breast cancer is the most common malignancy in women , and metastasis is the number one cause of mortality in breast cancer . invasion depends in part on degradation of extracellular matrix ( ecm ) and interaction with molecules formed in the process . recently , increasing attention has been paid to chemokines that may modify breast cancer cells and the surrounding matrix to facilitate metastasis . furthermore , there is evidence that chemokines , vegf - c , and heparanase are interrelated in the process of invasion [ 46 ] . increased expression of the lymphangiogenesis factors and heparanase has been correlated with progressive disease in certain cancers . peritumoral lymphangiogenesis involves the secretion of specific glycoproteins designated vascular endothelial growth factor c ( vegf - c ) and ( vegf - d ) that act on lymphatic endothelium , and are components of an established signaling system for tumor lymphangiogenesis . increased lymph node metastases are correlated with increased expression of vegf - d and vegfr3 by immunohistochemistry in invasive breast cancer . heparanase is an endo--glucuronidase that cleaves heparan sulfate ( hs ) side chains of heparin sulfate proteoglycan ( hspg ) . heparanase activity has been correlated with cell invasion associated with breast cancer metastasis , a consequence of structural modification of hs that alters the extracellular matrix [ 9 , 10 ] .    however , classical mammalian heparanase is an intracellular enzyme and is not specific to metastatic sites [ 1113 ] . cxcl7 is a member of the cxc subfamily of chemokines which can be further subdivided on the basis of the presence of the tripeptide motif glutamate - leucine - arginine ( elr ) . all elr - cxc chemokines act through cxc chemokine receptor type1 or type2 ( cxcr1 and cxcr2 ) . cxcl7 , has dual functions of heparin binding and is a ligand to the g - protein linked receptor cxcr2 . until recently , cxcl7 gene expression was thought to be restricted to cells within the megakaryocytic lineage [ 16 , 17 ] , as well as to neutrophils and lymphocytes . recent reports have suggested that other cell types may produce this chemokine as well [ 1823 ] .   despite studies on increased expression of cxcr2 in breast cancer , earlier studies from our laboratory have shown that the malignant breast cancer cells express more cxcl7 than premalignant mcf10at cells . cxcl7-transfected mcf10at breast cells have much higher heparanase activity than premalignant mcf10at cells . cxcl7-transfected mcf10at cells are as invasive as malignant breast cancer cells . in addition , the expression of vegf c and d is increased in these transfectants . heparanase and the lymphangiogenesis factors vegf - c and vegf - d are two important markers closely related to the metastasic capabilty of breast cancer . in this paper , we hypothesize that cxcl7   increases   the expression of vegf - c , vegf - d ,   and   heparanase , and increases cell invasion via cxcr2 signaling , all   linked to tumor lymphangiogenesis and metastasis . the mcf10at cells   were cultured in dulbecco 's modified eagle 's medium ( dmem)/f 12 ( 1  :  1 )   containing 5% horse serum , supplemented with 10  g / ml bovine insulin , egf ( 20  ng / ml ) ,   hydrocortisone ( 0.5  g / ml ) , and cholera toxin ( 100  ng / ml ) . all cell culture reagents were obtained from gibco ( grand island , ny ) . approximately , 1  10 cells / well were plated in 6-well plates in medium containing 5% horse serum   to grow overnight to 60%70% confluency . transfection of the plasmid was performed by using lipofectamine 2000 ( invitrogen , usa ) . the cells were divided into blank control group , negative control group , and the test group . the cells were transfected with the mixture of plasmid and lipofectamine 2000 ( 1 :  3 ) in 2  ml serum - free medium . at 24 hours after transfection , the medium was replaced by normal medium containing 5% fbs and antibiotics up to 72 hours post - transfection . since the mcf10at cells were transfected cells , g418 could not be used for the selection of the stably transfected cell line . therefore , we submitted the cells for the flow cytometry sorting by using the egfp antibody . briefly , biocoat matrigel invasion chambers ( becton - dickinson , bedford , ma ) were rehydrated according to the manufacturer 's instructions . cell suspensions ( 2.5  10 cells per 2  ml serum - free medium ) were added to the top chamber , and complete medium in the lower chamber .   for control , the cells were allowed to invade the matrigel at 37c in 5% co2 for 48 hours . the noninvading cells on the upper surface of membrane were removed from the chamber by gentle scrubbing with a cotton swab , and the invading cells on the lower surface of the membrane were stained with the quick - diff stain kit ( becton - dickinson ) . the number of invading cells was expressed as a percentage by the following :   the mean number of the cells invading through the matrigel insert membrane divided by the mean number of cells migrating under control insert membrane conditions multiplied by 100 . 74,804 ; qiagen , valencia , ca ) , including treatment with dnase i to prevent genomic dna contamination using rurbo dna - free kit ( catalog no . am1907 ; ambion , foster city , ca ) according to the manufacturer 's instructions . total rna ( two micrograms ) was reverse - transcribed to cdna by using the superscript iii first - strand synthesis system ( catalog no . 18080 - 051 ; invitrogen , carlsbad , ca ) , according to the manufacturer 's protocol . the primer sequences used for the reactions are in table 1 along with expected products and genbank accession numbers . the thermocycler parameters were as follows : an initial step at 95c for 10  min . , 40 cycles of 95c for 20  sec . , 58c for 30  sec . , and 72c for 20 seconds . the cycle threshold values were used to calculate the normalized expression of vegfc / d against -actin . qrt - pcr was performed in abi 7500 sequence detection system using a sybr green detection system ( catalog no . 170- 8880 ; bio - rad laboratories ) . by identifying the threshold cycle ( ct ) for expression of mrna , the ct for vegf - c / d was calculated and compared to vector transfected control cells ; cxcl7 transfected cells and sb225002-treated cxcl7 transfected cells . the ct was converted into a ratio ( target a / target b = 2 ) describing the comparison of the relative expression of vegf - c / d ( target a ) to -actin ( target b ) for each of the lines . the delta - delta model was used for comparison of relative expression rt - pcr results for vegf - c / d . vegf - c and vegf - d protein concentrations in conditioned media were measured by enzyme - linked immunosorbent assay ( elisa ) using human vegf - c or vegf - d elisa development system ( catalog no . measurements were done at least in duplicate for 2 dilutions . the optical density at 570  nm and 450  nm was determined for each well using the plate reader . then the reading at 570  nm was subtracted from the reading at 450  nm for each well . the protein concentration of the serum - free conditioned medium was measured by the bradford assay ( biorad , richmond , ca , usa ) . the heparanase activity of the conditioned medium was assessed as heparan sulfate degrading enzyme activity using the heparan degrading enzyme assay kit ( catalog no . one unit of heparanase activity defined the activity which degraded 0.063  ng of biotinylated heparin sulfate in 1  min . at 37c and the detection limit of this assay was 0.1  u / ml . each value of heparanase activity was normalized by protein concentration ( u / g protein ) . briefly , the cells were harvested in pbs , counted , and lysed in the ripa buffer ( catalog no . protein concentration was determined for all samples using the bio - rad protein assay ( catalog no . the equal - volume samples ( 50  g ) were separated by sds - page on a 10% polyacrylamide gel and transferred onto nitrocellulose membrane ( catalog no . 162 - 0114 , bio - rad , richmond , ca ) using transfer tank . 9101 , cell signaling technology ) and erk1/2 ( catalog no.9102 , cell signaling technology ) , then developed by ecl ( catalog no . prn 2106 , ge healthcare , waukesha , wi ) and was photodocumented . results are expressed as mean  standard error of the mean ( sem ) , unless indicated otherwise . for statistical analysis , one - way anova was used , and significance was defined as p < .05 . graphs were generated using graphpad prism ( graphpad software , san diego , ca ) . to further verify cxcl7 function in cell invasion , a selective nonpeptide cxcr2 antagonist sb225002   was used to check whether it can inhibit the invasive ability of mcf10at cells transfected with cxcl7 . , cells were added into the matrigel invasion chamber ; meanwhile , sb225002 ( 1.1  m ) was also added into the chamber . several concentrations of sb225002 were tested ( 0.5 , 0.7 , 0.9 , 1.1 , 1.3 , and 1.5  m ) and 1.1  m had the best inhibitory effect similar to that reported by levashove et al . . the decrease in the percent invasion of matrigel by the cells is shown in figure 1 . treatment with sb225002 resulted in 18.06%  0.76% invasive cells compared with 55.7%  1.4% in nontreated cxcl7 stable transfected mcf10at cells . thus , the cxcr2 antagonist blocked the invasive ability of cxcl7 stably transfected mcf10at cells . to   investigate the effect of sb225002 on expression and secretion of vegf - c and vegf - d by stable cxcl7-transfected mcf10at cells ,   we cultured the cells with and without sb225002 ( 1.1  m ) . expression of mrna for lymphangiogenic factors vegf - c and vegf - d was determined by quantitative real - time polymerase chain reaction ( qrt - pcr ) assay . the expression levels of mrnas for vegf - c , vegf - d , are shown in figure 2(a ) . compared with vector - transfected mcf10at cells , cxcl7-transfected mcf10at cells showed 11-fold higher vegf - c and 18-fold higher vegf - d expression . administration of sb225002 antagonist resulted in a significant ( p < .01 ) inhibition of vegf - c ( 3.5-fold ) and vegf - d ( 3-fold ) mrna expression . next , we examined the secretion of the vegf - c and vegf - d by elisa assay . the secretion levels of vegf - c , vegf - d are shown in figure 2(b ) . both vegf - c and vegf - d secretion from the cxcl7-transfected cells were significantly ( p < .01 ) increased compared with the vector - transfected control group ( vegf - c , 3-fold ; vefg - d , 2.5-fold ) . our data also showed a significant ( p < .05 ) inhibition in vegf - c and vegf - d secretion by sb225002-treated cells compared with control treated cells . thus the cxcr2 antagonist decreases vegf - c and vegf - d mrna and protein expression . next , we examined whether cxcl7 sirna inhibited the elevated heparanase activity of cxcl7-stably transfected cells . cells were transfected with cxcl7 sirna or control sirna , and after 48 hours , the cm was collected . compared with control sirna treatment , cxcl7 sirna - transfected cells showed significant ( p < .01 ) inhibition of heparanase enzymatic activity ( figure 3(a ) ) . the same results were obtained after using a microcon filter to remove molecules less than 30  kd ( which includes cxcl7 ) from cm , verifying by elisa that cxcl7 is not present after filtering . and then , retesting for heparanase activity were performed . to further confirm the increased invasion after transfection by cxcl7 the decrease in the percent invasion of matrigel by the cells is shown in figure 3(b ) . treatment with sirna resulted in 16%  1.2% invasive cells compared with 37%  2.5% by mcf10at cells transfected with cxcl7 . thus , cxcl7 sirna inhibits the invasion of mcf10at cells transfected with cxcl7 . to further confirm the increased expression and secretion of lymphangiogenesis factors vegf - c and vegf - d after transfection by cxcl7 , we used the sh - ppbp to silence the cxcl7 signal . to   investigate the effect of sh - ppbp on expression and secretion of vegf - c , vegf - d of stable cxcl7-transfected mcf10at cells ,   we cultured the cells transfected with sh - ppbp or control ( sh - con ) . expression of the mrna of lymphangiogenic factors vegf - c and vegf - d was determined by quantitative real - time polymerase chain reaction ( qrt - pcr ) assay . the expression levels of mrnas for vegf - c , vegf - d , are shown in figure 4(a ) . compared with vector - transfected mcf10at cells , cxcl7-transfected mcf10at cells showed 11-fold higher vegf - c and 18-fold higher vegf - d expression . the sh - ppbp transfection resulted in a significant ( p < .01 ) silencing of vegf - c ( 66% ) and vegf - d ( 68% ) mrna expression . next , we examined the secretion of the vegf - c and vegf - d by elisa assay . the secretion levels of vegf - c , vegf - d are shown in figure 4(b ) . both vegf - c and vegf - d secretion from the cxcl7-transfected cells were significantly ( p < .01 ) increased compared with the vector - transfected control group ( vegf - c , 3.3-fold ; vefg - d , 4.4-fold ) . our data also showed a significant ( p < .05 ) silencing in vegf - c ( 65% ) and vegf - d ( 68% ) secretion by sh - ppbp transfected cells compared with sh - con transfected cells . the map kinase pathway is important for growth , differentiation , and migration , and is considered a dominant signaling pathway for cxcr2 [ 27 , 28 ] . to investigate the mechanism by which the cxcl7-cxcr2 axis is involved in the stimulation of lymphangiogenic factors vegf - c , vegf - d toward invasion by human breast cancer cells , we investigated the involvement of erk1/2 map kinases by western blotting . stable cxcl7-transfected mcf10at cells did not induce erk1/2 phosphorylation in comparison with the vector transfected control cells , and there is no difference in erk1/ 2   map kinase expression in stable cxcl7 transfected mcf10at cells compared to vector controls ( figure 5 ) . although many molecules have been implicated in cancer metastasis , the detailed mechanism of tumor metastasis is still not completely understood . recently , the interest in chemokines in cancer research has been increasing as new chemokines are being identified and investigated . working along with many other molecules , the chemokines and their receptors expressed by both the cancer and its stroma influence growth , dormancy , angiogenesis , and invasion [ 3 , 6 , 29 ] . the cxcr4/cxcl12 ( sdf-1 ) axis was the most common interaction that has been shown to be involved in many different human malignancies , including breast cancer , ovarian cancer , and prostate cancer [ 30 , 31 ] . however , cxcr4 interactions alone did not completely explain the pattern of metastasis of cancer . there are 4 families of chemokines ( c , cc , cxc , and cx3c ) based on the arrangement of cysteine residues near the n terminus [ 32 , 33 ] . the cxc group can be further subdivided on the basis of presence of the tripeptide motif glutamate - leucine - arginine ( elr ) adjacent to the cxc motif . all elr - cxc chemokines act through cxc chemokine receptor type 1 or type 2 ( cxcr1 or cxcr2 ) , which are transmembrane g - protein - coupled receptors . secretion of stromal cell - derived factor-1 ( sdf-1)/cxcl12 and expression of cxcr4 have been identified to be associated with breast cancer metastasis . although cxcr4 was more highly expressed in the breast cancer cells tested bymller et al . than the other cxcrs , the expression of cxcr2 was also increased compared with the remaining receptors . cxcl7 , which is a member of the elr - cxc chemokines , binds with cxcr2 receptors , stimulating angiogenesis and association with neutrophils and other immune components . inhibition of cxcr2 function on endothelial cells has been shown to inhibit tumor growth , for lung cancer and renal cell cancer models [ 39 , 40 ] . the use of a small selective antagonist for cxcr2 ( sb225002 ) represents an attractive targeted therapeutic approach . previous work in our laboratory showed that breast cancer cells coexpress cxcl7 and cxcr2 , which may act as a potential autocrine mechanism in breast cancer . the malignant cell line mcf10ca1a.cl1 strongly expressed cxcl7 and has much higher invasive ability than mcf10at . thus , cxcl7-transfected mcf10at cells were as invasive as malignant cells , suggesting that cxcl7 may have a role in the invasion process . therefore , targeting its receptor ,   cxcr2 , seemed an obvious choice .    by using cxcl7-stably transfected mcf10at cells treated by sb225002 , invasion was decreased significantly compared with cxcl7-transfected cells , which might involve cxcl7 autocrine activity with cxcr2 . the present study did not examine whether sb225002 could block the invasive ability of isogenic malignant cells ( mcf10ca1a.cl1 ) or other malignant cell lines , which would be an interesting focus for future study . lymphangiogenesis refers to the formation of new lymphatic vessels that may occur in normal developing tissues or in tumors . overexpression of vegf - c or vegf - d can lead to lymphangiogenesis , intralymphatic tumor growth and formation of lymph node metastases [ 41 , 42 ] . vegf - c and vegf - d are ligands for vegfr-3 ( also termed fms - like tyrosine kinase 4 , flt-4 ) , a tyrosine kinase receptor that is expressed predominantly in lymphatic endothelial cells . the breast cancer cells secrete vegf - c , vegf - d which directly interact with the receptor . this paracrine relationship may lead to further changes in the breast cancer cells leading to invasion . chemokine and vegf - c interactions have been reported in a model of cross - talk for lymphatic endothelial cells and melanoma cells . in our study , both the selective cxcr2 antagonist and sh - ppbp suppresses the elevated expression and secretion of vegfc / d by the cxcl7-transfected mcf10at cells . these results support the notion that the cxcl7/cxcr2 axis plays an important role in cancer cell lymphangiogenesis . the chemokine connective tissue - activating peptide ( ctap - iii ) , which is an n - truncated derivative of cxcl7 , has been reported to have heparanase activity [ 15 , 44 ] . heparanase activity is also responsible for the egress of metastatic tumor cells and other blood - borne cells from the vasculature . recently , expression of vegf - c was shown to be induced by heparanase in prostate cancer cells , epidermoid cancer cells , breast cancer cells , and melanoma .    in this study , heparanase activity in the cm of cxcl7-transfected mcf10at cells was tested , and it was determined that the cxcl7-transfected cells demonstrated increased secreted heparanase activity . in addition , silencing cxcl7 inhibited the invasive ability of cxcl7-transfected mcf10 at cells which further elucidates its role in invasion . the expression of vegf - c and vegf - d mrna and protein was also decreased in these transfectants by shrna , thus linking the expression of the chemokine cxcl7 to vegf - c and vegf - d , heparanase expression , and invasive ability . the mcf10 model of progressive breast disease provides isogenic cell lines with increasing malignant potential to study the steps of metastasis . additional breast cancer cell lines and breast cancer tissue can be tested specifically for cxcl7 expression and effects on lymphangiogenesis , heparanase expression and invasion in the future . lymphangiogenesis in human breast cancer samples can be correlated with clinical parameters and cxcl7/cxcr2 staining . to explain the mechanism by which the cxcl7-cxcr2 axis is involved in the stimulation of lymphangiogenic factors vegf - c , vegf - d , increased invasion and heparanase expression in human breast cancer cells . we demonstrated that cxcl7-stable transfected mcf10at cells do not activate erk1/2 map kinase signaling . we did not observe a difference of erk1/2 kinase in cxcl7-stable transfected mcf10at cells compared with control cells . this result suggests the involvement of different pathways other than map kinase signaling for the cxcl7-cxcr2 axis activation . in general , the activation of g - protein coupled receptors ( gpcrs ) like cxcr2 leads to the dissociation of the  subunit from the ,-dimer when gdp is replaced by gtp . both subunits can activate many signaling pathways including phospholipase c and adenyl cyclase . when specifically considering cxcr2 , information from cxcl8 might shed some light on other potential pathways for cxcl7 since these two chemokines have 48% identity in their amino acid sequences and both bind cxcr2 . cxcl8 binding to cxcr2 activates the rac / pi3k , rho and ras pathways . therefore , the cxcl7/cxcr2 axis may activate pathways other than the map kinase pathway , including rac / pi3k , rho and ras pathways . in the present paper we showed that both the selective cxcr2 antagonist sb225002 and sh - ppbp suppress vegf - c , vegf - d expression and secretion in cxcl7-transfected mcf10at cells . furthermore , we also observed that both sb225002 and cxcl7 sirna reduced the invasion of cxcl7-stably transfected mcf10at cells , confirming the role of the cxcl7/ cxcr2 axis in cell invasion , possibly through the receptor 's signaling mechanism . however , this does not involve the map kinase signaling pathway as has been described for other elr cxc chemokines like il-8 . it has also been shown that cxcl7 sirna knocked down heparanase activity in transfected mcf10at cells , suggesting an important role for cxcl7 in heparanase expression . taken together , these data would support that the cxcl7/cxcr2 axis may be important in breast cancer metastasis . therapeutics aimed at antagonizing cxc chemokine action , including cxcl7 , may be beneficial in preventing invasion and thus the spread of breast cancer .