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Anti-angiogenic activity and mechanism of kaurane diterpenoids from Wedelia chinensis.

ABSTRACT

Background: Wedelia chinensis is a traditional medicinal herb used in Asia and it has been reported to possess various bioactivities including anti-inflammatory and anticancer effects. However, its anti-angiogenic activity has never been reported.

Purpose: To determine the most potent anti-angiogenic component in W. chinensis and its molecular mechanism of action.

Study design: Initially, the active fraction of the plant was studied. Then, we determined the active components of the fraction and explored the mechanism of the most active compound.

Methods: The ethanol extract of W. chinensis and its four fractions with different polarities were evaluated for their anti-angiogenic activity in the Zebrafish model using quantitative endogenous alkaline phosphatase (EAP) assay. The molecular mechanism of the most active compound from the active fraction was studied using the real-time polymerase chain reaction (PCR) assay on Zebrafish embryos. The inhibitory effect of the most active compound on the proliferation, invasion and tube formation steps of angiogenesis was evaluated using the vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cells (HUVECs) model, and the influences of the active compound on tyrosine phosphorylation of VEGF receptor (VEGFR-2) and its downstream signal pathway were evaluated by western blotting assay. Moreover, its anti-angiogenic effect was further evaluated by the VEGF-induced sprouts formation on aortic ring assay and the VEGF-induced vessel formation of mice on matrigel plug assay, respectively.

Results: Petroleum ether (PE) fraction of the plant displayed potent anti-angiogenic activity. Twelve kaurane diterpenoids (1-12) isolated from this fraction showed quite different effects. Compounds 9-12 could dose-dependently inhibit vessel formation in the Zebrafish embryos while the others showed little inhibitory effect. Among the active diterpenoids, compound 10, 3a-cinnamoyloxy-9/5-hydroxy-ent-kaura-16en-19-oic acid (CHKA), possessed the strongest effect, and it affected multiple molecular targets related to angiogenesis including VEGF and angiopoietin in Zerbrafish. Moreover, CHKA significantly inhibited a series of VEGF-induced angiogenesis processes including proliferation, invasion, and tube formation of endothelial cells. Besides, it directly inhibited VEGFR-2 tyrosine kinase activity and its downstream signaling pathways in HUVECs. CHKA also obviously inhibited sprouts formation of aortic ring, and block vessel formation in mice.

Conclusion: Our findings demonstrate that kaurane diterpenoids is one of anti-angiogenic components in W. chinensis, and CHKA may become a promising candidate for the development of anti-angiogenic agent.

Keywords:

Wedelia chinensis

Kaurane diterpenoid

Anti-angiogenesis

Zerbrafish

Mechanism

Introduction

Angiogenesis is the process of sprouting new blood capillaries from pre-existing vasculature. It is essential in many physiological processes including embryonic development and pathological responses (Folkman 1995). However, unregulated angiogenesis would cause angiogenic diseases such as diabetic retinopathy, tumor growth and metastasis, rheumatoid arthritis, and inflammatory diseases. Since cancers are closely linked with angiogenesis, inhibition of angiogenesis has been considered as a promising therapeutic strategy in cancer treatment (Bar and Onn 2008; Cao 2010). Meanwhile, evidences revealed that chronic and persistent inflammation could promote carcinogenesis and might be responsible for a substantial portion of tumor vascularization in "inflammatory angiogenesis". This led to the possibility of combating inflammation with appropriate reagents to prevent "inflammatory angiogenesis" in carcinogenesis (Allavena et al. 2008; Kobayashi and Lin 2009).

Wedelia chinensis (Compositae) is a medicinal plant that widely distributes in southern China and other Southeast Asia countries. It is traditionally used for the treatment of diphtheria, chincough, hemorrhoids, injuries due to falls, and faucitis in China (Institue of Materia Medica 1984) and often serves as a major component of folk herbal teas in Taiwan (Lin et al. 2012). Phytochemical studies have indicated that the plant contains triterpenoids, diterpenoids, sesquiterpenes, flavonoids, organic acids and steroids (Li et al. 2012; Qiu et al. 2014). Pharmacological studies reported that W. chinensis possessed anti-inflammatory (Manjamalai et al. 2012), anticancer (Tsai et al. 2015), antioxidant (Talukdar et al. 2013; Manjamalai and Grace 2012), and anti-microbial (Manjamalai et al. 2012) effects. Because of its anti-inflammatory and anticancer effects, W. chinensis was further investigated for its potential anti-angiogenic property in the present study. In this paper, the antiangiogenic activity and anti-angiogenic constituents of W. chinensis were studied using Zebrafish model, and the preliminary mechanism of the active component was also explored using the model. Moreover, both in vitro and in vivo anti-angiogenic activities of the most active compound was further investigated through HUVECs model, aortic arch model, and the model of matrigel implantation in mice.

Materials and methods

Plant material

Wedelia chinensis was purchased from a Chinese medicine pharmacy in Guangzhou, China. The species was identified by Professor Guangxiong Zhou of Jinan University. A voucher specimen (No. 20120135) was deposited in the Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University (Guangzhou, China).

Extraction, fractionation, isolation and structural identification

The preparation of ethanol extract of W. chinensis and its fractions were described previously (Qiu et al. 2014). Briefly, the dried and cut W. chinensis was extracted using 95% ethanol, then the extract was suspended in distilled water and partitioned with PE, ethyl acetate (EtOAc) and n-butanol (BuOH) successively. After evaporation under reduced pressure, the PE, EtOAc, BuOH, and the residual aqueous (Aq) fractions were obtained. Twelve kaurane diterpenoids (Fig. 2) were isolated from the PE fraction with chromatographic methods and their chemical structures were characterized by spectroscopic techniques (Qiu et al. 2014).

Quantitative EAP assay

Wild-type Zebrafish (AB strain) was used in this experiment. During the development of Zebrafish embryos, as the stage between 24 and 72 h post-fertilization (hpf) has the highest angiogenic activity, drugs are treated and evaluated using quantitative EAP assay (phosphatase substrate kit; Pierce, USA) at this stage according to the method of Huang et al. (2014). Semaxanib (SU5416; Sigma Corporation, St. Louis, MO, USA), a known effective inhibitor of angiogenesis, was used as a positive control in this experiment (He et al. 2012).

Microscopic imaging on Zebrafish embryo

For documentation of the development of the blood vessels, the embryos of the Tg (fiila:EGFP) yl-type Zebrafish were used. As the cells of this Zebrafish contain green fluorescent proteins, the endothelial cells of the vasculature in both the intersegmental blood vessels (ISVs) at 48 hpf and subintestinal vessels plexus (SIVs) at 72 hpf can be easily observed using a fluorescent microscope (He et al. 2012). The embryos (24 hpf) were arrayed in 96-well plate, and incubated with 100 [micro]l of embryo water per well containing various concentrations of drugs (treatment groups) or embryo water containing 0.2% DMSO (control group). After further incubated for 24 h or 48 h, embryos were observed for their blood vessel development using an Olympus 1X71S8F-2 inverted microscope.

Total RNA isolation, reverse transcription and real-time PCR

Briefly, twenty embryos were lysed in 500 ml TRIzol reagent. The RNA of embryos was extracted according the method of He et al. (2012). Reverse transcription was performed according to the Maloney murine leukemia virus reverse transcriptase kit (Invitrogen, Carlsbad, CA, USA). The expression level of the target genes and the internal control gene (elongation factor 1 alpha, Efla) in the embryos were examined by real-time PCR. The sequences of the primers used are listed in Table 1. The real-time PCR was performed in a total volume of 10 [micro]l containing 0.05 [micro]l Takara Taq (5 units/[micro]l), 0.5 [micro]l EvaGreen, 0.02 [micro]l of each primer (10 [micro]l), and diluted cDNA solution on the CFX96 Real-Time PCR Detection System (Bio-Rad, Hong Kong). The reaction protocol including 42 cycles at 95[degrees]C for 20 s, 61[degrees]C for 20 s, and 72[degrees]C for 20 s was preformed. The expression levels of the tested genes were normalized to that of Ef1a and presented as the fold change compared to the control. All the experiments were performed in triplicates.

Cell culture

HUVECs were purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in the Dulbecco's modified Eagle's medium with nutrient mixture F-12 (MEM/F12) containing 20% fetal bovine serum (FBS, Gibco, Gaithersburg, MD, USA), 0.1 mg/ml heparin (Sigma, MO, USA), 5ng/ml basic fibroblast growth factor (bFGF, PeproTech, NJ, USA) and 10 ng/ml epidermal growth factor (EGF, Invitrogen, CA, USA), and incubated at 37[degrees]C with 5% C[O.sub.2] in air.

Lactate dehydrogenase (LDH) toxicity assay

HUVECs were seeded in the 96-well plates at a density of 1 x [10.sup.4] cells per well at 37[degrees]C and 5% C[O.sub.2] for overnight attachment. The growth medium (MEM/F12 containing 20% FBS) was replaced with fresh medium containing various concentrations of drugs. After further incubation for 48 h, cell supernatants were collected and analyzed for LDH activity using Cytotoxicity Detection Kit (Roche Diagnostics GmbH, Germany) according to the manufacturer's instructions.

Cell counting assay

The inhibitory effects of drugs on the viability of HUVECs as well as VEGF-induced cell viability were measured using a cell counting kit (CCK)-8 assay (Dojindo, Kumamoto, Japan) according to the method of Li et al. (2012).

Invasion assay

The invasion assay was performed according to the method of Li et al. (2012). Briefly, the Transwell (8 mm pore; Corning, MA, USA) was pre-coated with matrigel (BD Bioscience Company, NJ, USA) for 8 h. The top chambers were filled with HUVECs (5 x [10.sup.4] cells per chamber) in 100 ml DMEM/F12 with 1% FBS while the bottom ones filled with 600 ml DMEM/F12 with 1% FBS containing VEGF (20ng/ml). Both top and bottom chambers contained the same concentrations of drug. Cells were allowed to invade for 24 h. Then the invaded cells were fixed and stained. Cells incubated with the medium only were used as a negative control. In the end, the invaded cells were photographed and counted.

Tube formation assay

The HUVECs (4~5 x [10.sup.4]) were seeded on the matrigel precoated wells in 48-well culture plates and incubated with growth medium containing with 50 ng/ml of VEGF in the presence or absence of various of drugs for 8 h. Cell tubular structures were photographed and the number of tubular structures was calculated. Cells incubated with growth medium only were used as a negative control (Li et al. 2012).

Aortic ring assay

Aortas isolated from Sprague-Dawley rats were cut into 1-1.5 mm long rings. The rings were placed in the matrigel precoated wells and covered with matrigel (Li et al. 2012). They were incubated at 37[degrees]C in the growth medium containing VEGF (50 ng/ml) either in the presence or absence of drugs at various concentrations. After 4-days incubation, the microvessel growth was observed and the number of branching sites was quantified. Rings incubated with growth medium only were set as a negative control.

Matrigel plug assay

Matrigel (0.5 ml/plug) containing 250 ng VEGF, 150 units of heparin and various concentrations of drugs was injected into the ventral area of the C57/BL/6 mice (five mice per group) following the method of Li et al. (2012). Matrigel containing neither VEGF nor drugs was used as a negative control. After 21 days of incubation in vivo, the matrigel plugs were removed, fixed and embedded with paraffin.

Western blotting

HUVECs were incubated for 4 h in medium containing 4% FBS with various concentrations of CHKA after 6h of serum starvation. Then, the medium was replaced with DMEM/F12 containing 50 ng/ml of VEGF and incubated for 1 h. cell lysates were separated by 8% SDS-PAGE and then transferred to polyvinylidene difluoride membranes. Membranes were incubated with primary antibodies (Cell Signalling Technology, MA, USA) for overnight incubation at 4[degrees]C. After incubation, membranes were washed and then incubated with secondary antibodies (Cell Signalling Technology, MA, USA) for 2h at room temperature. Immunoreactive bands were then visualized by the enhanced chemiluminescence detection system.

Statistical analysis

Data were expressed as means [+ or -] standard deviation. Statistical analysis was performed using one-way ANOVA followed by Dunnett's test, at p = 0.05 level of significance.

Results

Anti-angiogenic activity of ethanol extract of W. chinensis and its four fractions

By EAP staining method on Zebrafish embryo, W. chinensis displayed potent anti-angiogenic activity. In Fig. 1, PE fraction showed dose-dependent inhibition on vessel formation in the embryos. After the embryos were treated with PE fractions at concentrations of 10, 15 and 20[micro]g/ml, the vessel formations were reduced to 79.3, 67.2, and 52.4% of that of the control, respectively. Both ethanol and EtOAc fractions at 20 [micro]g/ml reduced vessel formation to 92.0 and 82.1% of that of the control, respectively. However, both BuOH and Aq fractions showed little effect at any of the concentrations tested. Overall, PE fraction possessed the strongest anti-angiogenic effect, and it was the most active fraction in W. chinensis.

Anti-angiogenic activity of kaurane diterpenoids from PE fraction

Twelve kaurane diterpenoids (compounds 1-12, Fig. 2) previously isolated from the PE fraction of W. chinensis (Qiu et al. 2014) were subjected to evaluate for their anti-angiogenic activity using the Zebrafish model by the EAP assay. Fig. 3 shows compounds 9, 10, 11 and 12 inhibited vessel formation dose-dependently. In fact, after the embryos were treated with them at the concentration of 10 [micro]M, vessel formations were reduced to 90.2, 50.4, 73.4 and 84.3% of that of the control, respectively. Also, compound 10 (CHKA) possessed the best anti-angiogenic effect with similar effect at 10 [micro]M with that of SU5416 at 3 [micro]l. However, the remaining eight compounds had little anti-angiogenic activity at any of the concentrations tested.

Microscopic imaging

Microscopic imaging of Tg (flila:EGFP)yl Zebrafish embryos was used to confirm the anti-angiogenic effect of CHKA at a concentration of 10 [micro]l. Treatment containing 0.2% DMSO served as a control because of its weak inhibition in the vessel formation (Fig. 4A and 4B). Compared to the control, CHKA blocked part of the ISVs formation after treated with CHKA for 24 h (Fig. 4A'), and inhibited most of SIVs formation after treated with CHKA for 48 h (Fig. 4B).

Molecular mechanism of CHKA on Zebrafish angiogenesis

The molecular mechanism of CHKA on Zebrafish angiogenesis was further investigated because of its strong anti-angiogenic activity. VEGF-VEGF receptor 2 (Kdr) and angpiopoietin (Ang)-Ang receptor (Tie) are two major signaling pathways in angiogenesis (Hicklin and Ellis 2005; Thomas and Augustin 2009; Ellis and Hicklin 2008). Six main angiogenic genes (VEGF165, Kdr, Angl, Ang2, Tiel and Tie2) participating in these two signaling pathways were selected to study. Fig. 5 showed that the mRNA expressions of VEGF165, Ang2, and Tie2 were down-regulated after both 24- and 48-h treatment with CHKA, and their expressions were reduced in the dose-dependent manner. The expressions of the other three genes were not affected after the 24-h drug treatment but their expressions were significantly down-regulated after 48-h drug treatment. Apparently, CHKA could significantly reduce the gene expressions of VEGF, angiopoietin and their receptors.

CHKA inhibited VEGF-induced viability of HUVECs

To evaluate whether or not CHKA (compound 10) would cause toxicity in the HUVECs, LDH cytotoxicity assay was carried out. As shown in Fig. 6a, CHKA brought no significant toxic effect to HUVECs at all the tested concentrations (0, 6.25, 12.5, 25, 50, 75 and 100 [micro]M) (p > 0.05). Cell-counting assay was then used to evaluate its inhibition on the proliferation of HUVECs. For dosages ranging from 0 to 100 [micro]M, no significant difference in the cell proliferation was observed (p > 0.05) (Fig. 6b), further suggesting little toxic effect of CHKA on HUVECs. Next, we studied whether CHKA inhibited the proliferation of HUVECs induced by VEGF. In the presence of VEGF for 48 h, the cell number of HUVEC increased by 1.4 folds of the control (Fig. 6c). However, CHKA significantly suppressed VEGF-induced cell proliferation in a dose-dependent manner (p < 0.05). Overall, CHKA at non-cytotoxic dosages could significantly inhibit the proliferation of VEGF-stimulated endothelial cells.

CHKA inhibited VEGF-induced invasion and tube formation of HUVECs

Next, we evaluated the potential of CHKA in blocking the invasion and tube formation abilities on the HUVECs induced by VEGF, and SU5416 was used as a positive control. Results showed that in the transwell assay, a large number of cells migrated to the lower side of the membrane in the transwell chamber after stimulation with VEGF, and the number of invasive cells was significantly increased to 159.1% of the control. However, in the presence of both CHKA and VEGF, the number of invasive cells was dramatically reduced in a dose-dependent manner. For example, when the cells were treated with CHKA at concentrations of 12.5 and 25 [micro]M, the number of invasive cells decreased significantly to 132.4 and 109.1% of the control, respectively (Fig. 7A). Similarly, in the absence of CHKA, VEGF-induced HUVECs showed well-formed tubular structure (Fig. 7C). But when the VEGF-treated cells were treated with increasing concentrations of CHKA, tubes were gradually interrupted. Taken together, CHKA suppressed the invasion process and tube formation activities of HUVECs in the presence of VEGF.

CHKA inhibited VEGF-induced vessel sprouts formation

The in-vitro experiments demonstrated that CHKA could inhibit angiogenesis. To further determine whether CHKA inhibited angiogenesis ex vivo, the effect of CHKA on the sprouting of microvessels from the aortic rings was carried out As shown in Fig. 8, compared to the control, sprouts around the ring treated with VEGF only was longer with more cells migrated into the matrix. When the rings treated with both VEGF and increasing concentrations of CHKA (6.25, 12.5 and 25 [micro]M), the sprouts around the rings were shorter. These data suggested that CHKA could inhibit the sprout length and density at the dose-dependent manner.

CHKA inhibited VEGF-induced blood vessel formation in mice

The in-vivo anti-angiogenic activity of CHKA was studied using the matrigel implantation assay. In Fig. 9A, compared to the control, the matrigel plugs containing VEGF only were dark red in color, and were filled with blood vessels, suggesting the functional vasculatures had formed. On the contrary, when the matrigel plugs containing both VEGF and increasing concentrations of CHKA (6.25, 12.5 and 25 [micro]M), lighter color was observed, suggesting that the formation of blood vessels was weak when CHKA was present. Furthermore, after staining with hematoxylin and eosin, fewer vessels in the matrigel plugs were observed for those treated with both VEGF and CHKA than those that were treated with VEGF alone (Fig. 9B). These results suggested that CHKA inhibited VEGF-induced blood vessel formation in vivo.

CHKA attenuated VEGFR-2 tyrosine kinase activity and VEGFR-2 signaling pathway

The above experimental results suggested that CHKA effectively inhibited VEGF-induced angiogenesis both in vitro and in vivo. We first examined the influences of CHKA on tyrosine phosphorylation of VEGFR-2 (p-VEGFR-2, the active form of VEGFR-2) stimulated by VEGF. The expressions of p-VEGFR-2 (Tyr1175) and total VEGFR-2 were evaluated by western blotting assay with their specific antibodies in the presence of VEGF. As shown in Fig. 10, the expression of p-VEGFR-2 was reduced in the cells treated with CHKA in dose-dependent manner, while the total levels of VEGFR2 had little changes. The result indicated that CHKA affected the interaction between VEGF and VEGFR-2. In addition, phosphorylation of VEGFR-2 could subsequently trigger multiple downstream signals that induced proliferation and differentiation activities of endothelial cells (Gingras et al. 2000). It has been reported that the protein kinase B (AKT), mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) play important roles in endothelial cell proliferation, adhesion, migration, invasion, metabolism and survival (Karar and Maity 2011; Xu et al. 2008). Then, we assessed multiple essential downstream signaling molecules involved in VEGFR-2 activation by western blotting assay. Results showed that CHKA could inhibit VEGF-stimulated phosphorylation levels of ERK, AKT and mTOR (p-ERK, p-AKT and p-mTOR), whereas the total expressions of them were almost unaffected.

Discussion

Wedelia chinensis was reported to possess various pharmacological effects including anti-inflammation and anticancer, but most studies focused on the active constituents of the plant particularly on flavonoids, such as wedelolactone, apigenin and luteolin (Yuan et al. 2013; Li et al. 2007). Kaurane diterpenoids are one of the main chemical constituents of W. chinensis, but they have never been reported as bioactive components of the plant. This study firstly demonstrated both anti-angiogenic activity and anti-angiogenic constituents of the plant. After screening for the anti-angiogenic activity of crude extract and the four fractions from the plant, PE fraction showed better effect. Kaurane diterpenoids isolated from this fraction had potent anti-angiogenic activity suggesting they are one of active components in this fraction.

So far, studies on the anti-angiogenic activity of diterpenoids are limited. Only Huang et al. (2014) reported a derodane-type diterpenoid (Crassifolin H) isolated from Croton crassifolius could reduce vessel formation in Zerbrafish embryo to 59.3% of that of the control at the concentrations of 15 [micro]g/ml, and He et al. (2012) found a labdane-type diterpenoid (Zerumin A) isolated from Alpinia caerulea inhibiting vessel formation in Zerbrafish embryo by about 30% in the range of 10-20 [micro]M. Yet, their anti-angiogenic molecular mechanisms are still not clear. Our experiments showed four kaurane diterpenoids (9-12) dose-dependently inhibited vessel formation in Zebrafish embryos in the range of 2.5-10 [micro]M. Among them, CHKA (10) possessed the strongest effect. CHKA was firstly reported in W. chinensis in our previous study (Qiu et al. 2014), and it was also found in Wedelia trilobata in recent years (Bohlmann et al. 1981; Ma et al. 2013). Yet, its bioactivity has never been reported. Our results firstly reported its anti-angiogenic activity on Zebrafish model. Zebrafish angiogenic model is an in vivo model for anti-angiogenic drug screening and target discovery, and increasing evidence suggested that anti-angiogenic compounds effective in mammals would elicit similar effects in Zebrafish (Taraboletti 2004).

Because of no anti-angiogenic activity reported on CHKA and its stronger effect than other kaurane diterpenoids, we decided to monitor its molecular targets using the Zebrafish model. CHKA could reduce multi-target expression, including VEGF165, Kdr, Angl, Ang2, Tiel, and Tie2, in time- and dose-dependent manners during Zebrafish angiogenesis. In the VEGF-Kdr pathway, the ligand of predominant isoform-VEGF165 binds to and activates its receptor (Kdr) to regulate the angiogenesis. Kdr expression is restricted primarily to the vasculature and is the key mediator of VEGF-induced angiogenesis (Ferrara et al. 2003). On the other hand, the Ang-Tie system, consists of two main ligands (Angl and Ang2) and two receptors (Tiel and Tie2), is essential for blood vessel formation. Angl and Ang2 are specific ligands of Tie2 with similar affinity and the activation of Tie2 would promote vessel assembly and maturation. Ang2 could induce vascular destabilization and stimulate tumor growth. However, the function of Angl in tumor-associated angiogenesis remains controversial because its promoting or inhibiting functions are dependent on the tumor cell type, the dosage, and possibly on the amount of Ang2 in the tumor. Therefore, compared to Angl, Ang2 is often a more appealing therapeutic target. For Tiel, it is critical for vascular development. Nevertheless, it is still considered as an orphan receptor, and its function is not clear (Thomas and Augustin 2009). VEGF-Kdr and Ang-Tie cooperatively increase angiogenesis, and co-blocking these two pathways is superior in inhibiting tumor angiogenesis, metastasis, and leakage.

As VEGF is the most potent angiogenesis stimulator, we further evaluated the inhibitory effects of CHKA on VEGF-induced angiogenesis both in vitro and in vivo. It was found that CHKA could markedly inhibit several steps of angiogenesis including proliferation, invasion, and capillary formation on VEGF-stimulated HUVECs. Nevertheless, the in vitro models lack the biological complexity of vascular system in vertebrate animals (Wang et al. 2012). Supporting evidences concerning ex-vivo and in-vivo anti angiogenic effects of CHKA then came from the models of sprouts formation of rat aorta and matrigel implantation in mice. Results demonstrated that CHKA significantly inhibited sprouts formation ex vivo and blood vessels formation in vivo.

Furthermore, we verified the intrinsic mechanisms for the above inhibition of VEGF-induced angiogenic activities of CHKA. Inhibition of VEGFR-2 has been considered as a prosperous strategy for angiogenesis therapeutic intervention and VEGFR-2 would undergo auto-phosphorylation mainly at Tyr1175 sites within its intracellular kinase domain and then initiate a series of downstream signal transductions to endothelial cells (Gingras et al. 2000). Our findings showed that CHKA could suppress Tyr1175 phosphorylation of VEGFR-2 stimulated by VEGF. Multiple VEGFR-2 downstream signaling mediators such as mTOR, ERK, and AKT were also involved in regulation of endothelial cells survival and proliferation, and CHKA dose dependently inhibited the phosphorylation of mTOR, ERK, and AKT. Results showed that CHKA treatment in HUVECs suppressed the VEGFR-2 pathways. Meanwhile, the in-vitro findings were also consistent with our in-vivo results, indicating that the inhibition of blood vessel formation in mice may owe to the suppressed activity of CHKA on the expression of phosphorylated VEGFR-2 and its downstream signaling molecules. Notably, we found that CHKA possessed little cytotoxicity to HUVECs at any tested concentrations based on LDH assay, indicating that its inhibitory effects was not likely due to toxicity at the cellular level.

Conclusion

Overall, CHKA may become a promising candidate for the development of anti-angiogenic agent. Meanwhile, our findings may offer new insights for the application of W. chinensis in the treatment of cancer and angiogenic diseases.

Conflict of interest

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

http://dx.doi.org/10.1016/j.phymed.2015.12.021

ARTICLE INFO

Article history:

Received 1 September 2015

Revised 10 November 2015

Accepted 12 December 2015

Acknowledgments

This work was supported financially by the Natural Science Foundations of China (81402805), the Guangdong Natural Science Foundation (2014A030310240), and partially by the National Major Scientific Program of Introducing Talents of Discipline to Universities (B13038).

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Abbreviations: CHKA, 3a-cinnamoyloxy-9/5-hydroxy-ent-kaura-16-en-19-oic acid; VEGF, vascular endothelial growth factor; Kdr, VEGF receptor 2; Ang, angpiopoietin; Tie, angpiopoietin receptor; HUVECs, human umbilical vein endothelial cells; PCR, polymerase chain reaction; PE, petroleum ether; EtOAc, ethyl acetate; BuOH, n-butanol; Aq, residual aqueous; EAP, endogenous alkaline phosphatase; LDH, lactate dehydrogenase; CCK, cell counting kit; ISVs, intersegmental blood vessels; SIVs, subintestinal vessels plexus; AKT, protein kinase B; mTOR, mammalian target of rapamycin; ERK, extracellular signal-regulated kinase.

Weihuan Huang (a), (1), Yeyin Liang (a) (1), Jiajian Wang (3), Guoqiang Li (3), Guocai Wang (3), Yaolan Li (a),**, Hau Yin Chung (b), *

(a) Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, P. R. China

(b) Food and Nutritional Sciences Programme, School of Life Sciences. The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China

* Corresponding author. Tel.: +852 39436149; fax: +852 26037246.

** Co-corresponding author. Tel.: +86 20 85221728; fax: +86 20 85221559.

E-mail addresses: tliyl@jnu.edu.cn (Y. Li), anthonychung@cuhk.edu.hk, 035489005@qq.com (H.Y. Chung).

(1) Co-first authors: Weihuan Huang and Yeyin Liang contributed equally to this study.

Table 1
Primers used in real-time PCR.

Gene       Forward primer

Efla       5'-GGCTGACTGTGCTGTGCTGATTG-3'
VEGF165    5'-TGCTCCTGCAAATTCACACAA-3'
Kdr        5'-GCCTGATCCACAACTGCTTCC-3'
Angl       5 '-ACAGCAGTGGAACCGAACAG-3'
Ang2       5'-AGGTGGAGGCTGGACTGTC-3'
Tiel       5'-CAAGAGGCACGGAAGGCTTA-3'
Tie2       5'-CTACCCAGTGACCAACGC-3'

Gene       Reverse primer                   Expected size (bp)

Efla       5'-CTTGTCGGTGGGACGGCTAGG-3'      410
VEGF165    5'-ATCTTGGCTTTTCACATCrGCAA-3'    84
Kdr        5'-CTCTCCTCACACGACTCAATGC-3 '    150
Angl       5'-AGCCrCCGCCAGCAGAC-3'          169
Ang2       5'-GTGGTGAGCAGGTGGATGAC-3'       141
Tiel       5'-AGTGACAGTAACGCAGAGCC-3'       166
Tie2       5'-GCTCTACAGCTCCTGACGAT-3'       118
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Author:Huang, Weihuan; Liang, Yeyin; Wang, Jiajian; Li, Guoqiang; Wang, Guocai; Li, Yaolan; Chung, Hau Yin
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Mar 15, 2016
Words:5310
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