Syzygium campanulatum korth methanolic extract inhibits angiogenesis and tumor growth in nude mice
© Aisha et al.; licensee BioMed Central Ltd. 2013
Received: 11 November 2012
Accepted: 10 June 2013
Published: 11 July 2013
Syzygium campanulatum Korth (Myrtaceae) is an evergreen shrub rich in phenolics, flavonoid antioxidants, and betulinic acid. This study sought to investigate antiangiogenic and anti-colon cancer effects of S.C. standardized methanolic extract.
Betulinic acid was isolated from methanolic extract by crystallization and chromatography techniques. S.C. methanolic extract was analyzed by UV-Vis spectrophotometry, FTIR, LC-MS, and HPLC. Antiangiogenic effect was studied on rat aortic rings, matrigel tube formation, cell proliferation and migration, and expression of vascular endothelial growth factor (VEGF). Antitumor effect was studied using a subcutaneous tumor model of HCT 116 colorectal carcinoma cells established in nude mice.
Analysis by HPLC, LC-MS and FTIR confirm presence of betulinic acid in S.C. methanolic extract. Quantitative analysis by HPLC indicates presence of betulinic acid in S.C. extract at 5.42 ± 0.09% (w/w). Antiangiogenesis study showed potent inhibition of microvessels outgrowth in rat aortic rings, and studies on normal and cancer cells did not show any significant cytotoxic effect. Antiangiogenic effect was further confirmed by inhibition of tube formation on matrigel matrix that involves human endothelial cells (IC50 = 17.6 ± 2.9 μg/ml). S.C. extract also inhibited migration of endothelial cells and suppressed expression of VEGF. In vivo antiangiogenic study showed inhibition of new blood vessels in chicken embryo chorioallantoic membrane (CAM), and in vivo antitumor study showed significant inhibition of tumor growth due to reduction of intratumor blood vessels and induction of cell death.
Collectively, our results indicate S. campanulatum as antiangiogenic and antitumor candidate, and a new source of betulinic acid.
Angiogenesis is a critical process in various physiological conditions such as wound healing, female reproductive system, and embryonic development. It also plays an important role in various pathological conditions including growth and metastasis of solid tumors, rheumatoid arthritis, proliferative diabetic retinopathy, and psoriasis [2, 3]. Inhibition of angiogenesis, which was suggested by Judah Folkman in 1971 , is now considered one of the most promising strategies to combat cancer. Recently, there has been a great interest in angiogenesis modulators for therapy of several angiogenesis related disorders. In this context, several plant-derived compounds have shown promising antiangiogenic and antitumor effects such as ursolic acid , oleanolic acid , lupeol , betulinic acid , green tea catechins , resveratrol from grapes , quercetin , rosmarinic acid , genistein  and curcumin .
Cell lines and reagents
HCT 116 human colorectal carcinoma, MCF-7 human breast cancer, CCD-18Co human normal fibroblasts, and MCF-10A human normal epithelial cell lines were purchased from American Type Culture Collection (ATCC; Manassas, Virginia). Human umbilical vein endothelial cells (HUVECs) and endothelial cell medium (ECM) supplied with endothelial cell growth supplements (ECGS) were obtained from ScienCell (Carlsbad, California). RPMI 1640, DMEM, MEM and M199 cell culture media, fetal bovine serum (FBS), MEM vitamins, and non-essential amino acids were obtained from Bio-Diagnostics (Petaling Jaya, Selangor, Malaysia). Human VEGF assay kit was obtained from IBL (Aramachi, Takasaki-Shi, Gunma, Japan). Betulinic acid, ursolic acid (UA), oleanolic acid (OA), penicillin/streptomycin (PS) solution, XTT reagent with phenazine methosulfate (PMS), suramin, amphotericin B, aprotinin, 6-aminocaproic acid, L-glutamine, thrombin, human insulin, epidermal growth factor, and gentamicin were obtained from Sigma–Aldrich (Kuala Lumpur, Malaysia). Fibrinogen was obtained from Calbiochem (Shah Alam, Selangor, Malaysia). Matrigel matrix (10 mg/ml) was obtained from SABiosciences (Frederick, Maryland). Analytical and HPLC grade solvents were obtained from Avantor Performance Materials (Petaling Jaya, Selangor, Malaysia).
Plant material and extraction
S.C. leaves were collected from the main campus of Universiti Sains Malaysia (USM) during November 2008. The plant was authenticated by the Herbarium of School of Biological Sciences, USM, where a voucher specimen was deposited (Ref. No. 11047). Leaves were washed, oven-dried at 40°C for 4 days, and grinded into fine powder. The powder (550 g) was macerated in 2L methanol for 48 h in water bath at 50°C with intermittent shaking. The extract was filtered and methanol was evaporated at 50°C using rotavapor, and further dried at 50°C for 48 h.
HUVECs were cultured in ECM containing 5% FBS, 1% PS and 1% ECGS; HCT 116 cells were cultured in RPMI 1640 containing 10% FBS and 1% PS; MCF-7 and CCD-18Co cells were cultured in DMEM supplemented with 10% FBS and 1% PS. MCF-10A cells were cultured in MEM enriched with 10% FBS, 1% PS, 1% sodium pyruvate, 1% nonessential amino acids, 1% L-glutamine, 1% MEM vitamins, human insulin (5 μg/ml), and epidermal growth factor (EGF) (100 ng/ml). Cells were propagated as monolayer at 37°C and 5% CO2.
Estimation of total phenolics, total flavonoids and antioxidant effect
Total phenolics content was estimated using a colorimetric assay . Extract (100 μl at 1.0 mg/ml in methanol) was added to 750 μl Folin-Ciocalteau reagent and incubated for 5.0 min in the dark at RT. Subsequently, 750 μl sodium bicarbonate (60 g/L) was added, incubated in the dark at 30°C for 90 min, and absorbance was measured at 725 nm. Gallic acid was used as a standard (50–1600 μg/ml), and the results are expressed as %w/w gallic acid equivalents.
Total flavonoids content was determined using quercetin as a standard . The standard and extracts (500 μl) were added to 0.1 ml 10% (w/v) of aluminium chloride, 0.1 ml of 1.0 M potassium acetate, 1.5 ml methanol and 2.8 ml water. Potassium acetate and aluminium chloride were replaced with water in the blank reaction. The reaction mixture was incubated for 30 min at RT, and absorbance was taken at 415 nm. The results are presented as %w/w of total flavonoids.
The results are presented as mean percentage inhibition ± SD (n = 3).
Isolation and characterization of betulinic acid
BA was isolated from S.C. methanolic extract as described previously with some modifications . In brief, 50 g of the methanolic extract was dissolved in 150 ml methanol and was kept at 2–8°C for overnight. Subsequently, the resulted solid was collected by filtration, and the filtrate was concentrated again and kept at 2–8°C for overnight. The solid from both cycles was pooled, and washed 3× with ice-cold methanol to give 2.5 g of BA-rich fraction. BA was further purified from this fraction by column chromatography as the following; 1 g of the fraction was dissolved in 10 ml of 1:1 methanol: chloroform and mixed with 5 g silica gel 60 (0.063–0.200 mm), the solvent was evaporated in a fume hood and the mixture was further dried for 30 min at 50°C. The column (20” × 1”) was packed with 50 g silica gel 60 (0.063–0.200 mm) prepared in the mobile phase. Elution was performed with n-hexane: ethyl acetate at 8:2 (v/v). Fractions (10 ml) were collected and tested by HPLC versus BA standard as described previously . The fraction of highest purity (100 mg) was further characterized by FTIR and liquid chromatography-mass spectrometry (LC-MS).
LC-MS analysis of the BA reference compound, isolated BA, BA-rich fraction and the S.C. methanolic extract was performed using a Micro TOF-Q ESI Mass Spectrometer (Bruker) coupled with a Dionex U3000 HPLC system. Liquid chromatography was carried out on a reverse phase HPLC using Acclaim RSLC C18 column (2.2 μm, 2.1 × 50 mm). The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B), and a gradient elution was used (5–95%) of B in 15 min at a flow rate of 0.3 ml/min. Injection volume was 10 μl, and mass analysis was carried out in the negative ion mode within the range 100–1000 m/z.
Quantification of betulinic acid in S.C. methanolic extract
Analytical chromatography was carried out using Agilent 1100 HPLC system, on ZORBAX Eclipse Plus C18 column (5 μm, 4.6 × 250 mm). The mobile phase consisted of A (Acetonitrile), B (0.1% H3PO4 in water). The elution program was isocratic at 86% (A) and 14% (B) for 20 min, at a flow rate of 1 ml/min. Injection volume was 10 μl, and the wavelength was 210 nm. BA, ursolic acid (UA) and oleanolic acid (OA) reference compounds were also analyzed. Linear regression equation of BA calibration curve (y = 0.3708x + 9.2599, R 2 = 1.0) was then applied to calculate BA concentration in the S.C. extract, and the results are presented as %w/w.
Rat aortic rings angiogenesis model
Antiangiogenesis effect was firstly investigated using the 3-dimensional rat aortic rings model as described previously [15, 20, 21]. Basically, aortic rings were seeded in 48-well plate containing 500 μl M199 medium containing fibrinogen (3.0 mg/ml), aprotinin (5.0 μg/ml) and L-glutamine (1.0% w/v), followed by addition of 10 μl thrombin (50 U/L). After 90 min incubation, 500 μl M199 medium was added; this medium was supplemented with FBS (20% v/v), L-glutamine (2.0 mM), 6-aminocaproic acid (1.0 mg/ml), amphotericin B (2.5 μg/ml), gentamicin (60 μg/ml), and the treatment compounds. After 4-days incubation at 37°C and 5% CO2, the upper layer medium was replaced with a fresh one. On day 5, the distance of outgrowth of the sprouting microvessels was measured [15, 22], and the mean percentage growth inhibition was calculated (n = 3).
The median inhibitory concentrations (IC50s) were calculated using the dose response curves (n = 3).
HUVECs tube formation on matrigel matrix
Matrigel tube formation of HUVECs was investigated as previously described with minor modifications [24, 25]. In brief, 2 × 105 treated HUVECs (150 μl) were added to 48-well plate containing 150 μl solidified matrigel and incubated for 6 h. Subsequently, the tube-like structures were visualized and photographed under light microscopy at 4× magnification. The images were analyzed by measuring the area occupied by the tube-like structures using ScnImage software package (available free online). The results are presented as percentage inhibition to untreated cells, and the IC50 was calculated using the dose response curve (n = 4).
Determination of VEGF concentration in HUVECs lysates
Concentration of human VEGF-165 in HUVEC cell lysates was determined by human VEGF ELISA kit according to manufacturer’s instructions. HUVECs were treated for 6 h, washed with PBS, and cell lysates were prepared. Calibration curve of VEGF standard was prepared simultaneously, and concentration of VEGF in cell lysates was then determined by applying the VEGF linear regression equation, y = 0.0021x + 0.0585, R 2 = 0.999 (n = 3).
Where x refers to the treatment time in hours.
Chicken embryo chorioallantoic membrane assay
The chicken embryo chorioallantoic membrane (CAM) assay was performed as described previously [27, 28]. Fertile eggs were incubated for 5 days at 37°C in a humidified incubator with intermittent manual rotation. On day 5, the large blunt edge was covered with a small piece of adhesive tape, where a small hole was made and 2–5 ml albumin was withdrawn and the eggs were incubated horizontally for 2 h. Subsequently, the eggs were covered with adhesive tape and a circular window was made. Treatments were prepared in ethanol at 20 mg/ml and applied on Whattman filter paper discs at 200 and 100 μg/disc; discs for negative control received the same volume of ethanol. Ethanol was evaporated and the discs were applied directly onto the CAM through the window (n = 12). After 24 h, CAMs were illuminated and photographed under dissecting microscope.
In vivo antitumor effect
Where W is the width and L is the length.
After 28-days treatment, animals were euthanized and the tumors were collected, cross sectioned, and stained with eosin/hematoxylin for microscopic examination.
Athymic NCR nu/nu nude mice were obtained from Taconic Farms Inc., USA. Mice were housed in specific pathogen free (SPF) cages, and supplied with autoclaved food, water and bedding. The procedures were approved by USM Animal Ethics Committee (Ref. PPSG/07(A)/044/(2010)(59)).
Sprague Dawley male rats were obtained from animal breeding facility at USM. Animals were euthanized by CO2 and the thoracic aortas were collected. Experiments were performed according to the guidelines of USM Animal Ethics Committee and had their approval (Ref. USM/PPSF/50 (084) Jld.2).
The results are presented as mean ± SD. Differences between groups were analyzed by the student t-test or One-way ANOVA and were considered significant at P < 0.05.
Results and discussion
The methanolic extract of S.C. was obtained at relatively high percentage yield (16.4%, w/w). Preliminary phytochemical screening showed presence of high concentration of total phenolics (38 ± 1.3%), total flavonoids content (30 ± 3.7%), and also showed a potent DPPH scavenging activity, IC50 33.0 ± 1.0 μg/ml.
FTIR and UV-Vis spectroscopy fingerprints
S.C. extract was analyzed by FTIR and UV-Vis spectroscopy (Additional file 1: Figures S1 and S2). In FTIR the strong and broad band at 3314 cm-1 corresponds to stretching vibration of O–H group, the peak at 2930 cm-1 refers to stretching vibration of aliphatic chains, peaks at 1615 cm-1, 1521 cm-1, and 1449 cm-1 correspond to C–C stretching in aromatic rings, the peak at 1697 cm-1 corresponds to stretching vibration of the carbonyl group C = O, the bands at 1351 cm-1 and 1232 cm-1 refer to –C–OH deformation vibrations, and the band at 1044 cm-1 refers to –C–OH stretching vibrations. The UV-Vis spectrum showed absorption maxima at 273.1 nm.
Isolation and characterization of betulinic acid
Identity of BA was then confirmed by FTIR analysis; the dominant absorbance bands are located at 3450, 2939, 2872, 1689, 1642, 1455, 1377, 1232, 1190, 1142, 1036, and 884 cm-1. By comparing the band positions of BA standard with isolated BA, identical spectra were obtained which confirm the identity of isolated BA (Additional file 1: Figure S3). BA identity was further confirmed by MS analysis; BA reference and isolated compound was eluted at the same retention time (10.56 min), and the mass spectral isotopic pattern of isolated BA matches that of reference BA (455.35, 456.35 and 457,35 m/z). LC-MS analysis of methanolic extract also showed presence of compound with a mass of 455.35 m/z (Additional file 1: Figure S5). Taken together these results confirm presence of BA in S.C. methanolic leaf extract. HPLC quantitative analysis of BA in the S.C. extract indicates presence of the compound at 5.42 ± 0.09% (w/w).
In vitro antiangiogenesis effect
VEGF is a key growth factor overexpressed in most solid tumors; it initiates angiogenesis process that is required for tumor growth and metastasis by inducing proliferation, migration, sprouting and tube formation of endothelial cells [36–39]. Therefore, suppression of VEGF expression is considered as a good target in treatment of angiogenesis dependent diseases. In this study effect on VEGF expression in endothelial cells was investigated as a possible mechanism of S.C. extract antiangiogenic effect; the results showed significant suppression of VEGF expression in HUVECs lysates at 100 μg/ml (12 ± 1.8 pg/ml) compared to untreated cells (26 ± 2.7 pg/ml), P = 0.001.
S.C. methanolic extract Inhibits HUVECS migration
% Wound Closure
Suramin 25 μg/ml
74 ± 10%
36 ± 6%
50 ± 7%
53 ± 5%
87 ± 6%
41 ± 4%
64 ± 7%
84 ± 6%
In vivo antiangiogenesis effect
Taken together, inhibition of microvessels outgrowth in rat aortic rings, inhibition of HUVECs migration, inhibition of tube formation on matrigel matrix and interference with vascularization of chicken embryo CAMs provide evidence about antiangiogenic effect of S.C. extract, which can be explained due to suppression of VEGF expression in endothelial cells.
Phytochemical analysis of S.C. extract showed presence of high phenolics, flavonoids and BA content, which may explain the antiangiogenic effect of S.C. extract. Antiangiogenic effect of phenolic compounds such as flavonoids has been reported by several research groups worldwide, with different mechanisms of action such as suppression of VEGF and HIF-1α expression . Recent studies that explored the mechanism of BA antiangiogenic effect in different types of cancer concluded that the compound inhibits tumor angiogenesis by suppressing expression of the signal transducer and activator of transcription 3 (STAT3), hypoxia inducible factor-1α and VEGF [41, 42].
In vivo antitumor effect
Collectively, our data showed that S.C. methanolic extract is rich in phenolics, flavonoids and betulinic acid. The extract suppressed expression of VEGF in endothelial cells, and inhibited angiogenesis and tumor growth in nude mice. A possible mechanism of the anti-colon cancer activity of S.C. extract is the inhibition of tumor angiogenesis. Antiangiogenic and antitumor effects of S.C. can be explained, at least partly, due to the high flavonoids and betulinic acid content. Thus, our findings suggest that S.C. extract could be an interesting antiangiogenic candidate that targets the VEGF signaling pathway. S. campanulatum may have applications in colon cancer adjuvant therapy and other angiogenesis related diseases.
This work was financially supported by Universiti Sains Malaysia (USM) Research University Grant [1001/PFARMASI/81144]. The project was also supported by the research chair funded by King Saud University on drug targeting and treatment of cancer using nanoparticles.
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