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Acta Histochemica 114 (2012) 94– 100

Contents lists available at ScienceDirect

Acta Histochemica

j o ur nal homepage: www.elsev ier .de /ac th is

ffects of lycopene and apigenin on human umbilical vein endothelial cellsn vitro under angiogenic stimulation

ehmet S ahina, Emel S ahinb, Saadet Gümüs lüc,∗

Health Sciences Research Centre, Faculty of Medicine, Akdeniz University, 07070 Antalya, TurkeyCentral Laboratory, Clinical Biochemistry Unit, Faculty of Medicine, Akdeniz University, 07070 Antalya, TurkeyDepartment of Biochemistry, Faculty of Medicine, Akdeniz University, 07070 Antalya, Turkey

r t i c l e i n f o

rticle history:eceived 28 December 2010eceived in revised form 4 March 2011ccepted 6 March 2011

a b s t r a c t

Angiogenesis is the formation process of new blood vessels from preexisting vessels. Solid tumors needangiogenesis for growth and metastasis. The suppression of tumor growth by inhibition of neoangiogenicprocesses represents a potential approach to cancer treatment. Lycopene has powerful antioxidant capac-ities and anticarcinogenic properties. The aim of this study was to investigate the effects of lycopene onangiogenesis in vitro. For this reason, we measured in vitro angiogenesis in human umbilical vein endothe-

eywords:ngiogenesisycopenepigeninancer

lial cells including parameters of cell proliferation, tube formation, cell migration. Lycopene and apigeninwere observed to block the endothelial cell proliferation in a dose-dependent manner. In addition, theysignificantly decreased the capillary-like tube lengths, tube formation and endothelial cell migration.This study provides indications that apigenin and lycopene, which are considered as chemopreventiveagents, to be effective in vitro on endothelial cells and angiogenesis.

UVEC

ntroduction

Angiogenesis, the formation of new blood vessels, plays severaloles in various human pathologies including: rheumatoid arthritis,iabetic retinopathy, atherosclerosis, psoriasis, and chronic airway

nflammation. One of the most important roles of angiogenesis is to

upport tumor growth, which is dependent on nutritional supporterived from the local blood supply (Sahin et al., 2009).

Targeting inhibition of angiogenesis represents a potentialpproach in the treatment of solid tumors and such antiangio-

Abbreviations: BHT, butylated hydroxytoluene; DMSO, dimethyl sulfoxide;BM-2, endothelial basal medium; EGM-2, endothelial growth medium; EGF, epi-ermal growth factor; FBS, fetal bovine serum; FGF, fibroblast growth factor; Hep3B,

human hepatoma cell line; HUVEC, human umbilical vein endothelial cells; IGF-, insulin-like growth factor; KB-1, a cell line derived from a human oral cavityumor; LNCaP, an androgen-sensitive human prostate adenocarcinoma cell line;

CF-7, a breast cancer cell line; MDA-MB-231, an estrogen-independent humanreast cancer cell line; MMP, matrix metalloproteinase; MTT, (3′(4,5-dimethyl--thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide); PC3, a metastatic cell lineerived from advanced androgen independent prostate cancer; RPMI, Roswell Parkemorial Institute cell culture medium; SK-Hep1, an immortal and metastatic cell

ine derived from a liver adenocarcinoma; THF, tetrahydrofuran; VEGF, vascularndothelial growth factor.∗ Corresponding author. Tel.: +90 2422496896; fax: +90 2422274495.

E-mail addresses: msahin@akdeniz.edu.tr (M. S ahin), esahin@akdeniz.edu.trE. S ahin), sgumuslu@akdeniz.edu.tr (S. Gümüs lü).

065-1281/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.acthis.2011.03.004

© 2011 Elsevier GmbH. All rights reserved.

genic strategies inhibiting the growth of endothelial cells maybe more advantageous than targeting cancer cells. The inhibitionof tumor growth requires a chronic inhibition of vascular orga-nization, defined as ‘angiostasis’ (Hanahan and Folkman, 1996).Long-term treatment is necessary to achieve angiostasis and it isnecessary to identify and characterize angiostatic molecules show-ing low or no toxicity. Current dietary guidelines to combat chronicdiseases including cancer, recommend increased intake of plantfoods that are rich in antioxidants. The role of dietary antioxidants,including carotenoids and flavonoids in disease prevention hasreceived much attention (Kim et al., 1998; Agarwal and Rao, 2000).Apigenin is a flavonoid with anti-inflammatory, anticarcinogenicand free radical scavenger properties (Kim et al., 1998). Lycopene,one of more than 600 carotenoids, is a natural pigment synthesizedby plants and photosynthetic microorganisms. Several epidemio-logical studies have strongly implied that consumption of foodscontaining high concentrations of lycopene may reduce the riskof certain types of cancer (Gann et al., 1999; Giovannucci et al.,2002).

The mechanism of lycopene action is still under investigation.Lycopene was shown to induce apoptosis in a dose-dependentmanner in prostate cancer cells (Hantz et al., 2005). Similarly, treat-ment of human colon carcinoma cells with lycopene at 2.0 or 4.0 �M

was found to be able to induce apoptosis (Salman et al., 2007). Therehave been several reports that lycopene can induce cell cycle arrest.It has been reported that the growth of Hep3B human hepatomacells was inhibited 20–50% by lycopene (Park et al., 2005). A simi-

M. S ahin et al. / Acta Histochemica 114 (2012) 94– 100 95

Fig. 1. Lycopene inhibited tube formation produced by human umbilical vein endothelial cells. Representative micrographs of HUVECs cultured for 24 h on Matrigel in thepresence of the indicated compound are illustrated. Serum-starved cells were seeded to each well of 24-well plate as ∼2.5 × 104 cells in EGM-2 complete medium. HUVECc �M. S

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ells were incubated in (A) THF (Control), (B) Lyc 1 �M, (C) Lyc 5 �M and (D) Lyc 10

ar study with the human prostate cancer cell lines, LNCaP and PC3,lso found that lycopene induced mitotic arrest at the G0/G1 phaseIvanov et al., 2007).

With respect to anti-invasive and anti-metastatic activities,ycopene (5–10 �M) has been shown to decrease the activities ofhe gelatinolytic matrix metalloproteinases, MMP-2 and MMP-9,nd to inhibit adhesion, invasion and migration of SK-Hep1 cells,hich is a highly invasive human hepatoma cell line (Hwang and

ee, 2006). Lycopene at similar concentrations was also found tonduce the metastasis suppressor gene nm23-H1 (Huang et al.,005).

Although there have been several studies based on the antiox-dant and anticarcinogenic activities of lycopene, its role onndothelial cells and angiogenesis has remained unclear. The aimf this study was to investigate whether lycopene is effective onndothelial cells and angiogenesis inhibition at different concen-rations, and to compare the effects of lycopene with apigenin withegard to antiangiogenic activities.

aterials and methods

eagents

Human umbilical vein endothelial cells (HUVECs) andatrigelTM were purchased from BD Biosciences (BD-354234;

edford, MA, USA). EGM-2® BulletKit® (CC-3162, EBM- + supplements) for HUVEC culture was purchased from LonzaWalkersville, MD, USA). Lycopene and apigenin were purchasedrom Sigma–Aldrich (St. Louis, MO, USA).

cale bar = 250 �m.

Cell culture

Human umbilical vein endothelial cells (HUVECs) were cul-tured in EGM-2 complete medium consisting of EBM-2 basalmedium supplemented with hydrocortisone, ascorbic acid, hep-arin, GA-1000 and 2% FBS plus growth factors (epidermal growthfactor (EGF), vascular endothelial growth factor (VEGF), fibroblastgrowth factor (FGF), insulin-like growth factor (IGF-1)) except forserum starvation in which FBS concentration was reduced to 0.1%and no growth factors were added. Cell culture flasks or wellplates, pre-coated with gelatin, were used for culture of HUVECs(Sigma–Aldrich, Irvine, Ayrshire, UK). All cells were grown in ahumidified atmosphere, 95% air and 5% CO2 at 37 ◦C and passagedevery 4–6 days. Cells from the fourth to the sixth passage were usedfor experiments. Lycopene was dissolved in tetrahydrofuran (THF;stabilized with 0.025% BHT) and applied to the cells at 0 �M, 1 �M,5 �M and 10 �M concentrations. Lycopene was prepared accordingto methods reported by Martin et al. (2000) and Lin et al. (2007) toincrease its stability and to facilitate its uptake by cells. Apigeninwas dissolved in dimethyl sulfoxide (DMSO) and added at 5 mg/l(18.5 �M) and 10 mg/l (37 �M) concentrations. The final concen-tration of DMSO and THF did not exceed respectively 0.2% (v/v)and 0.1% (v/v) in any case.

In vitro angiogenesis assay

MatrigelTM (BD-354234, Bedford, MA, USA) was thawed at 4 ◦C,and 250 �l were quickly added to each well of a 24-well plate andallowed to solidify for 1 h at 37 ◦C. Once solid, HUVECs, serum-

9 tochemica 114 (2012) 94– 100

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Fig. 2. Inhibition of the in vitro angiogenesis of human umbilical vein endothe-lial cells by apigenin. Representative micrographs of HUVECs cultured for 24 h onMatrigel as mentioned in material and methods section in the presence of the indi-

6 M. S ahin et al. / Acta His

tarved for 24 h, were added to each well (∼2.5 × 104 cells/well) inGM-2 complete medium. After cells had adhered to the Matrigel,MSO, THF, apigenin or lycopene was added, and cells were incu-ated at 37 ◦C for 24 h. The tube formation of HUVECs was recordedith an Olympus IX81 Photomicroscope (Olympus, Tokyo, Japan)ith a ×4 objective. Tube lengths of three different fields of imagesere assayed with the UTHSCSA ImageTool Version 3.0 softwarerogramme (University of Texas Health Science Center, San Anto-io, TX, USA). Experiments were repeated five times and the resultsere expressed as mean ± standard deviation (SD).

ell proliferation assay

The effects of lycopene and apigenin on the viability of cells wereetermined by MTT (3′(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-H-tetrazolium bromide) assay as described previously withome modifications (Mosmann, 1983). MTT was purchased fromppliChem (Darmstadt, Germany). MTT stock solution (5 mg/ml)

n RPMI-1640 without phenol red (Sigma–Aldrich, St. Louis, MO,SA) was diluted (1:10) for experiments. The cells were platedt ∼2.5 × 104 cells per well. 70–80% confluent HUVECs in 24-ell plates were serum-starved for 24 h. After 24 h, HUVECs were

reated with DMSO, THF, apigenin, or lycopene and incubated at7 ◦C for 24 h. After medium was exchanged with 500 �l MTTolution, cells were incubated at 37 ◦C for 3 h. At the end of the incu-ation period, the converted dye was solubilized with 500 �l acidic

sopropanol (0.04 M HCl in absolute isopropanol). Absorbance ofhe converted dye was measured at 570 nm with background sub-raction at 650 nm by colorimetric plate reader (Model No.: 1500;hermo Labsystem, Finland). Experiments were repeated five timesnd the results were expressed as mean ± SD.

ell migration assay

We used a Millipore QCMTM Endothelial Migration Assay Kitontaining fibronectin coated 24-well Boyden Chamber Systemith 3 �m pore width (Millipore ECM201, Temecula, CA, USA).

xperiments were carried out according to manufacturer’s kit assayrocedures. Briefly, migrated cells at the bottom plate were dyedith CyQUANT® GR fluorescent dye (Millipore, Temecula, CA, USA).

his green-fluorescent dye was assayed with a fluorescence plateeader using 480/520 nm filter set (Type: 374; Thermo Fluoroskanscent FL, Finland). Experiments were repeated five times andesults were expressed as mean ± SD of five different experiments.

ound healing assay

For another cell migration assay, a wound healing assay waspplied as horizontal migration. The experiment was appliedsing methods of Schleef and Birdwell (1982) with some mod-

fications. After full-confluent HUVECs were starved of serumnd growth factors for 24 h, cells in 12-well plate werecratched with a sterile 200 �l plastic pipette tip across mono-ayer of cells. Cells were treated with DMSO, THF, apigenin orycopene. When HUVECs in control groups had almost com-letely migrated (approximately 12–14 h), cells were dyed with,1′-dioctadecyl-3,3,3′,3′-tetramethylene indocarbocynanine per-hlorate (Dil) (Aldrich 468495) and photographed by integratedlympus DP70 digital camera (Olympus, Japan) on an invertedicroscope (Olympus IX81S1F-2, Japan) with x40 magnification.il were prepared as stock solution (1 mg/ml) in DMSO and diluteds 1:200 in growth medium for experiments. Migration distances

f ten different fields at photographs were assayed with UTHSCSAmageTool Version 3.0 software programme (University of Texasealth Science Center, San Antonio, TX, USA). Experiments were

epeated five times.

cated compound are illustrated. HUVECs were incubated with (A) DMSO (Control),(B) Apigenin (5 �g/ml) and (C) Apigenin (10 �g/ml). Scale bar = 250 �m.

Statistical analysis

Data were expressed as mean ± standard deviation (SD). Com-parison of groups according to the parameters was performed usingthe Student’s t-test and test of one-way ANOVA (Tukey’s MultipleComparison) in Prism Program of GraphPad Software (San Diego,CA). P values less than 0.05 were considered statistically significant.

Results

Lycopene and apigenin inhibit capillary-like tube formation

In this study, we found that 1 �M, 5 �M and 10 �M concen-

trations of lycopene have significantly suppressed the endothelialtube formations (7102 ± 497, 4448 ± 426, 2718 ± 287, respectively)on Matrigel matrix as compared with controls (14574 ± 575) inwhich the cells were treated with THF (p < 0.001) (Fig. 1, Fig. 3A).

M. S ahin et al. / Acta Histochemica 114 (2012) 94– 100 97

the do

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Fig. 3. Tube lengths of capillary-like structures are affected by

e found that the inhibition of tube lengths and organization wasose-dependent. In apigenin-treated HUVECs, capillary-like tubeormation was also dramatically inhibited at 5 �g/ml (18.5 �M)r 10 �g/ml (37 �M) concentrations (8098 ± 479, 3842 ± 461,espectively) as compared with control (16431 ± 502, p < 0.001)Figs. 2 and 3 Figs. 2 and 3B).

ycopene and apigenin suppress cell proliferation

Treatment of cells with three different concentrations ofycopene (Fig. 4A) and two different concentrations of apigeninFig. 4B) decreased the proliferation of endothelial cells in a dose-ependent manner. As compared with controls, percentage of theroliferating cells in groups treated with 1 �M, 5 �M and 10 �Moncentrations of lycopene (63.34 ± 2.34, 41.53 ± 2.17, 30.24 ± 1.96xpressed as the percentage of control cells, respectively) wasound to be significantly decreased (p < 0.001). Percentage of prolif-ration was seen to be dose-dependent. Difference between dosesas found significant (p < 0.001) (Fig. 4A). Apigenin was also shown

o be effective against endothelial proliferation at 5 �g/ml and0 �g/ml concentrations (54.24 ± 2.33 and 38.15 ± 2.38, respec-ively) compared with control groups (expressed as 100) (p < 0.001).n addition, there was a significant difference among the doses, as

ell (p < 0.001) (Fig. 4B).

ycopene and apigenin block endothelial cell migration

Endothelial cells migrate towards the chemo-attractants dur-ng angiogenesis. In this system endothelial cells migrate throughhe pores and move to the bottom of the plate containing EGM-2

edium and serum. Lycopene at 1 �M, 5 �M and 10 �M con-entrations suppressed endothelial cell migration through poresompared with controls (56.02 ± 3.11, 52.10 ± 2.44, 42.06 ± 3.24,espectively) (Fig. 5A). Values of fluorescence intensity in controlroups were expressed as 100%. We did not find any significantifference between 1 and 5 �M lycopene. However, differencesmong other doses were found as significant (p < 0.001). It washown 5 �g/ml and 10 �g/ml apigenin concentrations decreaseduorescence intensity of cells (57.98 ± 3.22, 49.72 ± 2.84, respec-ively) as compared with control groups (expressed as 100%)p < 0.001). 10 �g/ml apigenin was more effective than 5 �g/mlp < 0.01) (Fig. 5B).

ycopene and apigenin decrease directional migration

Additionally, we have also used another migration assay methodamed “wound healing assay” showing directional migration. Wenly assayed 10 �M lycopene and 10 �g/ml apigenin for this assay.

ses of lycopene in (A) and apigenin in (B). *** Shows p < 0.001.

Similarly, migration of cells towards each other was found to besuppressed (or migration length increase) both group treated withlycopene (610.17 ± 36.17) and apigenin (670.62 ± 39.39) as com-pared to controls THF (68.41 ± 15.36) and DMSO (99.46 ± 19.39)(Fig. 6A–D) (p < 0.001).

Discussion

The critical role of tumor angiogenesis in cancer progression waspostulated about 40 years ago in pioneering studies by Folkmanet al. (1971). However, only in recent years has the knowledge ofendothelial cell physiology and tumor angiogenesis provided thenecessary background to develop effective antiangiogenic strate-gies. Developing antiangiogenic strategies against the growth ofendothelial cells may be more advantageous than directly target-ing cancer cells in shrinkage of tumors. The endothelial cell, whichis a cell type common to all solid tumors, represents a preferentialtarget for therapy. Even though every cancer is virtually a uniquedisease, the tumor endothelium is a relatively uniform, normal celltype (Tosetti et al., 2002). Another importance of this approach isthe apparent inability by the endothelial cells to counteract ther-apy through development of multi-drug resistance mechanisms,due to the low mutagenesis rate of this normal cell type (Boehmet al., 1997).

Chronic inhibition of vascular recruitment may be required toblock tumor growth; thus, long-term treatment may be necessary(Hanahan and Folkman, 1996). Therefore, we need to identify andcharacterize angiostatic molecules endowed with low or no toxi-city. Substantial efforts have been dedicated to identifying naturaland synthetic compounds that can be used to either prevent insur-gence of primary tumors in subjects at high risk to develop canceror prevent tumor relapse after surgical removal. Cancer chemo-prevention involves the use of agents to slow the progression ofcarcinogenesis, reverse, or inhibit it, with the aim of lowering therisk of developing invasive or clinically significant disease. Chemo-preventive drugs must be devoid of toxicity and well tolerated sincethey must be used over extended periods (Hanahan and Folkman,1996).

In this study, we investigated the effects of lycopene on endothe-lial cells. Because of its chemical structure, lycopene is a moleculethat has very powerful antioxidant activity. As described earlier,growth of endothelial cells occur under tight regulation. However,conditions around the cancerous tissue change the regulation to

constitute new vessels. Under these conditions, endothelial cellsincreasingly proliferate, migrate towards chemoattractants pro-duced by cancer cells, and finally generate a new capillary network.In this study, we have investigated whether these steps are affected

98 M. S ahin et al. / Acta Histochemica 114 (2012) 94– 100

Fig. 4. Percentage proliferation of endothelial cells. HUVECs were plated at ∼2.5 × 104 cells per well. 70–80% confluent HUVECs in 24-well plate were serum-starved for 24 h.After this period, cells in growth medium were treated with DMSO, THF, apigenin and lycopene. After incubated at 37 ◦C for 24 h, cells were treated with MTT solution andt osed

( yc (1 �e g/ml)

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he converted dye was measured at 570 nm. (A) Percentage of proliferating cells exp1 �M), (b) C vs. Lyc (5 �M), (c) Lyc (1 �M) vs. Lyc (5 �M), (d) C vs. Lyc (10 �M), (e) Lxposed to apigenin: (a) C vs. Api (5 �g/ml), (b) C vs. Api (10 �g/ml) and (c) Api (5 �

y lycopene. MatrigelTM matrix used in our study is an extracellu-ar matrix provided from a cancer type and containing an agentssential for angiogenesis. In addition, we added EGM containingrowth factors such as VEGF and FGF important for the angiogen-sis process of cells. Under these environmental conditions, whilee observed widespread tube formation in cells treated with DMSO

r THF alone, capillary-like tubular formation appeared to be sup-ressed in cells treated with lycopene. In our experiment, lycopeneas applied at concentrations of 0, 1, 5 and 10 �M. We observed

hat lycopene significantly inhibited in vitro angiogenesis in a dose-ependent manner.

Apigenin which is used for positive control is known to displayntiangiogenic effects in a variety of studies (Trochon et al., 2000;im, 2003; Erdogan et al., 2007). We also demonstrated that api-enin inhibited tube formation in a dose-dependent manner. Theroliferation rate of endothelial cell increases during angiogene-is. Therefore, we investigated the effects of lycopene via the MTTethod on cells in medium containing serum and growth factors

nd observed lycopene significantly suppressed cell proliferationor all doses used. While lycopene at the most effective dose 10 �Mecreased proliferation by 69.76%, 5 �M and 1 �M lycopene inhib-

ted proliferation by 58.47% and 36.66%, respectively. Differencesmong the concentrations are statistically significant. Ivanov et al.2007) suggested treatment of LNCaP prostate carcinoma cells with

ig. 5. Lycopene (A) and apigenin (B) treatments suppress the migration of endothelial

erum and growth factors. Migrated cells were dyed and assayed with fluorescence plaontrols (DMSO or THF). No significant difference between 1 and 5 �M lycopene. ***p < 0.

to lycopene. Values represent mean ± SD of five different experiments: (a) C vs. LycM) vs. Lyc (10 �M) and (f) Lyc (5 �M) vs. Lyc (10 �M). (B) Percentage of living cells

vs. Api (10 �g/ml). (C) Control; Lyc: lycopene; Api: apigenin.

lycopene decreased the cell proliferation. In another study, it wasshown that lycopene suppressed the proliferative capacity of sev-eral malignant cell lines (Salman et al., 2007). Moreover, in cellculture system, lycopene was demonstrated to strongly inhibit IGF-1-mediated proliferation of human endometrial, breast and lungcancer cells (Karas et al., 2000). Lycopene is also known to inhibitproliferation of human oral cavity tumor (KB-1) (Livny et al., 2002)and human breast cancer cells (MCF-7 and MDA-MB-231) (Prakashet al., 2001). Even if the types of the cells are different, the generalsuppressive effects of lycopene on proliferation are consistent withour study. We revealed that apigenin at both 5 �g/ml and 10 �g/mlalso significantly inhibited cell proliferation 45.76% and 61.85%,respectively.

As regards the migration assay, another important parameterfor in vitro angiogenesis, we used two different methods to assayendothelial cell migration. One of the methods is based on cellmovements towards the chemoattractant. In this experiment, weassayed migration of cells from the upper micro-pore (3 �m) platecoated with fibronectin to the bottom plate containing serum andgrowth factors. Control of the experiment involves using albumincoated plates instead of fibronectin. Consequently, we observed

all concentrations of lycopene, as compared with THF, inhibitedcell migration. Similarly, apigenin also significantly suppressed themigration. Another method, named wound healing assay, is based

cells from fibronectin-coated plates with pores (3 �m) to bottom plate containingte reader. Data were given as relative percent fluorescence intensity according to001, **p < 0.01.

M. S ahin et al. / Acta Histochemica 114 (2012) 94– 100 99

Fig. 6. The fluorescence microphotographs of wound healing assayed cells treated with (A) THF (Control), (B) lycopene (10 �M), (C) DMSO (Control) and (D) Apigenin(10 �g/ml). After full-confluent HUVECs were starved with serum and growth factors for 24 h, cells in 12-well plate were scratched with sterile 200 �l plastic pipette tip.C lmostm icroscfi ar = 1

oAm

istcal

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ells were treated with the indicated compound. When HUVECs in control groups aaterial and methods section. Microphotographs were taken under fluorescence m

elds of each of five independent experiments was calculated as mean ± SD. Scale b

n mimicking the cell migration during wound healing in vivo.pigenin and lycopene in this assay significantly decrease theovement of cells.Hwang and Lee (2006) suggested that lycopene at 5 and 10 �M

nhibited the adhesion, invasion and migration of a highly inva-ive human hepatoma cell line, SK-Hep1. Moreover, it was foundhat the metastasis suppressor gene nm23-H1 was induced in theseells (Huang et al., 2005). The studies performed with SK-Hep1re similar to our study in terms of anti-migration properties ofycopene.

Our study is consistent with other reported studies defend-ng the protective effects of flavonoids and carotenoids againsthronic diseases (Kim et al., 1998; Agarwal and Rao, 2000). In ourtudy, it was revealed apigenin and lycopene showed similar effectsnder the same experimental conditions. It may be concluded that

ycopene is more effective than apigenin when comparing theironcentrations.

Various epidemiological and experimental studies haveevealed that lycopene is an important agent to prevent fromancer, however, effects of lycopene on endothelial cells or angio-enesis could not be found in the literature. In conclusion, the

resent results suggest that lycopene has some antiangiogenicroperties, though additional studies are needed to demonstratehe effects of lycopene on molecular mechanisms in endothelialells.

completely migrated, cells were stained with Dil fluorescence dye as mentioned inope at ×40 magnification. Reciprocal distance (pixel) between cells at ten different00 �m. ***p < 0.001.

Conflict of interest

The authors have no conflict of interest.

Acknowledgements

Funding for this study was provided by grants from the Akd-eniz University Scientific Research Project Unit (Project Number:2006.03.0122.005). Also this study was supported by Akdeniz Uni-versity Health Sciences Institute.

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