a function of any tumor cell that allows for propagation of diseased cells is the ability for that tumor cell to invade the surrounding tissue .
one family of proteins involved in this pathological process is the plasminogen activator ( pa ) system     [ 1 , 2 ] .
in addition to the role of the upa / upar complex in the degradation of the ecm , this complex plays a role in cell adhesion .
upar is able to engage cell surface integrins , allowing for attachment of cells expressing the upa / upar complex to other surrounding cells .
another key component is plasminogen activator inhibitor-1 ( pai-1 ) , the physiological inhibitor of upa activity     [ 1 , 2 ] .
pai-1 binds upa bound to the cell surface , forming a pai-1/upa / upar complex that is then recognized by the scavenger protein low - density lipoprotein receptor - related protein ( lrp ) , which internalizes the tertiary complex     [ 3 , 4 ] .
paradoxically , elevated levels of pai-1 in breast cancer patients are associated with decreased patient survival     .  
peroxisome proliferator - activated receptor - gamma ( ppar- ) is a transcription factor that is considered the master regulator of adipogenesis     [ 6 , 7 ] .
however , ppar- has been found in numerous cell lines , including endothelial cells     [ 8 , 9 ] , normal and malignant prostate epithelium     [ 10 , 11 ] , and normal and malignant breast epithelium     .
ppar- is a ligand - activated nuclear transcription factor and the target of the thiazolidinedione ( tzd ) class of insulin sensitizing drugs     [ 6 , 13 ] .
drugs in this family bind ppar- , resulting in the activation of the ppar-/retinoid x receptor ( rxr ) heterodimer .
ppar- then binds the ppar response element ( ppre ) in the promoter of target genes , recruits coactivators , and then the gene is transcribed .
in addition to tzd drugs , ppar- has been shown to be activated by the naturally occurring 15-deoxy--prostaglandin j2 ( 15d - pgj2 )     .
although 15d - pgj2 is a potent agonist for ppar-  in vitro , there is data suggesting 15d - pgj2 is not found at a high enough concentration to act as an in vivo ligand for ppar-     .  
in addition to the tzd class of drugs and 15d - pgj2 , ppar- has also been shown to be activated by a number of dietary fatty acids , specifically omega-3 ( -3 ) and omega-6 ( -6 ) fatty acids . a diet high in fat
is associated with the development of a number of diseases , including cardiovascular disease , type 2 diabetes mellitus , and a variety of cancers .
dietary fat intake has been linked to prostate cancer risk     , colon cancer     [ 1719 ] , and breast cancer .
thoennes , et al . , showed differential transcriptional activity by ppar- following treatment of mcf-7 cells with -3 and -6 fatty acids     .
treatment with -3 fatty acids inhibited levels of ppar- activation , while -6 fatty acids increase ppar- activity over control     .  
the goal of this study was to investigate the effect of ppar- ligands on breast cancer cell motility and the plasminogen activator system .
the tzd ciglitazone decreased cell motility , independent of ppar-. pai-1 levels were lower following ciglitazone treatment .
the naturally occurring ppar ligand 15d - pgj2 also reduced wound - induced cell migration . interestingly , treatment with the -6 fatty acid arachidonic acid ( ara ) increased cell motility , while the -3 fatty acid docosahexanoic acid ( dha ) had no significant effect .
our collective results suggest that the ppar- ligand ciglitazone decreases cell motility , in a ppar- independent manner , potentially though the down - regulation of pai-1 ; alternatively , the ppar- ligand ara promotes migration in a ppar- dependent manner that increases upa .
f. miller , wayne state university , detroit , mich , usa ) were cultured as previously described     [ 22 , 23 ] .
all cell lines were cultured in dmem : f12 ( gibco , invitrogen , carlsbad , calif , usa ) containing 5% horse serum ( hyclone , logan , ut ) , 1% psf ( gibco , invitrogen , carlsbad , ca ) , 20  mg / ml egf ( invitrogen , carlsbad , calif , usa ) , 50  ng / ml hydrocortisone , 100  ng / ml cholera toxin ( calbiochem , san diego , calif , usa ) , and 10  mg / ml insulin ( gibco , invitrogen , carlsbad , calif , usa ) .
cells were grown in a humidified atmosphere of 5% co2 at 37c as previously described .
cells were plated at 1.0  10 cells per well in a 12-well tissue culture treated plate as detailed previously     [ 24 , 25 ] . at confluence , cells were serum - starved overnight .
cells were then scratched with the tip of a sterile yellow pipet tip and serum - free media containing various concentration of 15d - pgj2 ( calbiochem , san diego , calif , usa ) or ciglitazone ranging to 10  m ( cayman chemical , ann arbor , mich , usa ) from ethanol stocks were added to each well .
migration was monitored at 0 , 6 , and 12-hours using a kodak mds290 camera .
wound closure was quantified by measuring distance as pixels between each leading edge of the wound ( 10  lines / wound ) at each time point using the measuring tool in adobe photoshop , with a grid superimposed on image to guide measurements .   following serum starvation ,
cells were treated with ppar- ligands ranging to 10  m of ciglitazone , ara ( ara - sodium salt , sigma , st .
louis , mo , usa ) , or dha ( dha - sodium salt , sigma , st .
lower wells of chamber contained dmem : f12 plus 1  mg / ml fatty - acid - free bovine serum albumin ( bsa , sigma , st .
louis , mo , usa ) with or without 5  ng / ml egf ( invitrogen , carlsbad , calif , usa ) .
cells ( 1  10 ) were plated in upper wells in dmem : f12 containing 1  mg / ml fatty - acid free bsa , above a collagen iv coated , 10  mm porated membrane .
cells were fixed and stained with diff - quick ( dade - behring , newark , de ) .
cells that migrated to the undersurface of the membrane were examined microscopically at 200x magnification .
each condition was done in triplicate , with 4 fields counted per well . in experiments with gw9662
, serum - starved cells were pretreated for 30 minutes with gw9662 ( 5  m ) ( calbiochem , san diego , calif , usa ) , then ciglitazone or fatty acid treatment was added to cells for 24-hours .
gw9662 is an irreversible ppar- antagonist and it was used at a concentration where it is selective for ppar- in cells     [ 26 , 27 ] .
cells were plated at 1.0  10 cells per well in a 96-well tissue culture plate .
confluent cells were serum - starved 24 hours , then mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ) reagent was added to cells and incubated at 37c for 3-hours .
absorbance was measured ( abs = 595  nm ) on a spectramax microplate reader ( molecular devices , sunnyvale , calif , usa ) .
conditioned media from treated cells was collected and concentrated with centrifugal concentrators ( amicon ultracel 30  kd , millipore , billerica , mass , usa ) .
protein concentration was determined using biorad protein dc assay ( biorad , hercules , calif , usa ) .
proteins were separated by sds - page in 10% polyacrylamide and electrotransferred to pvdf membrane .
phosphate buffered saline/0.1% tween-20 ( pbs / tween ) buffer was used in all steps of immunoblot analysis .
nonspecific binding was blocked by 5% nonfat dry milk for 30 minutes at room temperature .
membrane was incubated at 4c overnight with primary antibody diluted 1  :  1000 ( unless otherwise noted ) in 1% nonfat dry milk .
membrane was exposed for 1-hour at room temperature to horseradish peroxidase conjugated secondary antibody diluted 1  :  5000 in 1% nonfat dry milk in pbs / tween .
membrane was exposed to luminal substrate for 1 minute , covered in plastic wrap then exposed to x - ray film .
primary antibodies were : rabbit antihuman pai-1 ( 1  :  2000 dilution ) ( molecular innovations , novi , mi ) and rabbit anti - human upa ( no . 389 , american diagnostica , stamford , conn , usa ) as described previously . mcf-10a and mcf-10ca1 cells ( 1  10 ) were plated in a 96-well plate . following 24-hour serum starvation ,
cells were pretreated with ppar- antagonist gw9662 or vehicle control for 30 minutes at 37c .
cells were then treated with various concentrations ( up to 10  m ) of ciglitazone or arachidonic acid for 24 hours at 37c .
after treatment , cells were washed with pbs and plasminogen was added to cells and incubated at room temperature .
the supernatant was removed and added to another 96-well plate containing buffer amiloride to inhibit any residual upa activity .
chromogenic substrate is then added to the well and hydrolyzed by plasmin generated by plasminogen cleaved by upa on the cell surface .
rate of chromogenic substrate cleavage by plasmin was measured at 405  nm for 90 minutes .
as previously reported , mcf-10a cells express less upa and upar but more pai-1 than mcf-1ca1 breast cancer cells .
both cell lines express ppar- and rxrs ( data not included ) . based on these findings
, we performed a study with some ppar- ligands on upa / pai-1-mediated cell migration processes comparing near normal mcf-10a cells to oncogenic ras - transformed metastatic mcf-10ca1 cells .
ciglitazone decreased wound closure dose dependently ( figure 1(a ) ) , with 5  m ciglitazone reducing cell closure by 39% compared to no ciglitazone .
15d - pgj2 also decreased cell closure dose dependently , with 10  m 15d - pgj2 reduced cell closure by 50% compared to no 15d - pgj2 ( figure 1(b ) ) .
these results show that ppar- ligands decrease wound closure of mcf-10a cells , and they further support the literature that ppar- activation inhibits migration of cancer cells in vitro .
ciglitazone decreased cell chemotaxis to egf in a dose - dependent manner ( figure 2 ) . to determine if these effects were mediated by ppar- , we pretreated the cells with the ppar- specific antagonist gw9662 .
interestingly , blocking ppar- activation with gw9662 ( 5  m ) pretreatment did not reverse the effect of ciglitazone ( 5  m ) in either cell line .
control experiments with 5  m gw9662 showed neither detrimental effect on cell viability nor changes in cell motility .
the data suggest ciglitazone is working in a ppar- independent manner to reduce cell migration .
treatment of mcf-10a and mcf-10ca1 cells with 5  m ciglitazone partially reduced cell viability ( abs 595  nm of cells with no and 5  m ciglitazone was 0.331   .014 and 0.292   .003 for mcf-10a cells and
0.304   .006 and 0.279   .002 for mcf-10ca1 cells , resp . ) .
there was a substantial loss of cell viability at 10  m ciglitazone for both cell lines ; thus , all further experiments used 5  m ciglitazone . in additional control experiments , there was no loss of cell viability with the ppar- ligands 15d - pgj2 or ara when tested up to 10  m ( data not included ) .
these results imply that the effect of ciglitazone in mcf-10a and mcf-10ca1 cell motility is not due to a substantial reduction in cell viability .   in both mcf-10a and mcf-10ca1 cell lines
, ciglitazone treatment resulted in decreased pai-1 protein expression ( figure 3 ) . to determine if this decrease in pai-1 expression was mediated by ppar- , we pretreated with gw9662 prior to ciglitazone treatment .
we did not see a reversal of ciglitazone - mediated reduction in pai-1 expression ( figure 3 ) suggesting ciglitazone is affecting pai-1 levels independently of ppar-.   in mcf-10a cells , ciglitazone treatment alone or in conjunction with gw9662 pretreatment increases upa activity on the cells surface ( figure 4(a ) ) .
ciglitazone treatment in mcf-10ca1 cells did not significantly alter upa activity although it seems gw9662 treatment in these cells results in more plasmin generation ( figure 4(b ) ) . with 10  m ara
, we see a significant increase in cell motility compared to control cells ( figure 5(a ) ) .
when we pretreated the mcf-10a cells with the ppar- antagonist gw9662 , we see a reduction in cell motility ( figure 5(a ) ) .
ara treatment increases upa activity on the cell surface though gw9662 did not seem to fully reduce upa activity ( figure 5(b ) ) .
additionally , treatment with the -3 fatty acid dha , up to 10  m , had no effect on either cell motility or cell viability ( data not shown ) .
these results suggest ara is able to activate ppar- , resulting in increased cell motility and upa activity .
pai-1 and upa protein expression have been used as strong independent prognostic indicators for breast cancer     [ 5 , 2830 ] .
in addition to cancer , pai-1 overexpression is linked to a variety of disease states .
morbidly obese individuals have elevated circulating pai-1 levels , likely due to an increase in pai-1 expression from adipose tissue     . in rats with streptozocin - induced diabetes ,
pai-1 levels are increased 6080% over control     . in humans , elevated pai-1 levels have been reported in patients with t2 dm      and is related to cardiovascular dysfunction     [ 33 , 34 ] . while the literature on ppar- activation and pai-1 alterations is conflicting , it has been shown in a number of cell types and in vivo that ppar- does modulate pai-1 expression     [ 3437 ] .
we treated cells with ciglitazone , 15d - pgj2 , and ara acid to investigate effects of ppar- activation on migration and pai-1 expression following treatment .
based on previous literature , we expected to see differential effects of ppar- activation , specifically with ara treatment     . 
 in vitro ,
treatment of tumor cells with tzds results in a number of antitumor effects . in prostate cancer cells ,
ppar- ligands reduced proliferation , induced terminal differentiation , and downregulated e - cadherin and c - myc expression     .
pioglitazone , in combination with valproic acid , upregulates e - cadherin and reduced invasion and migration in prostate cancer cells     .
we found that treatment with either ciglitazone or 15d - pgj2 resulted in a significant decrease in wound closure of mcf-10a cells .
gw9662 is a specific ppar- antagonist , which binds ppar- and blocks ligand binding and subsequent activation of the receptor     .
surprisingly , pretreatment with gw9662 did not reverse the effects of ciglitazone , which suggests that ciglitazone mediates this reduction in migration through a ppar--independent mechanism .
emery et al . showed rosiglitazone and pioglitazone inhibited proliferation of pituitary tumors ; however , ppar- antagonists did not reverse these effects , suggesting the antiproliferative effect was independent of ppar- activation     .
another study found ciglitazone and 15d - pgj2 induced apoptosis in normal and malignant b cell , independent of ppar- .
finally , ciglitazone and 15d - pgj2 have been shown to activate p38 mapk signaling , which were reported to be independent of ppar- activation     [ 43 , 44 ] .
these effects were reversed in cells pretreated with gw9662 , suggesting ara is acting in a ppar--dependent manner . since -3 and -6 fatty acids have been shown to have differential effects on ppar- activation     , we also investigated if -3 fatty acids had an effect on cell migration in our system .
we saw no change in migration in mcf-10a cells treated with dha , which agrees with past studies that -6 , but not -3 , fatty acids promote cell motility .
gw9662 pretreatment did not fully reverse ara - induced upa activity ; one possibility for this is ara also signals through pi3k      to upregulate upa expression     .
it is also possible ara is engaging ppar- intracellularly , resulting in increased cell migration , while independently initiating the pi3k signaling cascade and then upregulating upa activity .
one limitation of our study was the exclusive use of gw9662 for its irreversible ppar- antagonist effect     [ 26 , 27 ] .
future studies with mcf-10a and mcf-10ca1 cells would benefit from either silencing ppar- expression or expressing a dominant negative ppar- to investigate any possible differences in cell motility or proliferation following treatment with ciglitazone or other ppar- agonists . another limitation to our study was the absence of reporter studies for ppar-   gene regulation .
one clinical trial in phasetwo investigated the effect of pioglitazone in conjunction with a cox-2 inhibitor in glioma patients and saw moderate results in patients with high - grade glioma , suggesting pioglitazone treatment may be beneficial to a subset of patients     .
a phase - i trial of a non - tzd ppar- agonist ly29311 studied maximum tolerated dose in a combination regimen in patients with advanced solid tumors and determined there was no limiting toxicity and no disease progression     . to date , these advances have not been realized with ppar- agonists in contrast to their preventative benefits in diabetic patients .
this study shows ciglitazone treatment reduces both normal and malignant epithelial cell migration in vitro , independently of ppar- activation .
additionally , we found ciglitazone treatment reduces pai-1 protein levels , and this effect was not reversed by antagonism of ppar-. we hypothesize that the antimigratory effects of ciglitazone are mediated by the alteration of the pa system in these cells .
we know pai-1 inhibits apoptosis , can promote cell motility , and plays a role in intracellular signaling     [ 1 , 2 ] .
given the role of pai-1 in these tumor processes , the in vivo data showing fda - approved tzds decrease pai-1 in diabetic patients , and our results and those of others , one could draw the conclusion that tzd therapies may eventually prove to be a valid adjuvant therapy for some breast cancer patients .
 we investigated peroxisome proliferator - activated receptor- ( ppar- ) ligands effect on cell motility and the plasminogen activator system using normal mcf-10a and malignant mcf-10ca1 cell lines . 
 ciglitazone reduced both wound - induced migration and chemotaxis . 
 however , the effect was not reversed with pretreatment of cells with the ppar--specific antagonist gw9662 . 
 immunoblot analysis of conditioned media showed ciglitazone decreased plasminogen activator inhibitor-1 ( pai-1 ) in both cell lines ; this effect was also unaltered by ppar- antagonism . alternatively , treatment with the -6 fatty acid arachidonic acid ( ara ) , but not the -3 fatty acid docosahexanoic acid , increased both mcf-10a cell migration and cell surface upa activity . 
 pretreatment with a ppar- antagonist reversed these effects , suggesting that ara mediates its effect on cell motility and upa activity through ppar- activation . 
 collectively , the data suggest ppar- ligands have a differential effect on normal and malignant cell migration and the plasminogen activation system , resulting from ppar--dependent and ppar--independent effects . 