Anti-angiogenic activity of water extract from Euphorbia pekinensis Rupr

Journal of Ethnopharmacology 206 (2017) 337–346

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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep

Anti-angiogenic activity of water extract from Euphorbia pekinensis Rupr a

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Wenting Zhang , Bin Liu , Yaru Feng , Jie Liu , Zhiqiang Ma , Jian Zheng , Qing Xia , ⁎ Yuanyuan Nia, Farong Lic, Ruichao Lina, a

Beijing Key Lab for Evaluation of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China Department of Ethnodrug, National Institute of Traditional Chinese Medicine, National Institutes for Food and Drug Control, Beijing 100050, China Key Laboratory of Ministry of Education for Medicinal Resources and Natural Pharmaceutical Chemistry, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shanxi Normal University, Xi'an 710062, China b c

A R T I C L E I N F O

A BS T RAC T

Keywords: Euphorbia pekinensis Rupr. Anti-angiogenesis Transgenic zebrafish HUVEC Quantitative real-time PCR

Ethnopharmacological relevance: Euphorbia pekinensis Rupr. (EP) is a Euphorbia species of Euphorbiaceae, which is widely used in traditional Chinese medicine. It has been reported to exhibit therapeutic effects on solid tumors, leukemias, and malignant ascites although underlying molecular mechanisms are poorly delineated. Anti-angiogenic therapy is a recognized strategy for treating cancer-based solid tumors, and is also associated with malignant ascites treatment. Study aim: To study the anti-angiogenic properties of the water extract of EP vinegar preparation (WEVEP). Materials and methods: Following WEVEP treatment, intersegmental blood vessels were assessed during the development of transgenic Tg (flk: mCherry) zebrafish as was the proliferation, migration and network formation of HUVECs in vitro. mRNA expression of specific angiogenic-related genes including VEGF family members, Met, and NRP2 was also measured using quantitative real-time PCR (Q-PCR). Results: Data demonstrated that angiogenesis was inhibited by the WEVEP in zebrafish (from 100 µg/mL to 250 µg/mL, p < 0.0001) and in the HUVEC model (from 100 µg/mL to 400 µg/mL, p < 0.0001). In the zebrafish model, the mean vessel numbers of administered groups were 26.00 ± 1.29 (100 µg/mL), 24.54 ± 2.20 (150 µg/mL), 22.66 ± 2.68 (200 µg/mL), 20.80 ± 1.75 (250 µg/mL), compared to 27.67 ± 0.96 of control group. Relative quantitative gene expression in zebrafish treated with WEVEP demonstrated that only VEGFR3 was significantly increased and other 23 genes including Met, VEGFA, Flt-1 were significantly decreased. Conclusion: WEVEP can positively modulate angiogenesis via multiple targeting mechanisms. Our novel results contribute towards the discovery of a possible mechanism(s) of the traditional use of EP in the treatment of cancer and malignant ascites.

1. Introduction Euphorbia pekinensis Rupr. (EP) is often used to treat edema and ascites, but it is documented to be toxic, depending upon the manner in which it is prepared (Zeng et al., 2013; Zhang et al., 2006). The anticancer properties of EP were previously described when it was demonstrated to inhibit synthesis of leukemic cells in a L615 mouse model of leukemia and prolong their survival time (Zhang et al., 2006). Based on data with regards to cellular proliferation and DNA measurements in the KY821 leukemia cell line, it was confirmed that EP had the ability to block S phase cells by inhibiting DNA in this cell line which may provide a basis for an anti-leukemic mechanism (Shang et al., 2000). Pekinenal, which is isolated from extract of EP, demonstrated anti-hepatoma effects by inhibiting cancer cell DNA synthesis and ⁎

arresting tumors in the S phase (Chen et al., 2016). However, the mechanism of action for pekinenal is currently unknown. Two compounds derived from Eupborbia plants Euphorbia microsciadia Boiss and Euphorbia fischeriana, 3-O-propionyl-5, 10, 14-O-triacetyl-8-O(20 -methyl- butanoyl)-cyclomyrsinol and 12-deoxyphorbol 13-palmitate (G), were reported to have anti-angiogenic activity (Mendonà et al. 2009; Xu et al., 2013). It is recorded in the “Compendium of Materia Medica” that EP can dredge the retention of water which may form such pathological substances as water, dampness and phlegm retention, and usually seen in edema, tympanites and phlegm-retention. EP has been shown to be involved in the elimination of potent excessive heat, abdominal masses and extravasated blood. This purging process has previously been used as a therapeutic strategy for tumor shrinkage or eradication

Correspondence to: No. 6, Wangjing Central South Road, Chaoyang District, Beijing 100102, China. E-mail addresses: [email protected] (W. Zhang), [email protected] (R. Lin).

http://dx.doi.org/10.1016/j.jep.2017.05.033 Received 22 December 2016; Received in revised form 11 May 2017; Accepted 27 May 2017 Available online 07 June 2017 0378-8741/ © 2017 Published by Elsevier Ireland Ltd.

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as recorded in “Huangdi Neijing.” Angiogenesis is a normal physiological process that occurs during embryonic and fetal development (Schuermann et al., 2014; Carmeliet and Jain, 2011). During adulthood, angiogenesis is associated with wound healing, skeletal growth, the menstrual cycle, and pregnancy. Abnormal angiogenesis is also associated with specific disease states including intraocular neovascular disorders, immunogenic rheumatoid arthritis, psoriasis, and tumorigenesis (Folkman, 1971; Carmeliet and Jain, 2011). Pathological angiogenesis is often enduring and is associated with deleterious effects, as seen in the chronic progression of diabetic retinopathy and solid tumors (Carmeliet and Jain, 2011). In the case of solid tumors, oncogenic transformation has been shown to play a pivotal role in promoting tumor vascular growth through a number of independent and redundant mechanisms (Chung and Ferrara, 2011). In contrast to normal vessels, tumor vessels are highly disorganized, as well as tortuous and dilated, with uneven diameter, excessive branching and shunts. This may be due to an imbalance of angiogenic regulators, such as VEGF and angiopoietins. Consequently, tumor blood flow is chaotic and variable and leads to hypoxic and acidic regions in tumor (Carmeliet and Jain, 2000). Meanwhile, hypoxia has emerged as a primary physiological regulator of the angiogenic switch (Liao and Johnson, 2007). With alterations of local pH, the hypoxia can upregulate angiogenesis-related genes to promote angiogenesis (Muz et al. 2015). Angiogenesis is important to disease, but in cancer, it is executed in a poorly controlled manner during solid tumor development, leukemia and malignant ascites (Amano et al., 2007; Eskander and Tewari, 2012; Folkman, 1971). Angiogenesis has been identified as a signal of cancer development, and anti-angiogenic therapy is a promising avenue for cancer treatment. Since, tumor growth and proliferation depend on tumor vessel activity, angiogenesis may be used to evaluate tissue pathology classification, radiation therapy, and prognosis (Weis and Cheresh 2011). Therefore, identifying anti-angiogenesis targets and the underlying pharmacology of EP may point to potential cancer treatments as well as delineate a potential mechanism of action. Zebrafish have become a widely used vertebrate model organism for genetic, developmental research and drug discovery, because of its fecundity and genetic similarity to mammals (Golling et al., 2002). In addition, zebrafish embryos are transparent, and all internal organs and structures can be observed microscopically without injuring the organism. The vascular system includes major and sprout vessels become functional in day 3 during development of zebrafish embryos. Zebrafish are similar to humans with respect to blood vessel structure, physiology and molecular aspects of angiogenesis (Stern and Zon, 2003; Tobia et al., 2011). Therefore, zebrafish is a good model for vascular system analyses in vivo. As demonstrated in other vertebrate systems, the primary vasculature in zebrafish involves the differentiation of hemangioblasts from the mesoderm which then becomes segmented into angioblasts and subsequently into endothelial cells. This stage is generated via vasculogenesis (Tobia et al., 2011). By the 12-somite stage (approximately 12 h post fertilization, hpf), cells of the lateral mesoderm have begun to express hemangioblast markers such as SCL/Tal-1 and Flk1. By 24 hpf, the formation of the dorsal aorta (DA) and axial vein (AV), as well as the blood circulation over the yolk sac through the Ducts of Cuvier (DC) is complete (Serbedzija et al., 1999) while trunk circulation of zebrafish embryo begins at approximately 24–26 hpf. Subsequent sprouting and branching of blood vessels is formed by angiogenesis (i.e., the formation of new blood vessels from pre-existing vessels). During embryonic angiogenesis, the intersegmental vessels are subsequently formed by angiogenic sprouting from dorsal aorta to the dorsal side of the trunk (Tobia et al., 2011), and then the dorsal longitudinal anastomotic vessels (DLAVs) were consequently formed. We chose to analyze the number of intersegmental vessels following WEPEV treatment to determine its effects on vessel growth and density. The clinical use of EP has been reported after vinegar preparation

conformed to the criterion of Chinese Pharmacopoeia. Within the context of this study, we chose to utilize the water extract of EP's vinegar preparation to explore its potential anti-angiogenic activity. 2. Materials and methods 2.1. Plant and extraction The raw herb of EP was purchased from Anguo (HeBei Province, China), and authenticated by Professor Yaojun Yang (School of Chinese Materia Medica, Beijing University of Chinese Medicine). The specimen was deposited in the herbarium in School of Chinese Materia Medica. EP was then processed according to Chinese Pharmacopoeia 2010 before extraction. A vinegar preparation of EP (VEP) was crushed and sieved through a 40-mesh sieve. The powder (30 g) was extracted with water (300 mL) by refluxing twice, 1.5 h each time. After merging and filtering, the supernatant was collected for concentration under vacuum, and then dried in a drying oven at 60 °C for 48 h. This yielded a dry extract powder of 3.28 g (extraction efficiency 10.94%). The dried extract powder was kept in a desiccator before use. 2.2. Cell culture and reagents Primary human umbilical vein endothelial cells (HUVECs) were purchased from the China Infrastruture of Cell Line Resources (Beijing, China). HUVECs used in all experiments were between passages 10–15 and were cultured in 6-cm tissue culture dishes with Dulbecco's Modified Eagle Medium (DMEM, Genview, China) containing 10% Fetal Bovine Serum (GIBCO, NY, USA) according to manufacturer's instructions. Cells were incubated at 37 °C with 5% CO2 in air. Matrigel (BD Biosciences, CA, USA) was used in HUVEC network formation assays. Vatalanib (PTK787) 2HCl (Selleck Chemicals, Houston, TX), a selective VEGFR2/KDR inhibitor, was used as positive control (Wood et al., 2000; Murakami et al., 2011; Cross et al., 2003). 2.3. Zebrafish culture The transgenic (Tg) zebrafish line (flk: mCherry) with red fluorescent expression in blood vessels was purchased from the laboratory of Bo Zhang at Peking University and utilized according to published methods (Xia et al., 2013). Zebrafish were maintained at 28 °C on a 14 h/10 h light/dark photoperiod and were fed with brine shrimp three times daily. 2.4. Inhibition of vessel formation in zebrafish embryos During embryonic development, the most prevalent angiogenic activity occurs between 24 and 72 hpf, which can be evaluated by counting fluorescent intersegmental blood vessels (ISV) (Camus et al., 2011). Following fertilization, the embryos were collected and raised in embyro water, which consisted of deionized water containing 5 mmol/ L NaCl, 0.17 mmol/L KCl, 0.4 mmol/L CaCl2, 0.16 mmol/L MgSO4. The LD50 of VEP water extract was 250–300 µg/mL as measured in previous experiments (unpublished data). Zebrafish embryos at 6 hpf were arrayed in 24-well plates with 20 embryos per well and incubated at 28 °C until 72 hpf in 3 mL embryo water containing drugs (100– 250 µg/mL). We chose the embryo water containing 0.1% DMSO as the vehicle control and the embryo water containing 100 ng/mL PTK787 2HCl as the positive control. 10 larvae were photographed in each group using an Axio Zoom V16 fluorescence microscope (Zeiss, Germany). Due to the uncontrolled changes of concentration and pH of the original solution, the media of each well was refreshed every 24 h and the debris was removed to keep the media clean and oxygenated. Since the media of each well was refreshed every 24 h, the drug solutions were prepared at one time then divided into three parts in order to 338

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Table 1 Angiogenesis-related gene in test. Gene categories

Gene names

Angiogenesis-related growth factors and receptors

EGFR (Epidermal growth factor receptor a), Met (MET proto-oncogene, receptor tyrosine kinase), IGF1 (Insulin like growth factor 1), CTGF (Connective tissue growth factor), NRP2 (Neuropilin 2), VEGFA, Flt-1 (Vascular endothelial growth factor receptor 1), KDR (Kinase insert domain receptor), VEGFR3 (Vascular endothelial growth factor receptor 3) Ets-1 (v-ets avian erythroblastosis virus E26 oncogene homolog 1), HIF1A (Hypoxia inducible factor 1 alpha subunit) MMP-2 (Matrix metallopeptidase 2), MMP-9 (Matrix metallopeptidase 9), TIMP2 (TIMP metallopeptidase inhibitor 2), PLG (Plasminogen) ITGAV (Integrin alpha V), ITGβ3 (Integrin β-3), β -catenin (cadherin associated protein), PECAM1 (Platelet endothelial cell adhesion molecule) Tie-2 (TEK tyrosine kinase), PDGFR-β (Platelet derived growth factor receptor-β), CDH5 (vascular endothelium Type 2 cadherin 5), S1PR1 (Sphingosine-1-phosphate receptor 1), FGF2 (Fibroblast growth factor 2), FGFR2 (Fibroblast growth factor receptor 2), Shh (Sonic hedgehog), TGFβ1 (Transforming growth factor β 1), TGFRβ1 (Transforming growth factor 1 receptor β) Ephrin B2, EPHB4 (Ephrin type B receptor 4)

Transcription factors Matrix degradation/endothelial cell migration factors Cell adhesion factors Tubule formation and morphogenesis/smooth muscle cell recruitment and differentiation factors

Blood vessel maturation/formation factors

after 24 h. The total tubule length was measured using Image-J software as previously described.

maintain consistency of drug action. After administration, the remaining two parts were stored at −20 °C and thawed before use. 2.5. Cell proliferation assay

2.8. mRNA expression in zebrafish determined by qRT-PCR

Cell proliferation was measured using a CCK-8 kit (Greiner, Germany) according to the manufacturer's instructions (Wang et al., 2016b). HUVECs (1 × 104 per well) were seeded in a 96-well plate in growth medium (DMEM containing 10% FBS) for 12 h. Cells were treated for 24 h in the growth medium containing 0.1% DMSO and VEP water extract at different concentrations of 25, 50, 100, 200, 300, 400 µg/mL. Cells were treated for 24 h in the growth medium containing 0.1% DMSO as controls. Subsequently, CCK-8 solution (10 µL of a stock solution) was added to each well, and then the plate was incubated at 37 °C for 2 h. Viable cells were counted (450 nm) and expressed as percent of controls.

Zebrafish embryos at 6 hpf were placed in 24-well plates with 20 embryos per well and incubated at 28 °C until 72 hpf in 3 mL embryo water containing VEP water extract (150 µg/mL). Embryos incubated in normal embryo water served as a control group. Zebrafish larvae were kept in the Sample Protector for RNA/DNA extraction (Takara, Tokyo, Japan) per manufacturer's instructions. Total RNA from zebrafish larvae were extracted using a MiniBEST Universal RNA Extraction Kit (Takara, Tokyo, Japan) according to the manufacturer's instructions. Quantitative real-time PCR was employed utilizing an Applied Biosystems 7500 Fast Real Time System (Thermo Fisher Scientific, MA) using One Step SYBR PrimeScript RT-PCR Kit II. We chose the quantitation-comparative CT (△△CT) method as previously described (Huang et al., 2015) and cycling conditions were as follows: 42 °C for 5 min, 95 °C for 10 s, then 40 cycles of 95 °C for 3 s and 60 °C for 30 s. Genes undergoing PCR testing are provided in Table 1 and the primer pairs used for PCR are provided in Table 2. Primers were synthesized by Beijing Dingguo Changsheng Biotechnology Co. Ltd. (Beijing). βactin was a house-keeping gene. All experiments were performed three independent times.

2.6. Cell migration assay HUVEC migration was measured using the wound healing method (Liu et al., 2013). Briefly, HUVECs (1 × 106 per well) were seeded into 6-cm cell culture dishes and incubated with DMEM at 37 °C and 5% CO2 in air. HUVECs were scraped vertically with a 200 µL pipette tip (Eppendorf, Hamburg, Germany) and the “scratch” was photographed in each well using a bright-field microscope (100×). Media was replaced with fresh media in two different concentrations of VEP water extracts and 0.1% DMSO as control. After 12 and 24 h incubation, a second and third set of images were obtained. Then the media was replaced with complete DMEM media and incubated for another 24 h. A fourth set of images was subsequently photographed. To measure migration of HUVECs, images were analyzed using Image-J software (Fiji version) as described (Outeda et al., 2014). We then measured the area which were surrounded by cells as the closed area. The percent of the closed area was measured and compared with values obtained before treatment. Increases in the percent of closed areas (% control) indicated cell migration.

2.9. Statistical methods All analyses in this study were carried out by GraphPad Prism 7 Version 7.0a software (GraphPad Inc., CA, USA). A Kruskal-Wallis test was used for the non-parametric data, and each experimental group was compared with the control group followed by Mann-Whitney testing (Liu et al., 2014). One- way ANOVA followed by Dunnett's multiple comparisons test was used to analyze the statistical significance of other results. P values < 0.05 were considered statistically significant.

2.7. Cell network formation assay The effects of VEP water extract on HUVEC differentiation and vascular formation were assessed by tube formation in Matrigel (Merchan et al., 2003). HUVECs were seeded onto 24-well plates at 1.5 × 105 cells per well over 400 µL Matrigel (BD Biosciences, CA) which was pre-cooled at −20 °C. Fresh media in two different concentrations of VEP water extracts (100 µg/mL and 200 µg/mL) and 0.1% DMSO as control were subsequently added. Each group was arrowed in three parallel wells. Tubular structures were photographed

2.10. Ethics statement The zebrafish used in this study were utilized in accordance with the Regulation on the Administration of Laboratory Animals (2013 Revision, Document Number: Order No. 638 of the State Council) for experimental care and use of animals. 339

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Table 2 The primers of tested genes. Genes name

Forward primer

Reverse primer

EGFR Met CTGF HIF1A MMP-2 IGF1 NRP2a VEGFAa Flt-1 KDR VEGFR3a Ets1 TIMP2 PLG MMP9 ITGβ3a ITGAV PECAM1a Tie2 β-catenina PDGFRβa CDH5a S1PR1a FGF2a FGFR2a Shha TGFβ1a TGFRβ1a Ephrin B2a EPHB4a actina

5′-TGGCTATGTTCTTATCGCGGT-3′ 5′-ATGTGTGGTCGTTTGGTGTTTTG-3′ 5′-CTGGACGGTGCTGTAGGTTGC-3′ 5′-TCTCACCTGGACAAAGCCTCCATT-3′ 5′-GCTGGTGATGAGATGTGGGTATA-3′ 5′-GTCTAGCGGTCATTTCTTCCA-3′ 5′-ATACACACACTCAGACTCGCGCTT-3′ 5′-GAACTTGGTTGTTTATTTG-3′ 5′-TGTAAAGGACGGTTGACGAGTGT-3′ 5′-GATGGAGATACACACCTTCAG-3′ 5′-AAATACATCCCAGTCAAAGCAA-3′ 5′-AGCAGAGGTAAGCTGGGTGG-3′ 5′-ATAAGCATGCGCTGAGGAAGAGGA-3′ 5′-CATGCAAAGGCCTTGATGGGAACT-3′ 5′-AACCACCGCAGACTATGACAAGGA-3′ 5′-TGGGTTTGGCTGCTCTCTTG-3′ 5′-ACGGCTCTCTGCTTTACATCACCA-3′ 5′-CAGGAGGTCAAGAGTGAAGTT-3′ 5′-TGAGCTACCTGAGCCAGAAACAGT-3′ 5′-ATGAGGGCATGCAGATACCTTCCA-3′ 5′-TGGGTCCTCACATCAACATCGTCA-3′ 5′-TGGACACCAATGGCTACGATGTCT-3′ 5′-GCATCGACAGCATGAACAACTGCT-3′ 5′-GTCGGCCAAATGCTGATGGAGAAA-3′ 5′-AGGACAAACCGAAAGAGGCTGTGA-3′ 5′-GCAAGATAACGCGCAATTCGGAGA-3′ 5′-TCTGGGAACTCGCTTTGTCTCCAA-3′ 5′-GTCATCGCCTCCTTCTACTG-3′ 5′-AGGTGGTTCAATGGAAGGAGTGGA-3′ 5′-ATTCCTCGTCTCCGCTGCTTGTTA-3′ 5′-TCCCCTTGTTCACAATAACC-3′

5′-TCATTCTCTGGCACTTGTCGG-3′ 5′-TGATATGAGAGAGGGGTAGGGTG-3′ 5′-AGACTCGTGGTGTGCGGATGC-3′ 5′-AAGCCATTCAGCTGACTTTCCAGC-3′ 5′-TTCTTGGTCTTGTGGAAGGAGTA-3′ 5′-ATAGTTTCTGCCCCCTGTGTT-3′ 5′-AGGAATTGGTGTTGCGCACAGAAG-3′ 5′-TACTCCTGGATGATGTCTA-3′ 5′-GTTGTTGTTGGAGAGCAGGATGT-3′ 5′-TGCGTACCGATGACACATTTC-3′ 5′-AGAAGAAGGACCCAGAAAAAGA-3′ 5′-TGAAGGCTGTTGAAGGACGA-3′ 5′-AGCTGCAACAATCCAACTCCATGC-3′ 5′-TTGATCTCCACAACTAGGCACGCT-3′ 5′-GTGCTTCATTGCTGTTCCCGTCAA-3′ 5′-GGTGGTGGTTGCTCCTTTGT-3′ 5′-TTCAGAGGGTTGAGCTCCCTGTTT-3′ 5′-TGAGGCTGTATGTAAAGGATG-3′ 5′-TCTTCGCCACAAAGTTCTCTCCCA-3′ 5′-TTGACCACGGCATGTTTGAGCATC-3′ 5′-TGTTTCTGTGCAGGTAGTCCACCA-3′ 5′-TGTCACCCTTACAAGCAGGAGGTT-3′ 5′-ACAGGATGACGATGGCCATGAGAA-3′ 5′-TGTGTGTTGGGCTCCTGTGACATA-3′ 5′-GTTCTTGTGCCGGCCAATCATCTT-3′ 5′-TGCATCTCTGTGTCATGAGCCTGT-3′ 5′-TCTTCTGAACCCTGCAGCCATTCT-3′ 5′-ATGTCCTTCTCGTTCTTCCA-3′ 5′-ATTTGACGTCCTGGTCTGGCTTGA-3′ 5′-TGTCGCTCAGCTCTGGATCTTTGT-3′ 5′-TCTGTTGGCTTTGGGATTC-3′

a

The primers were obtained from Liu et al. (2014), other primers were designed by the authors.

Fig. 1. Anti-angiogenic views of water extract of vinegar E. pekinensis Rupr. in transgenic zebrafish, and photographed under fluorescence microscope (magnification: 40×). The vessel numbers were counted as shown in the Control group. We counted the vertical vessels which connect the DLAV and DA shown in the A. (A) Control: embryos were treated with embryo water containing 0.1% DMSO. All the vertical vessels were complete and it presents no inhibition in control group. (B) Positive control: 100 ng/mL PTK787·2HCl. Only 17 vessels were complete in B. (C) 100 µg/mL: treated with VEP water extract solution at concentration of 100 µg/mL. (D) 150 µg/mL: treated with VEP water extract solution at concentration of 150 µg/mL. (E) 200 µg/mL: treated with VEP water extract solution at concentration of 200 µg/mL. (F) 250 µg/mL: treated with VEP water extract solution at concentration of 250 µg/ mL. These images are representative of data obtained during the course of this study. In these 4 images which with water extract of vinegar E. pekinensis Rupr., the numbers of complete vessels were reduced along with concentrations.

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Fig. 2. Anti-angiogenic activity of water extract of vinegar E. pekinensis Rupr. in a doseresponse manner. Each experiment was performed in triplicate and data are means ± SD (n = 10). This data representation is consistent with the literature. The experimental group ****p < 0.0001 versus control, the data were analyzed followed by Mann-Whitney test.

Fig. 3. Inhibitory effect of water extract of vinegar E. pekinensis Rupr. on HUVEC proliferation. Each experiment was performed for three times and data are means ± SD. ****p < 0.0001 analyzed with a one-way ANOVA test followed by Dunnett's method for multiple comparisons.

Fig. 1A demonstrates that complete vessels extend from the dorsal aorta (DA) and connected to the dorsal longitudinal anastomotic vessel (DLAV). This has been previously described by Vollmer et al. (2011). Only complete vessels were counted as ISV number. Fig. 1A shows that there was no anti-angiogenic effect (Fig. 1A) following treatment with vehicle control 0.1% DMSO. The mean vessel number of control group was 27.67 ± 0.96, while the mean vessel numbers of administered groups were 26.00 ± 1.29 (100 µg/mL), 24.54 ± 2.20 (150 µg/mL), 22.66 ± 2.68 (200 µg/mL), 20.80 ± 1.75 (250 µg/mL). Fig. 2 shows that, like the positive control PTK787 (100 ng/mL), VEP water extract significantly inhibited vessel formation (100, 150, 200, and 250 µg/ mL) compared to controls, but not at lower concentrations. Vessel number was concentration-dependent.

degradation of basement membrane of blood vessels followed by vascular endothelial cell activation. The endothelial cells then proliferate into the surrounding matrix and form solid sprouts connecting neighboring vessels. With migration of endothelial cells, these sprouts then form networks to become a full-fledged vessel lumen as cells migrate to the site of angiogenesis (Folkman, 1971; Carmeliet and Jain, 2011). Vessel formation encompasses the ability of endothelial cells to form a lumen which is closely associated with the differentiation and maturation of blood vessels. In this experiment, HUVECs seeded on Matrigel can become elongated and form capillary-like structures mimicking the in vivo angiogenesis process (Taraboletti and Giavazzi, 2004). HUVECs at 24-h post-seeding had peak network formation in control group (Fig. 5). VEP at 100 and 200 µg/mL significantly blocked network formation at 12-h post-seeding (Fig. 5), which revealed that VEP water extract possessed anti-angiogenic properties by mediating suppression of HUVEC tubule network formation.

3.2. Cell proliferation assay

3.5. mRNA expression in zebrafish determined by qRT-PCR

Proliferation of HUVECs is directly related to angiogenesis. Cell viability assays showed that after 24 h treatment, VEP water extract significantly inhibited cell viability in a concentration-response manner (100–400 µg/mL, p < 0.0001) (Fig. 3). The cell viability was 72.0% ± 0.5%, 41.1% ± 3.1% and 28.0% ± 1.8% at concentrations of 100 µg/ mL, 200 µg/mL and 400 µg/mL, respectively. Thus, VEP water extract could inhibit endothelial cell proliferation.

Fig. 6 demonstrates that VEP water extract down-regulated angiogenic-related genes responsible for proliferation, adhesion, migration and vessel formation in endothelial cells. There was only one gene, VEGFR3, that was significantly increased, while other genes such as Met, IGF1, CTGF, VEGFA were all significantly decreased.

3.3. Cell migration assay

This study is the first to describe the anti-angiogenic effects of EP. We offer evidence that EP potently inhibited angiogenesis in vitro demonstrated by HUVEC proliferation, migration, network formation and within the context of our in vivo zebrafish model. VEP water extract is a mixture of chemicals that can inhibit multiple signaling pathways of angiogenesis in zebrafish in vivo as determined by Q-PCR. This was shown by the down-regulation of angiogenesis associated genes including VEGFA, KDR, HIFA, and a number of other genes (Fig. 6). Angiogenesis may contribute to the growth of solid tumors, but also the pathogenesis of leukemia and other "liquid" tumors. Accordingly, leukemia cells are filled with micro- vessels, forming grape-like clumps, and have high expression of angiogenic-related genes (Ma and

3. Results 3.1. Inhibition of vessel formation in zebrafish embryos

4. Discussion

Inhibition of cell migration is a committed step required for antiangiogenesis. Migration assays demonstrated that water extract inhibited HUVEC migration (Fig. 4). HUVEC migration was significantly decreased after the cells were treated with VEP water extract at concentrations of 100 µg/mL and 200 µg/mL. When media was changed and the treatment was removed, migration was partially restored 24 h later. 3.4. Cell network formation assay The development and formation of new vessels in vivo start form 341

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Fig. 4. (A) Quantitative analysis of the VEP inhibited HUVEC migration (magnification: 100×). ****P < 0.0001 for differences in wound closure (% control) from baseline cultures without treatment. Data are expressed as mean ± SD from three individual experiments analyzed with a one-way ANOVA test. (B) VEP inhibited HUVEC migration after 12, 24, and 24 h after treatment removal. Images captured when 100 or 200 µg/mL of VEP water extract were added to the wells at different times.

Future experiments will include the division of the water extract of E. pekinensis into different fractions and use UPLC-Q-TOF/MS to analyze the effective components. The molecular mechanisms and signaling pathways of angiogenicrelated genes were discussed as following and their proposed interaction was described in Fig. 7. The anti-angiogenic effect induced by VEP was analyzed by measuring expression of angiogenic-related genes. Expression of VEGFR3 (7.4-fold, p < 0.0001) was significantly increased. Met (0.02-fold, p < 0.001), IGF1 (0.01-fold, p < 0.001), CTGF (0.08-fold, p < 0.001), NRP2 (0.05-fold, p < 0.001), VEGFA (0.02-fold, p < 0.001), and Flt-1 (0.23-fold, p < 0.05) were significantly decreased (Fig. 6). Met, a receptor of tyrosine kinase, can activate multiple signal transduction pathways including the Src/FAK, p120/ STAT 3 pathway, and the PI3K/Akt pathway. These multiple signaling pathways can modulate the migration and proliferation of endothelial cells (You and Mcdonald 2008). Activation of the Akt in PI3K/Akt pathway is known to play a key role in numerous cellular functions including proliferation, adhesion, migration, invasion, metabolism, and survival (Karar and Maity, 2011). Furthermore, activated Akt phosphorylates and inhibits forkhead transcription factor (Foxo1), thus down-regulating

Waxman, 2008). Increased vessel permeability due to tumors can also lead to angiogenesis and cause malignant ascites (Amălinei et al., 2010; Gershtein et al., 2011). These diseases are associated with the abnormal blood vessel formation, so that anti-angiogenic therapy is increasingly being viewed as a strategy for malignant ascites. VEP has been reported to have a curative effect in leukemia and malignant and its mechanism may be anti-angiogenic. Two compounds derived from Euphorbia plants have been reported that display activities related to angiogenesis. 3-O-propionyl-5, 10, 14O-triacetyl-8-O-(20 -methyl- butanoyl)-cyclomyrsinol, a mirsinanetype diterpene, which is isolated from the methanolic extract of Euphorbia microsciadia Boiss (Mendonà et al. 2009). Its anti-angiogenic activity was demonstrated on vascular endothelium growth factor (VEGF)-induced angiogenesis in cultured HUVECs in vitro by assessing capillary-like tube network formation (Mendonà et al. 2009). 12deoxyphorbol 13-palmitate (G) is a toxic compound isolated from Euphorbia fischeriana. It can inhibit vascular endothelial growth factor (VEGF)-induced angiogenic processes in vitro, and inhibit the in vivo growth of MCF-7 cells in a grafted mouse model via the suppression of VEGFR-2-signaling pathway (Xu et al., 2013). These two compounds are lipophilic whereas the raw extract of E. pekinensis. is hydrophilic.

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Fig. 5. Effect of network formation of VEP-treated HUVEC on Matrigel (magnification: 100×). (A) Quantitative analysis of the tubule length in VEP-treated HUVEC. Data are expressed as tubule length (% control) ± SD from three individual experiments analyzed with a Students t test. (B) Tubule length was quantified based upon the number of closed circles indicated in the images. Differentiation of HUVEC morphological changes were observed in tubule network formation following treatment.

activity directly and it is associated with uncontrolled angiogenesis as seen in the evolution of solid tumor vascularization (Seo et al., 2003). Similarly, PLG is activated by proteolysis and regenerate to plasmin and angiostatin which inhibits angiogenesis by decreasing stimulation of mitogen-activated protein kinases (Redlitz et al., 1999). Combined with up-regulation of TIMP2 and PLG present in pro-angiogenic effects of Flos carthami water extract (Zhou et al., 2014), perhaps TIMP2 and PLG might provide negative feedback regulation for anti-angiogenesis. Cell adhesion factors participate in angiogenesis and adhesion factors are less expressed, as shown by ITGAV (0.01-fold, p < 0.001) and β-catenin (0.35-fold, p < 0.01) which blocks endothelial cell adhesion, within the context of our study. ITGAV is the receptor of vitronectin, which mediates adhesion, migration and angiogenesis of HUVECs (Liu et al., 2011). β-catenin, which builds and maintains epithelial cell layers, is a subunit of adherens junctions, and can regulate cell growth and adhesion (Lilien and Balsamo, 2005). Downregulation of these two genes has anti-angiogenic effects. We demonstrated the down-regulation of Tie-2 (0.15-fold, p < 0.01), PDGFR-β (0.06-fold, p < 0.001), CDH5 (0.16-fold, p < 0.01), FGF2 (0.14-fold, p < 0.01), FGFR2 (0.27-fold, p < 0.05), Shh (0.04fold, p < 0.001), TGFβ1 (0.08-fold, p < 0.001), and TGFRβ1 (0.04fold, p < 0.001) inhibits tubule formation and morphogenesis or smooth muscle cell recruitment and differentiation. Trans-association of Tie2 was induced by angiopoietin-1 at cell-cell junctions, which lead to the preferential activation of the PI3K/AKT pathway. Trans-associated Tie2 combined with vascular endothelial protein tyrosine phosphatase enhanced endothelial cell adhesion (Fukuhara et al., 2010). The down-regulation of PDGF/PDGFR-β inhibited cell migration with the deactivation of sphingosine kinase and the reduced expression of endothelial differentiation gene-1. In studies conducted by other laboratories, the inhibition of PDGFR-β was also shown to decrease endothelial cell proliferation and migration due to the binding to ITGβ3 (Moreno et al., 2013). FGF2 on the other hand can interact with TGFβ1 to further promote cell proliferation and differentiation. FGF2/FGF receptors in the FGFR2 signaling pathway have been

the expression of Foxo1 target genes involved which are involved in endothelial cell apoptosis and vascular remodeling (Puigserver et al., 2003). Finally, Akt also activates eNOS, contributing to vascular maturation (Zheng et al., 2007). The down-regulation of IGF1 inhibits the Akt signaling that is required for endothelial cell proliferation (Piecewicz et al., 2012) concurrent with the down-regulation of TGF β1 due to a positive association with IGF1 (Rosendahl and Forsberg, 2006). Phosphatidylinositol 3-kinase (PI3K)-Akt-dependent pathways induce CTGF, the down-regulation of which can inhibit cell adhesion, proliferation, and migration (Suzuma et al., 2001). NRP2 and Flt-1 (or VEGFR1) are co-receptors of VEGF, and their down-regulation suppresses endothelial cell survival and migration (Ferrara et al., 2003). VEGFR3 is the main receptor of VEGFC (Bridenbaugh, 2005). The morphological and anti-angiogenesis observations may be explained by significant up-regulation of VEGFR3 via modulation by the Notch pathway (Rui et al., 2012). Notch upregulates VEGFR-3 in primary developmental angiogenesis (Benedito et al., 2012; Pytowski et al., 2005; Zhang et al., 2010; Shawber et al., 2007). VEGFA was down-regulated, and up-stream HIF1A was also down-regulated, and this was associated with low expression of other angiogenic factors leading to the formation of unstable or leaky vasculature (Hadjipanayi and Schilling, 2013). In addition, as a connection between the PI3K pathway and angiogenesis, hypoxia leads to HIF-1α stabilization and is a major stimulus for increased vascular endothelial growth factor (VEGF) production by tumor cells (Karar and Maity, 2011; Chai et al., 2013). The down-regulation of specific angiogenesis-related proteins and associated structural proteins coincided with the matrix degradation and endothelial cell migration-related factors within our study. Inhibition of MMP-9 (0.10-fold, p < 0.01) is known to decrease cell migration (Zhao et al., 2008), which seems to restrain angiogenesis. However, TIMP2 (0.04-fold, p < 0.001) and PLG (0.27-fold, p < 0.05), which inhibit angiogenesis, were down-regulated. TIMP2 can suppress endothelial cell proliferation and MMP2 (0.16-fold, p < 0.01) 343

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Fig. 6. Effects of VEP on the expression of angiogenic-related genes by quantitative RT-PCR analysis with β-actin as house-keeping gene. Proposed gene interaction down-regulated by VEP water extract in zebrafish in vivo. Data are expressed as means ± SD. of relative mRNA (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 present significant differences compared to control group.

Fig. 7. Proposed gene interaction in anti-angiogenesis by VEP water extract in zebrafish in vivo.

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References

known to contribute to tumor angiogenesis and subsequent metastases. Inhibition of the bFGF/FGFR2 pathway has been shown to negatively affect endothelial cell proliferation and vessel formation (Stepanova et al., 2013). Dissociation of the adherens junction protein from the TGFβ1 receptor was associated with increased β-catenin tyrosine phosphorylation and decreased threonine phosphorylation. β-catenin became associated with the TGFβ1-signaling molecules Smad3 and Smad4 after receptor-ligand binding (Tian and Phillips, 2002). βcatenin can also bind to the CDH5 junction complex associated with the cellular cytoskeleton, promoting endothelial cell adhesions and subsequent vessel formation (Guo et al., 2008). The downregulation of Shh possibly decreased ANGPT1-mediating reciprocal interactions with TIE-2/Akt pathway between the endothelium and surrounding matrix and mesenchyme, and destroyed blood vessel maturation and stability (Fiedler et al., 2003). Low Shh expression can inhibit cell proliferation, migration and tube formation, while concomitantly downregulating VEGFA and Notch1 expression (Wang et al., 2016a), thereby discrupting the angiogenic mechanism. Down-regulation of EPHB4 (0.19-fold, p < 0.01) and Ephrin B2 (0.12-fold, p < 0.01) indicated that VEP water extract decreased vessel formation and/or maturation. The function of Flt-1 is directly regulated by ephrinB2 in developmental and tumor angiogenesis (Sawamiphak et al., 2010). Associated with PECAM1 for endothelialmesenchymal interactions, the down-regulation of Ephrin B2 suggests that our isolated VEP water extract inhibited blood vessel loop formation (Korff et al., 2006). This relationship between the EPHB4 and Ephrin B2 enhances blood vessel formation and remodeling (Noren et al., 2004). Finally, the inhibition of EPHB4 and Ephrin B2 binding represents potent anti-angiogenic activity.

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5. Conclusion Through in vitro and in vivo experiments, we have demonstrated that E. pekinensis has anti-angiogenic properties. This was supported by our data through Q-PCR studies in which mRNA expression levels revealed that the E. pekinensis can inhibit most expression of angiogenesis tumor-related genes. The regulatory mechanism and proposed gene interaction are shown in Fig. 7. Funding This work was in-part supported by the Beijing Joint Project of Science Research with postgraduate Education-Key technology research and application of safety evaluation of toxic Chinese medicinal materials based on the chemical composition and the characteristics of zebrafish (grant number: 2050205). This work was also supported by special funding from the Beijing Municipal Science and Technology Commission for innovation environment and platform construction (grant number: Z16111000500000). Conflicts of interest The authors declare no conflicts of interest. Author contributions Wenting Zhang, Zhiqiang Ma and Ruichao Lin conceived and designed this study; Wenting Zhang, Yaru Feng, Qing Xia, Yuanyuan Ni, Jie Liu, and Jian Zheng performed the experiments; Jie Liu, and Jian Zheng analyzed the data; Bin Liu, and Farong Li revised the paper. Acknowledgments We thank ACCDON for editorial assistance during the preparation of this manuscript. 345

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