)


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Fig. 4 (A)

(B)

* *

(C)


*

*


Fig. 5 (A)

(B)

(C)


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Fig. 6 (A)

(B)

(C)



Fig. 7


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TOC graphic


PI3K Complex and

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