In vitro and in vivo mechanistic study of a novel proanthocyanidin, GC-(4→8)-GCG from cocoa tea (Camellia ptilophylla) in antiangiogenesis

Available online at www.sciencedirect.com

ScienceDirect Journal of Nutritional Biochemistry 25 (2014) 319 – 328

In vitro and in vivo mechanistic study of a novel proanthocyanidin, GC-(4→8)-GCG from cocoa tea (Camellia ptilophylla) in antiangiogenesis Kai-kai Li a, 1 , Cheuk-lun Liu b, c, 1 , Jacqueline Chor-wing Tam b, c , Hin-fai Kwok b, c , Ching-po Lau b, c , Ping-chung Leung b, c, d , Chun-hay Ko b, c, d,⁎, Chuang-xing Ye a,⁎ a

Department of Biology, School of Life Sciences, Sun Yat-sen University, Guangzhou, China b Institute of Chinese Medicine, The Chinese University of Hong Kong, Hong Kong, China c State Key Laboratory of Phytochemistry and Plant Resources in West China, The Chinese University of Hong Kong, Hong Kong, China d The Chinese University of Hong Kong Shenzhen Research Institute

Received 2 May 2013; received in revised form 29 October 2013; accepted 9 November 2013

Abstract Angiogenesis, the process of blood vessel formation, is critical to tumor growth. Ant-angiogenic strategies demonstrated importance in cancer therapy. Cocoa tea (Camellia ptilophylla), a naturally decaffeinated tea commonly consumed as a healthy drink in southern China, had recently been found to be a potential candidate for antiangiogenesis. A novel proanthocyanidin, GC-(4→8)-GCG, which consisted of gallocatechin and gallocatechin 3-O gallate moieties, was discovered and thought to be one of the effective candidates for antiangiogenesis. Hence, the present study aimed to evaluate the antiangiogenesis activities of GC-(4→8)-GCG in vitro and in vivo, and SU5416 was applied as a positive control. The inhibitory effects of GC-(4→8)-GCG on three important processes involved in angiogenesis, i.e., proliferation, migration and differentiation, were examined using human microvascular endothelial cell line HMEC-1 by MTT assay, scratch assay and tube formation assay, respectively. Using transgenic zebrafish embryos TG(fli1:EGFP)y1/+(AB) as an animal model of angiogenesis, the antiangiogenic effect of GC-(4→8)-GCG was further verified in vivo. Our results demonstrated that GC-(4→8)-GCG significantly inhibited migration (Pb.001) and tubule formation (Pb.001–.05) of HMEC-1 in dose-dependent manner. Regarding intracellular signal transduction, GC-(4→8)-GCG attenuated the phosphorylation of ERK, Akt and p38 dose-dependently in HMEC-1. In zebrafish embryo, the formation of new blood vessels was effectively inhibited by GC(4→8)-GCG in a dose-dependent manner after 3 days of treatment (Pb.001–.05). In conclusion, these results revealed that our novel proanthocyanidin, GC(4→8)-GCG might be a potential and promising agent of natural resource to be further developed as an antiangiogenic agent. © 2014 Elsevier Inc. All rights reserved. Keywords: Antiangiogenesis; GC-(4→8)-GCG; HMECs; Transgenic TG(fli1:EGFP)y1/+(AB) zebrafish embryo; Camellia ptilophylla

1. Introduction Angiogenesis, the formation of new blood vessel, is essential in normal development of embryo and fetus as well as in normal physiological processes, including wound healing [1,2]. However, dysregulated angiogenesis is pathological and often associated with tumor growth, rheumatoid arthritis, diabetic retinopathy and hemangiomas. Initiation, growth and development of new blood vessels through angiogenesis are crucial for tumor growth. In order to maintain growth and achieve metastasis, tumor masses require access to blood vessels for a sufficient supply of oxygen and nutrients. New blood vessel formation might favor the transition from ⁎ Corresponding authors. C.-H. Ko is to be contacted at E302, Institute of Chinese Medicine, Science Centre East, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China. Tel.: +852 3163 4134; fax: +852 2603 5248. C.-X. Ye, Department of Biology, School of Life Sciences, Sun Yat-sen University, Guangzhou, China. E-mail address: [email protected] (C. Ko). 1 Authors with equal contribution. 0955-2863/$ - see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jnutbio.2013.11.006

hyperplasia to neoplasia which marks the onset of uncontrolled tumor growth [3]. Thus, angioprevention is a good therapeutic approach in cancer therapy. Over the last few decades, various inhibitors of angiogenesis had been developed to inhibit or slow down the growth of tumors by blocking blood vessel formation, including TNP-470 and SU5416. TNP-470 is a synthetic analogue of fumagillin, a natural compound secreted by the Aspergillus fumigatus; it blocks endothelial cell cycle progression in the late G1 phase and activates p53 in endothelial cells [4]. Moreover, TNP-470 could inhibit vascular endothelial growth factor (VEGF)-induced endothelial function and inhibit the proliferation of many types of tumor cells [5]. SU5416 is a selective inhibitor of the tyrosine kinase activity of the VEGF receptor Flk-1/KDR. SU5416 demonstrated potent antiangiogenic effects [6]. These inhibitors possess strong antiangiogenesis effects, and these chemical compounds are currently in clinical trials as a tumor vascular-targeting agent for the treatment of advanced malignancies. However, the efficacy of antiangiogenesis treatment in a number of cases offered was a transient effect, and drug resistance was developed after several months of treatment [7]. Besides, there was also increasing evidence

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showing that these drugs have the potential to cause a number of adverse effects upon long-term administration. For instance, the angiogenic inhibitor TNP-470 was found to be associated with various neurological problems including motor coordination, short-term memory and concentration, dizziness, confusion, anxiety and depression [8–10]. Another antiangiogenic agent, combretastatin A-4 phosphate (CA4P), may cause hypertension and tachycardia [11]. The use of plants in medicine is widely applied to both preventive and curative purposes. Generally speaking, most of the natural products are relatively nontoxic and could be consumed as dietary supplement. Therefore, in recent years, clinicians and scientists had attempted to mine natural resources for antiangiogenesis agents. Increasing evidences showed that a number of dietary bioactive phytochemicals, such as resveratrol, curcumin and proanthocyanidins, possessed antiangiogenic potential [12]. Brakenhielm et al. demonstrated that resveratrol was effective in inhibiting angiogenesis in vivo through blocking Src-dependent tyrosine phosphorylation of VE-cadherin [13]. On the other hand, Yoysungnoen et al. reported that curcumin could inhibit tumor angiogenesis through the reduction of COX-2 and VEGF protein expression [14]. Apart from those phytochemicals, green tea polyphenols had been recognized for their antiangiogenic properties. Oral administration of green tea polyphenols in drinking water was found to inhibit the protein expression and activity of both angiogenic protease MMP-2 and MMP-9 in the tumors [15]. Among the tea polyphenols, epigallocatechin-3-gallate (EGCG) had been considered as the major constituent in chemopreventive effect [16]. Mantena et al. found that EGCG could inhibit photocarcinogenesis through inhibition of angiogenic factors and activation of CD8+ T cells in UV-B-induced skin tumors [17]. Jung and Ellis observed that EGCG not only inhibits new blood vessel formation and tumor growth but also induces tumor cell apoptosis [18]. Piyaviriyakul et al. reported that 10 μM EGCG could significantly inhibit the invasion and tube formation of HUVECs in vitro and angiogenesis in vivo in dorsal air sac model mice; however, other tea polyphenols such as catechin (C), epicatechin (EC), epicatechin-3-gallate (ECG) and epigallocatechin (EGC) did not inhibit the motility and invasion of HUVECs at this concentration [19]. A naturally decaffeinated tea plant, Camellia ptilophylla, which was named cocoa tea, was discovered in 1981. This was an endemic tree growing on the highlands in the Longmen area of southern China. Cocoa tea has a similar chemical profile as traditional tea (Camillia sinensis). However, because of the deficiency in caffeine synthase, cocoa tea contained theobromine instead of caffeine, and the major catechin was (−)-gallocatechin gallate (GCG) instead of (−)epigallocatechin gallate EGCG [20]. Proapoptotic effects of cocoa tea had been demonstrated previously [21]. In our previous studies, we found that cocoa tea extract could exert inhibitory ability against prostate cancer carcinoma both in vitro and in vivo [22]. In addition, in vivo studies also indicated that cocoa tea was effective in reducing the tumor size of human hepatocarcinoma xenografts in nude mice without apparent toxicity to the host [23]. Apart from proapoptotic effects on tumor cells, our preliminary results also indicated that cocoa tea could inhibit angiogenesis in human microvascular endothelial cell line (HMEC-1) in vitro tube formation and migration scratch assay in a dose-dependent manner (our unpublished data). Given that cocoa tea was effective in antiangiogenesis and its chemical profile is different from traditional green tea, we hypothesize that the main bioactive constituent responsible for antiangiogenesis in cocoa tea is different. In this study, we firstly identified a novel proanthocyanidin, compound GC-(4→8)-GCG, which consisted of gallocatechin and gallocatechin 3-O gallate moieties, in cocoa tea. The antiangiogenic potential of GC-(4→8)-GCG was also explored using the HMEC-1 for in vitro studies of cell proliferation, migration and tube formation. Moreover, the potential inhibition of angiogenesis by GC-(4→8)-GCG was

further studied in vivo using a transgenic zebrafish embryos TG(fli1: EGFP)y1/+(AB) model. 2. Material and methods 2.1. Extraction and isolation procedures of GC-(4→8)-GCG The cocoa tea water extract was adsorbed with XAD-7HP and then subjected to LH20 gel column chromatography eluted with 55% ethanol. Fractions that contained GC-(4→8)-GCG were combined and subjected to a C18 gel with 5% ethanol. The identification of the purified compound was based on the high-performance liquid chromatography (HPLC)–mass spectrometry (MS) and 1H and 13C nuclear magnetic resonance (NMR). 2.2. Cell culture Human microvascular endothelial cell line HMEC-1 was purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were maintained in MCDB131 medium supplemented with 10% fetal bovine serum (Invitrogen, CA, USA), 1 μg/ml hydrocortisone, 100 U/ml penicillin and streptomycin. The cells were incubated at 37°C in a humidified atmosphere of 5% CO2. When the cells reached 80% confluence in the culture flask, trypsin–EDTA was used to remove the cells. 2.3. Measurement of cell viability by MTT assay Cell viability was determined by 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. HMEC-1 cells (3×103 cells/well) were seeded in 96well culture plates and incubated overnight. Subsequently, various concentrations (12.5–400 μg) of GC-(4→8)-GCG were added into the wells. After 24-h treatment, 20 μl of MTT solution (5 mg/ml in PBS) was added into the wells. The cells were then incubated for 2 h at 37°C. Then, MTT solution was removed, and 100 μl of dimethyl sulfoxide (DMSO) was added. Absorbance was measured at 540 nm using a microplate reader. The percentage of cell viability was calculated against control. 2.4. Cell migration assay Cell migration assay, also known as scratch assay, was carried out as described previously [21]. HMEC-1 (1×105 cells) cells were seeded in 24-well plate and incubated with complete medium at 37°C and 5% CO2. After 24 h of incubation, the cells were starved in medium with 0.5% fetal bovine serum for 16 h. HMEC-1 cells were scrapped horizontally and vertically with a P100 pipette tip, and the wells were washed with phosphate-buffered saline (PBS) to remove detached cells. Two views on the cross were photographed on each well attached to the microscope at 40× magnification. The medium was replaced with fresh medium in the absence or presence of GC-(4→8)GCG. After 6 h of incubation, the second set of images was photographed. To determine the migration of HMEC, the images were analyzed using Tscratch software [24]. Percentage of the open wound area was measured and compared with the value obtained before treatment. An increase of the percentage of open wound area indicated the inhibition of cell migration. In this study, SU5416 was applied as a positive control. Four replicates were done in each individual experiment, and the images are representatives of three experiments with similar outcome. 2.5. Tubule formation assay The effect of GC-(4→8)-GCG on HMEC-1 differentiation and vascular formation was assessed by tubule formation on Matrigel [25]. Briefly, a 96-well plate was firstly coated with Matrigel and was allowed to solidify at 37°C for 1 h. Cells (1.5×104 in 100 μl of medium) were added to each well, and 100 μl of medium containing different concentrations of GC-(4→8)-GCG or SU5416 was added and incubated for 6 h. Each treatment was performed in triplicate. The tubules were photographed under a microscope (Nikon Eclipse TS100). The total tubule length formation was measured for quantification of angiogenesis by the Image-Pro Plus version 6.0 (Media Cybernetics, Bethesda, MD, USA). Inhibition of tubule formation was calculated as tubule length (treated) over tubule length (control) [26,27]. 2.6. Western blot assay Human endothelial cells HMEC-1 (1.25×105 cells/ ml) were seeded in 80-mm culture dish and incubated for 24 h to allow attachment. Various concentrations (50– 200 μg/ml) of GC-(4→8)-GCG were added to the dishes and incubated for 8 h. After treatment, cells were harvested and washed twice with PBS. The cell pellets were then lysed with whole cell extraction buffer [2% sodium dodecyl sulfate (SDS), 10% glycerol, 625 mM Tris–HCl, pH 6.8] for 30 min on ice and then centrifuged at 14,000 rpm for 20 min at 4°C. Protein concentration was measured using Bio-Rad DC Protein Assay kit (Bio-Rad, Hercules, CA, USA). Equal amount (20 μg) of protein samples was separated on a 12% SDS polyacrylamide gel and electrophoretically transferred (100 V, 2 h) onto a nitrocellulose membrane (Pall Gelman Laboratory, Ann Arbor, MI, USA). Afterwards, the membranes were blocked for 1 h using 5% nonfat dry milk. The following primary antibodies were used: anti-phospho-ERK1/2, anti-phospho-Akt, anti-phospho-p38 and

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anti-β-actin (Cell Signaling Technology, Beverly, MA USA; 1:2000). The membranes were incubated with the indicated antibodies at 4°C overnight. After incubation with the secondary horseradish-peroxidase-conjugated antibodies (Invitrogen, Carlsbad, CA, USA) for 1 h, detection was performed using enhanced chemiluminescence assay kit (GE Healthcare, UK). 2.7. Angiogenesis study of zebrafish embryos The transgenic zebrafish line TG(fli1:EGFP)y1/+(AB) with endothelial cells expressing enhanced green fluorescent protein was purchased from the Zebrafish International Resource Centre, University of Oregon, USA, and cultured with reference to a previous report [28]. The zebrafish were maintained at 28°C on a 10-h (light):14-h (dark) photoperiod and were fed with brine shrimp and tropical fish food twice daily. Healthy and regular embryos were selected at their 1–4 cell stage and distributed into a six-well microplate with 20 embryos per well depending on the assay [29]. Then, the embryos were treated with various concentrations of GC-(4→8)-GCG and SU5416. Embryos receiving 0.1% DMSO served as negative controls. After 72 h postfertilization (hpf), the embryos were examined using an Olympus IX71S8F-2 inverted microscope (Olympus, Tokyo, Japan) for the length of vessels in the subintestinal vessel plexus (SIVs) region to assess angiogenesis [30]. The vessel length formation was measured for quantification of angiogenesis by the Image-Pro Plus version 6.0 (Media Cybernetics, Bethesda, MD, USA). 2.8. Statistical analyses One-way analysis of variance with Dunnett’s multiple comparison test was used to analyze the statistical significance of the results. All experimental results were expressed as mean±standard deviation (S.D.) unless otherwise specified. For all assays, the significance of difference was calculated between GC-(4→8)-GCG-treated samples and control. The results were considered as statistically significant when P values were b.05.

3. Results 3.1. Structural details GC-(4→8)-GCG The negative mode of ESI–MS spectrum showed the m/z of 761 [M–H]−, suggesting that the molecular weight (MW) of GC-(4→8)GCG was 762. The 13C NMR spectrum showed that there were 30 aromatic carbon signals at δ157.355–94.175, five methenyl carbon signals at δ37.142–82.724 and one methylene carbon signals at δ25.876, one carbonyl carbon (δ 165.084). The 1H nuclear magnetic resonance (NMR) and heteronuclear multiple quantum correlation (HMQC) spectra showed two aromatic proton signals from B-ring at

Fig. 1. The structure of GC-(4→8)-GCG.

321

Table 1 H NMR and 13C NMR data for GC-(4→8)-GCG

1

Position

δC type

δH(J in Hz)

HMBC a

C2 C3 C4 A5 A6 A7 A8 A9 A10 B1’ B2’ B3’ B4’ B5’ B6’ F2 F3 F4

82.724, CH 70.943, CH 37.142, CH 155.533,qC 96.039, CH 155.979, qC 94.175, CH 157.355, qC 106.088, qC 130.535, qC 107.027, CH 145.432, qC 132.573, qC 145.432, qC 107.027, CH 78.519, CH 69.555, CH 25.876, CH2

4.105, d (8.8) 4.263, m 4.263, m

C3, C4, B1’, B2’, B6’ A10 C3, A5, A9, A10

5.705, d (2.4)

A5, A8, A10

5.633, d (2.4)

A6, A7, A10

6.373, s

C2, B1’, B3’, B4’, B6’

6.373, s 4.762, d (8.4) 5.274, ddd (6.0, 7.2, 8.4) α 2.940, dd (6.0, 16.4) β 2.570, dd (7.2, 16.4)

C2, B1’, B2’, B4’, B5’ F3,D9, E1’, E2’, E6’ D10 D10

D5 D6 D7 D8 D9 D10 E1’ E2’ E3’ E4’ E5’ E6’ G1” G2” G3” G4” G5” G6” G7”

153.268, qC 96.268, CH 153.852, qC 108.891, qC 154.466, qC 97.709, qC 128.544, qC 106.250, CH 145.601, qC 132.803, qC 145.601, qC 106.250, CH 119.251, qC 108.718, CH 145.432, qC 138.652, qC 145.432, qC 108.718, CH 165.084, qC

5.901, s

D5, D7, D8, D10

6.434, s

F2, E1’, E3’, E4’, E6’

6.434, s

F2, E1’, E2’, E4’, E5’

6.885, s

G1”, G3”, G4”,G6”,G7”

6.885, s

G1”, G2”, G4”, G5”, G7”

a

HMBC correlations are from proton(s) stated to the indicated carbon.

δ6.373 (2H, s, H-2', 6'), two aromatic proton signals from E-ring at δ6.434 (2H, s, H-2', 6'), two aromatic proton signals from G-ring at δ6.885 (2H, s, H-2', 6') and two meta-coupled doublets from A-ring at δ5.705 (1H, d, H6) and 5.633 (1H, d, H-8), revealing that this compound had four benzene rings with four substituents. The 1H NMR and HMQC spectra showed an aromatic proton signal from D-ring at δH 5.901 (H, s, H-6), indicating that this compound have a benzene ring with five substituents. The HMBC correlation between H-2'', 6'' (δ 6.885) and the carbonyl carbon at δ 165.084 indicated the presence of one galloyl group in the structure. Taken from the above analysis, the compound GC-(4→8)-GCG was the interconnection of two catechins and a gallic acid. One oxygenated methenyl proton signal at δ 4.105 (1H, d, J=8.8 Hz, H-2) and two methenyl proton signals at δ 4.263 (2H, m, H-3) and 4.263 (2H, m, H-4) from C-ring. The nuclear overhauser enhancement spectroscopy (NOESY) spectra showed no interrelation between these methenyl protons, suggesting that H-2 was on the opposite side of the H-3 and H-3 was on the opposite side with H-4, which indicated that this part of the compound was GC (A,B,C-rings). The results showed that two methenyl proton signals at δ 4.762(1H, d, J=8.4 Hz, H-2) and δ 5.274 (1H, ddd, J=6.0, 7.2, 8.4Hz, H-3), one methylene proton signals at 2.940 (1H, dd, H-3a), and 2.570 (1H, dd, H-3b) from F-ring. The NOESY spectra showed no interrelation between them, and H-2 was on the opposite side of the H-3; so this part of the compound was also GC (D,E,F-rings). And they were connected through C-4 of A-ring to C-8 of F-ring. In the spectrum of NOESY, the signal of dH6.885 was correlated with dH5.274, dH 4.762, dH 2.570 and 2.940.The above NMR data indicated

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Cell viability (%)

322

150

HMEC-1. By MTT cell viability assay, we found that it would be safe to use GC-(4→8)-GCG at or below 400 μg/ml for the subsequent functional assays as shown in Fig. 2.

100

3.3. Inhibition of endothelial cell migration by GC-(4→8)-GCG

50

0 0

6.25

12.5

25

50

100

200

400

800

Concentration(µg/ml) Fig. 2. Effects of GC-(4→8)-GCG on cell viability of HMEC-1 cells as determined by MTT assay. Data represent mean±S.D. for three independent experiments.

that the position of the galloyl ester group was located at C-3 of F-ring. Hence, this compound was identified as GC-(4→8)-GCG (Fig. 1), and the 1 H NMR (400 MHz) and 13C NMR (100 MHz) data were shown in Table 1. However, the absolute configuration of it needs further research.

Angiogenesis involved the acquisition of endothelial cells to degrade the basement membrane and to migrate through the extracellular matrix followed by subsequent adhesion and tubule network connection for new blood vessel formation. Cell migration assay (or scratch assay) allowed the testing of the effect of treatments on the migratory capabilities of cells growing in culture as monolayers, as in the case of endothelial cells. Fig. 3 shows the inhibitory effect of GC-(4→8)-GCG (12.5–100 μg/ ml, Pb.001) on endothelial cell migration in a dose–response manner. With the treatment of 1 μM SU5416, the cell migration was inhibited, and the open wound area was 91% at 6 h compared with 0 h. The open wound area (% 6 h/0 h) of the GC-(4→8)-GCG treated (12.5–100 μg/ ml, Pb.001) was significantly increased to around 90 to 100% when compared to that in control with around 75% only. These indicated that GC-(4→8)-GCG demonstrated inhibitory effect in endothelial cell migration. 3.4. Suppression of tubule formation by GC-(4→8)-GCG

3.2. Effects of GC-(4→8)-GCG on HMEC-1 cell viability In our study, we firstly evaluated the noncytotoxic range of GC-(4→8)-GCG for our further functional angiogenesis assay with

HMEC-1 cells without GC-(4→8)-GCG treatment formed a mature tubular network after 6 h of incubation. Upon treatment with various concentrations of GC-(4→8)-GCG, the network formed was less

A

B Open would area ( 6h/0h %)

150

***

100

***

***

***

25

50

100

***

50

0 0

12.5

SU 5416

Concentration(µg/ml) Fig. 3. Effects of GC-(4→8)-GCG on endothelial cell migration. (A) Representative photographs of 1 μM SU5416 and GC-(4→8)-GCG-treated HMEC-1 at times 0 h and 6 h. (B) Quantitative analysis of effects of GC-(4→8)-GCG on endothelial cell migration. Data represent mean±S.D. for three independent experiments (***Pb.001).

K.-K. Li et al. / Journal of Nutritional Biochemistry 25 (2014) 319–328

323

A

B

Tube length(% Control)

150

*

100

*** ***

***

50

*** 0 0

25

50

100

200

SU 5416

Concentration(µg/ml) Fig. 4. Effects of GC-(4→8)-GCG on tubule formation of endothelial cells with Matrigel. (A) Representative photographs of control and treatment with different concentrations of GC-(4→8)-GCG and 10 μM SU5416 on Matrigel after 6 h of treatment. (B) Quantitative analysis of effects of GC-(4→8)-GCG and SU5416 on tubule formation. Data represent mean± S.D. for three independent experiments. Symbols indicate significant differences between control-untreated and treated cells (*Pb.05; ***Pb.001).

complete with shorter tubule length (Fig. 4A). The total length of all tubules formed in each sample is summarized in Fig. 4B. As the results show, SU5416 (10 μM) inhibited the tubule formation with a

percentage of 53% compared with the control group. GC-(4→8)GCG inhibited the tubule formation of HMEC-1 cells in a dosedependent and significant manner, when the concentration of

324

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Fig. 5. Western blot analyses of effect of GC-(4→8)-GCG on signaling kinases. GC-(4→8)-GCG inhibits ERK, p38 and Akt phosphorylation in HMEC. Immunoblotting was performed three to four times using independently prepared cell lysates, and representative blots of HMEC-1 were shown.

GC-(4→8)-GCG was 25 μg/ml, the tubule formation was inhibited (Pb.05) with a significant suppression at concentrations of 50 to 200 μg/ml (Pb.001–.05). 3.5. Effect of GC-(4→8)-GCG on mitogen-activated protein kinase (MAPK) and Akt signaling Angiogenesis required the coordinated activation of various signaling pathways, including Akt and MAPK signaling pathways. To evaluate the effects of GC-(4→8)-GCG on intracellular signal transduction, the phosphorylation level of ERK, Akt and p38 was examined in endothelial cell lines. The results showed that 50, 100 and 200 μg/ml GC-(4→8)-GCG inhibited the phosphorylation of ERK, Akt and p38 in a dose-dependent manner in HMEC-1 (Fig. 5). 3.6. In vivo antiangiogenesis effect of GC-(4→8)-GCG in zebrafish embryos The potential antiangiogenic effect of GC-(4→8)-GCG was further translated and confirmed in zebrafish angiogenesis assays. At 72 hpf, the SIVs developed as a smooth basket-like structure with approximately five to six arcades (asterisks, Fig. 6A, B) in the control group. As the results show, treatment with SU5416 at a concentration of 2 μM caused a complete loss of SIVs. In control group, complete SIV formation was observed in most treated embryos. However, as shown in Fig. 6C, when the embryos were treated with GC-(4→8)-GCG, the SIV was inhibited when the concentration was 3.125 μg/ml (Pb.05), when the concentration of GC-(4→8)-GCG was 6.25 μg/ml or more, the length of SIV was significantly reduced when compared to that of the control (3.125–25 μg/ml, Pb.001–.05). 4. Discussion Aberrant angiogenesis is closely associated with a number of severe diseases including tumor metastasis, rheumatism and inflammatory diseases [31]. Antiangiogenic therapy has been increasingly considered as a promising anticancer therapeutic strategy [32,33]. Angiogenesis involves a series of orchestrated processes, including endothelial cell proliferation, migration, tube assembly and remodeling. Suppression at any step might inhibit angiogenesis [34]. An increasing interest was devoted to naturally occurring cancer chemopreventive agents, including flavonoids, which are rich in fruits, soybeans, herbs, roots and leaves [35]. These polyphenolic compounds display markable spectrum of biological activities. Those might be able to influence processes that were dysregulated during

cancer development, suggesting their suppressing ability in angiogenesis [36]. In analyzing the chemical profile of cocoa tea, a novel compound, GC-(4→8)-GCG, was firstly identified. The compound belonged to the proanthocyanidins class. Proanthocyanidins are oligomeric and polymeric end products of the flavonoid biosynthetic pathway. They are present in the fruits, bark, leaves and seeds of many plants. Proanthocyanidins are especially rich in tea, grape seed and cranberry [37]. Recently, they received recognition for possessing beneficial effects on human health supported by several lines of evidence; these studies stated that proanthocyanidins showed antioxidant [38], antimutagenic [39], anticancer [36] and anti-inflammatory activities [40,41]. In the present study, the antiangiogenic activities of GC-(4→8)GCG isolated from cocoa tea were studied for the first time using human microvascular endothelial cells, HMEC-1, and zebrafish embryo angiogenesis model in vivo. In the human body, majority of endothelial cells belong to the microvasculature [39]. HMEC-1, which carried a majority of traits to that of primary microvascular endothelial cell, have been widely used for endothelial research [42,43]. Using human endothelial cell HMEC-1, our MTT assay showed that GC-(4→8)-GCG did not have a toxic effect on the cell viability of HMEC-1 cells when the concentration was lower than 400 μg/ml for 24 h; this indicated that the antiangiogenesis effect of GC-(4→8)-GCG was not due to cytotoxicity. In the cell migration assay, the open wound area (% control) of the GC-(4→8)-GCG treated (12.5–100 μg/ ml, Pb.001) was significantly augmented to around 90% to 100% when compared to that in control with around 75%. These showed that GC-(4→8)-GCG demonstrated inhibitory effect in endothelial cell migration. From the results of tubule formation, GC-(4→8)-GCG could significantly inhibit tubule formation of HMEC-1 cell (25–200 μg/ml, Pb.001 to .05). Indeed, when the concentration reached 200 μg/ ml, the tubule formation was almost totally inhibited (Pb.001). The antiangiogenesis effects of SU5416 were also demonstrated in these above assays. This result indicated that GC-(4→8)-GCG, as the SU5416, has strong antiangiogenesis effects on the HMEC-1 cells. In parallel, our classic angiogenesis assays were validated by the wellknown VEGFR-2 inhibitor SU5416. Our in vitro angiogenesis assays suggested that GC-(4→8)-GCG possessed strong antiangiogenesis effect in microvascular endothelial cells HMEC-1. GC-(4→8)-GCG is a relatively big dimeric structure (MW: 762), which consists of GC and GCG. In order to investigate whether GC-(4→8)-GCG can enter the cells, the cellular uptake of GC-(4→8)GCG amount was performed previously using HPLC. It was found that the intracellular content of GC-(4→8)-GCG was 233.3±47.1 nmol/μg protein in the HMEC-1 cells after 6 h of GC-(4→8)-GCG treatment. A similar finding was reported in the other complex tea constituent, theaflavin-3,3'-digallate (TFDG). TFDG is a potent scavenger of superoxide with MW 868.7. Although TFDG is larger than GC-(4→8)-GCG, it was found that TFDG was dose-dependently absorbed by liver cells and did not reach plateau via oral administration in vivo, suggesting that TFDG uptake mainly occurs by a passive diffusion process [44]. MAPK and Akt activation participated in different stages of angiogenesis [45,46]. Extracellular signal-regulated kinases (ERKs) phorsphorylation had been shown to be involved in cell migration [46,47] and tubule formation [48,49]. Phorsphorylated ERK (p-ERK) activated myosin light chain kinase, which in turn promoted myosin light chains phosphorylation that regulated the actin–myosin interaction for the increase in cell migration. Akt was demonstrated in mediating angiogenesis [50] via cell migration [51]. In fact, one of the main pathways leading to the activation of Akt was via the activation of PI3K, and the downstream effector of Akt could be endothelial nitric oxide synthase, producing nitric oxide (NO) [51]. Morales-Ruiz et al. demonstrated that microvascular endothelial cells

K.-K. Li et al. / Journal of Nutritional Biochemistry 25 (2014) 319–328

325

A

B

C Length of SIV ( µm )

2000

1500

*

***

1000

***

***

500

*** 0 0

3.125

6.25

12.5

25

SU 5416

Concentration(µg/ml) Fig. 6. (A) Lateral view of TG(fli1a:EGFP)y1 zebrafish embryos at 72 hpf treated with GC-(4→8)-GCG at various concentrations. (B) Lateral view of TG(fli1a:EGFP)y1 zebrafish embryos at 72 hpf in control group and SU5416 group. (C) The vessel length of the SIVs with different concentrations of GC-(4→8)-GCG. Data represent mean±S.E.M of tube formation (% control) (*Pb.05; ***Pb.001).

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infected with adenoviruses encoding activation-deficient Akt hindered VEGF-induced cell locomotion [51]. As demonstrated in our Western blot analysis (See Fig. 4), GC-(4→8)-GCG could attenuate the activation of p-ERK, p-p38 and p-Akt in a dose-dependent manner, suggesting that GC-(4→8)-GCG might mediate through the MAPK and Akt pathway for its inhibition in angiogenesis. In addition, MAPK and Akt activation demonstrated significance in tubule formation in angiogenesis. Inhibitors of ERK and p38 exhibited blockade in the bFGF-mediated tubule formation [49]. On the other hand, VEGF-induced tubule formation was blocked by PI3K inhibitor in endothelial cells [50], while NO was also shown to be involved in endothelial cell tubule formation [52]. Hence, the suppression of p-ERK, p-p38 and p-Akt protein expression could be responsible for the antiangiogenic effect of GC-(4→8)-GCG in endothelial cell migration and/or endothelial cell differentiation [supported by our tubule formation assay (25–200 μg/ml GC-(4→8)-GCG, Pb.001 to .05)] in angiogenesis. Angiogenesis was controlled by multiple signaling pathways; indeed, various aberrant pathways were regulated in tumor angiogenesis. Thus, inhibition of a single pathway might not be greatly effective, and that could even trigger the tumor cells to develop resistance upon the other angiogenic pathways [53]. As such, targeting multiple signaling pathways in angiogenesis might help us to alleviate or delay the drug resistance development [7,53]. The next suitable candidate for the development of antiangiogenesis drugs should therefore possess potency in targeting multiple signaling pathways [7]. Our study presented the first report that green tea, cocoa tea and their constituents could inhibit angiogenesis through suppression of p-ERK, p-p38 and p-Akt protein expression together. The antiangiogenic effects of GC-(4→8)-GCG are comprehensive since most of the studies of EGCG were focused on its inhibition of Akt and ERK pathways [54]. It was suggested that phytochemicals might aid through acting upon multiple cell signaling pathways to reduce the side effects and resistance development of cancer cells [55]. Indeed, our novel compound GC-(4→8)-GCG from cocoa tea demonstrated blockage of cell migration, tubule formation and SIV formation of zebrafish, as well as the inhibition upon the activation of both ERK and Akt pathways. These suggested that GC-(4→8)-GCG might act through multiple pathways in suppressing vessel formation. To illustrate the overall picture of angiogenesis, in vivo angiogenesis models should be used. Among them, chick chorioallantoic membrane assay (CAM) [56] was a simple and inexpensive assay of which the angiogenesis effects are observed by counting the number of blood vessels formed. However, several limitations of CAM assay include the fact that since vascular network existed before the treatment, it was hard to observe the newly formed blood capillaries. Besides, the slides placed on the embryos could induce immune response that masked the newly formed capillaries. Other in vivo models, such as rabbit corneal pocket [57], were rather invasive, required heavy surgical experimental manipulations and were expensive, and data were difficult to quantify [56,58]. Tackling these setbacks, the use of a noninvasive real-time observation of transgenic zebrafish TG (fli1:EGFP)y1 with fluorescent vasculature by the expression of enhanced green fluorescent protein marker in the endothelial cells [28] for quantitative analysis of the drug possessing angiogenesis effect could be a favorable choice [28,30]. The zebrafish model has been applied in many biology and pharmacology fields, especially in angiogenesis. Blood vessel patterning is highly characteristic in the developing zebrafish embryo, and the SIVs can be visualized microscopically as a primary screen for compounds that affect angiogenesis. Small molecules added directly to the fish culture media diffuse into the embryo and induce observable, dose-dependent effects. In the present study, a positive control, SU5416, was used to evaluate the model establishment and quality control. As shown in our result, SU5416 could effectively inhibit new blood vessel formation, in

parallel with those previous reports [59]. Our group also adopted this transgenic zebrafish to study the antiangiogenic and proangiogenic effects of some small molecules and herbal agents [60–63]. The quantification of the average length of the SIVs is one of the reliable and consistent parameters to quantify angiogenesis, and therefore, our in vivo data were presented in length of SIV. Indeed, more and more evidences showed that antiangiogenic compounds effective in mammals elicit similar effects in zebrafish [64,65]. Furthermore, in this study, the in vivo antiangiogenic effect of GC-(4→8)-GCG was studied using a zebrafish model. GC-(4→8)-GCG showed high inhibition effect on the SIVs formation. The in vivo study substantiated the inhibitory effects of GC-(4→8)-GCG, and this was consistent with the results of the in vitro studies. Further investigation is required to study the antiangiogenesis effects of GC-(4→8)-GCG using the other in vivo models such as chick embryo model and mouse tumor model. As our results show, being a candidate of tumor vascular-targeting agent, the antiangiogenic effects of GC-(4→8)-GCG may not be as strong as the well-known drugs, such as TNP-470 and CA4P. However, GC-(4→8)-GCG has its own advantages. GC-(4→8)-GCG belongs to the group of proanthocyanidins, which have been reported to show various beneficial properties; bioactivities of proanthocyanidins include having strong free radical scavenging and antioxidant activity [66]. Chemoprotective properties of proanthocyanidins against oxygen free radicals and oxidative stress, anti-inflammatory, anticancer, anti-inflammatory, antiallergic and cardioprotective activity have also been reported [67–69]. Besides, our novel compound GC-(4→8)-GCG demonstrated inhibition of endothelial cell migration, tubule formation and zebrafish SIV formation, as well as the inhibition upon the activation of ERK and Akt pathways. These suggested that GC-(4→8)-GCG might play through multiple pathways in blocking vessel formation. More importantly, our GC-(4→8)-GCG existed in cocoa tea leaf which is a new functional food. It can be an integral part of the human diet, so we can easily obtain GC-(4→8)-GCG. In our further study, biological activities such as antioxidative, antitumor and cardioprotective effects of GC-(4→8)-GCG will also be studied. In conclusion, we demonstrated for the first time the in vitro and in vivo antiangiogenic activity of a novel proanthocyanidin, GC-(4→8)-GCG, isolated from cocoa tea. GC-(4→8)-GCG showed potential antiangiogenic potency upon tubule formation and cell migration of endothelial cell. It also inhibited SIV formation on the zebrafish model. The antiangiogenic effects of GC-(4→8)-GCG might be exerted through inhibiting the activation of ERK1/2 and Akt for suppressing endothelial cell migration and tubule formation. These findings might provide support for the potential use of GC-(4→8)GCG in antiangiogenesis regimen. Acknowledgments This study was supported by the Ming Lai Foundation, The International Association of Lions Clubs District 303 and Hong Kong & Macau Tam Wah Ching Chinese Medicine Resource Centre. References [1] Bussolino F, Mantovani A, Persico G. Molecular mechanisms of blood vessel formation. Trends Biochem Sci 1997;22:251–6. [2] Dvorak HF. Angiogenesis: update 2005. J Thromb Haemost 2005;3:1835–42. [3] Gordon MS, Mendelson DS, Kato G. Tumour angiogenesis and novel antiangiogenic strategies. Int J Cancer 2010;126:1777–87. [4] Ingber D, Fujita T, Kishimoto S, Sudo K, Kanamaru T, Brem H, et al. Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumour growth. Nature 1990;348:555–7. [5] Miura S, Emoto M, Matsuo Y, Kawarabayashi T, Saku K. Carcinosarcoma-induced endothelial cells tube formation through KDR/Flk-1 is blocked by TNP-470. Cancer Lett 2004;203:45–50. [6] Mendel DB, Laird AD, Smolich BD, Blake RA, Liang C, Hannah AL, et al. Development of SU5416, a selective small molecule inhibitor of VEGF receptor

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