Cultivated Orostachys japonicus extract inhibits VEGF-induced angiogenesis via regulation of VEGFR2 signaling pathway in vitro and in vivo

Journal Pre-proof Cultivated Orostachys japonicus extract inhibits VEGF-induced angiogenesis via regulation of VEGFR2 signaling pathway in vitro and in vivo Hyun-Dong Cho, Kwan-Woo Lee, Yeong-Seon Won, Jeong-Ho Kim, Kwon-Il Seo PII:

S0378-8741(19)33809-7

DOI:

https://doi.org/10.1016/j.jep.2020.112664

Reference:

JEP 112664

To appear in:

Journal of Ethnopharmacology

Received Date: 24 September 2019 Revised Date:

2 January 2020

Accepted Date: 7 February 2020

Please cite this article as: Cho, H.-D., Lee, K.-W., Won, Y.-S., Kim, J.-H., Seo, K.-I., Cultivated Orostachys japonicus extract inhibits VEGF-induced angiogenesis via regulation of VEGFR2 signaling pathway in vitro and in vivo, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/ j.jep.2020.112664. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

1

Abstract

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Ethnopharmacological relevance: Orostachys japonicus A. Berger (O. japonicus), so-called

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Wa-song in Korea, a traditional food and medicine that grows on mountain rocks and roof

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tiles.

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Wa-song containing various phenolic compounds have been reported as a medicinal plant for

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prevention of fibrosis, cancer, inflammation, and oxidative damage.

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Aim of the study: The present study was designed to examine the anti-angiogenic effects of

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cultivated Orostachys japonicus 70% ethanol extract (CE) in vascular endothelial growth

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factor (VEGF)-stimulated human umbilical vein endothelial cells (HUVECs).

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Materials and methods: CE was prepared with 70% ethanol. HUVECs, rat aortic rings, and

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matrigel plug in mice were treated with CE (10-20 µg/mL) and VEGF (20-50 ng/mL), and

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the anti-angiogenic activities of CE were analyzed by SRB, wound healing, trans-well

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invasion, capillary-like tubule formation, rat aortas, western blot, and matrigel plug assay.

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Phenolic compounds in CE were analyzed using a high-performance liquid chromatography

15

(HPLC)-PDA system.

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Results: Treatment of CE (10-20 µg/mL) markedly suppressed proliferation of HUVECs in

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the presence (from 136.5% to 112.2%) or absence of VEGF (from 100.0% to 92.1%). The

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proliferation inhibitory effect of CE was caused by G0/G1 cell cycle arrest, and the decrease

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of CDK-2, CDK-4, Cyclin D1 and Cyclin E1. Furthermore, CE treatment showed significant

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angiogenesis inhibitory effects on motility, invasion and micro-vessel formation of HUVECs,

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rat aortic rings and subcutaneous matrigels under VEGF-stimulation condition. In HUVECs,

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CE-induced anti-angiogenic effect was regulated by inhibition of the PI3K/AKT/mTOR,

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MAPK/p38, MAPK/ERK, FAK-Src, and VEGF-VEGFR2 signaling pathways.

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Conclusion: This study demonstrated that CE might be used as a potential natural substance,

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multi-targeted angiogenesis inhibitor, functional food material.

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Keywords

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Angiogenesis; Cultivated Orostachys japonicus; Vascular endothelial growth factor receptor

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2

30 31

Abbreviations: BBE, Bovine Brain Extract; BSA, bovine serum albumin; CE, cultivated O.

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japonicus 70% ethanol extract; CDK, cyclin dependent kinase; ECGM, endothelial cell

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growth medium; EnGS, Endothelial Growth Supplement; ERK1/2, extracellular signal-

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related kinase 1/2; FAK, focal adhesion kinase; FBS, fetal bovine serum; GA,

35

Gentamicin/Amphotericin-B; HUVECs, human umbilical vein endothelial cells; hEGF,

36

human Epidermal Growth Factor; MMP-9, matrix metallopeptidase 9; mTOR, mammalian

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target of rapamycin; PI3K, phosphatidylinositol 3-kinases; SRB, sulforhodamin B; TIMP-1,

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tissue inhibitor of metalloproteinases-1; VEGF, vascular endothelial growth factor; VEGFR,

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vascular endothelial growth factor receptor

Angiogenesis inhibition in Human umbilical vein endothelial cells (HUVECs) Cell cycle arrest

VEGF

Migration inhibition

VEGF + CE (20 µg/mL)

VEGF

Cultivated O. Japonicus 70% ethanol extract (CE)

Invasion inhibition

VEGF + CE (20 µg/mL)

VEGF

VEGF + CE (20 µg/mL)

Tube-formation inhibition

VEGF

VEGF + CE (20 µg/mL)

Angiogenesis inhibition in rat aortic rings VEGF

VEGF + CE (20 µg/mL)

Angiogenesis inhibition in mouse subcutaneous matrigels 100 µm

100 µm

VEGF

VEGF + CE (20 µg/mL)

100 µm

100 µm

VEGF

VEGF + CE (20 µg/mL)

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Cultivated Orostachys japonicus extract inhibits VEGF-induced angiogenesis via

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regulation of VEGFR2 signaling pathway in vitro and in vivo

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Hyun-Dong Choa1, Kwan-Woo Leeb1, Yeong-Seon Wonb, Jeong-Ho Kimc, Kwon-Il Seob*

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a

7

b

8

c

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41566, Republic of Korea

Industry-Academy Cooperation, Dong-A University, Busan 49315, Republic of Korea Department of Biotechnology, Dong-A University, Busan 49315, Republic of Korea

Department of Food Science and Biotechnology, Kyungpook National University, Daegu

10 11 12 13 14 15 16 17

*Corresponding author. Department of Biotechnology, Dong-A University, 37, Nakdong-

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daero 550 street, Saha-gu, Busan, 49315, Republic of Korea. E-mail address: [email protected]

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1

These authors contributed equally to this work.

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1. Introduction

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Angiogenesis is a series of programs to form new blood vessels with critical roles in normal

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organs. Under normal conditions, angiogenesis is vitally regulated during wound healing,

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tissue regeneration, embryonic growth, and fetal development (Greaves et al., 2013; Ferrara,

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2001). On the other hand, the blood supply by angiogenesis can exacerbate specific

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pathologies, such as cancer growth and metastasis (Greaves et al., 2013; Folkman, 2002).

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Owing to the additional supply of oxygen and nutrient through the blood vessel, most solid

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tumors overcome the limitation of growth caused by hypoxia and nutrient starvation

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(Gimbrone et al., 1972). To attract endothelial cells toward tumor tissue, hypoxic tumor

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tissues trigger angiogenesis by secreting chemotactic substances, including fibroblast growth

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factor (FGF), and vascular endothelial growth factor (VEGF) family, such as VEGF-A, -B, -

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C, -D, hypoxia-inducible factors, and angiopoietins (Roussos, Condeelis and Patsialou, 2011;

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Cao and Liu, 2007).

34

VEGF as a well-known cancer-derived proangiogenic mediator that activates diverse inter-

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cellular signaling mechanisms through relations with VEGF receptors (VEGFR) in blood

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vessel cells. On the membrane of endothelial cells, VEGF-VEGFR binding results in the

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proliferation, migration, invasion, and microtubule formation through the activation of

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diverse proangiogenic factors, including Src family kinase, focal adhesion kinase (FAK),

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mammalian target of rapamycin (mTOR) kinase, phosphoinositide 3-kinase (PI3K), Akt

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kinase, extracellular signal-related kinases 1/2 (ERK1/2), p38 kinase, and matrix

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metalloproteinases (MMPs) (Carmeliet and Jain, 2011a). Furthermore, a range of growth

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factor receptors associated with angiogenesis may be highly linked with blockage of cell

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cycle process in endothelial cells (Xiao et al., 2015; Mehta et al., 2011). The binding of

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VEGF on VEGFR2 can promote the cell cycle progress in HUVECs with the upregulation of

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cyclin D1, cyclin A, cyclin E, and mitogen-activated protein kinases (MAPKs) (Yasui et al.,

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2006; Yoshihara et al., 2010). Therefore, a range of chemo-preventive approaches to

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inhibiting the multiple steps of angiogenesis on VEGF-induced proliferation, invasion,

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migration, and microtubule formation are considered effective strategies in cancer patients.

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Because standard first line angiogenesis inhibitors clearly show anticancer properties,

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several anti-angiogenic synthetic drugs, including bevacizumab (Avastin®), sunitinib malate

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(Sutent®), and sorafenib (Nexavar®) have been approved by the U.S. Food and Drug

52

Administration (Kamba and McDonald, 2007). Although synthetic chemotherapeutics

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effectively inhibit angiogenesis targeting the downstream pathways in endothelial cells,

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unexpected adversary effects, such as, heart failure, hypertension, proteinuria, thrombosis,

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impaired wound healing, endocrine dysfunction, reversible posterior leukoencephalopathy,

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and gastrointestinal perforation have been reported (Ribeiro et al., 2018). In addition,

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chemotherapeutic resistance and toxic effects on normal tissues have been pointed out as key

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hurdles to overcome in cancer therapies. Considering the serious problems of existing drugs,

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several studies have made continuous efforts to find safe and efficient novel angiogenesis

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inhibitors given as a single-treatment drug or in co-treatment with some chemotherapeutics.

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Recently, the interest in natural sources to inhibit angiogenesis has been increasing because

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of their safety benefits and cost effectiveness (Grimstein and Huang, 2018). Orostachys

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japonicus A. Berger (Herbal medicine Latin name, Orostachys Herba), so-called Wa-song in

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Korea, is a large annual broadleaf species and a native plant that grows on mountain rocks

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and roof tiles (Lee et al., 2009). O. japonicus has been consumed as a food material and

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medicinal plant for a long time in Korea and East Asia, and the ethnopharmacological

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application of O. japonicus was reported to contain diseases like hepatitis, pneumonia,

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hepatotoxicity and malaria (Korea Food and Drug Administration, 2003; Park, 2017).

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Furthermore, recent studies reported that O. japonicus contains a range of phytochemical

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compounds, such as sterols (Yoon et al., 2005), flavonoids (Lee et al., 2011), phenolic acid

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and triterpenoid (Lee et al., 2004), and species show potential as a medicinal herb with anti-

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oxidant (Lee et al., 2008), anti-fibrosis (Koppula et al., 2017), anti-inflammatory (Lee et al.,

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2012) and anticancer (Lee et al., 2018; Kim et al., 2012; Ryu et al., 2014) activities. Although

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angiogenesis has been reported as an essential process in the steps of viral hepatitis to

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develop cirrhosis (Medina et al., 2004) and carcinogenesis (Mazzanti et al., 1997), anti-

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angiogenic effect of O. japonicus and its molecular mechanisms are not fully understood in

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terms of evidence-based herbal medicine.

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Along with a wide range of uses of Wa-song-based food and traditional medicine, O.

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japonicus has recently begun to be cultivated in some Asian countries. These cultivated

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agricultural products have many advantages over wild plants, including lower cost with

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similar quality and productivity. In our previous studies, compared to wild O. japonicus

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extract, cultivated O. japonicus extract showed more effective anticancer activity against

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prostate cancer cells (Kim et al., 2012), anti-angiogenic activities in human umbilical vein

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endothelial cells (HUVECs) as well as contained higher phenolic compounds (Cho et al.,

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2019). Moreover, the optimal solvent condition of cultivated O. japonicus extract to inhibit

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angiogenesis was identified as 70% ethanol extract (Cho et al., 2019). Interestingly, non-toxic

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concentration of cultivated O. japonicus 70% ethanol extract (CE) significantly blocked

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VEGF-induced angiogenesis in HUVECs (Cho et al., 2019). However, the anti-angiogenic

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mechanisms of CE in HUVECs are not completely understood.

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Therefore, the present study evaluated the role of CE against the inhibition of angiogenesis

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in HUVECs. In addition, the inhibitory mechanisms involved in the anti-angiogenesis activity

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of CE were investigated both in vitro and in vivo. These observation suggests that CE can be

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used as a promising anti-angiogenic natural medicine with many industrial and toxicological

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advantages.

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2. Material and Method

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2.1. Sample Preparation & chemicals

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Cultivated O. japonicus was obtained from Depart. of Development in Oriental Medicine

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Resources, Sunchon National University (Suncheon, Republic of Korea). The dried herb

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resource was collected from Sunchon area of Korea during the July, 2017, and authenticated

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by Prof. Ji-Wook Jung. A voucher specimen was deposited in the Daegu Hanny University

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Herbarium (No. 805221). Cultivated O. japonicus was dried in dry oven (63°C), and cut into

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small pieces. The extraction condition was similar with our previous study. Briefly, 10 g of

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dried O. japonicus was mixed with 100 mL of 70% ethanol (Junsei Chemical Co., Tokyo,

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Japan), and extracted for 3 days at room temperature. Then, CE was centrifuged at 3000 ×g,

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and filtered by 0.45 µm of membrane filter. Finally, the filtrate was concentrated by using

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rotary vacuum evaporator (N-1000, Eyela, Tokyo, Japan) up to dryness, and dried extract

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(yield of CE: 11.42%) was stored in -80°C refrigerator for further experiments on anti-

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angiogenesis.

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ECBM, EGM™-PLUS SingleQuots™ kit (Lonza Inc., Basel, Switzerland), FBS, trypsin-

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EDTA, penicillin, and antibiotic-antimycotic were bought from LONZA Inc. Recombinant

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human VEGF (VEGF165) was purchased from the Peprotech (Rocky Hill, NJ, USA).

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Matrigel was bought from BD Bioscience (Franklin Lakes, NJ, USA). Antibodies against

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anti-PI3K, -AKT, -mTOR, -p38, -ERK 1/2, -Src, -FAK, -TIMP-1, -MMP-9, -CDK-2, -CDK-

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4, -cyclin D1, -cyclin E1, -β-actin, -phosphorylated AKT (Ser473), -phosphorylated FAK

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(Tyr407), -phosphorylated p38 (Tyr182), -phosphorylated ERK 1/2 (Tyr202/204), -CD34, -

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phosphorylated mTOR (Ser2448), -phosphorylated Src (Tyr530), and VEGF were obtained

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from Santa Cruz Biotechnology (Dallas, CA, USA). Anti-VEGFR2 and -phosphorylated

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VEGFR2 (Tyr1175 and Tyr951) were bought from Cell Signaling Technology (Danvers, MA,

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USA).

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To measure phenolic compounds in CE, Kaempferol-3-rutinoside (Cat. No. 17650-84-9,

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purity >98%) were purchased from Chengdu Biopurify Phytochemicals Ltd (Sichuan, China,

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purity >98%). Kaempferol-3-D-glucopyranoside (Cat. No. 79851, purity >97%), kaempferol

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(Cat. No. 60010, purity >97%), quercetin (Cat. No. Q4951, purity >95%), quercetin-3-

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glucose (Cat. No. 16654, purity >98%), rutin (Cat. No. R5143, purity >94%), epicatechin

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(Cat. No. E4018, purity >98%), epicatechin-gallate (Cat. No. E3893, purity >98%), catechin

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(Cat. No. 43412, purity >99%), and gallic acid (Cat. No. 27645, purity >99%) were

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purchased from Sigma-Aldrich Inc.

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2.2. Cell culture

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HUVECs were bought from Lonza Inc., and only 2-5 of passage number cells were used

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in vitro experiments. HUVECs were cultured in ECGM including EGM™-PLUS

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SingleQuots™ Kit (EnGS, L-Glutamine, Ascorbic Acid, Hydrocortisone Hemisuccinate,

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hEGF, Heparin, FBS, GA and BBE) at 37 °C under 5% of CO2 circumstance.

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2.3. SRB analysis

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Growth of HUVECs was analyzed by SRB assay (Skehan et al., 1990). In brief, cells

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were attached (2.5 × 104 cells per well) in 48-well plates, and stabilized in CO2 incubator for

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24 h. After 1 day, 10-80 µg/mL of CE was treated for 24 h. Cells was fixed on plate by 12%

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trichloroacetic acid, after which 0.4% SRB agent (Sigma-Aldrich) was treated to each group.

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After 1 h at room temperature, the cells were rinsed with 1% acetic acid, and SRB dye were

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extracted with Tris buffer (10 mM). Then, the UV spectra was analyzed at 540 nm through a

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microplate reader (Molecular Devices, Sunnyvale, CA, USA).

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2.4. Cell cycle assay

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Cultured HUVECs were analyzed by a Muse® Cell Cycle Assay Tool (Merck Millipore,

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USA), following the protocol methods. Briefly, HUVECs were detached with 0.25% trypsin-

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EDTA, and centrifuged (300 × g, 5 min). The pellets were rinsed with 1× PBS and strongly

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chilled in 1mL of ice-cool 70% ethanol (-20℃, 3 hours). Then, fixed-HUVECs were reacted

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with Muse Cell Cycle Tool (Merck Millipore, USA) for 40 min and then measured with

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Muse® Analyzer (Merck Millipore, USA).

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2.5. Wound healing migration assay

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Scratch-healing motility assay was assessed as following previous method (Cho et al.,

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2019). The endothelial cells were spread on 48-well plates at a concentration of

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2.5×104cells/well and incubated until culture plate was fully filled with cells. Surface of

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HUVECs were scratched using yellow micropipette tips to make cross lines and removed

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floating cells via PBS washing. After washing step, ECGM with low FBS (0.5%) were

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treated for 4 h, followed by 20 ng/mL of VEGF and 10-20 µg/mL of CE were added for 12 h.

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Images of lines were taken through an inverted microscope (×200). Numbers of moved

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HUVECs were compared with control group cells.

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2.6. Trans-well invasion assay

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Trans-well invasion analysis was assessed using 24-well trans-well insert plate (8-µm

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pore size, Corning, NY, USA) as following previous protocol (Cho et al., 2019). The

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endothelial cells were attached on the upper space of plates with a cell number of 5 × 104 per

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wells, and 10-20 µg/mL of CE were treated simultaneously. In the bottom space, 1 mL of

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ECGM supplemented with 50 ng/mL of VEGF was filled to induce cell invasion. After 12 h

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of VEGF-stimulation, invaded HUVECs were strongly fixed by 5% paraformaldehyde

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solution, and stained by 1% crystal violet dye. Invaded cell images were obtained by an

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inverted microscope (×200). The percentages of invaded cell numbers were compared to

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control group.

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2.7. Capillary-like tube formation assay

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The formation of capillary-like tubule was performed as following previous protocol

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(Cho et al., 2019). HUVECs were incubated in 48-well plate, and cultured with 10% FBS

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supplemented ECGM for 24 h. Then, the HUVECs were starved under 0.5% FBS condition

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for 4 h, followed by CE was treated for 2 h. To set thin collagen layer, 40 µL of matrigel

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reagent was polymerized in the 96-well plates, after which endothelial cells were detached

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from 48 well plates and moved to matrigel-coated 96-well plates at a density of 2.5 × 104 per

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well. To formulate building of capillary-like tubule, HUVECs were incubated in CO2

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incubator for 6 h, and images were captured by an inverted microscope (×200).

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2.8. Rat aortic ring assay

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Following previous protocol, rat aorta ring assay was analyzed to identify the ex vivo anti-

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angiogenic properties of CE (Cho et al., 2019). Liquid state matrigel reagent were placed on

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the bottom of 96-well plates, and solidified in CO2 incubator for 30 min. Aortic rings were

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extracted from 5 weeks old male Sprague-Dawley rats, and periadventitial fat were carefully

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eliminated using tweezers. After PBS washing step, aortas were cut into rings of 1-1.5 mm in

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circumference. The aortic rings were then embedded on matrigel in 96 well plates, and

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covered with matrigel reagent in CO2 incubator (37 °C, 1 h). Next, 100 µL of ECGM (0.5%

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FBS) mixed with VEGF (20 ng/mL) in the presence or absence of CE (10-20 µg/mL) was

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treated into the wells. After 6 days, picture of sprouting of microvessel was taken by an

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inverted microscope (×200). The score of angiogenesis was determined from 0 (most

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negative) to 5 (most positive) in a double-blind manner. Each data point was analyzed 3 times,

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respectively.

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2.9. Animal study

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All in vivo process using mice were approved by the Dong-A University Committee for

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the Care and Use of Laboratory Animals (DIACUC-18-22). Four-week-old male C57BL/6

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mice were obtained from Hyo-Chang Science (Busan, Korea), and cared in pathogen-free

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cage. All mice were fed an AIN-93G diet, and maintained under barrier room condition at

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room temperature (22 ℃) with 12 h light-dark cycle.

204 205

2.10. Angiogenesis assessment in vivo

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In vivo anti-angiogenic activity of CE was assessed by matrigel plug assay. Matrigels (0.5

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mL/plug) in liquid form containing 25 units of heparin with or without VEGF (150 ng/mL)

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and CE (10-20 µg/mL), were subcutaneously injected into the ventral area of C57BL/6 mice

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(n=5 per group). Injected matrigel was quickly polymerizing in subcutaneous tissue with

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body temperature of mice, then finally formed tumor-like prosthesis containing VEGF and/or

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CE. After 7 days, mice were sacrificed, and the solidified matrigel plugs were harvested from

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mice.

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2.11. H&E staining & immunohistochemistry

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Isolated matrigel plugs were immediately soaked in 37% formaldehyde to fix the tissue.

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After then, the matrigels were embedded in paraffin, and cut into sections (thickness of 5 µm).

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To observe infiltrating blood vessels, matrigel sections were stained with haematoxylin and

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eosin (H&E) for histochemical analysis, and processed by immunohistochemical staining for

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CD34 antibody (Santa Cruz Biotechnology). Tissue pictures were taken by Olympus BX41

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photo microscope (Olympus Co., Tokyo, Japan).

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2.12. Western blot anaylsis

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Firstly, endothelial cells were starved in FBS-free ECGM for 4 h, followed by pretreated

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with or without 10-20 µg/mL of CE for 2 h. CE-treated cells were stimulated with 20 ng/mL

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of VEGF for 2 h. In the case of proteins related with migration events and cell cycle,

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HUVECs were stimulated with 20 ng/mL of VEGF for 12 h and 24 h, respectively. After

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treatment, cellular proteins were lysed using cell lysis buffer, and analyzed by loading in 2D-

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SDS polyacrylamide gel electrophoresis. Loaded proteins were transferred to polyvinylidene

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fluoride membranes. Specific immune-dominant proteins were found by using above

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described primary and secondary antibodies. ECL kit (Santa Cruz Biotechnology) was used

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for chemo-luminescence detection. The protein density was quantified by Image studioTM

232

(Li-COR Inc., NE, USA), and the change of protein was expressed by comparing with β -

233

actin.

234 235

2.13. Statistical analysis

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All analysis assays were performed at least three times. Data are expressed as means ±

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SEM. Significance of statistic was detected by One-Way ANOVA, followed by the Tukey-

238

Kramer Multiple Comparisons test using Graphpad prism 5 program. A significant level was

239

described as *P < 0.05, **P < 0.01, and ***P < 0.001.

240 241

3. Results

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3.1. CE induces cell cycle arrest at G1 phase in HUVECs

243

The proliferation inhibitory effects of CE were identified by examining the cell viability

244

in various concentrations of CE in HUVECs. As indicated in Fig. 1A and B, high

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concentrations of CE (40-80 µg/mL) induced cellular damage in HUVECs, but incubation

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with low concentrations of CE (5-20 µg/mL) did not cause significant cytotoxicity or

247

morphological changes. Flow cytometry showed that incubation with 40-80 µg/mL of CE in

248

HUVECs enhanced the population of cells in the sub-G1 group, from 23.2 to 31.3%, whereas

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a low concentration CE treatment (20 µg/mL) enhanced the population of cells in the G0/G1

250

phase significantly from 54.1 to 68.3% compared to the control (Fig. 1C). The CE treatment

251

also suppressed the expression of CDK-2, -4, cyclin D1, and cyclin E1 significantly (Fig. 1D).

252

These results show that low concentrations of CE (20 µg/mL) induced cell cycle arrest at the

253

G1 phase, whereas high concentrations of CE (40-80 µg/mL) increased the sub G1 phase

254

significantly leading to apoptosis.

255 256

3.2. CE suppresses the VEGF-induced proliferation of HUVECs through cell cycle arrest at

257

the G1 phase.

258

The proliferation and death of endothelial cells are highly involved in the VEGF-

259

VEGFR2 signaling pathway (Ou et al., 2014). To confirm that CE inhibits cell proliferation

260

under VEGF stimulation conditions, this study investigated the cell viability with a CE

261

treatment in VEGF-stimulated HUVECs. Compared to VEGF-treated HUVECs, CE-treated

262

cells inhibited cell proliferation markedly from 136.5 to 35.5% in a dose- and time-dependent

263

manner (Fig. 2A and B). Similar to Fig. 1A and B, a treatment with high concentrations of

264

CE (40-80 µg/mL) produced significant cytotoxicity in VEGF-induced HUVECs. Compared

265

to the control group, VEGF stimulation facilitated cell cycle entry with upregulation of the S

266

phase and G0/G1 phase from 14.6 to 21.9% and from 48.9 to 51.1%, respectively, in

267

HUVECs. On the other hand, HUVECs incubated with CE and VEGF showed remarkable

268

cell cycle interruption in the G0/G1 phase from 51.1 to 65.5% compared to the VEGF-treated

269

cells (Fig. 2C). Furthermore, the VEGF stimuli enhanced the cell cycle progression proteins,

270

such as CDK-2, -4, cyclin D1 and cyclin E1 in HUVECs, but CE reduced the expression of

271

CDK-2, -4, cyclin D1, and cyclin E1 significantly in VEGF-treated HUVECs (Fig. 2D).

272

Therefore, CE effectively suppressed VEGF-stimulated proliferation and growth in HUVECs

273

through cell cycle arrest at the G1 phase.

274 275

3.3 CE inhibits VEGF-induced endothelial cell migration, invasion and tube formation in

276

vitro.

277

The chemotactic motility, invasion, and tube formation of HUVECs are important events

278

during the vascular tube formation process (Folkamn, 2002; Carmeliet and Jain, 2011b).

279

Compared with the untreated control cells, the VEGF-treated HUVECs indicated a significant

280

increase in cell motility, invasion, and capillary-like tube formation (Fig. 3A-C). On the other

281

hand, the CE treatment significantly suppressed VEGF-stimulated HUVECs migration,

282

invasion, and microtubule development in a dose-dependent manner. For further molecular

283

understanding of the anti-angiogenic property of CE, expression of MMP-9 and TIMP-1,

284

which are major proteins in the angiogenesis process, was analyzed in HUVECs. Treatment

285

with VEGF produced considerable enhancement of TIMP-1 and MMP-9 compared to

286

untreated cells. On the other hand, CE caused the upregulation of anti-angiogenic TIMP-1,

287

and down-regulation of proangiogenic MMP-9 in VEGF-stimulated HUVECs (Fig. 3D).

288

These findings suggest that a CE treatment can block VEGF-stimulated migration, invasion,

289

and capillary-like tubule development through the suppression of MMP-9 expression and

290

enhancement of TIMP-1 expression.

291 292

3.4 CE inhibits VEGF-induced angiogenic sprouting ex vivo.

293

As a response to VEGF stimulation, the sprouting of primary microtubules gradually

294

builds up highly organized vascular networks to supply nutrients, gases, macromolecules, and

295

oxygen towards the site of cancer tissues (Risau, 1997). A rat aortic ring assay was performed

296

to examine the anti-angiogenic activity of CE ex vivo. Compared to the untreated group,

297

VEGF induced the notable sprouting of new blood vessels from the rat aortic ring (Fig. 4). In

298

contrast, a co-treatment of VEGF and CE suppressed microvessel sprouting in rat aortic rings

299

considerably. These results suggest that CE effectively inhibited VEGF-induced angiogenesis

300

both in vitro and ex vivo.

301 302

3.5 CE inhibits the phosphorylation of VEGFR2

303

To determine if the CE-induced antiangiogenic effect was involved in VEGFR2

304

inactivation, the phosphorylation of VEGFR2 was further measured in VEGF-treated

305

endothelial cells. As shown in Fig. 5, the HUVECs incubated with VEGF showed

306

significantly increased phosphorylation of VEGFR2 towards the site of p951 and p1174, but

307

the CE treatment markedly suppressed phosphor-VEGFR2 in VEGF-treated HUVECs.

308

Moreover, HUVECs treated with CE indicated statistically reduced-VEGF expression

309

compared to VEGF-activated HUVECs. Hence, CE effectively inhibits angiogenesis in

310

HUVECs by suppressing the VEGF-VEGFR2 signaling pathways.

311 312

3.6. CE inhibits suppression of intracellular proangiogenic kinases in HUVECs.

313

The stimulation of VEGF activates VEGFR2 followed by a range of signaling proteins in

314

HUVECs. PI3K/AKT/mTOR, MAPK/p38, MAPK/ERK 1/2, FAK, and SRC proteins are

315

involved in the proliferation, permeability, invasion, and actin reorganization of HUVECs

316

(Abhinand et al., 2016). This founding focused on the anti-angiogenesis activity of CE via the

317

regulation of several intracellular proangiogenic kinases, and analyzed the protein expression

318

associated with angiogenesis in VEGF-treated HUVECs. A single treatment of VEGF

319

increased the expression of phosphor-PI3K, AKT, mTOR, p38, ERK 1/2, FAK, and SRC

320

significantly in HUVECs (Fig. 6). On the other hand, the simultaneous treatment of VEGF

321

and CE inhibited the VEGF-facilitated phosphorylation of PI3K, AKT, mTOR, p38, ERK 1/2,

322

FAK, and SRC in HUVECs. These findings suggest that CE suppresses VEGF-stimulated

323

angiogenic property in HUVECs by blocking VEGFR2 and its downstream pathways.

324 325

3.7. CE inhibits VEGF-induced angiogenesis in vivo.

326

A matrigel plug analysis was performed in C57BL/6 mice to further examine the anti-

327

angiogenesis effects of CE in vivo. H&E staining and immune-histochemical (IHC) staining

328

with CD34 antibody were also performed to assess the permeability of HUVECs, and the

329

development of microvessels (Fig. 7). Compared to matrigel in the untreated mice, the

330

VEGF-treated group showed an obvious red color filled with red blood cells and

331

microvascular vessels (Fig. 7A). On the other hand, the matrigel treated simultaneously with

332

VEGF and CE showed a significant intensity of red color compared to the VEGF group.

333

Furthermore, the accumulations of H&E stained (dark blue or violet color) and CD34 positive

334

(dark brown) endothelial cells were enhanced dramatically in the VEGF-treated matrigel than

335

in the untreated group (Fig. 7B). These H&E stained- and CD34 positive-endothelial cell

336

accumulation was attenuated significantly in the matrigel co-treated with VEGF and CE (10-

337

20 µg/mL) (Fig. 7B). These results suggest that CE effectively blocked VEGF-induced

338

endothelial cell accumulation in the pathological in vivo model of angiogenesis.

339 340

4. Discussion

341

The aim of this study was to develop a natural medicine for cancer-related angiogenesis

342

using a cultivated O. japonicus extract. The O. japonicus extract using 70% EtOH

343

demonstrated potent angiogenesis inhibition effects without significant toxicity in vitro, ex

344

vivo, and in vivo. Although further studies should be needed to identify the relevance of

345

cancer metastasis inhibition and CE, these results suggest that CE is a potential source of

346

natural medicine with anti-angiogenesis effects.

347

Numerous chronic diseases, including cancer species, Alzheimer’s disease, obesity, and

348

diabetes, are commonly caused by multifactorial elements via complex molecular

349

mechanisms. In particular, tumors are related to a range of genetic mutations in multiple

350

stages, and can rarely be treated with single-target chemotherapies (Cesca et al., 2013). As a

351

result, multi-target agents have attracted significant interest because they may simultaneously

352

block diverse signaling pathways related to cancer growth, angiogenesis, and metastasis

353

(Talevi, 2016; Jia et al., 2009). Several studies have reported that O. Japonicus is a safe and

354

effective natural product with multi-target anti-cancer activity and low cytotoxicity to normal

355

cells (Ryu et al., 2014; Lee, Kim, Lee, 2018; Shin et al., 2013). Previous studies have

356

indicated that cultivated O. Japonicus extracts effectively inhibit the vascularization of

357

HUVECs (Cho et al., 2019) and proliferation of human colorectal cancer (Kim et al., 2012) at

358

concentrations of 20 µg/mL and 100 µg/mL, respectively. In the current study, high

359

concentrations of CE (40-80 µg/mL) showed significant cytotoxicity to HUVECs, whereas

360

low concentration of CE (20 µg/mL) did not have a significant proliferation inhibitory effect

361

on HUVECs. On the other hand, 20 µg/mL of CE displayed strong cell cycle arrest, including

362

the reduction of the cell cycle check point proteins such as, CDK2, CDK4, cyclin E1 and

363

cyclin D1, at G0/G1 phase. Since regulation of cell cycle is closely involved with

364

angiogenesis and endothelial cell growth (Ingber et al., 1995), these results suggest that CE

365

can regulate the mitogenesis of HUVECs at lower concentrations (<20 µg/mL) without cell

366

death.

367

A range of proangiogenic factors, such as VEGF, MMPs, receptor tyrosine kinase (RTK),

368

hypoxia-inducible factor (HIF-1) and cell cycle checkpoint proteins, are closely related to

369

growth, motility, invasion, and tubule development of endothelial cell (Ribeiro, 2018).

370

Among these proangiogenic factors, VEGF plays a critical role against the stimulation of

371

endothelial cells. Moreover, VEGFR2 is a major receptor in the VEGF-induced angiogenesis

372

process (Holmes, 2007). MMPs, extracellular matrix proteases, are associated mainly with

373

tumor metastasis and endothelial cell invasion (Yoon et al., 2003; Zheng et al., 2006).

374

Although there is some controversy regarding TIMP-1 as an angiogenesis inducer, MMP-2

375

and -9 can be regulated systematically by TIMP-1 in HUVECs (Reed et al., 2003; Akahane et

376

al., 2004; Baker, Edwards, and Murphy, 2002; Kopitz et al., 2007; Cui et al., 2014). In the

377

present study, HUVECs proliferation and cell cycle progression were enhanced by VEGF,

378

but a treatment with CE (20 µg/mL) reduced the VEGF-facilitated proliferation dramatically

379

in HUVECs with G0/G1 phase cell cycle arrest. Furthermore, 10-20 µg/mL of CE effectively

380

blocked the VEGF-induced chemotactic migration, invasion, and capillary-like tube

381

formation via a decrease in MMP-2 and an increase in TIMP-1. Lee et al. (2017) reported that

382

bioconverted O. japonicus 70% MeOH extracts inhibited the migration, adhesion, and tube

383

formation of mouse endothelial cells significantly at concentrations ranging from 50 to 100

384

µg/mL. On the other hand, these results suggest that even lower concentrations of CE can

385

effectively inhibit the chemotactic angiogenesis of HUVECs. Because the initial treatment of

386

tumors using cytotoxic agents may lead to undesirable side effects to the normal organs, a

387

combination strategy with a non-toxic angiogenesis inhibitor will allow more synergic

388

activity to improve the drug efficacy without significant adverse reactions (Byrne, Bouchier-

389

Hayes, and Harmey, 2005). These studies suggest that lower concentrations of CE have a

390

broad-spectrum of potential for angiogenesis inhibition without significant cytotoxicity in

391

HUVECs.

392

VEGFR2 is activated in response to VEGF stimulation, resulting in phosphorylation of

393

the Tyr801, Tyr951, Tyr996 Tyr1054, Tyr1059, Tyr1175, and Tyr1214 sites (Smith et al.,

394

2015). Subsequently, cytoskeletal changes, extracellular matrix degradation, and focal cell

395

adhesion in HUVECs for migration are regulated by the assembly or disassembly of FAK-Src

396

at the c-terminal of VEGFR2 (Rousseau, Houle, and Huot, 2000; Schlessinger, 2000).

397

Previous evidence suggests that VEGFR2 activation triggered by exogenous and/or

398

endogenous VEGF promotes cell proliferation, migration, invasion, and tube formation by

399

regulating the PI3K/AKT/mTOR, MAPK/ERK 1/2, MAPK/p38 signaling pathways (Karar

400

and Maity, 2011; Abhinand, 2016; Zhang et al., 2010). In the present study, CE (10-20

401

µg/mL) inhibited autocrine/paracrine of VEGF and the VEGF-promoted phosphorylation of

402

VEGFR2 at its auto-phosphorylation sites (Tyr 951 and Tyr1175). Moreover, the downstream

403

signaling pathways of VEGF-VEGFR2, such as PI3K/AKT/mTOR, MAPK/ERK 1/2,

404

MAPK/p38, FAK, and Src, were suppressed by the CE treatment in VEGF-treated HUVECs.

405

Sorafenib (Nexavar, BAY 43-9006) suppressed VEGF-stimulated endothelial cell migration,

406

invasion and capillary-like tubule formation at concentrations of 1.5-3 µM without significant

407

cytotoxicity (Supplementary data 3), as well as various types of tumors-related angiogenesis

408

by targeting the VEGFR-related MAPK/ERK and MAPK/p38 pathways (Wilhelm et al.,

409

2004). In addition, sunitinib malate inhibits the tyrosine kinases of VEGFRs or platelet-

410

derived growth factor receptors (PDGFRs) that then blocks the downstream signaling

411

pathways (Christensen, 2007). Although further work will be needed to determine if CE

412

inhibits the VEGFR2 activity directly as an anti-VEGFR2 agent or specific phenolic

413

compounds in CE bind competitively to VEGFR as a VEGF antagonist (Multhoff, Radons,

414

and Vaupel, 2014), these results clearly suggest an antiangiogenic strategy to reduce

415

autocrine and/or paracrine-enhanced VEGFR2 signaling pathways using a low toxic natural

416

medicine.

417

A number of natural products and food materials, including green teas, soy beans, parsley,

418

onions, berries, apple peels, ginseng radix, Silybum marianum, Lithospermum erythrorhizon,

419

Ginkgo biloba, Artemisia annua, and Capsicum annum, have provided clear evidence of the

420

beneficial effect on cancer-related angiogenesis inhibition (Wahl et al., 2011). Previous

421

studies have shown that CE contains numerous types of phytochemicals, such as gallic acid

422

(21.84 mg/g), epicatechin-gallate (6.58 mg/g), quercetin-3-glucose (2.74 mg/g), kaempferol-

423

3-D-glucopyranoside (5.11 mg/g), kaempferol-3-rutinoside (3.09 mg/g), kaempferol (6.23

424

mg/g) and quercetin (8.55 mg/g) (Cho et al., 2019; Supplementary data 1, 2). Based on our

425

HPLC data, 2.29 µM of gallic acid, 0.29 µM of epicatechin-gallate, 0.11 µM of quercetin-3-

426

glucose, 0.22 µM of kaempferol-3-D-glucopyranose, 0.10 µM of kaempferol-3-rutinoside,

427

0.42 µM of kaempferol, and 0.56 µM of quercetin were contained in 20 µg of CE. In addition,

428

major secondary metabolites in CE, including gallic acid (Zhao and Hu, 2013), epicatechin-

429

gallate (Tang, Nguyen, and Meydani, 2003), kaempferol (Kim, 2017), and quercetin

430

(Pratheeshkumar et al., 2012), have already been identified as effective phenolics on

431

angiogenesis suppression on VEGF-stimulated endothelial cells at the concentrations of 0.5-

432

30 µM. Therefore, the abundant phenolic compounds in CE have a possible role in inhibiting

433

angiogenesis by regulating the PI3K/AKT/mTOR, MAPK/ERK 1/2, MAPK/p38, FAK-Src,

434

and VEGFR2 pathways.

435

In conclusion, CE regulated the angiogenic factors in vitro, ex vivo and in vivo. CE

436

inhibited cell migration, invasion, and capillary-like tube formation in VEGF-stimulated

437

HUVECs. In addition, it suppressed microvessel sprouting from VEGF-induced rat aortic

438

rings and matrigel in C57BL/6 mice. The anti-angiogenic activity of CE was related to the

439

inhibition of PI3K/AKT/mTOR, MAPK/ERK, MAPK/p38, FAK-Src, and VEGFR2

440

signaling pathways in HUVECs. Overall, these findings suggest that CE has a potential as

441

multi-targeting herbal medicine to prevent cancer-involved angiogenesis.

442 443

Conflicts of interest

444

The authors declare that there are no conflicts of interest.

445

446

Acknowledge

447

This work was supported by the National Research Foundation of Korea (NRF) grant funded

448

by the Korea government (MEST; 2017R1A2B4009097).

449 450

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611

612

Figure legends

613

Fig. 1. Effect of cultivated O. japonicus 70% EtOH extract (CE) on proliferation and cell

614

cycle interruption in HUVECs. HUVECs were incubated with CE (5-80 µg/mL) for 24 h.

615

After CE treatment, (A) cell viability was determined by SRB assay, and (B) cell morphology

616

was visualized by inverted microscope (×200). (C) Cell cycle population of HUVECs was

617

measured using flow cytometer. (D) Expression of cell cycle related proteins was detected by

618

western blotting analysis. Total cell lysates were loaded by SDS-PAGE gel electrophoresis,

619

and each protein band was analyzed with respective antibodies. Data values were expressed

620

as mean ± SD of triplicate determinations. Significant of difference was compared with the

621

control at *P<0.05,

622

comparison.

623

Fig. 2. Effect of CE on VEGF-induced cell proliferation and cell cycle progression in

624

HUVECs. Cells were pretreated with VEGF-A (20 ng/mL) for 2 h, followed by treatment of

625

CE (5-80µg/mL) for 12-24 h. (A) After incubation in CO2 incubator for 12-24 h, cell viability

626

was determined by SRB assay. (B) Morphological pictures were visualized by inverted

627

microscope (×200). (C) Cell cycle distribution in HUVECs was measured with flow

628

cytometer. (D) Expression of cell cycle related protein was detected by western blotting

629

analysis. Total cell lysates were separated by SDA-PAGE gel electrophoresis, and each

630

protein band was analyzed with respective antibodies. Data values were expressed as mean ±

631

SD of triplicate determinations. Significant of difference were compared with the control at

632

##

633

way ANOVA and Tukey’s multiple comparison.

634

Fig. 3. Effect of CE on VEGF-induced chemotactic migration, invasion, and capillary-like

635

tubule structure development in HUVECs. (A) HUVECs were scratched by a pipette and co-

636

treated with or without 20 ng/mL of VEGF and 10-20 µg/mL of CE. After 12 h incubation,

P<0.01,

###

**

P<0.01 and

***

P<0.001 by one-way ANOVA and Tukey’s multiple

P<0.001 and with VEGF group at *P<0.05,

**

P<0.01, and

***

P<0.001 by one-

637

moved cells were photographed by microscope (magnification, ×200), and quantified by cell

638

number counting. (B) HUVECs were seeded on the upper surface of plates with the 10-20

639

µg/mL of CE, and the bottom chambers were filled with 1 mL of ECGM supplemented with

640

50 ng/mL of VEGF-A. After incubation for 16 h, invaded cells through the trans-well

641

membrane was photographed by microscope (magnification, ×200), and quantified by cell

642

counting. (C) HUVECs were cultured in ECGM medium containing 0.5% FBS for 6 h, and

643

pretreated with CE (10-20 µg/mL) for 1 h. After gathering HUVECs, cells were seeded on the

644

thin collagen layer (matrigel), and cultured for 6 h in ECGM medium containing 2% FBS and

645

50 ng/mL of VEGF-A. Capillary structure formation of endothelial cells was photographed

646

by microscope, and quantified by measuring the number of branching points per unit area. (D)

647

The expression level of MMP-9 and TIMP-1 were determined by western blotting analysis.

648

Total cell lysates were separated by SDA-PAGE gel electrophoresis, and each protein band

649

was analyzed with respective antibodies. Data values were expressed as mean ± SD of

650

triplicate determinations. Significant of difference were compared with the control at

651

###

652

Tukey’s multiple comparison.

653

Fig. 4. Effect of CE on VEGF-induced ex vivo microvessel sprouting. Aortic rings isolated

654

from Sprague-Dawley rats were incubated on matrigel-coated 96-well plates, and treated with

655

VEGF in the presence or absence of CE. After incubation for 6 days, representative aortic

656

rings were photographed by microscope (magnification, ×200). Sprouts from rings were

657

scored from 0 to 5 in a double-blind manner. Data values were expressed as mean ± SD of

658

triplicate determinations. Significant of difference were compared with the control at

659

###

660

comparison.

661

Fig. 5. Effect of CE on the activation of VEGF and VEGFR2 in HUVECs. HUVECs were

P<0.001, and with VEGF group at

P<0.001, and with VEGF group at

**

P<0.01 and

***

P<0.001 by one-way ANOVA and

***

P<0.001 by one-way ANOVA and Tukey’s multiple

662

pretreated with or without CE for 2 h, followed by VEGF treatment for 2 h. Total cell lysates

663

were subjected to detect expression levels of proteins. Data values were expressed as mean ±

664

SD of triplicate determinations. Significant of difference were compared with the control at

665

###

666

Tukey’s multiple comparison.

667

Fig. 6. Effect of CE on expression of PI3K/AKT/mTOR, MAPK/p38, FAK-Src, and

668

MAPK/ERK 1/2 pathway in HUVECs. Cells were treated with or without CE for 2 h,

669

followed by VEGF-A treatment for 1 h. Total cell lysates were subjected to detect expression

670

levels of proteins. Data values were expressed as mean ± SD of triplicate determinations.

671

Significant of difference were compared with the control at

672

VEGF group at ***P<0.001 by one-way ANOVA and Tukey’s multiple comparison.

673

Fig. 7. Effect of CE on VEGF-induced angiogenesis in vivo. Five-weeks-old C57BL/6 mice

674

were injected with 0.5 mL of matrigel containing 10-20 µg/mL of CE, 150 ng/mL of VEGF,

675

and 20 units of heparin into the ventral area. (A) After 6 days, representative matrigel plugs

676

extracted from each group of mice were photographed. (B) Matrigel plugs obtained from

677

each group of mice were embedded in paraffin, and stained with H&E staining and

678

immunohistochemical staining (CD34) to visualize blood vessels and endothelial cells. Scale

679

bar, 100 and 2000 µm.

P<0.001, and with VEGF group at *P<0.05 and

***

P<0.001 by one-way ANOVA and

##

P<0.05,

###

P<0.001, and with

680

Authors contribution

681

Cho HD and Lee KW mainly performed all the experiments, and wrote this manuscript. Won

682

YS and Kim JH assisted experiments, and reviewed manuscript. Seo KI supervised the

683

experiments, and participated in study design and coordination, and material support for

684

funding obtaining. All authors reviewed and approved the final manuscript.

685

▶ CE blocked cell cycle progression in G0/G1 phase. ▶ CE inhibited VEGF-induced endothelial cell migration, invasion, and differentiation into capillary-like structure in vitro ▶ CE inhibited phosphorylation of PI3K/AKT/mTOR and MAPK signaling pathways in HUVECs ▶ CE inhibited activation of VEGFR2 ▶ CE suppressed VEGF-induced angiogenesis in vivo