angiogenesis is the process of new blood vessel generation and is involved in the pathophysiology of a wide variety of diseases such as cancer ( 1 , 2 ) . anti - angiogenic therapy represents a new approach to the early intervention and prevention of malignant disease ( 3 ) . it is highly desirable to find dietary sources of anti - angiogenic molecules that have the ability to reduce cancer risk as well . punica granatum l. ( pomegranate ) has been known for its several beneficial effects including prevention and treatment of several types of cancer through its various metabolites ( 4 ) . pomegranate peels are characterized by large amounts of phenolic compounds , and their great antioxidant potential ( 5 ) . we previously showed that pomegranate peel extract ( ppe ) inhibits human umbilical vein endothelial cells ( huvecs ) angiogenesis , vefg mrna expression and secretion ( 6 ) . previous studies have shown that ppe inhibited cell proliferation and angiogenesis marker expression ( 7 , 8) . recent research demonstrated that some parts of pomegranate inhibit angiogenesis via reduction of vascular endothelial growth factor ( 9 ) . also experimental studies have shown that the skin and arils of pomegranate extract inhibit tumor angiogenesis ( 10 ) . although previous studies have proved the effectiveness of anti - angiogenic effect of pomegranate compartments but still the mechanisms underlying its anti - angiogenic activity and specially ppe remain unknown . some studies have shown that pomegranate carries out its therapeutic effects by affecting the peroxisome proliferator - activated receptors ( ppars ) pathway ( 11 , 12 ) . ppars are ligand - activated transcription factors that perform diverse metabolic functions ( 13 ) . three subtypes of ppars (  ,  or  ,  ) have been recognized , each encoded by distinct genes and expressing in a different way in many parts of the body ( 14 ) . the ppar activators seem to inhibit tumor growth by several mechanisms including inhibition of angiogenesis ( 15 ) . ppar and ppar have a wide range of events involving the vasculature , including atherosclerotic plaque formation and stability , vascular tone , and angiogenesis ( 16 ) . recent studies showed that the ppar- pathway may be a therapeutic target for numerous diseases in which excessive angiogenesis is implicated , including cancer ( 17 , 18 ) . hence , in this study , we investigated whether the anti - angiogenic effect of ppe is partly attributable to ppars activation in the huvecs . for this purpose , we used t0070907 as a selective ligand for ppar , and gw6471 as a ppar alpha specific inhibitor(19 , 20 ) . pomegranate peels were dried and powdered and extracted with ethanol 70% containing 1% acetic acid at room temperature for 24 hr . high - performance thin - layer chromatography method for the determination of ellagic acid as the major component of pomegranate was done at 280 nm using a tlc scanner 3 ( camag , muttenz , switzerland ) ( 22 ) . the huvecs ( national cell bank of iran affiliated to pasteur institute , tehran , iran ) were cultured in dulbecco s modified eagle s medium ( dmem ) , supplemented with 1% antibiotics ( 100 units / ml penicillin and 100 g / ml streptomycin ) , and 10% fetal bovine serum . when the cells were 7080% confluent , they were treated with 0.25% trypsin and passaged to a new culture . a total of 100 l matrigel basement membrane matrix ( invitrogen , usa ) was coated for 30 min at 37c within a 24-well plate . the cells were detached by trypsin- edta , and after neutralization of trypsin , cells were resuspended in medium 200prf containing 10% fbs ; then , huvecs ( 110 ) in 4 groups were seeded on the matrigel . each well was treated with different treatments , which included ppe ( 10 g / ml ) , ppe + ppar- antagonist ( t0070907 ; 50 mol / l ) , ppe + ppar antagonist ( gw6471 ; 50 mol / l ) , and the control group ( dmso 0.1 % ) . after 24 hr of incubation , calcein am cell - permeable dye was added to each well and photographed with a fluorescent microscope . finally , tube length , size , and junction were quantified using the angioquant v1.33 software ( the mathworks , natick , ma , usa ) . total rna was extracted from huvec using an rneasy mini plus kit ( qiagen , valencia , ca , usa ) , following the manufacturer s protocols . cdna was synthesized using revertaidtm reverse transcriptase ( fermentas , vilnius , lithuania ) with oligo - dt primers as described before ( 6 ) . qrt - pcr was performed using specific primers for vegfa and gapdh ( as an internal control ) mrnas with the maxima sybr green / rox qpcr master mix ( fermentas , vilnius , lithuania ) and run on the rotor - gene 6000 ( qiagen , hilden , germany ) . the pcr cycling conditions for the genes included an initial denaturation step at 95 c for 10 min , followed by 45 amplification cycles consisting of denaturation at 95 c for 15 sec , annealing at 60 c for 30 sec and an extension at 72 c for 30 sec . gapdh : aatgcatcctgcaccaccaa gapdh : gtagccatattcattgtcata vegf - a : gcagaatcatcacgaagtgg vegf - a : gcatggtgatgttggactcc the experiments were performed in duplicate and replicated three times . at last , using spss 15 tests in one way anova and lsd as post hoc ( bonferroni ) analysis were performed . pomegranate peels were dried and powdered and extracted with ethanol 70% containing 1% acetic acid at room temperature for 24 hr . high - performance thin - layer chromatography method for the determination of ellagic acid as the major component of pomegranate was done at 280 nm using a tlc scanner 3 ( camag , muttenz , switzerland ) ( 22 ) . the huvecs ( national cell bank of iran affiliated to pasteur institute , tehran , iran ) were cultured in dulbecco s modified eagle s medium ( dmem ) , supplemented with 1% antibiotics ( 100 units / ml penicillin and 100 g / ml streptomycin ) , and 10% fetal bovine serum . when the cells were 7080% confluent , they were treated with 0.25% trypsin and passaged to a new culture . a total of 100 l matrigel basement membrane matrix ( invitrogen , usa ) was coated for 30 min at 37c within a 24-well plate . the cells were detached by trypsin- edta , and after neutralization of trypsin , cells were resuspended in medium 200prf containing 10% fbs ; then , huvecs ( 110 ) in 4 groups were seeded on the matrigel . each well was treated with different treatments , which included ppe ( 10 g / ml ) , ppe + ppar- antagonist ( t0070907 ; 50 mol / l ) , ppe + ppar antagonist ( gw6471 ; 50 mol / l ) , and the control group ( dmso 0.1 % ) . after 24 hr of incubation , calcein am cell - permeable dye was added to each well and photographed with a fluorescent microscope . finally , tube length , size , and junction were quantified using the angioquant v1.33 software ( the mathworks , natick , ma , usa ) . total rna was extracted from huvec using an rneasy mini plus kit ( qiagen , valencia , ca , usa ) , following the manufacturer s protocols . cdna was synthesized using revertaidtm reverse transcriptase ( fermentas , vilnius , lithuania ) with oligo - dt primers as described before ( 6 ) . qrt - pcr was performed using specific primers for vegfa and gapdh ( as an internal control ) mrnas with the maxima sybr green / rox qpcr master mix ( fermentas , vilnius , lithuania ) and run on the rotor - gene 6000 ( qiagen , hilden , germany ) . the pcr cycling conditions for the genes included an initial denaturation step at 95 c for 10 min , followed by 45 amplification cycles consisting of denaturation at 95 c for 15 sec , annealing at 60 c for 30 sec and an extension at 72 c for 30 sec . gapdh : aatgcatcctgcaccaccaa gapdh : gtagccatattcattgtcata vegf - a : gcagaatcatcacgaagtgg vegf - a : gcatggtgatgttggactcc the experiments were performed in duplicate and replicated three times . at last , using spss 15 tests in one way anova and lsd as post hoc ( bonferroni ) analysis were performed . using the ph differential method for freeze dried ppe , its anthocyanin content equivalent to cyaniding-3-glucoside was detected as % 1.771.80 . dried extract of the pomegranate peel was standardized to contain % 1.570.128 ( g/100 g ) ellagic acid . for the assessment of the anti - angiogenic activity of ppe the results showed that ppe ( 10 g / ml ) significantly suppressed the formation of tube - like structures ( size , length , junction ) compared the control ( p<0.05 ) ( figure 1 and table 1 ) . ppar antagonists completely antagonized the formation of the tube - like structures of huvec cells . there was no significant difference between the control group and ppe plus antagonists group ( p>0.05 ) . huvecs were plated on a well coated with 100 l of matrigel basement membrane matrix . after they were treated with ppe for 24 hr , the cells were dyed and photographed with a nikon camera attached to a fluorescent microscope at 10 magnification . a : huvecs were treated with ppe + t0070907 , b : huvecs were treated with ppe + gw6471 , c : treatment of the cells with ppe , d : control group endothelial tube size , length , and junction after treatment in different groups on matrigel significant decrease compared to the control group ( p<0.05 ) ; significant increase compared to the ppe group ( p<0.05 ) to examine whether ppe inhibits angiogenesis through ppars , we used ppar antagonists . for this purpose interestingly results have shown that treatment of the endothelial cells with ppe plus antagonists has led to significantly increased tube formation ( figure 1 , table 1 ) . after optimization of the qrt - pcr , expression of vegfa and gapdh genes was determined in the treated and control cells . relative expression of vegf gene was determined by dividing its expression amount to that of the gapdh gene . ppe ( 10 g / ml ) significantly inhibits vegf mrna expression ( p<0.05 ) . interestingly the cells that were treated by both of ppars antagonists suppressed the ppe activity and there was significant increase in vegf mrna expression compared to control and ppe groups ( p<0.05 ) . using the ph differential method for freeze dried ppe , its anthocyanin content equivalent to cyaniding-3-glucoside was detected as % 1.771.80 . dried extract of the pomegranate peel was standardized to contain % 1.570.128 ( g/100 g ) ellagic acid . for the assessment of the anti - angiogenic activity of ppe , huvecs were added on matrigel then treatment was started . the results showed that ppe ( 10 g / ml ) significantly suppressed the formation of tube - like structures ( size , length , junction ) compared the control ( p<0.05 ) ( figure 1 and table 1 ) . ppar antagonists completely antagonized the formation of the tube - like structures of huvec cells . there was no significant difference between the control group and ppe plus antagonists group ( p>0.05 ) . huvecs were plated on a well coated with 100 l of matrigel basement membrane matrix . after they were treated with ppe for 24 hr , the cells were dyed and photographed with a nikon camera attached to a fluorescent microscope at 10 magnification . a : huvecs were treated with ppe + t0070907 , b : huvecs were treated with ppe + gw6471 , c : treatment of the cells with ppe , d : control group endothelial tube size , length , and junction after treatment in different groups on matrigel significant decrease compared to the control group ( p<0.05 ) ; significant increase compared to the ppe group ( p<0.05 ) to examine whether ppe inhibits angiogenesis through ppars , we used ppar antagonists . for this purpose interestingly results have shown that treatment of the endothelial cells with ppe plus antagonists has led to significantly increased tube formation ( figure 1 , table 1 ) . after optimization of the qrt - pcr , expression of vegfa and gapdh genes was determined in the treated and control cells . relative expression of vegf gene was determined by dividing its expression amount to that of the gapdh gene . ppe ( 10 g / ml ) significantly inhibits vegf mrna expression ( p<0.05 ) . interestingly the cells that were treated by both of ppars antagonists suppressed the ppe activity and there was significant increase in vegf mrna expression compared to control and ppe groups ( p<0.05 ) . the present study demonstrates that when huvecs were treated simultaneously by ppe and ppar- or ppar-antagonist , the anti - angiogenic effect of ppe suppressed and ec tube formation increased . after treatment by ppars antagonists and ppe , increased vegf mrna expression raises the possibility that anti - angiogenic effect of pomegranate may be suppressed through the ppar pathway . we have recently demonstrated that ppe can suppress the formation of tube - like structures in ( 200 , 300 , and 400 g / ml ) doses(6 ) , therefore , we examined the ppe ( 10 g / ml ) to find the minimal effective dose and for assessment of ppe anti - angiogenic potential in lower doses and the second probable mechanism . pomegranate peel is rich in estrogenic flavonoids that have been shown to be anti - angiogenic . previous studies indicate that pomegranate compartments , inhibit angiogenesis in cancer and human umbilical vein endothelial cell lines via downregulation of vascular endothelial growth factor ( 23 ) . several in vitro and in vivo studies proposed that selective activation of ppar and ppar promotes a vigorous angiogenic process by a mechanism dependent on vegf stimulation ( 24 , 25 ) . we defined the role of ppar antagonist in suppressing the anti - angiogenic effect of ppe . the treatment of endothelial cells with ppe simultaneous ppar antagonists showed that anti - angiogenic effects of ppe were inhibited . further ppe anti - angiogenic properties may be involved by the ppars - dependent mechanism . ppar and ppar , which are both expressed in endothelial cells , have several well - characterized roles in endothelial cells , including anti - inflammatory , antiproliferative , and anti - angiogenic effects ( 26 , 27 ) . quantitative analysis of mrna expression of vascular endothelial growth factor in human umbilical vein endothelial cells by real - time rt - pcr . after treatment of huvecs by ppe alone or ppe with ppar antagonists , total rna was extracted . the relative expression of vegf was normalized to gapdh levels measured in the same rna preparation . significant increase compared to the control group ppar has been shown to regulate the expression of the vegf . biscetti et al have indicated that wy14643 ( ppar agonist ) promotes corneal angiogenesis in vivo and enhances endothelial tubulogenesis in vitro ( 28 ) . ppar ligands might act as potent anti- angiogenic factors , for example , endothelial cells proliferation was avoided by fenofibrate through inhibiting cyclooxygenase-2 expression ( 29 , 30 ) . ppar agonists were shown to inhibit endothelial vegfr2 expression by inhibiting sp1-dependent promoter binding and transactivation ( 31 ) . finally , we have shown that ppar and  agonists inhibit endothelial tube formation ( 32 ) . inhibition of vegf , and vegf receptor expression and mitogen - activated protein kinase - dependent activation are probable mechanisms of anti - angiogenic actions of ppar ( 33 , 34 ) . however , the anti - angiogenic mechanism of ppar activation in the endothelial cells remains currently unclear . hontecillas et al in their studies demonstrate that punicic acid , which is a conjugated linolenic acid isomer frequently found in pomegranate , binds and robustly activates ppar , increases ppar-responsive gene expression ( 12 ) , furthermore , other pomegranate compartments such as flowers , have been shown to activate ppars . these findings indicate for the first time that ppar- and  might be the molecular targets for ppe extract and provide a better understanding of the potential mechanism of the anti - angiogenic action of ppe . further in vivo and in vitro studies are needed to shed more light on the underlying mechanisms of ppe beneficial effects in cancer prevention and treatment . it seems that the anti - angiogenic effects of ppe could be mediated in part through ppar dependent pathway .
objective(s):herbal medicines are promising cancer preventive candidates . 
 it has been shown that punica granatum l. could inhibit angiogenesis and tumor invasion . in this study , we investigated whether the anti - angiogenic effect of pomegranate peel extract ( ppe ) is partly attributable to peroxisome proliferator - activated receptors ( ppars ) activation in the human umbilical vein endothelial cells ( huvecs).materials and methods : ethanol extract from ppe was prepared . 
 huvecs were treated in four groups ( with ppe ( 10 g / ml ) alone , ppe with or without ppar ( t0070907 ) and  ( gw6471 ) antagonists , and control group ) 
 . the possible effect of ppars on angiogenic regulation was checked by matrigel assay . 
 the mrna expression levels of vascular endothelial growth factor ( vegf ) was detected by quantitative reverse transcription - polymerase chain reaction ( qrt - pcr).results : ppe significantly inhibited both tube formation ( size , length , and junction of tubes ) and vegf mrna expression ( p<0.05 ) . 
 our results showed that the anti - angiogenic effects of ppe were significantly reversed by both ppar antagonists ( p<0.05 ) . 
 there was no difference between ppe plus antagonists groups and the control group.conclusion:in summary our results showed that the anti - angiogenic effects of ppe could be mediated in part through ppar dependent pathway .