the incidence rate of this 
 malignancy is steadily 
  rising in developing 
  countries .
it is expected 
  to 
 account for 31% of all new cancer 
  cases among women in usa   . in tunisia ,
the incidence of breast 
 cancer is approximately 19 new cases per 100  000 women per year .
invasive carcinomas where the 
 basement membrane is totally or partially destroyed represent 7080% of all breast 
 cancer and include more than 10 different types . among these , infiltrating 
 ductal carcinomas ( idca ) are the most aggressive forms and have a poor 
 prognosis . nowadays ,
improvement of 
 breast - cancer detection at its early stage through the identification of 
 selective diagnostic biomarkers and targets for therapeutic intervention is of 
 critical importance .
alterations that occur in disease versus normal tissues at 
 either the gene or protein level have been regarded as an appropriate way to 
 identify such markers of pathologies . in biomedicine ,
proteomics , with the 
 recent advances in mass spectrometry , is considered as a powerful analytical 
 method for deciphering protein expression alterations as a function of disease 
 progression .
recently , proteomics - based analyses of breast tissue lysates 
 have resulted in the finding of a number of potential tumor biomarkers 
 providing , therefore , a basis for a better understanding of the intracellular 
 signalling pathways leading to breast - cancer development and progression , 
 and eventually serving as diagnostic and prognostic markers .
probably the 
 most widely used proteomic technology is the identification of alterations in 
 protein expression between two different samples through comparative two - dimensional 
 gel electrophoresis ( 2-de ) which provides high - resolution separation of 
 proteins and offers a powerful method for their identification and 
 characterization .
actually , there is no universal 2-de method that can be 
 applied for all types of samples investigated , and protocols must be optimized 
 and adapted according to the types of tissues and proteins of interest .
it is 
 also crucial to minimize protein alterations and degradations that could result 
 in artifactual spots and to improve solubilization procedures in order to avoid loss of 
 proteins during extraction .
although 2-de is a laborious 
 method , it is still considered as the principal tool in proteomics since it is 
 able to separate proteins according to two independent physicochemical 
 parameters which are isoelectric point and size , and also it provides the 
 highest resolution in the separation of complex 
 protein mixtures .
2-de remains however , poor resolutive in detecting 
 proteins that are smaller than 10.000 da or are basic in charge .
an 
 alternative method for the analysis of basic proteins is found in the 
 application of nonequilibrium ph gradient electrophoresis ( nephge ) , which can 
 provide excellent resolution of basic proteins , offering thus a promising 
 approach for the discovery of tumor - related alterations . in a previous study , we have 
 shown using ief and mass spectrometry that the levels of a number of proteins 
 including fibrinogen - e , which is a potent antiangiogenic factor , were dysregulated 
 in idcas of the breast . in the current report , protein expression profiles issued 
 from breast idcas and their matched surrounding histologically normal tissues 
 were examined by nephge two - dimensional gel electrophoresis and maldi - tof mass 
 spectrometry . using this procedure , we established a nephge-2-de map of 
 proteins altered in idcas of the breast . by comparing 2-de protein profiles , we 
 report herein the identification of several proteins of interest dysregulated 
 in tumor tissues .
these altered proteins belong to multiple cellular pathways 
 and may have a pathological role in breast carcinogenesis .
breast 
 tumor samples ( n = 10 ) were selected from patients living in the middle coast of tunisia 
  ( median 
 age at diagnosis of 50 years ) and classified as infiltrating ductal carcinomas . 

breast tumors were obtained with informed consent according to protocols 
 approved by the institutional review boards of the respective hospitals . 

carcinomas were pathologically staged according to the tnm ( tumors , nodes , 
 metastasis ) classification system of uicc .
all the tumors 
 investigated were classified as t2/n0 and the grade of the tumors was g3 .
breast 
 tumors and surrounding histologically normal tissues were collected at the time 
 of surgery and analyzed by histopathology .
the first , middle , and last sections were 
 stained with hematoxylin and eosin , and examined by a pathologist to ensure 
 that at least 70% of the tissue sections used for the proteomic study contained 
 tumor cells .
the nontumor tissues were confirmed by cryostat section before 
 protein extraction . for 
 proteomic analyses ,
tissue sections verified histologically were suspended and 
 homogenized in 250 l of 2-de 
 analysis buffer : 7  m urea , 
 2 m thiourea , 4% chaps , 0.5% dtt , and 2% ampholytes ( 1 part ph 3/10 , 1 part ph 
 5/7 , 2 parts ph 6/8 ) .
the clear supernatant issued after centrifugation of the 
 homogenate at 12500  g for 15 minutes was transferred to a sterile microcentrifuge tube and 
 immediately subjected to 2-de analysis .
protein contents were determined according to the procedure described by bradford and modified by ramagli and rodriguez .
equal amounts of proteins ( 100 g ) issued from tumor or nontumor breast tissues were applied to the first dimension and at least three nephge gels were run for each sample .
extraction of proteins , solubilization , nephge , and sds - page were carried under similar conditions for the tumor and nontumor tissues .
the alterations reported in the current study were retained after 2-de analysis of protein extracts issued respectively from different frozen 
 sections of the 10 tumor and nontumor samples investigated .
the reproducibility was about 85% or more which reflected the small variances between samples or running .
the dye solution contained 17% ( w / v ) ammonium sulfate , 3% ( v / v ) phosphoric acid , 0,1% ( w / v ) coomassie g250 , and 34% ( v / v ) methanol .
the staining solution was changed once after 12 hours staining and the gel slabs subjected to a 24-hour cycle for increasing dye deposition on low - abundance proteins .
the detection was then increased by placing the gel into 1% v / v acetic acid for producing a better contrast between spots and gel .
coomassie stained gels were scanned and spot detection and quantification were carried out using pdquest 2-d software version 7.2 ( biorad ) .
proteins with significant and reproducible changes between tumor and nontumor tissues were considered as differentially expressed .
the individual spot volumes were normalized by dividing their optical density ( od ) volumes by the total od values of all the spots present in the gel and expressed as % vol .
the significance of expression between tumor and nontumor tissues was estimated by student 's t - test , p < .05 .
picked 
 spots were washed with 100 l of 25 mm nh4hco3 and dehydrated with 100 l of acetonitrile ( acn ) .
this operation was repeated twice and the pieces of 
 gel were dried under vacuum for 10 minutes .
reduction was achieved by 1-hour 
 treatment with 10  mm dtt in nh4hco3 buffer ( 100 l ) at 56c . after discarding the dtt 
 solution ,
alkylation reaction was performed by the addition of 100 l of 25  mm iodoacetamide in 25  mm nh4hco3 buffer for 1 hour at room temperature , protected from light . 
 finally , the excised gel pieces were again washed 3 times for 5 minutes with 25  mm nh4hco3 and acn alternately .
the dried gel volume was evaluated ( about 1 to 2 l ) and 3 volumes 
 of trypsin ( promega , v5111 ) , 12.5 ng/ l , in 25 mm nh4hco3 buffer ( freshly diluted ) , were added .
afterwards , 5 l of 35% h2o/60% acn/5% hcooh 
 were added and the mixture is sonicated for 30 minutes and centrifuged in order 
 to extract tryptic peptides .
mass 
 measurements were carried out on a biflex iii maldi - tof 
 ( bruker , daltonics , bremen , ge ) equipped with the scout high - resolution optics 
 with x - y multisample probe and gridless reflector .
this instrument was used at 
 a maximum accelerating potential of 19  kv ( in positive mode ) and was operated 
 in reflector mode . a saturated solution of -cyano-4-hydroxycinnamic acid 
 ( sigma , saint louis , mo ) in acetone
a first 
 layer of fine matrix crystals was obtained by the spreading and fast 
 evaporation of 0.5 l of matrix solution . on this fine layer of crystals ,
a 
 droplet of 0.5 l of aqueous hcooh ( 5% ) solution was deposited . afterwards
, 0.5 l tryptic digest were added and mixed to a second dried under vacuum .
the 
 sample was washed once by applying 0.7 l of aqueous hcooh ( 5% ) solution on the 
 target and then flushed after a few seconds . in positive mode ,
internal 
 calibration was performed with tryptic peptides coming from autodigestion of 
 trypsin , with respectively monoisotopic masses at m / z = 842.510 and m / z = 
 2211.105 .
for the mass measurements , up to 1 missed tryptic cleavage and 
 optional methionine oxidation were considered . in most cases ,
the mass accuracy 
 was less than 50 ppm , a value which is generally considered adequate for 
 achieving statistically significant results for protein identification .
these 
 files were then fed into the search engine mascot ( matrix science , london , uk ) . 

ihc analysis of -b crystallin was performed on formalin - fixed , 
 paraffin - embedded sections of idca breast tumors .
briefly , 
 five - micrometer sections were deparaffinized in toluene and subsequently 
 hydrated with graded ethanol , and rehydrated in water .
slides were microwaved 
 in 0.01  m citrate buffer at ph 6.0 for 20 minutes at 750  w. thereafter , sections were 
 rinsed thoroughly with water and placed in a tris  buffered saline 
 ( tbs ) solution ( 0.05  m tris - hcl ph 7.6 , 1.15  m nacl ) .
endogenous peroxidase activity was blocked with hydrogen peroxide 
 solution for 7 minutes and sections were rinsed gently with tbs .
sections were 
 then incubated at 4c overnight with the antibody against -b crystallin ( ab1546 , chemicon ) at a dilution of 1 : 1000 . 

after rinsing in tbs , sections were incubated with the secondary antibody , 
 dakocytomation envision+dual link system peroxydase ( dakocytomation , glostrup , denmark ) , 
 for 30 minutes .
sections were washed 2 times with tbs , followed by application 
 of the diaminobenzidine substrate pack according to the manufacturer 's 
 instructions ( dakocytomation ) , yielding a brown - colored signal .
standardization of 
 the incubation and development times allowed an accurate comparison of 
 expression levels in all cases .
cases were considered positive if 10% or 
 more of the tumor cells showed unequivocal immunoreactivity .
in 
 the current study , we have used an nephge-2-de protocol for the analyses of 
 basic proteins , and using this procedure established an nephge-2-de map of 
 proteins altered in idcas of the breast . since nephge is stopped before 
 equilibrium is attained , accurate experimental estimation of the pis of the 
 dysregulated proteins can not be made by this method .
table 1 depicts the 
 theoretical isoelectric points ( pis ) and molecular weights of altered proteins 
 in tumor tissues , many of which are basic . in our hands , while nephge-2-de 
 allowed the resolution of basic proteins , ief-2-de resulted as expected in the 
 stacking of most of basic proteins at the basic end , even if the ief ph 
 gradient has been extended to higher ph values . in 
 our hands , a comparison of the 2-de protein patterns obtained at constant 
 voltages of 400  v for 3 hours ( 1200 v - hr ) or 4 hours ( 1600  v - hr ) showed an 
 optimal resolution of basic proteins at 1200 v - hr while preventing basic proteins 
 from running off the basic end of the gels . by using the protein extraction 
 procedure listed in section 2 ,
an average amount of 100 g of total proteins 
 from each sample was used in 2-de investigations .
figure 1 displays electrophoresis 
 patterns of proteins from tumor and nontumor samples subjected to nephge-2-de 
 electrophoresis . by using this procedure , we have found that the majority of 
 the proteins displayed were not different in abundance between normal and 
 cancer tissues .
protein alterations occurring in the tumor and normal tissues 
 are highlighted with arrows ( figure 1 ) . among the proteins
identified , the 
 levels of 13 proteins were increased in the tumor samples , while 7 proteins 
 were rather observed at higher amounts in the nonneoplastic breast tissues .
these 
 alterations were regarded as serious alterations occuring between tumor and 
 nontumor tissues .
altered proteins were subsequently excised and subjected to 
 in - gel tryptic digestion and maldi - tof analyses .
figure 2 shows an 
 example of maldi - tof peptide mass fingerprints of the tryptic digests 
 corresponding to alpha b - crystallin .
a wealth of evidence suggests that most of these dysregulated 
 proteins are closely related to neoplastic transformation .
these proteins could 
 be classified on the basis of their functions into stress - related , chaperoning 
 proteins , cytoskeletal - related proteins , metabolic enzymes , and other molecules 
 related to immunological responses .
some of these alterations depicted in 
 figure 1 involve co - upregulations of glycolytic enzymes ( enolase1 , spot 8 ; 
 phosphoglycerate kinase 1 , spot 9 ) in tumor tissues , as well as , proteins 
 involved in antioxidation pathways including peptidylprolyl isomerase a ( spots 
 15 , 16 ) , which has been reported to protect cells against oxidative stress and 
 manganese superoxyde dismutase ( mn - sod , spot 12 ) .
two other newly identified 
 proteins related to immunologic responses present at lower levels in tumor 
 tissues include complement component c3c ( spot 1 ) and ighg1 ( spots 26 ) . more interestingly , 
 the levels of raf kinase inhibitor protein ( spot 14 ) were found to be 
 overexpressed in cancer tissues indicating a possible special pathological role 
 for this protein in breast carcinogenesis .
other alterations more represented 
 with respect to the idca tissues include heat shock proteins ( hsp27 , spots 18 , 
 19 ) and -b - crystallin ( spot 13 ) .
subsequent immunohistochemical staining 
 against -b - crystallin has also been performed ( figure 3 ) .
expression of -b - crystallin 
 was noted in all cases investigated and positivity has been retrieved in 80 to 
 90% of tumor cells .
the signals detected in tumor cells were strong whereas 
 epithelial cells of the normal tissues express lower levels of this protein .
proteomics - based 
 studies are increasingly used to examine cancer - related changes in protein 
 expression .
such protein alterations may eventually contribute to increase our 
 understanding of the disease pathogenesis as well as in the development of 
 effective strategies for diagnosis and treatment .
the present study aims to 
 investigate protein alterations in idcas of the breast using an nephge-2-de 
 procedure .
as shown in this comparative analysis , certain specific proteins 
 were dysregulated in idcas compared with their paired normal samples .
these 
 altered proteins were subsequently analyzed by mass spectrometry and unambiguously 
 assigned on the basis of maldi - tof spectra obtained after trypsin digestion 
 ( table 1 ) .
the proteins identified span a wide range of functions and may have 
 a value as diagnostic and prognostic biomarkers for breast cancer . among the 
 proteins identified , we established that there is a decreased expression of igg1 
 and complement component c3c proteins in breast - tumor tissues .
these 
 alterations related to immunologic responses constitute novel identifications 
 that were not previously reported as potential markers of idca breast tumors .
as 
 shown in figure 1 , the igg1 proteins were observed as multiple protein spots 
 with different isoelectric points and similar molecular weights . as displayed 
 in nephge-2-de gels
, these proteins appeared intact as judged by their apparent 
 molecular mass and were closely associated with nonmalignant tissues .
consistent 
 with our findings , several studies have shown that malignant diseases of 
 various tissue origin are associated with a shift in the igg subclass 
 distribution .
for example , the decrease of igg1 serum levels reported in 
 colorectal cancer has been considered as an early tissue nonspecific indicator 
 of malignant transformation that could be useful in the primary diagnosis of 
 colorectal tumors .
such alterations were recently reported to occur in 
 colorectal adenocarcinomas and normal colorectal tissues using suppression 
 subtractive hybridization techniques . a highly reduction of the percent of 
 igg1 in several gynecologic malignant diseases has also been correlated with 
 early carcinoma stages and was found as a reliable marker that discriminates 
 between cancer patients and controls with a specificity ranging between 90 and 
 100% .
the reduction in igg1 plasmatic levels has also been reported as a 
 highly sensitive tumor marker for detecting primary and metastatic 
 squamous - cell carcinomas of the head and neck , and was significantly 
 correlated with early stages of invasive breast cancer . up to now , such 
 alterations in igg1 levels were found with neoplastic diseases only and may 
 likely reflect changes in the host 's immune system linked to the presence of 
 malignant tumors .
recent data unveiled that malignant tumors of various tissues 
 lead to irregularity of the biosynthesis of igg1 and igg2 by affecting their 
 messenger rna levels in b - cell lymphocytes .
it has been hypothesized in 
 this context that cancer cells may affect the synthesis of such igg subclasses by acting on the t - helper 
 1/t - helper 2 ratio in the host , and , consequently , affect the level of 
 specific cell - derived cytokines implicated in human igg synthesis .
our 
 observation of underexpression of igg1 in idcas with regard to nontumor tissues 
 may therefore , indicate a similar tumor - associated mechanism by which cancer 
 cells affect igg1 synthesis .
further investigations are needed however , to 
 elucidate how malignant breast tissues may interfere with the t - cell mediated 
 regulation of igg suclasses .
interestingly , downregualtion of igg1 levels and 
 concomitant downregulation of complement component c3c were observed in our 
 results . to our knowledge ,
no studies have yet reported the decreased 
 expression of this protein in idcas of the breast .
the differential tissue 
 expression of this complement protein provides evidence for the involvement of 
 anti - inflammatory processes in idca tissues and may indicate a selective 
 adaptation by breast tumors to escape elimination by antitumor immune 
 responses . until recently ,
several studies with normal and malignant tissues 
 have disclosed the presence of several immune escape strategies by which cancer 
 cells protect themselves from the lytic action mediated by complement . several 
 evasion strategies to complement attack
have been reported in malignant cells 
 and include complement protective proteins ( cd46 , cd55 , cd59 ) , membrane 
 proteases reported to confer complement resistance by removing cell - bound 
 complement components such as c3 and tumor secreted complement inhibitors . 
 such inhibitory mechanisms , often upregulated in tumors ,
are thought to 
 increase resistance to complement in malignant tissues through inhibition of 
 serum complement activation upon cancer cells . a previous study using a rat 
 model of human breast cancer unveiled that inhibitors of complement activation 
 may facilitate tumor growth and enhance tumorigenicity of breast - cancer cells 
 by inhibiting c3 deposition .
the present downregulation of complement c3 
 component c3c in idcas may , therefore , indicate the involvement in breast 
 tumors of similar complement inhibitory mechanisms .
the 
 raf 
 kinase inhibitor protein ( rkip ) known also as phosphatidylethanolamine binding 
 protein-1 ( pebp-1 ) is another basic protein that has been described as being a 
 component of the important feedback inhibition in g - protein coupled signal 
 transduction .
it is also involved in the regulation of the nf - kappa b 
 pathway and is associated with resistance to tnf- by inhibiting activation of 
 the raf 1/mek / extracellular signal regulated kinase pathway , jnk , and 
 phosphatidylethanolamine ( pe ) externalization , which is an early symptom of 
 apoptotic cell death resulting in the total loss of asymmetric distribution of 
 aminophospholipids in the plasma membrane bilayer [ 24 , 25 ] .
it has also been 
 reported that overexpressing a new member of the pebp family ( pebp-4 ) correlates 
 with both a decreased tnf--induced pe externalization and 
 resistance to tnf--induced apoptosis . 

interestingly , recent data revealed that silencing of pebp-4 expression in 
 mcf-7 breast - cancer cells promotes tnf--induced apoptosis , as well as tnf--induced 
 erk and jnk activation making this pe binding protein as a promising target for 
 the treatment of breast cancer .
overall , the present findings in idcas of 
 the breast might reflect the previously reported results for the relation 
 between pe - binding proteins and tnf - related apoptosis pathway .
the present 
 increase in rkip may confer breast - cancer cells to an advantage for survival 
 and proliferation . from 
 our results ,
the high abundance of hemoglobin in breast tumors could not be 
 considered as a reliable parameter for distinguishing between tumor and 
 nontumor tissues .
indeed , the relationship between tissue hemoglobin levels and 
 breast tumors appears indirect and indicates rather an enhanced blood supply to 
 the tumor .
since tumor angiogenesis is a crucial arbiter of breast - cancer 
 growth / metastasis , this higher level of hemoglobin is consistent with the 
 higher vascular density of growing tumors compared with normal tissues . besides these alterations
, the 
 concomitant 
 upregulation of two other stress - related proteins ( peptidylprolylisomerase 
 a , mnsod ) was observed in idcas .
the peptidylprolylisomerase a also called 
 cyclophilina ( cypa ) is described as being involved in protein folding and 
 catalyzes the cis / trans - isomerization of peptide bonds preceding a proline 
 residue .
it is secreted by cells in response to both inflammatory stimulation and 
 oxidative stress .
our observation on the expression of cypa is consistent 
 with the published results from studies on other cancers .
overexpression of 
 cypa has been reported in colorectal carcinomas in comparison to normal mucosa 
 and is involved in lung - tumor growth by affecting proliferation and apoptosis 
 .
a recent study has shown that both cypa and its receptor cd147 are upregulated 
 in human pancreatic adenocarcinomas compared to normal pancreatic ductal epithelium 
 .
these authors pointed that cypa is a promoter of pancreatic tumor growth 
 and is involved in chemotaxis and cell signalling cascades .
the present finding 
 reveals a similar correlation , implying that breast carcinomas may share a 
 similar mechanism of cell proliferation mediated by cypa . the mnsod is a 
 mitochondrial enzyme that has been reported to protect cells against oxidative 
 stress by increasing the dismutation rate 
 of superoxide anion ( o2 ) 
 to hydrogen peroxide ( h2o2 ) which
the role of this antioxidant enzyme in carcinogenesis 
 is still however controversial and unclear .
in fact although , it has been 
 reported to suppress apoptosis and protect cells against several insults , under 
 some circumstances , the mnsod may prevent cell proliferation .
although 
 further studies are needed , the present elevation of mnsod may indicate that 
 the antioxidant defense system has been stimulated in invasive carcinomas of 
 the breast , highlighting the ability of tumor cells to prevent damage due to 
 reactive oxygen species .
interestingly , the 
 co - upregulation of two glycolytic enzymes , enolase 1 and phosphoglycerate 
 kinase 1 ( pgk-1 ) also occurred in idca tissues .
these enzymes are key 
 components of the glycolytic pathway that have shown a prior relationship to 
 cancer .
indeed , our observation of increased expression of enolase 1 and pgk-1 
 is supported in various tumor tissues and may relate to the increased requirement 
 of both energy and protein synthesis / degradation pathways during carcinogenesis 
 .
this increased glycolytic metabolism is also thought to regulate 
 hypoxia - inducible gene expression in solid tumor tissues and participates in 
 angiogenesis .
the protein cofilin-1 also was 
 identified as differentially expressed between tumor and normal tissues .
this 
 protein is recruited at the leading edge of cells where its activation is 
 essential for polarized migration .
cofilin-1 is directly implicated in severing 
 actin cytoskeleton filaments and regulates the polymerization / depolymerization 
 of actin during cell migration .
it has been recently demonstrated that 
 cofilin expression is required for tumor cell invasion since inhibition of 
 cofilin activity in carcinoma cells inhibits cell motility whereas increased 
 expression correlates with enhanced cell migration .
have demonstrated that overexpression of cofilin-1 is necessary for 
 the activation of the cofilin pathway resulting in cell motility , invasion and 
 metastasis .
this protein has also been associated with melanoma cell invasion 
 and migration via its interaction with the v3 integrin .
overall , the 
 current findings support the recently identified role for cofilin activity and highlight 
 its involvement in invasive lesions of the breast as a determinant factor 
 required for cell migration and invasion . the molecular chaperone hsp27 
 and alpha b - crystallin , which is a small heat shock protein ( hsp ) are two other 
 dysregulated proteins in tumor tissues . in a previous investigation ,
we have 
 reported that three other molecular chaperones ( grp78 , calreticulin , protein 
 disulfide isomerase ) are differentially expressed in breast tumors .
the 
 concomitant upregulation of these hsps together with alpha b - crystallin and 
 hsp27 is not surprising since chaperones are thought to work cooperatively to 
 fulfil their functions . due to the capacity of hsps to prevent 

stress - accumulated , unfolded , and nascent protein aggregation , their expression 
 has proven to have important pathological implications such as cell 
 proliferation and disease prognosis .
the hsp27 is a molecular chaperone 
 whose rate of synthesis increases many folds in response to environmental 
 stress and during malignant transformation . as shown in 
 figure 1 , this 
 protein exists as two distinct isoforms that were confirmed by mass 
 spectrometry as hsp27 ( table 1 ) .
although no evidence of posttranslational 
 alterations was pointed out , these isoforms as reported in renal cell 
 carcinomas might reflect phosphorylation or other posttranslational modifications 
 . in the last years
, the role of alpha b - crystallin in cancer pathology has 
 been widely discussed with regard to its potential oncogenic role .
previous 
 studies unveiled that this small hsp may constitute a good target for 
 modulating cell death pathways .
its expression has been shown to inhibit 
 both the mitochondrial and the receptor death activation pathways of caspase 3 
  and correlates with trail resistance in a panel of cancer cell lines . 

this protein may also be an interesting molecular target for exploring the 
 evolution and the origin of breast tumors since higher alpha b - crystallin 
 levels were reported in ductal carcinoma in situ , which is an earliest form of 
 detectable breast cancer .
although further studies are needed to answer how 
 this oncoprotein contributes to breast cancer , the data reported herein 
 highlight the importance of this molecular chaperone in invasive carcinomas as 
 a biomarker that may play a distinctive role in the process of 
 carcinogenesis .
in summary , our data have 
 shown the value of using an nephge 2-de based proteomic approach in identifying 
 potential molecular alterations and targets for breast - cancer therapeutics and 
 diagnostics .
we have identified a number of tumor - associated proteins including 
 chaperones , energy - producing enzymes , stress - related proteins , proteins 
 involved in apoptosis and cell motility , as well as alterations highlighting 
 changes in the host 's immune system linked to the presence of malignant 
 tissues .
these protein variations indicate that multiple cellular pathways are 
 involved in the process of breast carcinogenesis .
such alterations may help 
 to better understand the pathology of this disease and may have potential 
 clinical applications as cancer biomarkers .
 improvement of breast - cancer detection through the identification of potential cancer biomarkers is 
 considered as a promising strategy for effective assessment of the disease . 
 the current study has used nonequilibrium ph gradient electrophoresis with subsequent analysis by mass spectrometry to identify protein alterations in invasive ductal carcinomas of the breast from tunisian women . 
 we have identified multiple protein alterations in tumor tissues that were picked , processed , and unambiguously assigned identities by matrix - assisted laser desorption / ionization - time of flight mass spectrometry ( maldi - tof ) . 
 the proteins identified span a wide range of functions and are believed to have potential clinical applications as cancer biomarkers . 
 they include glycolytic enzymes , molecular chaperones , cytoskeletal - related proteins , antioxydant enzymes , and immunologic related proteins . among these proteins , 
 enolase 1 , phosphoglycerate kinase 1 , deoxyhemoglobin , mn - superoxyde dismutase , -b - crystallin , hsp27 , raf kinase inhibitor protein , heterogeneous nuclear ribonucleoprotein a2/b1 , cofilin 1 , and peptidylprolyl isomerase a were overexpressed in tumors compared with normal tissues . 
 in contrast , the ighg1 protein , the complement c3 component   c3c , which are two newly identified protein markers , were downregulated in idca tissues . 
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS
4. DISCUSSION
5. CONCLUSION