- Email: [email protected]
Inhibitory effects of quercetin on angiogenesis in larval zebrafish and human umbilical vein endothelial cells
European Journal of Pharmacology 723 (2014) 360–367
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Inhibitory effects of quercetin on angiogenesis in larval zebrafish and human umbilical vein endothelial cells$ Daxian Zhao a,c,n, Chuanjie Qin b, Xiaohui Fan c, Yuncong Li c, Binhe Gu c a
State Key Laboratory of Food Science and Technology, School of Life Science and Food Engineering, Nanchang University, Nanchang 330047, PR China College of Life Science, Neijiang Normal University, Neijiang 641112, PR China c Department of Soil and Water Science, Tropical Research and Education Center, University of Florida, Homestead, FL 33031, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 14 September 2013 Received in revised form 25 October 2013 Accepted 31 October 2013 Available online 13 November 2013
Angiogenesis plays an essential role in many physiological and pathological processes. Quercetin, a plant pigment and traditional Chinese medicinal herb, is an important flavonoid that has anti-cancer activity. However, the function of quercetin in blood vessel development in vivo and in vitro is still unclear. In this study, we investigated the anti-angiogenic activity of quercetin in zebrafish embryos and in human umbilical vein endothelial cells (HUVECs). Our results showed that quercetin disrupted the formation of intersegmental vessels, the dorsal aorta and the posterior cardinal vein in transgenic zebrafish embryos. In HUVECs, quercetin inhibited cell viability, the expression of vascular endothelial growth factor receptor 2 and tube formation in a dose-dependent manner. In inhibiting angiogenesis, quercetin was found to be involved in suppressing the extracellular signal-regulated kinase signaling pathway in vivo and in vitro. This study has shown that quercetin has potent anti-angiogenic activity and may be a candidate anti-cancer agent for future research. & 2013 Elsevier B.V. All rights reserved.
Keywords: Quercetin Flavonoids Angiogenesis Zebrafish Human umbilical vein endothelial cells Anti-cancer
1. Introduction The circulatory system in vertebrates is a network of arteries, veins and capillaries, and the formation of the vascular system includes vasculogenesis and angiogenesis (Larrivee et al., 2009; Risau, 1997). Angiogenesis, the emergence of new blood vessels via branching from an existing vascular system, plays an important role in embryonic vascular formation and development (Potente et al., 2011; Risau, 1997). Studies have shown that tumors promote the angiogenic process, including the proliferation and migration of endothelial cells. One novel strategy to suppress tumor development is the inhibition of angiogenesis (McMahon, 2000; Pratheeshkumar et al., 2012), and there is increasing evidence demonstrating that angiogenesis is involved in increased cellular infiltration and proliferation (Jackson et al., 1997). Vascular endothelial growth factor (VEGF) is the most important angiogenic
Abbreviations: HUVECs, human umbilical vein endothelial cells; ISV, intersegmental vessel; DA, dorsa aorta; PCV, posterior cardinal vein; VEGF, vascular endothelial growth factor; RTKs, receptor tyrosine kinases; DMSO, dimethylsulfoxide; hpf, hours postfertilization; PVDF, polyvinylidene difluoride; ERK, extracellular signal-regulated kinase ☆ Chemical compounds studied in this article. Quercetin (PubChem CID: 5280343). n Corresponding author. Tel./fax: þ 86 791 83969531. E-mail address: [email protected] (D. Zhao). 0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.10.069
factor that increases mitogenic activity and the survival of vascular endothelial cells (Roberts and Palade, 1997; Yu et al., 2010). VEGF exerts its activity on endothelial cells through two types of receptor tyrosine kinases (RTKs): vascular endothelial growth factor receptor 1 (VEGFR-1) and vascular endothelial growth factor receptor 2 (VEGFR-2). VEGFR-2 plays a critical role in mediating the mitogenesis and proliferation of endothelial cells (Pratheeshkumar et al., 2012), and activation of VEGFR-2 enhances proliferation, migration and tube formation of endothelial cells by activating the phosphorylation of multiple signaling pathways, including the extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), phosphatidylinositide 3-kinase (PI3K), protein kinase B (AKT) and p38 mitogen-activated protein kinase (p38MAPK) pathways (Ferrara et al., 2003). Quercetin, a bioactive flavonoid with a molecular weight of 302.24 g mol 1 is a Chinese herbal medicine found in various edible plants, such as red onions, apples, tea, broccoli, red grapes and a number of berries (Bischoff, 2008). It exhibits a broad range of pharmacological activities, and is considered to be an antiinflammatory, anti-oxidant, anti-tumor and anti-ulcer agent, as well as exerting immunomodulatory and vasodilatory effects (Ajay et al., 2006; Alvarez et al., 1999; Shoskes and Nickel, 2011). In our study, we chose transgenic zebrafish (Danio rerio; fli1: EGFP), which are a useful model for the high-throughput screening of drugs and compounds (Cheng et al., 2001; Peterson et al., 2001), to investigate the effect of quercetin on angiogenesis. We
D. Zhao et al. / European Journal of Pharmacology 723 (2014) 360–367
additionally studied the in vitro activity of quercetin in human umbilical vein endothelial cells (HUVECs). We found that quercetin exerts anti-angiogenic activity in zebrafish, and that it significantly inhibits endothelial cell proliferation, migration and tube formation in vitro. Our results show that the molecular mechanism for quercetin-mediated inhibition of angiogenesis involves the ERK signaling pathway and the expression of VEGFR-2.
2. Materials and methods 2.1. Cell lines and chemicals HUVECs were obtained from ATCC. Quercetin was purchased from Sinopharm Chemical Reagent Co. Ltd. (SCRC, Shanghai, China). HUVECs were cultured in Kaighn's modification of Ham's F-12 medium (F-12K) with 10% heat-inactivated fetal bovine serum (FBS) and 100 U ml 1 penicillin–streptomycin (Gibco). Cells were incubated at 37 1C in 5% CO2 (v/v). Quercetin was dissolved in dimethylsulfoxide (DMSO; Amersco) to give a 200 mM stock solution. The stock solution was diluted with cell culture medium in different concentrations for use.
361
2.5. Embryo collection, drug treatment and measurement of the toxic effects of quercetin on zebrafish Tg(fli1: EGFP) transgenic zebrafish embryos were generated by natural pair-wise mating and were raised at 28.5 1C in distilled water. Quercetin was diluted in DMSO as needed, and then transferred to the embryo water. Healthy, hatched zebrafish embryos were picked out 6 h postfertilization (6 hpf), and treated with DMSO (0.1%) or different concentrations of quercetin (50, 100, 200, 300, 500, 700 or 1000 mM). They were then incubated in sixwell plates (20–30 embryos per well) at 28.5 1C from 6 to 72 hpf. Embryos treated with DMSO (0.1%) alone served as a vehicle control. During the experiment, the embryos were observed for survival and morphology under an inverted microscope (Nikon, Japan). Data were analyzed using the statistical package SPSS 17.0 (SPSS Inc., Chicago, IL, USA) for non-linear regression, and the minimum lethal concentration was defined as 0% mortality of zebrafish treated with quercetin. The assay was repeated three times independently with 20–30 embryos per group. 2.6. Assessment of vascular changes in zebrafish embryos by microscopy
Transgenic zebrafish Tg(fli1: EGFP) expressing enhanced green fluorescent protein (EGFP) in the endothelial cells were kindly provided by ZFIN (Oregon) for use as the in vivo model. Zebrafish were maintained as described in Westerfield (1993). In brief, zebrafish were maintained at 28.5 1C in 14 h:10 h light/dark cycles. Zebrafish were fed twice daily with dry food in the morning and afternoon. Embryos were collected in the morning and cultured at 28.5 1C in distilled water. At 6 h after fertilization, embryos were distributed into a six-well cell culture plate with 4 ml distilled water containing different concentrations of quercetin. Embryos receiving DMSO (0.1%) only served as a control. All of the experiments were repeated at least three times with 20–30 embryos per group.
Tg(fli1: EGFP) transgenic zebrafish embryos were treated with DMSO (0.1%) or various concentrations of quercetin (50, 100 or 200 mM), and incubated in six-well plates (20–30 embryos per well) at 28.5 1C from 6 to 72 hpf. At 72 hpf, zebrafish were removed from the six-well plates, and anesthetized with a standard solution of 0.02% MS-222 for 10 s until the tail fins stopped moving. Then, the fish were transferred to slides, and observed for viability and morphological changes in blood vessels under a fluorescence microscope (Axio Imager Z1, Zeiss, Oberkochen, Germany). Images were taken with an epifluorescence microscope (Zeiss, Germany). The section of the zebrafish just below the yolk sac was chosen for the measurement of the number of complete intersegmental vessels (ISVs) and angiogenic sprouts by manual counting. Embryos receiving DMSO (0.1%) alone were used as vehicle controls. The assay was repeated three times independently with 20–30 embryos per group.
2.3. Cell proliferation assay
2.7. Endothelial cell capillary-like tube formation assay
HUVECs were seeded into 96-well plates at a density of 5 103 cells per well. In order to achieve a quiescent state, the complete medium was replaced after 24 h incubation with low serum (0.5% FBS) medium. After this, the medium was replaced with low serum (0.5% FBS) medium containing various concentrations of quercetin. Cells receiving DMSO (0.1%) only served as a vehicle control. Plates were incubated for an additional 48 h, and cell proliferation was assessed using Cell Proliferation Kit II (MTS, Promega) in accordance with the manufacturer's protocol. MTS test solution (20 ml) was added to each well, and the cells incubated for an additional 4 h at 37 1C. The spectrophotometric absorbance of each well was measured using a multilabel-counter fluorescent plate reader (Infinites M 1000, Tecan, Switzerland). The wavelength used to measure absorbance was 490 nm. The results are expressed as the percentage of proliferating cells.
BD Matrigel™ Basement Membrane Matrix (growth factor reduced; BD Biosciences, San Jose, CA, USA) was thawed at 4 1C, pipetted into pre-chilled 24-well plates and incubated at 37 1C for 45 min. HUVECs were first incubated in endothelial cell growth medium (ECGM) supplemented with 0.5% FBS for 6 h and then treated with DMSO (0.1%) or various concentrations of quercetin (50, 100 or 200 mM). Cells were collected and placed onto a layer of Matrigel™ (4 104 cells per well) in 1 ml ECGM supplemented with 0.5% FBS. After 6 h of incubation at 37 1C in a 95%:5% (v/v) mixture of air and CO2, the network-like structures of the endothelial cells were examined under an inverted microscope (Olympus, Center Valley, PA, USA). The tube-like structures were defined as endothelial cord formations that were connected at both ends. Branching points in three random fields per well were quantified by manual counting. Cells receiving only DMSO (0.1%) served as a vehicle control. The inhibition percentage is expressed as the percentage of the vehicle control (100%). The assay was repeated three times independently.
2.2. Maintenance of zebrafish and treatment of embryos
2.4. Morphological observation of zebrafish After drug treatment, embryos were anesthetized using 0.01% tricaine (Sigma-Aldrich) and observed for any morphological changes using an Olympus spinning disk confocal microscope system (IX81-ZDC motorized inverted microscope). Images were analyzed with ImageJ and Adobe Photoshop 7.0 software.
2.8. Quantitative real-time PCR The effects of quercetin on certain genes were determined by quantitative real-time polymerase chain reaction (qRT-PCR). HUVECs (5 105 cells per well) were seeded in 24-well plates,
362
D. Zhao et al. / European Journal of Pharmacology 723 (2014) 360–367
and starved with ECGM containing 0.5% FBS for 24 h. After the pre-incubation, cells were treated with various concentrations of quercetin (50, 100 or 200 mM) for another 48 h. Then, cells were harvested in TRIzolH reagent, and their RNA was extracted and assessed for integrity by agarose-gel electrophoresis. RNA samples were quantified at OD260/OD280, and RNA was introduced to reverse transcribe to single-stranded cDNA using a PrimeScript™ RT reagent kit (TaKaRa, Otsu, Shiga, Japan), followed by qTR-PCR using SYBRH Premix Ex Taq™ (TaKaRa, Otsu, Shiga, Japan). The reverse-transcribed RNA was primed with oligonucleotides specific for VEGFR-2 (Flk-1; forward: 5′-GACTGTGGCGAAGTGTTTTTGA3′ and reverse: 5′-GTGCAGGGGAGGGTTGGCGTAG-3′) and β-actin (forward: 5′-GTGCGGGACATCAAGGAGAA-3′ and reverse: 5′AGGAAGGAGGGCTGGAAGAG-3′; Applied Biosystems, Carlsbad, CA, USA). The PCR program was set in accordance with the manufacturer's instructions. The abundance of mRNA was normalized to β-actin levels and expressed as percentage of the vehicle control (100%) for statistical analysis. Three independent experiments were performed.
Fig. 1. Toxic effects of quercetin on zebrafish. Tg(fli1: EGFP) zebrafish embryos were treated with various concentrations of quercetin (control, 50, 100, 200, 300, 500, 700 or 1000 mM) from 6 to 72 hpf.
2.9. Western blotting analysis To determine the effects of quercetin on the signaling cascades, HUVECs were first starved in ECGM containing 0.5% FBS for 12 h. After being washed with fresh medium, cells were treated with DMSO (0.1%) or various concentrations of quercetin (50, 100 or 200 mM) for 48 h for ERK phosphorylation. Zebrafish embryos were treated with DMSO (0.1%) or various concentrations of quercetin (50, 100 or 200 mM) at 28.5 1C from 6 to 72 hpf. Then, cell extracts were prepared in radioimmunoprecipitation assay buffer supplemented with phenylmethylsulfonyl fluoride. Proteins were resolved by electrophoresis, transferred out of the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel onto polyvinylidene difluoride membranes and probed overnight with the following antibodies at 4 1C: anti-p-ERK (1:1000, CST), mouse anti-ERK (1:1000, CST) and rabbit anti-GAPDH (1:1000, Santa Cruz). Goat anti-rabbit IgG and goat anti-mouse IgG (1:1000, Jackson ImmunoResearch) conjugated to horseradish peroxidase (GE Healthcare, Bucks, UK) were used as secondary antibodies, incubated at 37 1C for 1 h at room temperature. The grey value of each band was measured by ImageJ software (NIH). 2.10. Statistical analysis Each experiment was performed at least three times, and all values are presented as the mean 7S.E.M. of the triplicates. One-way analysis of variance with Tukey's post-hoc test was used to analyze the statistical significance of the results. Values of P o0.05 were considered statistically significant.
3. Results 3.1. Quercetin-induced morphological abnormalities during the early embryonic developmental stages of zebrafish To investigate the toxicity of quercetin in the zebrafish embryo, we performed a lethality assay in zebrafish embryos. The zebrafish embryos were treated with different concentrations of quercetin (50, 100, 200, 300, 500, 700 or 1000 mM) from 6 to 72 hpf. We found that the zebrafish embryos began to die when the concentration was higher than 200 mM, suggesting that the minimum lethal concentration of quercetin was 200 mM (Fig. 1). For this reason, 200 mM was selected as the largest safe concentration for further experiments in zebrafish.
Fig. 2. Morphological changes caused by quercetin during early embryonic development in zebrafish. (A) Embryos treated with different concentrations of quercetin were imaged at 72 hpf. Scale bar, 300 mm. (B) The percentage of morphologically abnormal and normal zebrafish treated with different concentrations of quercetin compared to the control (n¼ 27 per group).
In order to assess the phenotype generated by quercetin in zebrafish embryogenesis, we took bright field photos at 72 hpf and found that embryos treated with DMSO (0.1%) or quercetin (50 and 100 mM) developed normally, while 54.05% of the embryos treated with 200 mM quercetin displayed mildly upward-curved tails and a light pigment; however, the survival ratio was not affected (Fig. 2A and B). 3.2. Quercetin inhibited the angiogenesis process To evaluate the anti-angiogenic effects of quercetin in vivo, we inspected the inhibitory effects of quercetin on zebrafish blood vessel formation. Tg(fli1: EGFP) transgenic zebrafish embryos were treated with DMSO (0.1%) or different concentrations of quercetin (50, 100 or 200 mM) from the shield stage 6 to 72 hpf (the time point at which all ISVs had stretched to form dorsal longitudinal anastomotic vessels). We then examined ISV, dorsal aorta (DA) and
D. Zhao et al. / European Journal of Pharmacology 723 (2014) 360–367
363
Fig. 3. Quercetin treatment caused defects in zebrafish vascular development in vivo. (A) Fluorescence images showing the gross morphology of 72-hpf Tg(fli1: EGFP) zebrafish embryos treated with DMSO alone or different concentrations of quercetin. The hexagon indicates the sites of complete ISVs in zebrafish embryos for all figures, and pentagons indicate the sites of angiogenic sprouts in zebrafish embryos for all figures. Red and yellow bars indicate the lumina of the DA and PCV, respectively. All images shown are lateral views with rostral right and dorsal up. Scale bar, 50 mm. (B) Quantification of the ISV length in 72-hpf embryos in the vehicle control group and quercetin-treated groups. (C) Quantification of the DA lumina diameter in 72-hpf embryos in the vehicle control group and quercetin-treated groups. (D) Quantification of the PCV lumina diameter in 72-hpf embryos in the vehicle control group and quercetin-treated groups. Data are expressed as mean 7S.E.M. from three independent experiments (nP o 0.05; #Po 0.01 versus control; one-way ANOVA with Tukey's post-hoc test). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
posterior cardinal vein (PCV) development at 72 hpf using in vivo fluorescence imaging. We found that the mean ISV length was 101.317 2.38 mm after treatment with 50 mM quercetin, 84.25 71.63 mm after treatment with 100 mM quercetin and 72.25 71.29 mm after treatment with 200 mM quercetin. The mean ISV length of the control group was 106.06 71.34 mm, and it is clear that treatment with quercetin resulted in shorter ISVs than the control, and the lengths were significantly shorter following treatment with 100 and 200 mM quercetin compared to the control group (Fig. 3A and B; nP o0.05; #P o0.01; n ¼20). We also found that the diameter of the DA and PCV narrowed following treatment with quercetin. The DA and PCV diameters were 28.69 70.41 mm and 35.72 70.32 mm, respectively, following treatment with 50 mM quercetin, 25.11 70.31 mm and 35.3970.35 mm following treatment with 100 mM quercetin and 20.91 70.39 mm and 33.30 70.30 mm following treatment with 200 mM quercetin. Akin to the changes observed in the mean ISV lengths, the DA diameter was significantly narrower for the groups treated with 100 or 200 mM quercetin compared to the control group (29.64 70.77 mm; Fig. 3A and C; nP o0.05; #P o0.01; n ¼20). With regards to the diameter of the PCV, a significant difference was found between the group treated with 200 mM quercetin (33.370.31 mm) and the control group (36.12 7 0.35 mm; Fig. 3A and D; nP o0.05; n¼ 20). These results
demonstrate that quercetin can serve as an inhibitor of angiogenesis in zebrafish embryos. Furthermore, we found that quercetin had an obvious effect on subintestinal vessel (SIV) formation at doses of 50, 100 and 200 mM (Fig. 4A). SIV branch points were reduced by 26% (nP o0.05; n ¼20) and 58% (#P o0.01; n ¼20) following treatment with 100 and 200 mM quercetin, respectively, compared to the control group (Fig. 4A and B). We also measured the SIV length following the administration of different doses of quercetin, and we found a significant change in length following treatment with 100 mM (1302.44 732.23 mm) and 200 mM (1086.44 742.13 mm) quercetin compared to the SIV length of the control group (1602.02 7 36.09 mm) by quantitative analysis (Fig. 4A and C). These data demonstrate that quercetin can impair angiogenesis. 3.3. Quercetin inhibited the ERK signaling pathway in vivo To determine whether quercetin inhibits anti-angiogenesis through downregulating the ERK signaling pathway, we performed immunoblotting of p-ERK expression in zebrafish embryos treated with quercetin at 72 hpf (Fig. 5A). No significant difference in the p-ERK expression levels could be found between fish treated with 50 mM quercetin and the control. However, the levels of p-ERK protein on Western blots were significantly reduced
364
D. Zhao et al. / European Journal of Pharmacology 723 (2014) 360–367
Fig. 4. Quercetin inhibited angiogenesis of SIVs in vivo. (A) Fluorescence images showing the SIV morphology of 72-hpf Tg(fli1: EGFP) zebrafish embryos treated with DMSO or different concentrations of quercetin. Boxed areas are the sites of the SIVs in zebrafish embryos for all figures. Scale bar, 150 mm. (B) Quantification of the number of SIV branch points in 72-hpf embryos in the vehicle control group and quercetin-treated groups. (C) Quantification of the SIV length in 72-hpf embryos in the vehicle control group and quercetin-treated groups. Data are expressed as mean 7 S.E.M. from three independent experiments (nP o 0.05; #Po 0.01 versus control; one-way ANOVA with Tukey's post-hoc test).
Fig. 5. Quercetin inhibited ERK activation in vivo. (A) Western blots of p-ERK and ERK expression in 72-hpf whole embryos treated with various doses of quercetin. (B) Densitometric analysis of the relative level of p-ERK to ERK. Experiments were repeated three times (nPo 0.05; #Po 0.01 versus control; one-way ANOVA with Tukey's post-hoc test).
following treatment with 100 and 200 mM quercetin compared to the controls (Fig. 5A). Densitometric analysis also showed a significant decrease in p-ERK protein levels following treatment with 100 mM (0.47 70.038-fold reduction, P o0.05; n ¼20) and 200 mM quercetin (0.21 70.031-fold reduction, Po 0.01; n ¼20) compared to the control (Fig. 5B).
3.4. Quercetin inhibited cell viability in endothelial cells To illustrate the anti-angiogenic effects of quercetin in vitro, we performed Cell Proliferation Kit II (MTS) assays to detect HUVEC survival and proliferation. We found that quercetin observably inhibited HUVEC proliferation, and cell numbers were significantly
different following treatment with 50, 100 and 200 mM quercetin (Fig. 6, nP o0.05, #Po 0.01). 3.5. Quercetin inhibits tube formation in endothelial cells We measured the ability of endothelial cells to form tubular structures, using Matrigel™. As shown in Fig. 7, quercetin dosedependently inhibited HUVEC tube formation (Fig. 7A–D, A′–D′). In this experiment, tube formation was quantified by measuring the branching points upon treatment with quercetin (Fig. 7E) and significant inhibition was noted at doses of 50, 100 and 200 mM quercetin (nP o0.05, #P o0.01, n ¼3) compared to the control, indicating that quercetin prevents endothelial cells from forming tubular structures on Matrigel™.
D. Zhao et al. / European Journal of Pharmacology 723 (2014) 360–367
3.6. Quercetin inhibited VEFGR-2 mRNA expression in endothelial cells VEGFR-2 is the most biologically important receptor for VEGF. It regulates endothelial cell proliferation, migration, differentiation,
365
tube formation and angiogenesis (Nakatsu et al., 2003). To dissect the molecular basis of the quercetin-mediated anti-angiogenic effects, we investigated how quercetin affected VEGFR-2 protein expression in HUVECs using qRT-PCR analysis. We found that 50, 100 and 200 mM quercetin dramatically inhibited the expression of VEGFR-2 (Fig. 8). Our results show that quercetin exerts its antiangiogenic activity through regulation of VEGFR-2 activation.
3.7. Quercetin inhibited the ERK signaling pathway in endothelial cells
Fig. 6. Quercetin inhibited cell viability in endothelial cells in a dose-dependent manner under normal culture conditions. HUVECs were cultured in ECGM supplemented with 0.5% FBS prior to treatment with DMSO (0.1%) or various concentrations of quercetin (50, 100 or 200 mM) for 48 h. Cell viability was quantified by MTS assay. Cells receiving only DMSO (0.1%) served as a vehicle control. Data are expressed as percentages of the vehicle control and as the mean 7 S.E.M. of triplicate experiments (nP o 0.05; #Po 0.01 versus control; oneway ANOVA with Tukey's post-hoc test).
To determine whether quercetin-mediated anti-angiogenesis involved the ERK signaling pathway, we performed immunoblotting of p-ERK expression in HUVECs treated with quercetin for 48 h. No significant differences in the p-ERK expression levels were observed following treatment with 50 mM quercetin (0.97 7 0.061-fold reduction) compared to the control. However, Western blotting showed that the levels of p-ERK protein following treatment with 100and 200 mM quercetin were significantly reduced compared to the control (Fig. 9A). Densitometric analysis also showed a significant decrease in p-ERK protein levels following treatment with 100 mM (0.38 70.035-fold reduction, P o0.05; n ¼3) and 200 mM quercetin (0.16 70.037-fold reduction, Po 0.01; n ¼3) compared to the controls (Fig. 9B).
Fig. 7. Anti-angiogenic effect of quercetin in HUVECs. (A)–(D) Tube formation assay showing the morphological features of quercetin-treated HUVECs on Matrigel™. (A′)–(D′) Higher magnification images of those shown in (A)–(D). The branching points decrease after treatment with quercetin. (E) Quantitative data of the branch points are presented as mean 7S.E.M. Experiments were repeated three times (nPo 0.05; #Po 0.01 versus control; one-way ANOVA with Tukey's post-hoc test).
366
D. Zhao et al. / European Journal of Pharmacology 723 (2014) 360–367
4. Discussion Quercetin, a bioactive flavonoid from Hypericum attenuatum Choisy, has been found to have various pharmacological activities, including anti-oxidant, anti-inflammatory and anti-tumor activities (Boots et al., 2008; Linsalata et al., 2010; Mahmoud et al., 2013; Rajendran et al., 2004; Ruiz et al., 2007; Russo et al., 2010). However, little is known about its functions and mechanism of action with regards to angiogenesis. In our study, we demonstrated that quercetin is a potent anti-angiogenesis agent in both zebrafish, which are known to be a good model for drug discovery (Lawson and Weinstein, 2002; Serbedzija et al., 1999), and HUVECs. Our study is the first to show that quercetin inhibited the development of the ISVs, DA and PCV in transgenic zebrafish embryos, particularly, at doses of 100 and 200 mM. These findings are in agreement with previous studies, which have shown that quercetin can inhibit the development of abdominal aortic aneurysms and reduce aortic size in mice (Wang et al., 2012). In a prostate xenograft mouse model, quercetin inhibited tumorigenesis by targeting angiogenesis (Pratheeshkumar et al., 2012). Studies have also shown that quercetin exerts a neuroprotective effect in zebrafish through the blood brain barrier (Richetti et al., 2011; Zhang et al., 2011). Although the embryos treated with 200 mM quercetin showed mildly upward-curved tails and a light pigment, no significant morphological or color changes were found when the zebrafish were treated with 50 or 100 mM quercetin. Angiogenesis is a complex process containing several important steps (Risau, 1997); however, endothelial cell lines can be
used as an in vitro model for angiogenesis studies (Chen et al., 2008). In the present work, we observed that quercetin effectively inhibited human endothelial cell migration, proliferation and tube formation in vitro, and these effects were dose dependent. Studies have reported that quercetin can suppress angiogenesis and inhibit tumor growth through the suppression of blood vessel growth in vitro and in vivo (Anand et al., 2011; Fotsis et al., 1997; Pratheeshkumar et al., 2012; Rodgers and Grant, 1998). Quercetin can also inhibit the migration and tube formation of the rhesus macaque choroid–retina endothelial cell line (Chen et al., 2008). All these data indicate that quercetin can inhibit angiogenesis. VEGF has been proven to promote the process of angiogenesis through regulating proliferation, migration and differentiation of endothelial cells through two receptors, namely, VEGFR-1 and VEGFR-2 (Tie and Desai, 2012). In our study, quercetin significantly inhibited the levels of VEGFR-2 mRNA in HUVECs compared to the control group. Studies have shown that quercetin suppresses VEGF-induced phosphorylation of VEGFR-2, AKT, mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (P70S6K) in HUVECs (Chen and Fang, 2002; Ferrara et al., 2003; Pratheeshkumar et al., 2012). ERK, which is a member of the MAPKs, is a key regulatory protein that mediates cell survival, proliferation, and differentiation (Olsson et al., 2006; Yu et al., 2010). The molecular mechanisms in endothelial cells that have previously been shown to be inhibited by free aglycone are the MAPK, JNK and focal adhesion kinase pathways (Kaneider et al., 2004; Kobuchi et al., 1999) as well as metalloproteinase-2 expression and activity (Tan et al., 2003). Our in vivo and in vitro studies showed that doses of 100 and 200 mM quercetin significantly suppressed ERK phosphorylation levels, indicating that the anti-angiogenesis activity of quercetin may involve the downregulation of the ERK signaling pathway. Consistent with this hypothesis, quercetin is known to reduce MAPK activation, which is an intracellular signal underlying endothelial activation (Donnini et al., 2006). Recently, some studies have shown that quercetin acts as a novel mediator of angiogenesis by downregulating the phosphorylation of the AKT/ mTOR/p70S6K signaling pathway (Chen and Fang, 2002; Igura et al., 2001; Pratheeshkumar et al., 2012).
5. Conclusions Fig. 8. Quercetin insignificantly inhibits the VEGFR-2 mRNA expression in HUVECs. HUVECs (5 105 cells per well) were treated with various concentrations of quercetin (50, 100 or 200 mM) for 48 h. Cells receiving only DMSO (0.1%) served as vehicle controls. The levels of VEGFR-2 mRNA were normalized by β-actin and expressed as percentages of the vehicle control (100%) and as the mean 7 S.E.M. Experiments were repeated three times (nPo 0.05; #Po 0.01 versus control; one-way ANOVA with Tukey's post-hoc test).
Altogether, our study suggests that quercetin can inhibit blood vessel development in zebrafish and prevent cell viability, proliferation and tube formation in HUVECs. We have shown that the mechanism of this anti-angiogenic activity is, at least in part, due to the inhibition of ERK phosphorylation in vivo and in vitro, or the suppression of the VEGFR-2-mediated signaling pathway in
Fig. 9. Quercetin inhibited ERK activation in HUVECs. (A) Western blots of p-ERK and ERK expression in HUVECs treated with various concentrations of quercetin for 48 h. (B) Densitometric analysis of the relative levels of p-ERK compared to ERK. Experiments were repeated three times (nP o 0.05; #P o0.01 versus control; one-way ANOVA with Tukey's post-hoc test).
D. Zhao et al. / European Journal of Pharmacology 723 (2014) 360–367
endothelial cells. Hence, our study may prompt further development of quercetin as a novel inhibitor of angiogenesis for the treatment of a number of diseases and cancers.
Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (No. 31260642), China Postdoctoral Science Foundation (No. 2012M521292, No. 2013T60650), the Natural Science Foundation of Jiangxi Province (No. 20132BAB214015), the Foundation of Jiangxi Educational Committee (No. GJJ12144), Jiangxi Postdoctoral Researchers Projects (No. 2013KY23), the Open Project Program of State Key Laboratory of Food Science and Technology of Nanchang University (No. SKLF-KF-201001) and Key Technologies R & D Program of Neijiang, Sichuan of China (No. 12108). References Ajay, M., Achike, F.I., Mustafa, A.M., Mustafa, M.R., 2006. Effect of quercetin on altered vascular reactivity in aortas isolated from streptozotocin-induced diabetic rats. Diabetes Res. Clin. Pract. 73, 1–7. Alvarez, A., Pomar, F., Sevilla, M.A., Montero, M.J., 1999. Gastric antisecretory and antiulcer activities of an ethanolic extract of Bidens pilosa L. var. radiata Schult Bip. J. Ethnopharmacol. 67, 333–340. Anand, K., Asthana, P., Kumar, A., Ambasta, R.K., Kumar, P., 2011. Quercetin mediated reduction of angiogenic markers and chaperones in DLA-induced solid tumours. Asian Pac. J. Cancer Prev. 12, 2829–2835. Bischoff, S.C., 2008. Quercetin: potentials in the prevention and therapy of disease. Curr. Opin. Clin. Nutr. Metab. Care 11, 733–740. Boots, A.W., Wilms, L.C., Swennen, E.L., Kleinjans, J.C., Bast, A., Haenen, G.R., 2008. In vitro and ex vivo anti-inflammatory activity of quercetin in healthy volunteers. Nutrition 24, 703–710. Chen, J., Fang, Y., 2002. A novel pathway regulating the mammalian target of rapamycin (mTOR) signaling. Biochem. Pharmacol. 64, 1071–1077. Chen, Y., Li, X.X., Xing, N.Z., Cao, X.G., 2008. Quercetin inhibits choroidal and retinal angiogenesis in vitro. Graefes Arch. Clin. Exp. Ophthalmol. 246, 373–378. Cheng, S.H., Han, P.K., Wu, R.S., 2001. The use of microangiography in detecting aberrant vasculature in zebrafish embryos exposed to cadmium. Aquat. Toxicol. 52, 61–71. Donnini, S., Solito, R., Giachetti, A., Granger, H.J., Ziche, M., Morbidelli, L., 2006. Fibroblast growth factor-2 mediates Angiotensin-converting enzyme inhibitorinduced angiogenesis in coronary endothelium. J. Pharmacol. Exp. Ther. 319, 515–522. Ferrara, N., Gerber, H.P., LeCouter, J., 2003. The biology of VEGF and its receptors. Nat. Med. 9, 669–676. Fotsis, T., Pepper, M.S., Aktas, E., Breit, S., Rasku, S., Adlercreutz, H., Wähälä, K., Montesano, R., Schweigerer, L., 1997. Flavonoids, dietary-derived inhibitors of cell proliferation and in vitro angiogenesis. Cancer Res. 57, 2916–2921. Igura, K., Ohta, T., Kuroda, Y., Kaji, K., 2001. Resveratrol and quercetin inhibit angiogenesis in vitro. Cancer Lett. 171, 11–16. Jackson, J.R., Seed, M.P., Kircher, C.H., Willoughby, D.A., Winkler, J.D., 1997. The codependence of angiogenesis and chronic inflammation. FASEB J. 11, 457–465. Kaneider, N.C., Mosheimer, B., Reinisch, N., Patsch, J.R., Wiedermann, C.J., 2004. Inhibition of thrombin-induced signaling by resveratrol and quercetin: effects on adenosine nucleotide metabolism in endothelial cells and platelet–neutrophil interactions. Thromb. Res. 114, 185–194. Kobuchi, H., Roy, S., Sen, C.K., Nguyen, H.G., Packer, L., 1999. Quercetin inhibits inducible ICAM-1 expression in human endothelial cells through the JNK pathway. Am. J. Physiol. 277, C403–411.
367
Larrivee, B., Freitas, C., Suchting, S., Brunet, I., Eichmann, A., 2009. Guidance of vascular development: lessons from the nervous system. Circ. Res. 104, 428–441. Lawson, N.D., Weinstein, B.M., 2002. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev. Biol. 248, 307–318. Linsalata, M., Orlando, A., Messa, C., Refolo, M.G., Russo, F., 2010. Quercetin inhibits human DLD-1 colon cancer cell growth and polyamine biosynthesis. Anticancer Res. 30, 3501–3507. Mahmoud, M.F., Hassan, N.A., El Bassossy, H.M., Fahmy, A., 2013. Quercetin protects against diabetes-induced exaggerated vasoconstriction in rats: effect on low grade inflammation. PLoS One 8, e63784. McMahon, G., 2000. VEGF receptor signaling in tumor angiogenesis. Oncologist 5, 3–10. Nakatsu, M.N., Sainson, R.C., Pérez-del-pulgar, S., Aoto, J.N., Aitkenhead, M., Taylor, K.L., Carpenter, P.M., Hughes, C.C., 2003. VEGF (121) and VEGF (165) regulate blood vessel diameter through vascular endothelial growth factor receptor 2 in an in vitro angiogenesis model. Lab. Invest. 12, 1873–1885. Olsson, A.K., Dimberg, A., Kreuge,r, J., Claesson-Welsh, L., 2006. VEGF receptor signalling-in control of vascular function. Nat. Rev. Mol. Cell Biol. 7, 359–371. Peterson, R.T., Mably, J.D., Chen, J.N., Fishman, M.C., 2001. Convergence of distinct pathways to heart patterning revealed by the small molecule concentramide and the mutation heart-and-soul. Curr. Biol. 11, 1481–1491. Potente, M., Gerhardt, H., Carmeliet, P., 2011. Basic and therapeutic aspects of angiogenesis. Cell 146, 873–887. Pratheeshkumar, P., Budhraja, A., Son, Y.O., Wang, X., Zhang, Z., Ding, S., Wang, L., Hitron, A., Lee, J.C., Xu, M., Chen, G., Luo, J., Shi, X., 2012. Quercetin inhibits angiogenesis mediated human prostate tumor growth by targeting VEGFR-2 regulated AKT/nTOR/P70S6K signaling pathways. PLoS One 7, e47516. Rajendran, M., Manisankar, P., Gandhidasan, R., Murugesan, R., 2004. Free radicals scavenging efficiency of a few naturally occurring flavonoids: a comparative study. J. Agric. Food Chem. 52, 7389–7394. Richetti, S.K., Blank, M., Capiotti, K.M., Piato, A.L., Bogo, M.R., Vianna, M.R., Bonan, C. D., 2011. Quercetin and rutin prevent scopolamine-induced memory impairment in zebrafish. Behav. Brain Res. 217, 10–15. Risau, W., 1997. Mechanisms of angiogenesis. Nature 386, 671–674. Roberts, W.G., Palade, G.E., 1997. Neovasculature induced by vascular endothelial growth factor is fenestrated. Cancer Res. 57, 765–772. Rodgers, E.H., Grant, M.H., 1998. The effect of the flavonoids, quercetin, myricetin and epicatechin on the growth and enzyme activities of MCF-7 human breast cancer cells. Chem. Biol. Interact. 116, 213–228. Ruiz, P.A., Braune, A., Holzlwimmer, G, Quintanilla-Fend, L., Haller, D., 2007. Quercetin inhibits TNF-induced NF-kappaB transcription factor recruitment to proinflammatory gene promoters in murine intestinal epithelial cells. J. Nutr. 137, 1208–1215. Russo, M., Spagnuolo, C., Volpe, S., Mupo, A., Tedesco, I., Russo, G.L., 2010. Quercetin induced apoptosis in association with death receptors and fludarabine in cells isolated from chronic lymphocytic leukaemia patients. Br. J. Cancer 103, 642–648. Serbedzija, G.N., Flynn, E., Willett, C.E., 1999. Zebrafish angiogenesis: a new model for drug screening. Angiogenesis 3, 353–359. Shoskes, D.A., Nickel, J.C., 2011. Quercetin for chronic prostatitis/chronic pelvic pain syndrome. Urol. Clin. North Am. 38, 279–284. Tan, W.F., Lin, L.P., Li, M.H., Zhang, Y.X., Tong, Y.G., Xiao, D, Ding, J., 2003. Quercetin, a dietary-derived flavonoid, possesses antiangiogenic potential. Eur. J. Pharmacol. 459, 255–262. Tie, J., Desai, J., 2012. Antiangiogenic therapies targeting the vascular endothelia growth factor signaling system. Crit. Rev. Oncog. 17, 51–67. Wang, L., Wang, B., Li, H., Lu, H., Qiu, F., Xiong, L., Xu, Y., Wang, G., Liu, X., Wu, H., Jing, H., 2012. Mice treated with quercetin exhibited a 32.7% reduction in aortic size compared with vehicle-treated controls. Eur. J. Pharmacol. 690, 133–141. Westerfield, M., 1993. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish. The University of Oregon Press, Eugene, OR. Yu, P.C., Gu, S.Y., Bu, J.W., Du, J.L., 2010. TRPC1 is essential for in vivo angiogenesis in zebrafish. Circ. Res. 106, 1221–1232. Zhang, Z.J., Cheang, L.C., Wang, M.W., Lee, S.M., 2011. Quercetin exerts a neuroprotective effect through inhibition of the iNOS/NO system and pro-inflammation gene expression in PC12 cells and in zebrafish. Int. J. Mol. Med. 27, 195–203.
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Inhibitory effects of quercetin on angiogenesis in larval zebrafish and human umbilical vein endothelial cells$ Daxian Zhao a,c,n, Chuanjie Qin b, Xiaohui Fan c, Yuncong Li c, Binhe Gu c a
State Key Laboratory of Food Science and Technology, School of Life Science and Food Engineering, Nanchang University, Nanchang 330047, PR China College of Life Science, Neijiang Normal University, Neijiang 641112, PR China c Department of Soil and Water Science, Tropical Research and Education Center, University of Florida, Homestead, FL 33031, USA b
art ic l e i nf o
a b s t r a c t
Article history: Received 14 September 2013 Received in revised form 25 October 2013 Accepted 31 October 2013 Available online 13 November 2013
Angiogenesis plays an essential role in many physiological and pathological processes. Quercetin, a plant pigment and traditional Chinese medicinal herb, is an important flavonoid that has anti-cancer activity. However, the function of quercetin in blood vessel development in vivo and in vitro is still unclear. In this study, we investigated the anti-angiogenic activity of quercetin in zebrafish embryos and in human umbilical vein endothelial cells (HUVECs). Our results showed that quercetin disrupted the formation of intersegmental vessels, the dorsal aorta and the posterior cardinal vein in transgenic zebrafish embryos. In HUVECs, quercetin inhibited cell viability, the expression of vascular endothelial growth factor receptor 2 and tube formation in a dose-dependent manner. In inhibiting angiogenesis, quercetin was found to be involved in suppressing the extracellular signal-regulated kinase signaling pathway in vivo and in vitro. This study has shown that quercetin has potent anti-angiogenic activity and may be a candidate anti-cancer agent for future research. & 2013 Elsevier B.V. All rights reserved.
Keywords: Quercetin Flavonoids Angiogenesis Zebrafish Human umbilical vein endothelial cells Anti-cancer
1. Introduction The circulatory system in vertebrates is a network of arteries, veins and capillaries, and the formation of the vascular system includes vasculogenesis and angiogenesis (Larrivee et al., 2009; Risau, 1997). Angiogenesis, the emergence of new blood vessels via branching from an existing vascular system, plays an important role in embryonic vascular formation and development (Potente et al., 2011; Risau, 1997). Studies have shown that tumors promote the angiogenic process, including the proliferation and migration of endothelial cells. One novel strategy to suppress tumor development is the inhibition of angiogenesis (McMahon, 2000; Pratheeshkumar et al., 2012), and there is increasing evidence demonstrating that angiogenesis is involved in increased cellular infiltration and proliferation (Jackson et al., 1997). Vascular endothelial growth factor (VEGF) is the most important angiogenic
Abbreviations: HUVECs, human umbilical vein endothelial cells; ISV, intersegmental vessel; DA, dorsa aorta; PCV, posterior cardinal vein; VEGF, vascular endothelial growth factor; RTKs, receptor tyrosine kinases; DMSO, dimethylsulfoxide; hpf, hours postfertilization; PVDF, polyvinylidene difluoride; ERK, extracellular signal-regulated kinase ☆ Chemical compounds studied in this article. Quercetin (PubChem CID: 5280343). n Corresponding author. Tel./fax: þ 86 791 83969531. E-mail address: [email protected] (D. Zhao). 0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.10.069
factor that increases mitogenic activity and the survival of vascular endothelial cells (Roberts and Palade, 1997; Yu et al., 2010). VEGF exerts its activity on endothelial cells through two types of receptor tyrosine kinases (RTKs): vascular endothelial growth factor receptor 1 (VEGFR-1) and vascular endothelial growth factor receptor 2 (VEGFR-2). VEGFR-2 plays a critical role in mediating the mitogenesis and proliferation of endothelial cells (Pratheeshkumar et al., 2012), and activation of VEGFR-2 enhances proliferation, migration and tube formation of endothelial cells by activating the phosphorylation of multiple signaling pathways, including the extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), phosphatidylinositide 3-kinase (PI3K), protein kinase B (AKT) and p38 mitogen-activated protein kinase (p38MAPK) pathways (Ferrara et al., 2003). Quercetin, a bioactive flavonoid with a molecular weight of 302.24 g mol 1 is a Chinese herbal medicine found in various edible plants, such as red onions, apples, tea, broccoli, red grapes and a number of berries (Bischoff, 2008). It exhibits a broad range of pharmacological activities, and is considered to be an antiinflammatory, anti-oxidant, anti-tumor and anti-ulcer agent, as well as exerting immunomodulatory and vasodilatory effects (Ajay et al., 2006; Alvarez et al., 1999; Shoskes and Nickel, 2011). In our study, we chose transgenic zebrafish (Danio rerio; fli1: EGFP), which are a useful model for the high-throughput screening of drugs and compounds (Cheng et al., 2001; Peterson et al., 2001), to investigate the effect of quercetin on angiogenesis. We
D. Zhao et al. / European Journal of Pharmacology 723 (2014) 360–367
additionally studied the in vitro activity of quercetin in human umbilical vein endothelial cells (HUVECs). We found that quercetin exerts anti-angiogenic activity in zebrafish, and that it significantly inhibits endothelial cell proliferation, migration and tube formation in vitro. Our results show that the molecular mechanism for quercetin-mediated inhibition of angiogenesis involves the ERK signaling pathway and the expression of VEGFR-2.
2. Materials and methods 2.1. Cell lines and chemicals HUVECs were obtained from ATCC. Quercetin was purchased from Sinopharm Chemical Reagent Co. Ltd. (SCRC, Shanghai, China). HUVECs were cultured in Kaighn's modification of Ham's F-12 medium (F-12K) with 10% heat-inactivated fetal bovine serum (FBS) and 100 U ml 1 penicillin–streptomycin (Gibco). Cells were incubated at 37 1C in 5% CO2 (v/v). Quercetin was dissolved in dimethylsulfoxide (DMSO; Amersco) to give a 200 mM stock solution. The stock solution was diluted with cell culture medium in different concentrations for use.
361
2.5. Embryo collection, drug treatment and measurement of the toxic effects of quercetin on zebrafish Tg(fli1: EGFP) transgenic zebrafish embryos were generated by natural pair-wise mating and were raised at 28.5 1C in distilled water. Quercetin was diluted in DMSO as needed, and then transferred to the embryo water. Healthy, hatched zebrafish embryos were picked out 6 h postfertilization (6 hpf), and treated with DMSO (0.1%) or different concentrations of quercetin (50, 100, 200, 300, 500, 700 or 1000 mM). They were then incubated in sixwell plates (20–30 embryos per well) at 28.5 1C from 6 to 72 hpf. Embryos treated with DMSO (0.1%) alone served as a vehicle control. During the experiment, the embryos were observed for survival and morphology under an inverted microscope (Nikon, Japan). Data were analyzed using the statistical package SPSS 17.0 (SPSS Inc., Chicago, IL, USA) for non-linear regression, and the minimum lethal concentration was defined as 0% mortality of zebrafish treated with quercetin. The assay was repeated three times independently with 20–30 embryos per group. 2.6. Assessment of vascular changes in zebrafish embryos by microscopy
Transgenic zebrafish Tg(fli1: EGFP) expressing enhanced green fluorescent protein (EGFP) in the endothelial cells were kindly provided by ZFIN (Oregon) for use as the in vivo model. Zebrafish were maintained as described in Westerfield (1993). In brief, zebrafish were maintained at 28.5 1C in 14 h:10 h light/dark cycles. Zebrafish were fed twice daily with dry food in the morning and afternoon. Embryos were collected in the morning and cultured at 28.5 1C in distilled water. At 6 h after fertilization, embryos were distributed into a six-well cell culture plate with 4 ml distilled water containing different concentrations of quercetin. Embryos receiving DMSO (0.1%) only served as a control. All of the experiments were repeated at least three times with 20–30 embryos per group.
Tg(fli1: EGFP) transgenic zebrafish embryos were treated with DMSO (0.1%) or various concentrations of quercetin (50, 100 or 200 mM), and incubated in six-well plates (20–30 embryos per well) at 28.5 1C from 6 to 72 hpf. At 72 hpf, zebrafish were removed from the six-well plates, and anesthetized with a standard solution of 0.02% MS-222 for 10 s until the tail fins stopped moving. Then, the fish were transferred to slides, and observed for viability and morphological changes in blood vessels under a fluorescence microscope (Axio Imager Z1, Zeiss, Oberkochen, Germany). Images were taken with an epifluorescence microscope (Zeiss, Germany). The section of the zebrafish just below the yolk sac was chosen for the measurement of the number of complete intersegmental vessels (ISVs) and angiogenic sprouts by manual counting. Embryos receiving DMSO (0.1%) alone were used as vehicle controls. The assay was repeated three times independently with 20–30 embryos per group.
2.3. Cell proliferation assay
2.7. Endothelial cell capillary-like tube formation assay
HUVECs were seeded into 96-well plates at a density of 5 103 cells per well. In order to achieve a quiescent state, the complete medium was replaced after 24 h incubation with low serum (0.5% FBS) medium. After this, the medium was replaced with low serum (0.5% FBS) medium containing various concentrations of quercetin. Cells receiving DMSO (0.1%) only served as a vehicle control. Plates were incubated for an additional 48 h, and cell proliferation was assessed using Cell Proliferation Kit II (MTS, Promega) in accordance with the manufacturer's protocol. MTS test solution (20 ml) was added to each well, and the cells incubated for an additional 4 h at 37 1C. The spectrophotometric absorbance of each well was measured using a multilabel-counter fluorescent plate reader (Infinites M 1000, Tecan, Switzerland). The wavelength used to measure absorbance was 490 nm. The results are expressed as the percentage of proliferating cells.
BD Matrigel™ Basement Membrane Matrix (growth factor reduced; BD Biosciences, San Jose, CA, USA) was thawed at 4 1C, pipetted into pre-chilled 24-well plates and incubated at 37 1C for 45 min. HUVECs were first incubated in endothelial cell growth medium (ECGM) supplemented with 0.5% FBS for 6 h and then treated with DMSO (0.1%) or various concentrations of quercetin (50, 100 or 200 mM). Cells were collected and placed onto a layer of Matrigel™ (4 104 cells per well) in 1 ml ECGM supplemented with 0.5% FBS. After 6 h of incubation at 37 1C in a 95%:5% (v/v) mixture of air and CO2, the network-like structures of the endothelial cells were examined under an inverted microscope (Olympus, Center Valley, PA, USA). The tube-like structures were defined as endothelial cord formations that were connected at both ends. Branching points in three random fields per well were quantified by manual counting. Cells receiving only DMSO (0.1%) served as a vehicle control. The inhibition percentage is expressed as the percentage of the vehicle control (100%). The assay was repeated three times independently.
2.2. Maintenance of zebrafish and treatment of embryos
2.4. Morphological observation of zebrafish After drug treatment, embryos were anesthetized using 0.01% tricaine (Sigma-Aldrich) and observed for any morphological changes using an Olympus spinning disk confocal microscope system (IX81-ZDC motorized inverted microscope). Images were analyzed with ImageJ and Adobe Photoshop 7.0 software.
2.8. Quantitative real-time PCR The effects of quercetin on certain genes were determined by quantitative real-time polymerase chain reaction (qRT-PCR). HUVECs (5 105 cells per well) were seeded in 24-well plates,
362
D. Zhao et al. / European Journal of Pharmacology 723 (2014) 360–367
and starved with ECGM containing 0.5% FBS for 24 h. After the pre-incubation, cells were treated with various concentrations of quercetin (50, 100 or 200 mM) for another 48 h. Then, cells were harvested in TRIzolH reagent, and their RNA was extracted and assessed for integrity by agarose-gel electrophoresis. RNA samples were quantified at OD260/OD280, and RNA was introduced to reverse transcribe to single-stranded cDNA using a PrimeScript™ RT reagent kit (TaKaRa, Otsu, Shiga, Japan), followed by qTR-PCR using SYBRH Premix Ex Taq™ (TaKaRa, Otsu, Shiga, Japan). The reverse-transcribed RNA was primed with oligonucleotides specific for VEGFR-2 (Flk-1; forward: 5′-GACTGTGGCGAAGTGTTTTTGA3′ and reverse: 5′-GTGCAGGGGAGGGTTGGCGTAG-3′) and β-actin (forward: 5′-GTGCGGGACATCAAGGAGAA-3′ and reverse: 5′AGGAAGGAGGGCTGGAAGAG-3′; Applied Biosystems, Carlsbad, CA, USA). The PCR program was set in accordance with the manufacturer's instructions. The abundance of mRNA was normalized to β-actin levels and expressed as percentage of the vehicle control (100%) for statistical analysis. Three independent experiments were performed.
Fig. 1. Toxic effects of quercetin on zebrafish. Tg(fli1: EGFP) zebrafish embryos were treated with various concentrations of quercetin (control, 50, 100, 200, 300, 500, 700 or 1000 mM) from 6 to 72 hpf.
2.9. Western blotting analysis To determine the effects of quercetin on the signaling cascades, HUVECs were first starved in ECGM containing 0.5% FBS for 12 h. After being washed with fresh medium, cells were treated with DMSO (0.1%) or various concentrations of quercetin (50, 100 or 200 mM) for 48 h for ERK phosphorylation. Zebrafish embryos were treated with DMSO (0.1%) or various concentrations of quercetin (50, 100 or 200 mM) at 28.5 1C from 6 to 72 hpf. Then, cell extracts were prepared in radioimmunoprecipitation assay buffer supplemented with phenylmethylsulfonyl fluoride. Proteins were resolved by electrophoresis, transferred out of the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel onto polyvinylidene difluoride membranes and probed overnight with the following antibodies at 4 1C: anti-p-ERK (1:1000, CST), mouse anti-ERK (1:1000, CST) and rabbit anti-GAPDH (1:1000, Santa Cruz). Goat anti-rabbit IgG and goat anti-mouse IgG (1:1000, Jackson ImmunoResearch) conjugated to horseradish peroxidase (GE Healthcare, Bucks, UK) were used as secondary antibodies, incubated at 37 1C for 1 h at room temperature. The grey value of each band was measured by ImageJ software (NIH). 2.10. Statistical analysis Each experiment was performed at least three times, and all values are presented as the mean 7S.E.M. of the triplicates. One-way analysis of variance with Tukey's post-hoc test was used to analyze the statistical significance of the results. Values of P o0.05 were considered statistically significant.
3. Results 3.1. Quercetin-induced morphological abnormalities during the early embryonic developmental stages of zebrafish To investigate the toxicity of quercetin in the zebrafish embryo, we performed a lethality assay in zebrafish embryos. The zebrafish embryos were treated with different concentrations of quercetin (50, 100, 200, 300, 500, 700 or 1000 mM) from 6 to 72 hpf. We found that the zebrafish embryos began to die when the concentration was higher than 200 mM, suggesting that the minimum lethal concentration of quercetin was 200 mM (Fig. 1). For this reason, 200 mM was selected as the largest safe concentration for further experiments in zebrafish.
Fig. 2. Morphological changes caused by quercetin during early embryonic development in zebrafish. (A) Embryos treated with different concentrations of quercetin were imaged at 72 hpf. Scale bar, 300 mm. (B) The percentage of morphologically abnormal and normal zebrafish treated with different concentrations of quercetin compared to the control (n¼ 27 per group).
In order to assess the phenotype generated by quercetin in zebrafish embryogenesis, we took bright field photos at 72 hpf and found that embryos treated with DMSO (0.1%) or quercetin (50 and 100 mM) developed normally, while 54.05% of the embryos treated with 200 mM quercetin displayed mildly upward-curved tails and a light pigment; however, the survival ratio was not affected (Fig. 2A and B). 3.2. Quercetin inhibited the angiogenesis process To evaluate the anti-angiogenic effects of quercetin in vivo, we inspected the inhibitory effects of quercetin on zebrafish blood vessel formation. Tg(fli1: EGFP) transgenic zebrafish embryos were treated with DMSO (0.1%) or different concentrations of quercetin (50, 100 or 200 mM) from the shield stage 6 to 72 hpf (the time point at which all ISVs had stretched to form dorsal longitudinal anastomotic vessels). We then examined ISV, dorsal aorta (DA) and
D. Zhao et al. / European Journal of Pharmacology 723 (2014) 360–367
363
Fig. 3. Quercetin treatment caused defects in zebrafish vascular development in vivo. (A) Fluorescence images showing the gross morphology of 72-hpf Tg(fli1: EGFP) zebrafish embryos treated with DMSO alone or different concentrations of quercetin. The hexagon indicates the sites of complete ISVs in zebrafish embryos for all figures, and pentagons indicate the sites of angiogenic sprouts in zebrafish embryos for all figures. Red and yellow bars indicate the lumina of the DA and PCV, respectively. All images shown are lateral views with rostral right and dorsal up. Scale bar, 50 mm. (B) Quantification of the ISV length in 72-hpf embryos in the vehicle control group and quercetin-treated groups. (C) Quantification of the DA lumina diameter in 72-hpf embryos in the vehicle control group and quercetin-treated groups. (D) Quantification of the PCV lumina diameter in 72-hpf embryos in the vehicle control group and quercetin-treated groups. Data are expressed as mean 7S.E.M. from three independent experiments (nP o 0.05; #Po 0.01 versus control; one-way ANOVA with Tukey's post-hoc test). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
posterior cardinal vein (PCV) development at 72 hpf using in vivo fluorescence imaging. We found that the mean ISV length was 101.317 2.38 mm after treatment with 50 mM quercetin, 84.25 71.63 mm after treatment with 100 mM quercetin and 72.25 71.29 mm after treatment with 200 mM quercetin. The mean ISV length of the control group was 106.06 71.34 mm, and it is clear that treatment with quercetin resulted in shorter ISVs than the control, and the lengths were significantly shorter following treatment with 100 and 200 mM quercetin compared to the control group (Fig. 3A and B; nP o0.05; #P o0.01; n ¼20). We also found that the diameter of the DA and PCV narrowed following treatment with quercetin. The DA and PCV diameters were 28.69 70.41 mm and 35.72 70.32 mm, respectively, following treatment with 50 mM quercetin, 25.11 70.31 mm and 35.3970.35 mm following treatment with 100 mM quercetin and 20.91 70.39 mm and 33.30 70.30 mm following treatment with 200 mM quercetin. Akin to the changes observed in the mean ISV lengths, the DA diameter was significantly narrower for the groups treated with 100 or 200 mM quercetin compared to the control group (29.64 70.77 mm; Fig. 3A and C; nP o0.05; #P o0.01; n ¼20). With regards to the diameter of the PCV, a significant difference was found between the group treated with 200 mM quercetin (33.370.31 mm) and the control group (36.12 7 0.35 mm; Fig. 3A and D; nP o0.05; n¼ 20). These results
demonstrate that quercetin can serve as an inhibitor of angiogenesis in zebrafish embryos. Furthermore, we found that quercetin had an obvious effect on subintestinal vessel (SIV) formation at doses of 50, 100 and 200 mM (Fig. 4A). SIV branch points were reduced by 26% (nP o0.05; n ¼20) and 58% (#P o0.01; n ¼20) following treatment with 100 and 200 mM quercetin, respectively, compared to the control group (Fig. 4A and B). We also measured the SIV length following the administration of different doses of quercetin, and we found a significant change in length following treatment with 100 mM (1302.44 732.23 mm) and 200 mM (1086.44 742.13 mm) quercetin compared to the SIV length of the control group (1602.02 7 36.09 mm) by quantitative analysis (Fig. 4A and C). These data demonstrate that quercetin can impair angiogenesis. 3.3. Quercetin inhibited the ERK signaling pathway in vivo To determine whether quercetin inhibits anti-angiogenesis through downregulating the ERK signaling pathway, we performed immunoblotting of p-ERK expression in zebrafish embryos treated with quercetin at 72 hpf (Fig. 5A). No significant difference in the p-ERK expression levels could be found between fish treated with 50 mM quercetin and the control. However, the levels of p-ERK protein on Western blots were significantly reduced
364
D. Zhao et al. / European Journal of Pharmacology 723 (2014) 360–367
Fig. 4. Quercetin inhibited angiogenesis of SIVs in vivo. (A) Fluorescence images showing the SIV morphology of 72-hpf Tg(fli1: EGFP) zebrafish embryos treated with DMSO or different concentrations of quercetin. Boxed areas are the sites of the SIVs in zebrafish embryos for all figures. Scale bar, 150 mm. (B) Quantification of the number of SIV branch points in 72-hpf embryos in the vehicle control group and quercetin-treated groups. (C) Quantification of the SIV length in 72-hpf embryos in the vehicle control group and quercetin-treated groups. Data are expressed as mean 7 S.E.M. from three independent experiments (nP o 0.05; #Po 0.01 versus control; one-way ANOVA with Tukey's post-hoc test).
Fig. 5. Quercetin inhibited ERK activation in vivo. (A) Western blots of p-ERK and ERK expression in 72-hpf whole embryos treated with various doses of quercetin. (B) Densitometric analysis of the relative level of p-ERK to ERK. Experiments were repeated three times (nPo 0.05; #Po 0.01 versus control; one-way ANOVA with Tukey's post-hoc test).
following treatment with 100 and 200 mM quercetin compared to the controls (Fig. 5A). Densitometric analysis also showed a significant decrease in p-ERK protein levels following treatment with 100 mM (0.47 70.038-fold reduction, P o0.05; n ¼20) and 200 mM quercetin (0.21 70.031-fold reduction, Po 0.01; n ¼20) compared to the control (Fig. 5B).
3.4. Quercetin inhibited cell viability in endothelial cells To illustrate the anti-angiogenic effects of quercetin in vitro, we performed Cell Proliferation Kit II (MTS) assays to detect HUVEC survival and proliferation. We found that quercetin observably inhibited HUVEC proliferation, and cell numbers were significantly
different following treatment with 50, 100 and 200 mM quercetin (Fig. 6, nP o0.05, #Po 0.01). 3.5. Quercetin inhibits tube formation in endothelial cells We measured the ability of endothelial cells to form tubular structures, using Matrigel™. As shown in Fig. 7, quercetin dosedependently inhibited HUVEC tube formation (Fig. 7A–D, A′–D′). In this experiment, tube formation was quantified by measuring the branching points upon treatment with quercetin (Fig. 7E) and significant inhibition was noted at doses of 50, 100 and 200 mM quercetin (nP o0.05, #P o0.01, n ¼3) compared to the control, indicating that quercetin prevents endothelial cells from forming tubular structures on Matrigel™.
D. Zhao et al. / European Journal of Pharmacology 723 (2014) 360–367
3.6. Quercetin inhibited VEFGR-2 mRNA expression in endothelial cells VEGFR-2 is the most biologically important receptor for VEGF. It regulates endothelial cell proliferation, migration, differentiation,
365
tube formation and angiogenesis (Nakatsu et al., 2003). To dissect the molecular basis of the quercetin-mediated anti-angiogenic effects, we investigated how quercetin affected VEGFR-2 protein expression in HUVECs using qRT-PCR analysis. We found that 50, 100 and 200 mM quercetin dramatically inhibited the expression of VEGFR-2 (Fig. 8). Our results show that quercetin exerts its antiangiogenic activity through regulation of VEGFR-2 activation.
3.7. Quercetin inhibited the ERK signaling pathway in endothelial cells
Fig. 6. Quercetin inhibited cell viability in endothelial cells in a dose-dependent manner under normal culture conditions. HUVECs were cultured in ECGM supplemented with 0.5% FBS prior to treatment with DMSO (0.1%) or various concentrations of quercetin (50, 100 or 200 mM) for 48 h. Cell viability was quantified by MTS assay. Cells receiving only DMSO (0.1%) served as a vehicle control. Data are expressed as percentages of the vehicle control and as the mean 7 S.E.M. of triplicate experiments (nP o 0.05; #Po 0.01 versus control; oneway ANOVA with Tukey's post-hoc test).
To determine whether quercetin-mediated anti-angiogenesis involved the ERK signaling pathway, we performed immunoblotting of p-ERK expression in HUVECs treated with quercetin for 48 h. No significant differences in the p-ERK expression levels were observed following treatment with 50 mM quercetin (0.97 7 0.061-fold reduction) compared to the control. However, Western blotting showed that the levels of p-ERK protein following treatment with 100and 200 mM quercetin were significantly reduced compared to the control (Fig. 9A). Densitometric analysis also showed a significant decrease in p-ERK protein levels following treatment with 100 mM (0.38 70.035-fold reduction, P o0.05; n ¼3) and 200 mM quercetin (0.16 70.037-fold reduction, Po 0.01; n ¼3) compared to the controls (Fig. 9B).
Fig. 7. Anti-angiogenic effect of quercetin in HUVECs. (A)–(D) Tube formation assay showing the morphological features of quercetin-treated HUVECs on Matrigel™. (A′)–(D′) Higher magnification images of those shown in (A)–(D). The branching points decrease after treatment with quercetin. (E) Quantitative data of the branch points are presented as mean 7S.E.M. Experiments were repeated three times (nPo 0.05; #Po 0.01 versus control; one-way ANOVA with Tukey's post-hoc test).
366
D. Zhao et al. / European Journal of Pharmacology 723 (2014) 360–367
4. Discussion Quercetin, a bioactive flavonoid from Hypericum attenuatum Choisy, has been found to have various pharmacological activities, including anti-oxidant, anti-inflammatory and anti-tumor activities (Boots et al., 2008; Linsalata et al., 2010; Mahmoud et al., 2013; Rajendran et al., 2004; Ruiz et al., 2007; Russo et al., 2010). However, little is known about its functions and mechanism of action with regards to angiogenesis. In our study, we demonstrated that quercetin is a potent anti-angiogenesis agent in both zebrafish, which are known to be a good model for drug discovery (Lawson and Weinstein, 2002; Serbedzija et al., 1999), and HUVECs. Our study is the first to show that quercetin inhibited the development of the ISVs, DA and PCV in transgenic zebrafish embryos, particularly, at doses of 100 and 200 mM. These findings are in agreement with previous studies, which have shown that quercetin can inhibit the development of abdominal aortic aneurysms and reduce aortic size in mice (Wang et al., 2012). In a prostate xenograft mouse model, quercetin inhibited tumorigenesis by targeting angiogenesis (Pratheeshkumar et al., 2012). Studies have also shown that quercetin exerts a neuroprotective effect in zebrafish through the blood brain barrier (Richetti et al., 2011; Zhang et al., 2011). Although the embryos treated with 200 mM quercetin showed mildly upward-curved tails and a light pigment, no significant morphological or color changes were found when the zebrafish were treated with 50 or 100 mM quercetin. Angiogenesis is a complex process containing several important steps (Risau, 1997); however, endothelial cell lines can be
used as an in vitro model for angiogenesis studies (Chen et al., 2008). In the present work, we observed that quercetin effectively inhibited human endothelial cell migration, proliferation and tube formation in vitro, and these effects were dose dependent. Studies have reported that quercetin can suppress angiogenesis and inhibit tumor growth through the suppression of blood vessel growth in vitro and in vivo (Anand et al., 2011; Fotsis et al., 1997; Pratheeshkumar et al., 2012; Rodgers and Grant, 1998). Quercetin can also inhibit the migration and tube formation of the rhesus macaque choroid–retina endothelial cell line (Chen et al., 2008). All these data indicate that quercetin can inhibit angiogenesis. VEGF has been proven to promote the process of angiogenesis through regulating proliferation, migration and differentiation of endothelial cells through two receptors, namely, VEGFR-1 and VEGFR-2 (Tie and Desai, 2012). In our study, quercetin significantly inhibited the levels of VEGFR-2 mRNA in HUVECs compared to the control group. Studies have shown that quercetin suppresses VEGF-induced phosphorylation of VEGFR-2, AKT, mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (P70S6K) in HUVECs (Chen and Fang, 2002; Ferrara et al., 2003; Pratheeshkumar et al., 2012). ERK, which is a member of the MAPKs, is a key regulatory protein that mediates cell survival, proliferation, and differentiation (Olsson et al., 2006; Yu et al., 2010). The molecular mechanisms in endothelial cells that have previously been shown to be inhibited by free aglycone are the MAPK, JNK and focal adhesion kinase pathways (Kaneider et al., 2004; Kobuchi et al., 1999) as well as metalloproteinase-2 expression and activity (Tan et al., 2003). Our in vivo and in vitro studies showed that doses of 100 and 200 mM quercetin significantly suppressed ERK phosphorylation levels, indicating that the anti-angiogenesis activity of quercetin may involve the downregulation of the ERK signaling pathway. Consistent with this hypothesis, quercetin is known to reduce MAPK activation, which is an intracellular signal underlying endothelial activation (Donnini et al., 2006). Recently, some studies have shown that quercetin acts as a novel mediator of angiogenesis by downregulating the phosphorylation of the AKT/ mTOR/p70S6K signaling pathway (Chen and Fang, 2002; Igura et al., 2001; Pratheeshkumar et al., 2012).
5. Conclusions Fig. 8. Quercetin insignificantly inhibits the VEGFR-2 mRNA expression in HUVECs. HUVECs (5 105 cells per well) were treated with various concentrations of quercetin (50, 100 or 200 mM) for 48 h. Cells receiving only DMSO (0.1%) served as vehicle controls. The levels of VEGFR-2 mRNA were normalized by β-actin and expressed as percentages of the vehicle control (100%) and as the mean 7 S.E.M. Experiments were repeated three times (nPo 0.05; #Po 0.01 versus control; one-way ANOVA with Tukey's post-hoc test).
Altogether, our study suggests that quercetin can inhibit blood vessel development in zebrafish and prevent cell viability, proliferation and tube formation in HUVECs. We have shown that the mechanism of this anti-angiogenic activity is, at least in part, due to the inhibition of ERK phosphorylation in vivo and in vitro, or the suppression of the VEGFR-2-mediated signaling pathway in
Fig. 9. Quercetin inhibited ERK activation in HUVECs. (A) Western blots of p-ERK and ERK expression in HUVECs treated with various concentrations of quercetin for 48 h. (B) Densitometric analysis of the relative levels of p-ERK compared to ERK. Experiments were repeated three times (nP o 0.05; #P o0.01 versus control; one-way ANOVA with Tukey's post-hoc test).
D. Zhao et al. / European Journal of Pharmacology 723 (2014) 360–367
endothelial cells. Hence, our study may prompt further development of quercetin as a novel inhibitor of angiogenesis for the treatment of a number of diseases and cancers.
Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (No. 31260642), China Postdoctoral Science Foundation (No. 2012M521292, No. 2013T60650), the Natural Science Foundation of Jiangxi Province (No. 20132BAB214015), the Foundation of Jiangxi Educational Committee (No. GJJ12144), Jiangxi Postdoctoral Researchers Projects (No. 2013KY23), the Open Project Program of State Key Laboratory of Food Science and Technology of Nanchang University (No. SKLF-KF-201001) and Key Technologies R & D Program of Neijiang, Sichuan of China (No. 12108). References Ajay, M., Achike, F.I., Mustafa, A.M., Mustafa, M.R., 2006. Effect of quercetin on altered vascular reactivity in aortas isolated from streptozotocin-induced diabetic rats. Diabetes Res. Clin. Pract. 73, 1–7. Alvarez, A., Pomar, F., Sevilla, M.A., Montero, M.J., 1999. Gastric antisecretory and antiulcer activities of an ethanolic extract of Bidens pilosa L. var. radiata Schult Bip. J. Ethnopharmacol. 67, 333–340. Anand, K., Asthana, P., Kumar, A., Ambasta, R.K., Kumar, P., 2011. Quercetin mediated reduction of angiogenic markers and chaperones in DLA-induced solid tumours. Asian Pac. J. Cancer Prev. 12, 2829–2835. Bischoff, S.C., 2008. Quercetin: potentials in the prevention and therapy of disease. Curr. Opin. Clin. Nutr. Metab. Care 11, 733–740. Boots, A.W., Wilms, L.C., Swennen, E.L., Kleinjans, J.C., Bast, A., Haenen, G.R., 2008. In vitro and ex vivo anti-inflammatory activity of quercetin in healthy volunteers. Nutrition 24, 703–710. Chen, J., Fang, Y., 2002. A novel pathway regulating the mammalian target of rapamycin (mTOR) signaling. Biochem. Pharmacol. 64, 1071–1077. Chen, Y., Li, X.X., Xing, N.Z., Cao, X.G., 2008. Quercetin inhibits choroidal and retinal angiogenesis in vitro. Graefes Arch. Clin. Exp. Ophthalmol. 246, 373–378. Cheng, S.H., Han, P.K., Wu, R.S., 2001. The use of microangiography in detecting aberrant vasculature in zebrafish embryos exposed to cadmium. Aquat. Toxicol. 52, 61–71. Donnini, S., Solito, R., Giachetti, A., Granger, H.J., Ziche, M., Morbidelli, L., 2006. Fibroblast growth factor-2 mediates Angiotensin-converting enzyme inhibitorinduced angiogenesis in coronary endothelium. J. Pharmacol. Exp. Ther. 319, 515–522. Ferrara, N., Gerber, H.P., LeCouter, J., 2003. The biology of VEGF and its receptors. Nat. Med. 9, 669–676. Fotsis, T., Pepper, M.S., Aktas, E., Breit, S., Rasku, S., Adlercreutz, H., Wähälä, K., Montesano, R., Schweigerer, L., 1997. Flavonoids, dietary-derived inhibitors of cell proliferation and in vitro angiogenesis. Cancer Res. 57, 2916–2921. Igura, K., Ohta, T., Kuroda, Y., Kaji, K., 2001. Resveratrol and quercetin inhibit angiogenesis in vitro. Cancer Lett. 171, 11–16. Jackson, J.R., Seed, M.P., Kircher, C.H., Willoughby, D.A., Winkler, J.D., 1997. The codependence of angiogenesis and chronic inflammation. FASEB J. 11, 457–465. Kaneider, N.C., Mosheimer, B., Reinisch, N., Patsch, J.R., Wiedermann, C.J., 2004. Inhibition of thrombin-induced signaling by resveratrol and quercetin: effects on adenosine nucleotide metabolism in endothelial cells and platelet–neutrophil interactions. Thromb. Res. 114, 185–194. Kobuchi, H., Roy, S., Sen, C.K., Nguyen, H.G., Packer, L., 1999. Quercetin inhibits inducible ICAM-1 expression in human endothelial cells through the JNK pathway. Am. J. Physiol. 277, C403–411.
367
Larrivee, B., Freitas, C., Suchting, S., Brunet, I., Eichmann, A., 2009. Guidance of vascular development: lessons from the nervous system. Circ. Res. 104, 428–441. Lawson, N.D., Weinstein, B.M., 2002. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev. Biol. 248, 307–318. Linsalata, M., Orlando, A., Messa, C., Refolo, M.G., Russo, F., 2010. Quercetin inhibits human DLD-1 colon cancer cell growth and polyamine biosynthesis. Anticancer Res. 30, 3501–3507. Mahmoud, M.F., Hassan, N.A., El Bassossy, H.M., Fahmy, A., 2013. Quercetin protects against diabetes-induced exaggerated vasoconstriction in rats: effect on low grade inflammation. PLoS One 8, e63784. McMahon, G., 2000. VEGF receptor signaling in tumor angiogenesis. Oncologist 5, 3–10. Nakatsu, M.N., Sainson, R.C., Pérez-del-pulgar, S., Aoto, J.N., Aitkenhead, M., Taylor, K.L., Carpenter, P.M., Hughes, C.C., 2003. VEGF (121) and VEGF (165) regulate blood vessel diameter through vascular endothelial growth factor receptor 2 in an in vitro angiogenesis model. Lab. Invest. 12, 1873–1885. Olsson, A.K., Dimberg, A., Kreuge,r, J., Claesson-Welsh, L., 2006. VEGF receptor signalling-in control of vascular function. Nat. Rev. Mol. Cell Biol. 7, 359–371. Peterson, R.T., Mably, J.D., Chen, J.N., Fishman, M.C., 2001. Convergence of distinct pathways to heart patterning revealed by the small molecule concentramide and the mutation heart-and-soul. Curr. Biol. 11, 1481–1491. Potente, M., Gerhardt, H., Carmeliet, P., 2011. Basic and therapeutic aspects of angiogenesis. Cell 146, 873–887. Pratheeshkumar, P., Budhraja, A., Son, Y.O., Wang, X., Zhang, Z., Ding, S., Wang, L., Hitron, A., Lee, J.C., Xu, M., Chen, G., Luo, J., Shi, X., 2012. Quercetin inhibits angiogenesis mediated human prostate tumor growth by targeting VEGFR-2 regulated AKT/nTOR/P70S6K signaling pathways. PLoS One 7, e47516. Rajendran, M., Manisankar, P., Gandhidasan, R., Murugesan, R., 2004. Free radicals scavenging efficiency of a few naturally occurring flavonoids: a comparative study. J. Agric. Food Chem. 52, 7389–7394. Richetti, S.K., Blank, M., Capiotti, K.M., Piato, A.L., Bogo, M.R., Vianna, M.R., Bonan, C. D., 2011. Quercetin and rutin prevent scopolamine-induced memory impairment in zebrafish. Behav. Brain Res. 217, 10–15. Risau, W., 1997. Mechanisms of angiogenesis. Nature 386, 671–674. Roberts, W.G., Palade, G.E., 1997. Neovasculature induced by vascular endothelial growth factor is fenestrated. Cancer Res. 57, 765–772. Rodgers, E.H., Grant, M.H., 1998. The effect of the flavonoids, quercetin, myricetin and epicatechin on the growth and enzyme activities of MCF-7 human breast cancer cells. Chem. Biol. Interact. 116, 213–228. Ruiz, P.A., Braune, A., Holzlwimmer, G, Quintanilla-Fend, L., Haller, D., 2007. Quercetin inhibits TNF-induced NF-kappaB transcription factor recruitment to proinflammatory gene promoters in murine intestinal epithelial cells. J. Nutr. 137, 1208–1215. Russo, M., Spagnuolo, C., Volpe, S., Mupo, A., Tedesco, I., Russo, G.L., 2010. Quercetin induced apoptosis in association with death receptors and fludarabine in cells isolated from chronic lymphocytic leukaemia patients. Br. J. Cancer 103, 642–648. Serbedzija, G.N., Flynn, E., Willett, C.E., 1999. Zebrafish angiogenesis: a new model for drug screening. Angiogenesis 3, 353–359. Shoskes, D.A., Nickel, J.C., 2011. Quercetin for chronic prostatitis/chronic pelvic pain syndrome. Urol. Clin. North Am. 38, 279–284. Tan, W.F., Lin, L.P., Li, M.H., Zhang, Y.X., Tong, Y.G., Xiao, D, Ding, J., 2003. Quercetin, a dietary-derived flavonoid, possesses antiangiogenic potential. Eur. J. Pharmacol. 459, 255–262. Tie, J., Desai, J., 2012. Antiangiogenic therapies targeting the vascular endothelia growth factor signaling system. Crit. Rev. Oncog. 17, 51–67. Wang, L., Wang, B., Li, H., Lu, H., Qiu, F., Xiong, L., Xu, Y., Wang, G., Liu, X., Wu, H., Jing, H., 2012. Mice treated with quercetin exhibited a 32.7% reduction in aortic size compared with vehicle-treated controls. Eur. J. Pharmacol. 690, 133–141. Westerfield, M., 1993. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish. The University of Oregon Press, Eugene, OR. Yu, P.C., Gu, S.Y., Bu, J.W., Du, J.L., 2010. TRPC1 is essential for in vivo angiogenesis in zebrafish. Circ. Res. 106, 1221–1232. Zhang, Z.J., Cheang, L.C., Wang, M.W., Lee, S.M., 2011. Quercetin exerts a neuroprotective effect through inhibition of the iNOS/NO system and pro-inflammation gene expression in PC12 cells and in zebrafish. Int. J. Mol. Med. 27, 195–203.