Microvascular Research xxx (2013) xxx–xxx

YMVRE-03347; No. of pages: 10; 4C:

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Microvascular Research

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Regular Article

Anti-angiogenic quassinoid-rich fraction from Eurycoma longifolia modulatesendothelial cell function

Omar Saeed Ali Al-Salahi a, Chan Kit-Lam b, Amin Malik Shah Abdul Majid c, Fouad Saleih R. Al-Suede c,Sultan Ayesh Mohammed Saghir b, Wan Zaidah Abdullah d,Mohamed B. Khadeer Ahamed c,⁎, Narazah Mohd Yusoff a,⁎⁎a Advanced Medical and Dental Institute (AMDI), Universiti Sains Malaysia (USM), 13200 Kepala Batas, Pulau Pinang, Malaysiab School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysiac EMAN Testing and Research Laboratories, School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysiad Haematology Department, School of Medical Sciences, USM, 16150 Kubang Kerian, Kelantan, Malaysia

⁎ Corresponding author. Fax: +60 4 641 0295.⁎⁎ Corresponding author. Fax: +60 4 5622349.

E-mail addresses: khadeer.nc@gmail.com (M.B.K. Ahamnarazah@amdi.usm.edu.my (N.M. Yusoff).

0026-2862/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.mvr.2013.07.007

Please cite this article as: Al-Salahi, O.S.A., efunction, Microvasc. Res. (2013), http://dx.d

a b s t r a c t

a r t i c l e i n f o

Article history:Accepted 22 July 2013Available online xxxx

Targeting angiogenesis could be an excellent strategy to combat angiogenesis-dependent pathophysiologicalconditions such as cancer, rheumatoid arthritis, obesity, systemic lupus erythematosus, psoriasis, proliferativeretinopathy and atherosclerosis. Recently a number of clinical investigations are being undertaken to assessthe potential therapeutic application of various anti-angiogenic agents. Many of these angiogenesis inhibitorsare directed against the functions of endothelial cells, which are considered as the building blocks of bloodvessels. Similarly, roots of a traditional medicinal plant, Eurycoma longifolia, can be used as an alternative treat-ment to prevent and treat the angiogenesis-related diseases. In the present study, antiangiogenic potential ofpartially purified quassinoid-rich fraction (TAF273) of E. longifolia root extract was evaluated using ex vivo andin vivo angiogenesis models and the anti-angiogenic efficacy of TAF273 was investigated in human umbilicalvein endothelial cells (HUVEC). TAF273 caused significant suppression in sprouting of microvessels in rat aortawith IC50 11.5 μg/ml. TAF273 (50 μg/ml) showed remarkable inhibition (63.13%) of neovascularization in chorio-allantoic membrane of chick embryo. Tumor histology also revealed marked reduction in extent of vasculariza-tion. In vitro, TAF273 significantly inhibited the major angiogenesis steps such as proliferation, migration anddifferentiation of HUVECs. Phytochemical analysis revealed high content of quassinoids in TAF273. Specially,HPLC characterization showed that TAF273 is enriched with eurycomanone, 13α(21)-epoxyeurycomanoneand eurycomanol. These results demonstrated that the antiangiogenic activity of TAF273may be due to its inhib-itory effect on endothelial cell proliferation, differentiation and migration which could be attributed to the highcontent of quassinoids in E. longifolia.

© 2013 Elsevier Inc. All rights reserved.

Introduction

Angiogenesis, a process of formation of new branches of blood ves-sels, is strongly implicated in several important physiological situationssuch as embryonic development, ovulation, menstrual cycle, formationof the placenta during pregnancy and wound healing (Chung andFerrara, 2011; Herbert and Stainier, 2011). Dysregulation of angiogene-sis is involved in several pathological conditions including atherosclero-sis, proliferative retinopathies, rheumatoid arthritis, psoriasis, tumorgrowth and metastasis (Goel et al., 2011). Although, it is well recog-nized that angiogenesis is essential for the growth and metastasis of

ed),

ghts reserved.

t al., Anti-angiogenic quassinooi.org/10.1016/j.mvr.2013.07

most solid malignancies, an increased body of evidence support the en-hancement of angiogenesis in hematologic malignancies as well (Frateret al., 2008). Angiogenesis is an intricate process involving several wellcontrolled steps. These steps involve the breakdown of the extracellularmatrix underlying the endothelium upon activation by some pro-angiogenic signals followed by, migration, adhesion, and proliferationof endothelial cells. The proliferative endothelial cells subsequentlyassemble into three-dimensional tubular structure and finallyestablished into new microvessels that support blood flow (Chungand Ferrara, 2011; Goel et al., 2011; Herbert and Stainier, 2011). Anyof these steps can act as a pharmacological target to block the formationof new blood vessels and thus, treating angiogenesis-related diseases(Papetti and Herman, 2002). Therefore, angiogenesis is currentlybecoming an important target for chemotherapeutic approaches incancer therapy (Cardenas et al., 2011).

Eurycoma longifolia Jack. (Simaroubaceae) is amedicinal plant widelydistributed in Southeast Asia (Bhat and Karim, 2010). The plant is

id-rich fraction from Eurycoma longifolia modulates endothelial cell.007

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commonly used as herbal tea, well known as “Tongkat Ali tea”. It is usedas a traditional medicine for treatment of a variety of angiogenesis relat-ed diseases including cancer, obesity, rheumatoid arthritis andpsoriasis (Kavitha et al., 2010; Perry, 1980). Studies have shown thatE. longifolia root extracts possess antioxidant, anti-inflammatory, anti-tumor, cytotoxic, anti-bacterial, aphrodisiac, antipyretic, antiamoebicand antimalarial properties (Mahfudh and Pihie, 2008; Patel et al.,2011; Wernsdorfer et al., 2009).

Kuo and his colleagues reported the isolation of about sixty five phe-nolic compounds from the root of this plant (Kuo et al., 2004). The plantis reported to be rich in various classes of bioactive compounds such as,quassinoids, canthin-6-one alkaloids, β-carboline alkaloids, triterpenetirucallane type, squalene derivatives and biphenyl-neolignan, eury-colactone, laurycolactone, and eurycomalactone, and bioactive steroids(Bhat and Karim, 2010; Kuo et al., 2004; Mahfudh and Pihie, 2008).Among these phytoconstituents, quassinoids share a major portion inE. longifolia root. The quassinoids are a group of nortriterpenoidswith dynamic pharmacological properties (Fiaschetti et al., 2011).Quassinoids are effective even in nanomolar and subnanomolar concen-trations to inhibit a cell growth-related plasma membrane-associatedNADH oxidase in cells (Morré et al., 1998). Several studies reportedthe strong inhibitory effects of quassinoids on Epstein–Barr virus activa-tion (Okano et al., 1995; Ping-Chung et al., 2004; Rahman et al., 1997).Quassinoids inhibit cellular proliferation, block cell cycle progression,induce apoptosis and down-regulate the expression of the oncogenec-MYC (von Bueren et al., 2007). Quassinoids are anticancer and anxio-lytic agents and exhibit a significant cytotoxic activity (Jiwajinda et al.,2002; Anderson et al., 1991; Arisawa et al., 1983; Ogura et al., 1977;Ozeki et al., 1998). The cell cycle arrest effect of quassinoids was corre-lated with a mechanism of protein synthesis inhibition (Beutler et al.,2009). Quassinoids possess inhibitory activities for nitric oxide produc-tion in LPS-activated macrophages with potent cytotoxicity againsthuman tumor cell lines (Liu et al., 2012).

The reports on remedial uses of the herb to alleviate mainly theangiogenesis-dependent diseases and the evidences that the plant isendowed with abundance of bioactive constituents, particularly thequassinoids, with significant anti-neovascularization and anti-tumorpromotional activities (Teh et al., 2010), have drawnattention toward hy-pothesizing that E. longifolia could be useful for treating angiogenesis-dependent diseases if its antiangiogenicity is investigated in vitro andscientifically provenusing an animalmodel of angiogenesiswith standardexperimental protocols. Moreover, there are little studies reported so faron the antiangiogenic activity of this herbal plant. Accordingly, the aimsof this study are to evaluate the antiangiogenic activity of partially puri-fied quassinoid-rich fraction derived frommethanol extract of E. longifoliaroots and to investigate and characterize the anti-angiogenic efficacy ofthe fraction in human umbilical vein endothelial cells (HUVECs). Thiswork is an attempt to verify its traditional use to treat the angiogenesisrelated ailments.

Materials and methods

Chemicals, cell culture and reagents

Eurycomanone, 13α(21)-epoxyeurycomanone, 13,21-dihydroeury-comanone, and eurycomanol were purchased from Sigma (Germany).Other chemicals used were either HPLC grade or analytical grade.

Phosphate buffered saline, trypsin, penicillin/streptomycin (PS),fibrinogen, aprotinin, thrombin, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) re-agent, suramin, and vincristine were purchased from Sigma, Germany.Matrigel matrix (10 mg/ml) was purchased from BD Bioscience, USA.HUVEC (ScienCell, USA) were maintained in endothelial cell medium(ECM) (ScienCell, USA) supplementedwith 5% heat inactivated fetal bo-vine serum (HIFBS) and 1% PS. K-562 (Human erythromyeloblastoidleukemia) cell line was purchased from American type culture

Please cite this article as: Al-Salahi, O.S.A., et al., Anti-angiogenic quassinofunction, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.07

collection (Rockville, MD, USA) and maintained in RPMI 1640 contain-ing 10% HIFBS and 1% PS.

Plant material and extraction

The plant and root samples of E. longifolia Jackwere identified by Pro-fessor Ahmad Latif from Universiti Kebangsaan Malaysia (UKM). Theplant samples were collected in February 2012 from Perak, Malaysia.The voucher specimen (no. 785-117) was deposited in the herbariumof Penang Botanical Garden, Penang, Malaysia.

The TAF273, TAF355 and TAF401 are partially purified sub-fractionsderived from resin chromatography of the crude methanol extract ofE. longifolia roots as previously reported (Teh et al., 2010). The detailedprocedure of collection of TAF273 and theother fractions is describedbyKavitha et al. (2012). Briefly, powdered roots of E. longifolia wereextracted with 95% methanol, using Soxhlet extractor at 60 °C for 6 h.The extract suspension was filtered and the filtrate was thenconcentrated to dryness under partial vacuum at room temperature(24–27 °C) to yield dark brown residue. The combined extract wasnext fractionated by column chromatography, using Diaion HP 20 col-umnwith aH2O–MeOH (1:0–0:1) gradient, to yield 4 fractions of the de-sired quassinoid-rich E. longifolia extract. Before the treatment, stocksolutions of TAF273, TAF355 and TAF401 were prepared at the concen-tration 10 mg/ml. TAF401 was dissolved in dimethyl sulfoxide (DMSO),while TAF273 and TAF355 were dissolved in water. All the fractionswere sterilized by filtration using 0.2-μm syringe filters (Sartorius,Germany).

High performance liquid chromatography (HPLC) analysis ofTAF273, TAF355 and TAF401

100 mg of each fraction was separately dissolved in 25 ml mixtureofmethanol and sonicated for 15 min. All sampleswere filtered through0.45 μm filter (nylon membrane filter). Similarly all reference com-pounds (5 mg) were dissolved in 5 ml of methanol and then filtered.Contents of eurycomanone, 13α(21)-epoxyeurycomanone, 13,21-dihydroeurycomanone and eurycomanol were determined by HPLCfingerprint analysis (Teh et al., 2011).

Experimental animals

For rat aortic ring assay, healthy male Sprague Dawley rats(8–10 week old) were obtained from the Animal House Facility,Universiti Sains Malaysia. For tumor xenograft studies, male athymicBALB/c nude mice (8 to 12-week-old) were used. The study protocolwas approved by Animal Ethics Committee, Universiti Sains Malaysia(USM), No.: USM/Animal Ethics Approval/2010/(60) (254).

Assessment of anti-angiogenic activity

Ex vivo rat aortic ring assay

This assay was carried out as previously described (Nicosia andOttinetti, 1990)withminormodifications. Thoracic aortaswere removedfrom euthanized male Sprague Dawley rates (12–14 week old), rinsedwith serum free medium and cleaned from fibroadipose tissues. Theaortaswere cross sectioned into small rings (approximately 1 mm thick-ness) and seeded individually in 48-wells plate in 300 μl serum freeM199 media containing 3 mg/ml fibrinogen and 5 mg/ml aprotinin.Ten microliters of thrombin (50 NIH U/ml in 1% bovine serum albuminin 0.15 M NaCl) was added into each well and incubated at 37 °C for90 min to solidify. A second layer (M 199 medium supplemented with20% HIFBS, 0.1% έ-aminocaproic acid, 1% L-glutamine, 2.5 μg/mlamphotericin B, and 60 μg/ml gentamicin) was added into each well(300 μl/well). TAF273, TAF355 and TAF401 were added at final concen-trations of 6.25, 12.5, 25, 50 and 100 μg/ml and the median inhibitory

id-rich fraction from Eurycoma longifolia modulates endothelial cell.007

Fig. 1. A: HPLC chromatograms of standard markers, eurycomanone (EN), eurycomanol(EL), 13α (21)-epoxyeurycomanone (EP) and 13,21-dihydroeurycomanone (ED). B, Cand D: HPLC chromatograms of fractions, TAF273, TAF355 and TAF401 showing differentproportions of respective markers. E: Chemical structures of marker compounds,quassinoids present in fractions of E. longifolia root extract. F: Illustration depicts the con-tents of quassinoids (% dry weight) present in the fractions of E. longifolia root extract. Thegraphical representation shows that TAF273 is enriched with eurycomanone (EN), 13α(21)-epoxyeurycomanone (EP) and eurycomanol (EL) but it is deprived of 13,21-dihydroeurycomanone (ED). The fraction TAF355 also contains these 3 quassinoids butin significantly low level. On the other hand, the fraction TAF401 is deprived of all testedquassinoids except 13,21-dihydroeurycomanone (ED) that too in negligible percentage.

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concentrations (IC50) were determined by nonlinear regression analysisof log-concentration–response curves. Similarly, serial concentrations(6.25 to 100 μg/ml) of suramin were used as standard reference(Nassar et al., 2011), whereas distilled water was used as negativecontrol. On day four, the medium was replaced with a fresh onecontaining the test materials. On day five, aortic rings werephotographed using an AMG EVOS fI inverted microscope (40× magni-fication) and subsequently the length of blood vessels outgrowth fromthe primary tissue explants was measured using Leica Qwin software.

The inhibition of blood vessel formation was calculated using theformula;

% bloodvessel inhibition ¼ 1− A0=Að Þ½ � � 100;

where; A0 = distance of blood vessel growth in treated rings in μm;and A = distance of blood vessel growth in the control in μm.

The results are presented asmeanpercent inhibition ± SD, (n = 8).% of inhibition was plotted against the concentrations and IC50 was

calculated.The significant difference between the microvessel outgrowth in

treated versus untreated aortic rings was calculated using Student's ttest. Based on the results of this assay, TAF273 was chosen for the sub-sequent investigations for the anti-angiogenic property.

Antiproliferative activity of TAF273 on HUVEC cell line

Antiproliferative effect of TAF273was evaluated against human um-bilical vein endothelial cells (HUVECs) using MTS assay (Mosmann,1983). Cells (70–80% confluency) were treated with various concentra-tions of TAF273 (1.56, 3.12, 6.25, 12.5, 25 and 50 μg/ml). Similarly,vincristine with serial concentrations (1.56 to 50 μg/ml) is used as stan-dard reference. The IC50 values were calculated using regression equa-tion as explained before (Premkumar et al., 2005). The cells were thenincubated at 37 °C and 5% CO2 in humid atmosphere for 48 h. After48 h, MTS solution was added into the wells and the plate wasreincubated for 2–4 h. The absorbance at 490 nm was measured andthe percentage inhibition was calculated using the formula:

% inhibition ¼ 1−absorbanceof treated=absorbanceof untreatedð Þ� 100% :

The % of inhibitionwas plotted against the concentration inMicrosoftexcel and the IC50 was calculated using the regression equation.

Cell migration assay

HUVECs were maintained in 6-well plate in ECM until 100% conflu-ent monolayer growth was obtained. As previously described (Lianget al., 2007), themonolayer was scratchedwith a sterile 200 μl micropi-pette tip and then washedwith PBS to remove the unattached cells andto smoothen the edges of the scratch. After that, 2 ml of ECM with lowserum concentration (2.0%) was added to each well followed by addi-tion of TAF273 at 2.5, 5 and 10 μg/ml final concentrations. Suramin(100 μg/ml) (Ahamed et al., 2012) and vehicle (distilled water) wereadded as positive and negative controls, respectively. Subsequently,6–8 microscopic fields per well were photographed at 0, 12 and 24 husing an AMG EVOS fI inverted microscope. The width of the cell-freearea was measured using Leica Qwin software. The percentage ofwound closure was then calculated relative to zero time using theformula:

%woundclosure¼ 1−thewidthattheindicatedtimes hð Þ=thewidth at zero timeð Þ

� 100% :

The results are displayed as average ± SD, (n = 6).

Please cite this article as: Al-Salahi, O.S.A., et al., Anti-angiogenic quassinoid-rich fraction from Eurycoma longifolia modulates endothelial cellfunction, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.07.007

Fig. 2.Anti-angiogenic effect of fractions of E. longifolia root extract. Photomicrographs show the inhibition ofmicrovessel outgrowth in rat aortic ring. The aortic ringswere photographedunder an inverted phase-contrast microscope at, after 5-days of treatment with TAF273, TAF355 and TAF401. A: Control (10×magnification), B: Suramin (100 μg/ml, 4× magnification),C: TAF273 (6.25 μg/ml, 10× magnification), D: TAF273 (12.5 μg/ml, 10× magnification), E: TAF273 (25 μg/ml, 4× magnification), F: TAF273 (50 μg/ml, 10× magnification), G: TAF273(100 μg/ml, 4× magnification), H: TAF355 (100 μg/ml, 4× magnification), I: TAF401 (100 μg/ml, 10× magnification). J: Dose dependent inhibitory effect of TAF273 on sprouting ofmicrovessels from rat aorta. K: Graphical comparison of the anti-angiogenic activities between the fractions and the standard reference suramin. All values are expressed as mean ±SD (n = 8). * = P b 0.1, ** = P b 0.005, *** = P b 0.005.

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Tube formation assay

The ability of HUVECs to form tube-like structures was investigatedon a matrigel matrix as described before (Ahamed et al., 2012). In brief,the matrigel (150 μl/well) was added to 48-well plate and allowed topolymerize for 45 min at 37 °C and 5% CO2. HUVECs were harvestedand seeded onto the matrigel coated plates. The cells were treatedwith various concentrations of TAF273 and incubated at 37 °C for24 h. Suraminwas used as a positive control at 100 μg/ml in the growthmedium. The cells were imaged under an AMG EVOS fI inverted micro-scope at low magnification and the network length and width wasquantified.

In vivo CAM assay

Anti-neovascularization effect of TAF273 was investigated in vivo inchorioallantoic membrane (CAM) assay using chicken embryos as

Please cite this article as: Al-Salahi, O.S.A., et al., Anti-angiogenic quassinofunction, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.07

previously reported (Nassar et al., 2011). Five day-old fertilized eggswere obtained from local hatchery. 5 ml of albumin was aspirated andthe eggs were incubated horizontally to allow the CAM to detachfrom the shell. TAF273 was prepared in agarose 1.2% disks at 50 and100 μg/disk. Disks containing the vehicle only (1.2% agarose) were usedas negative control. A small window opening was made in the egg shell,and the disks were directly applied onto the CAM. The square openingwas covered with sterilized surgical tape and the embryos were incubat-ed for 24 h. The CAMswere photographed under a dissectingmicroscopeand blood vessels in each CAMwere counted. The results are presented asa mean blood vessel count per CAM ± SD, (n = 6).

In vivo assessment of tumor angiogenesis in nude mousexenograft model

Subcutaneous tumors were induced in male BALB/c nude mice byinjecting K-562 cells (107 cells/150 μl RPMI) into the dorsal side. Mice

id-rich fraction from Eurycoma longifolia modulates endothelial cell.007

Fig. 3. Effect of TAF273 on proliferation and migration of HUVECs. A): Effect of TAF273 onHUVEC proliferation. TAF273 inhibited HUVEC proliferation in dose dependent mannerwith IC50 32.5 μg/ml (n = 6, values are in mean ± SD). B): Due to the successful migrationof endothelial cells in untreated group (distilled water), the wound is almost closed after18 h, whereas in TAF273 treated group, the wound remained open even after 24 h incuba-tion. TAF273 (5 μg/ml) caused significant inhibition of endothelial cell migration. At a con-centration of 10 μg/ml, TAF273 caused dislodgement of monolayer of endothelial cells(indicated by the arrows)with almost complete inhibition of migration. C): Graphical repre-sentation of the time and dose and time-dependent inhibitory effect of TAF273 onmigrationof HUVECs (values are inmean ± SD, n = 6, * = P b 0.1, ** = P b 0.005, *** = P b 0.005).

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were then treated orally with 200 mg/Kg of TAF273 (n = 6) and withvehicle as control (n = 6) every other day for 16 days. Tumor tissuesections were collected and fixed with 10% buffered formalin. Paraffinblocks were prepared and sections of 5 μm were cut on a microtome(Accu-Cut® SRMTM, SAKURA) and stained with hematoxylin andeosin (H&E). The number of blood vessels in tissue sections, from thetest and control groups, was then counted. Ten microscopic fields perslide were examined at 40× total magnification and the means ofblood vessels were compared (test versus control) using SPSS software.Student t-test was used to determine the P value (P value less than 0.05was considered statistically significant).

Results

HPLC analysis of E. longifolia fractions

HPLC chromatograms of fractions of E. longifolia root extract (absor-bance at 238 nm) along with the mixed standards are shown in Fig. 1.Results from this study are consistent with previous reports suggestingthat eurycomanone is one of themajor chemical constituents of TAF273along with other quassinoids (Darise et al., 1982; Teh et al., 2011).

The analytical plots of standards, eurycomanone, 13α(21)-epoxy-eurycomanone,13,21-dihydroeurycomanone and eurycomanol alongwith the fractions are shown in Figs. 1A to D. Chemical structures ofthe biomarkers are given in Fig. 1E. The quantitative result of bioactivemarkers (% dry weight) is illustrated in Fig. 1F.

TAF273 inhibits sprouting of microvessels in rat aortic explants

Fig. 2A shows a massive sprouting of microvessels in the untreatedaortic explants. The standard reference, suramin demonstrated potentinhibition of microvessel formation (Fig. 2B). Similarly, treatment withTAF273 (Figs. 2C–G) resulted in a significant inhibition of microvesselformation with IC50 11.5 μg/ml which can be compared to that of stan-dard reference, suramin (IC50 9.6 μg/ml). TAF355 and TAF401 showed amoderate inhibitory effect at 100 μg/ml and at lower concentrations noremarkable inhibitionwas noted (Figs. 2H and I). The length anddensityof microvessel outgrowth were affected by treatment with TAF273 in adose dependent manner (Fig. 2J). Fig. 2K graphically depicts the differ-ence between the effects of the different fractions of E. longifolia rootextract and suramin.

TAF273 inhibits proliferation and migration of HUVECs

In order to confirm the possible mechanism by which TAF273inhibits the growth of aortic blood vessels, an investigation on a seriesof in vitro angiogenesis models was conducted. Since, angiogenesis in-volves local proliferation of endothelial cells in response to an angiogenicstimulus, thus we first determinedwhether TAF273 inhibited endotheli-al cell proliferation. TAF273 significantly decreased the rate of prolifera-tion of HUVEC cells (IC50 32.5 μg/ml). The results showed that thestandard reference, vincristine exhibited strong anti-proliferative effectwith IC50 0.06 μg/ml. Fig. 3A shows the comparative effects of TAF273and vincristine in a graphical form of mean cell viability curves obtainedwith MTS assay.

HUVEC migration assay represents an important step in the forma-tion of new blood vessels and is a straightforward and economicalmethod to study the cell migration phenomenon (Nassar et al., 2011).A scratch wound was created on the monolayer of cells (Fig. 3B) andeffect of TAF273 on closure of thewoundwas studied. TAF273 inhibitedHUVECsmigration (P b 0.01) in a dose dependentmanner. As shown inFig. 3B, the percentage wound closure at 12 h was 38 ± 2% for controland 38 ± 7, 26 ± 2 and 17 ± 1% for TAF273 at the concentrations2.5, 5 and 10 μg/ml, respectively. After 24 h, the percentage woundclosure for control was 93 ± 2% whereas, for TAF273 it was 72 ± 1,53 ± 2 and 32 ± 5% at the concentrations 2.5, 5 and 10 μg/ml,

Please cite this article as: Al-Salahi, O.S.A., et al., Anti-angiogenic quassinoid-rich fraction from Eurycoma longifolia modulates endothelial cellfunction, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.07.007

Fig. 4. In tube formation assay, HUVECs (2 × 104 cells/well) were plated onmatrigel precoated 96-well plates and treatedwith different concentrations of TAF273 for 24 h. Phase contrastmicrographs showing the effects of TAF273 and suramin on differentiation of HUVECs. A) Control, shows the endothelial cells grown on the 3-dimentional matrigel media differentiatedinto branching morphogenesis to form capillary tube-like structures composed of multiple cells with intercellular spaces or lumens. The picture clearly shows prominently thick-cellcovered areas (brackets), tubes (arrows), loops (Lo) and branching points (b). B): Photomicrograph shows that the effect of treatment of suramin (100 μg/ml) caused weakening ofthe bridges in tube-like structure (arrow head) which is evident through the loss of cell covered area (brackets), thinned tubes (arrow head), and feeble branching points (b). C): Treat-ment with TAF273 (2.5 μg/ml) shows slight disruption of bridges and branching points. D): Photomicrograph reveals that, TAF273 at 5 μg/ml caused notable disorganization in the tubelike structure (arrow head), as the improper development of a lumen in the cell–cell connections can be seen. E): Photomicrograph shows the apparent inhibitory effect of TAF273 on thedifferentiation of HUVECs, as the affected tubes and network can be seen clearly. F): Treatment with 20 μg/ml TAF273 caused a complete abrogation of the network, branches and thebridges between the cells. G): Graphical depiction of dose-dependent inhibitory effect of TAF273 on the height and width of capillary like structures of HUVECs.

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respectively. Fig. 3C shows the dose and time dependent inhibitoryeffect of TAF273 on migration of endothelial cells.

TAF273 inhibits tube formation in human endothelial cells

To characterize the anti-angiogenic activity of TAF273, endothelialcell tube formation assay, a well-established 3-dimensional in vitro an-giogenesis assay was conducted. HUVECs cultured on matrigel formed

Please cite this article as: Al-Salahi, O.S.A., et al., Anti-angiogenic quassinofunction, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.07

tube like networks (Fig. 4A) within 8 h, which might, in part, reflectthe process of angiogenesis. The activity of TAF273 was more pro-nounced than that of the standard drug, suramin which showed 98.7%inhibition at a concentration of 100 μg/ml (Fig. 4B). TAF273 showeddose-dependent inhibitory effect with 53.9, 58.8, 72.9 and 95.8% at theconcentrations, 2.5, 5, 10 and 20 μg/ml, respectively (Figs. 4C–F). Note-worthily, TAF273 absolutely disorganized the integrity of endothelialtube network (Fig. 4G), reducing the tube-like structure both in width

id-rich fraction from Eurycoma longifolia modulates endothelial cell.007

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and in length at 20 μg/ml concentration, which is lower than the IC50value of TAF273 on HUVEC proliferation. It can be seen clearly that,the HUVECs lost their pseudopodial like cellular extensions and the net-work structures were broken in the presence of TAF273. Fig. 4G depictsthe dose-dependent inhibitory effect of TAF273 on width and length ofendothelial cell tube-network.

TAF273 inhibits in vivo neovascularization in chick embryo

Neovascularisation in chick embryo was significantly inhibited byTAF273 at 50 and 100 μg concentrations. Fig. 5A shows normal vascula-ture pattern in the untreated CAMs with clear visualization of primary,secondary and tertiary vessels and dendritic branching pattern(arrows).Whereas, TAF273-treated (50 and100 μg/disk) CAMdisplayeddistorted architecture (arrow heads) in the vasculature (Figs. 5B and C).The number of the blood vessels was reduced drastically in treatedCAMs. TAF273 inhibited the formation of new blood vessels significantly(Fig. 5D), as the total mean count of blood vessels is 66 ± 3 and 42 ±4 at 50 and 100 μg/disk, respectively, when compared to the control(179 ± 5).

TAF273 inhibits in vivo angiogenesis in xenograft tumor

Morphology of the excised tumors showed significant reduction indensity of blood vessels in the tumors harvested from treated animalscompared to the tumors from control group (Figs. 6A and B). Tumorsections from TAF273-treated (200 mg/kg) and untreated (control)mice were stained with hematoxylin and eosin stain and examined forquantification of the blood vessels, viability and necrotic or apoptoticfeatures. The histology of tumors from control mice showed abundanceof viable cells (V) arranged compactly due to the rapid proliferation of

Fig. 5. Inhibitory effect of TAF273 on neovascularization in chorioallantoicmembrane of chick emagarose as a negative control, shows enormous neovascularization with primary, secondary ashows inhibition of secondary and tertiary branches of blood vessels (arrow heads). (C) CAMThe treatment caused disruption of even main branches of blood vessels (arrow heads). (D) G(P b 0.005, n = 6, values are in mean ± SD).

Please cite this article as: Al-Salahi, O.S.A., et al., Anti-angiogenic quassinofunction, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.07

the cancerous cells. The tumor cells showed compact sheets of polygo-nal cells with abundant supply of blood vessels (arrows). Very fewnecrotic areas (pink color) were seen in the histology of control mice(Fig. 6C). In the animals treated with TAF273, many tumors showedhistological evidence of TAF273's antiangiogenic effect (Fig. 6D). The tu-mors from these animals showed significant loss of compactness of thecells in the tumor with moderate to severe necrosis (N) with areas ofdecreased viable cell density. Small foci of viable cells (V)with apoptoticfeature such as membrane blebbing, nuclear condensation etc. can beseen in the treated tumor sections (Fig. 6D). The most remarkablechange observed in tumor histology was the treatmentwith TAF273 in-duced reduction in tumor vascular density. The treatment with TAF273resulted in a significant reduction in blood vessel formation than com-pared to the control. The mean blood vessel count per low power field(Fig. 6E) in the tumor tissues from treated mice was 5.58 ± 1.5 and incontrol mice it was 10.72 ± 3 (P b 0.012).

Discussion

The critical limitation of chemotherapy is due to the lack of targetspecificity. Mostly, chemotherapy results in damage of both cancerousand normal cells. The administration of suboptimal doses of cancerchemotherapeutic agents was studied in order to reduce the side effectsthat often culminatewith incomplete tumor response and eventual fail-ure of therapy, early disease relapse, drug resistance, and metastaticdisease (Eatock et al., 2000). Recently, the newly emerging strategy ofcombinatorial therapy using anti-angiogenic agents with chemothera-peutic drugs has become a promising and effective methodology incancer treatment (Bocci et al., 2008; Mabuchi et al., 2010). The beautyof anti-angiogenic drugs relies on their multi-targeted capability. Anti-angiogenic agents may inhibit angiogenesis by suppressing the

bryo. The pictureswere captured under dissectingmicroscope. (A) CAM treatedwith 1.2%nd tertiary branches of blood vessels (arrows). (B) CAM treated with TAF273 (50 μg/ml)treated with TAF273 (100 μg/ml) shows significant suppression of neovascularization.

raphical representation of the effect of TAF273 on the mean blood vessels count per CAM

id-rich fraction from Eurycoma longifolia modulates endothelial cell.007

Fig. 6. Antiangiogenic effect of TAF273 on xenograft tumor derived from humanerythromyeloblastoid leukemia (K-562) cell line. A): Morphological features of tumorharvested from control animal (arrows indicate prompt and well developed blood vesselsin control). B): Morphological features of tumor harvested from animal treated with200 mg/kg TAF273 (arrowhead points towards the reduction in tumor vasculature causedby TAF273). Here the negligible density of tumor vascularization can be seen. C): An H&E-stained tumor section (original magnification of 4×) of the “vehicle control” group is com-posed of compact sheet of aggressively proliferating viable tumor cells (designated by “V”)with little necrotic regions (N) and abundance of blood vessels (BV, indicated by thearrows). D): The tumor section (original magnification of 4×) of TAF273 (200 mg/kg)treatment revealed notable changes in tumor histology, as significant loss of compactarrangement of tumor cells with less number of blood vessels can be seen. The islands(indicated by arrows) of live tumor cells (V) surrounded by necrotic regions (N)suggesting the decreased supply of blood vessels to the tissue. E): Graphical comparisonof the mean blood vessel count between the control and TAF273 groups. Values arepresented as mean ± SD (n = 10). (For interpretation of the references to color in thisfigure, the reader is referred to the web version of this article.)

8 O.S.A. Al-Salahi et al. / Microvascular Research xxx (2013) xxx–xxx

Please cite this article as: Al-Salahi, O.S.A., et al., Anti-angiogenic quassinofunction, Microvasc. Res. (2013), http://dx.doi.org/10.1016/j.mvr.2013.07

synthesis of angiogenic signals from cancer cells, inhibiting the angio-genic mitogens, blocking the receptors of endothelia for angiogenicsignals, directly inducing endothelial cell apoptosis, or inhibiting endo-thelial cell migration and differentiation (Ahamed et al., 2012;Wu et al.,2008).

Protein synthesis is emerging as an interesting, under-exploredtarget for chemotherapeutic intervention. A number of studies indicatethat deregulation of protein synthesis is a major contributor in angio-genesis, cancer initiation and metastatic progression (Graff andZimmer, 2003; Watkins and Norbury, 2002). More than a dozen differ-ent proteins, as well as several smaller peptides, have been identified asangiogenic mitogens that are released by tumors as signals to promoteangiogenesis. Inhibitors of angiogenesis are currently the main focusof intense cancer research. Recent studies have attested to theantiangiogenic effects of protein synthesis modulators such as, sodiumbutyrate, a short-chain fatty acid naturally present in the human coloninhibits angiogenesis by inhibiting the angiogenesis-related proteinsynthesis (Pellizzaro et al., 2002). Similarly, valproic acid has been re-ported to impair tumor-cell-induced angiogenesis (Zgouras et al.,2004).

In the present work, TAF273 demonstrated potent inhibitory effecton sprouting of microvessels from rat aorta and neovascularization infertilized chicken embryo. Further investigation revealed that, theanti-angiogenic efficacy of TAF273 was shown to be mediated viainterfering with the endothelial cell function. Antiangiogenic agentsact via direct or indirect inhibitory mechanism. In the direct anti-angiogenesis process, the angiogenesis inhibitors interfere with theendothelial cell functions such as proliferation, migration and differen-tiation. During the process of angiogenesis, a single layer of endothelialcells lining the inside of blood vessels divide and break off from thevessel membrane, forming the tubes and eventually become new capil-laries (Maeshima et al., 2002). TAF273 effectively inhibited the keyaspects of angiogenesis in endothelial cells, such as proliferation andmigration. After cells migrate into the perivascular space in bloodvessels, HUVECs undergo differentiation and the cells assume a charac-teristic shape to facilitate the adherence amongst the cells to form alumen or tube-like structure (Fan et al., 2006). TAF273 remarkablyinhibited the formation of such tube like structures in a dose dependentmanner (Fig. 4). The in vivo tumor xenograft model also supported theanti-angiogenic property of TAF273, where TAF273 significantlysuppressed the vascular supply to the growing tumor. When a tumorforms, the endothelial cells divide more rapidly and spread much morequickly than theywould during the normal course of blood vessel forma-tion (Maeshima et al., 2002). In the present study, histopathological anal-ysis of the extent of tumor necrosis and vascularization of the tumorrevealed that TAF273 caused moderate to severe necrosis in the tumorswhich is an obvious sign of lack of blood supply to the tumors(Morioka et al., 2003). In addition, the tumor histology of treated animalsrevealed that TAF273 induced apoptotic characteristics in the tumorcells whichwas quite apparent with the signs of cellular blebbing, nucle-ar condensation etc. These effects of TAF273 could be ascribed mainly toits quassinoids contents, 13α(21)-epoxyeurycomanone, 13,21-dihy-droeurycomanone, eurycomanol, and particularly eurycomanone.Eurycomanone is abundantly found in E. longifolia root and is one ofthe bioactive compounds with promising potencies to be developed asa new chemotherapeutic agent. Eurycomanone is reported to havestrong anti-angiogenic activity with 50% inhibition at 40 μg/ml infertilized chicken embryo (Salamah et al., 2009), whereas in thepresent study, TAF273 showed 73% inhibition of neovascularization at50 μg/ml. Eurycomanone is cytotoxic to HepG2 cells and inducesapoptosis through the up-regulation of p53 and Bax, and down-regulation of Bcl-2 (Mahfudh and Pihie, 2008; Zakaria et al., 2009).Eurycomanone exhibits pro-apoptotic, anti-proliferative and anti-clonogenic cell growth effects and suppression of the tumor markersand several known cancer cell growth-associated genes (Wong et al.,2012; Zakaria et al., 2009). In general, quassinoids and their derivatives

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possess promising antiangiogenic properties (Wong et al., 2012). Fur-thermore, quassinoids are capable of inducing an array of biological re-sponses (Fukamiya et al., 2005; Salamah et al., 2009) includinginhibition of protein synthesis (Cuendet, and Pezzuto, 2004; Liao et al.,1976). Such an inhibition has been shown to occur via interference atthe peptidyltransferase site, thus preventing peptide bond formation(Hall et al., 1983). It has been shown in several studies that quassinoidsare able to down-regulate c-MYC protein expression in a panel of leuke-mia, lymphoma, and myeloma cell lines (Fresno et al., 1976; Fukamiyaet al., 2005). Quassinoids have a great potential to target cell-survivalpathways that are essential for the angiogenesis as well as development,progression andmetastasis of the tumors (Mata-Greenwood et al., 2002;Morré et al., 1998).

Unlike TAF273, TAF355 and TAF401 showed insignificant anti-angiogenic activity. These findings may suggest that the anti-angiogenicactivity may be partly attributed to the quassinoids, which are abundantin TAF273 (Teh et al., 2011).

In conclusion, the present work provides good supporting evidencethat TAF273 inhibits the angiogenesis in various experimental models.This robustic antiangiogenic effect of TAF273 may be due to the collec-tive contribution of phytochemicals particularly, the quassinoids,eurycomanone, eurycomanol and 13α(21)-epoxyeurycomanone inTAF273. These results convinced that TAF273 exerts the antiangiogeniceffect by suppressing the key cellular pathways of endothelial cells.Thus, E. longifolia could be the potential source of promising therapeuticagents to treat angiogenesis related disorders.

Conflict of interests

The authors have no conflict of interests to declare.

Acknowledgments

This work was supported by the Short Term Grant, Universiti SainsMalaysia (USM). Grant number is 304/CIPPT/6311016. The authorswould like to thank Dr. Hasnah Hashim for statistical assistant. Authorsalso thank Dr. Nor Azlina Khalil and other members of IPPT animalhouse for their help. Authors also would like Dr. Othman Bin Abdullahfrom department of pathology, Sultan Abdul Halim hospital, Malaysiafor their assistance in the interpretation of the histopathology slides.

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