ovarian    cancer is 
 the eighth most frequent malignant neoplasia and the fifth most common cause of 
 death from malignant tumor growth in women in the us 
  . the frequency of ovarian 
 cancer increases in each decade of life , with the highest rate of diagnosis 
 occurring in women that are 75 years of age .  
early diagnosis of ovarian tumors is difficult since tumors 
 with diameters less than 5 cm can not be recognized by 
 bimanual pelvic examination [ 3 , 4 ] . 

however , pelvic examination , ultrasonography , detection of 
 tumor markers ( such as ca 125 , ca 15.3 , ca 72.4 , and ca 19.9 ) , 
 as well as color doppler imaging have been shown to be useful 
 in the diagnosis of ovarian cancer , despite the limitations of 
 these methods in differentiating between benign 
 and malignant tumors .
nitric oxide ( no ) is a biological messenger synthesized from 
 l - arginine by nitric oxide synthase ( nos ) .
the endothelial and 
 neuronal isoforms of nos ( enos and nnos , resp . )
are 
 constitutively expressed in many cell types , 
 however , inducible nos ( inos ) is only induced in leukocytes , 
 endothelial cells , and other specific cell types after 
 stimulation by bacterial endotoxins or 
 cytokines , resulting in higher concentrations of no  
 .
production of no is a mechanism by 
 which activated endothelium can lyse tumor cells 
 , however , it can also 
 regulate tumor growth and metastasis depending on its 
 concentration [ 8 , 9 ] .
data from previous 
 studies also suggest that no is both cytotoxic and cytostatic 
 against microorganisms and malignant cells 
 [ 10 , 11 ] with 
 synthesis of no by malignant cells causing no - mediated 
 apoptosis . as a free radical
, 
 no can react to produce peroxynitrites which can directly and 
 indirectly cause dna damage . 

if produced for a long period of time , excess no production can 
 lead to mutations and ultimately to cancer 
 [ 13 , 14 ] . 

in addition to increasing the metastatic potential of tumor cells via 
 mutations in the dna , no production by neoplastic cells 
 promotes angiogenesis , an essential process for the growth and 
 maintenance of tumors [ 15 , 16 ] .
understanding the role of inos in ovarian cancer would 
 provide valuable insight into the development of additional 
 therapeutic options .
the aim of the present study was to 
 identify differences between inos expression and the local 
 production of no in patients with varying stages and grades of 
 ovarian cystic tumors .
therefore , levels of 
 intracystic no metabolites and expression of inos were analyzed 
 in tumor sections from patients with nonneoplastic , benign , or 
 malignant ovarian tumors .
this study enrolled 63 randomly selected women who received 
 pelvic mass outpatient services from the discipline of 
 gynecology and obstetrics of the federal university of 
 tringulo mineiro ( uftm ) , uberaba , brazil .
these patients 
 underwent surgery for an adnexal mass between february 1996 and 
 february 2007 , and informed consent was obtained from patients 
 to allow their tissue to be used for examination and related 
 experiments .
candidates for exploratory surgery were characterized by one 
 or more of the following criteria : cysts with 1 thick septum 
 ( > 3 mm ) or 2 thin septa , a cyst diameter 7 cm , 
 persistence or increase in the cyst or ovarian volume over a 
 minimum of two follow - up periods , the presence of vegetation or 
 calcification , a solid or predominantly solid tumor , ascites , 
 elevated serum levels of tumor markers , or a resistance index 
 0.4 as detected by color doppler imaging 
 [ 3 , 5 ] . 

inclusion criterion was the anatomicopathological finding of an 
 ovarian tumor ( primary neoplastic or nonneoplastic tumor ) . 

exclusion criteria were adnexal torsion , cyst rupture , 
 metastasis of another primary tumor , or previous chemotherapy . 

the anatomicopathological evaluation and staging of all cases 
 were performed according to guidelines published by the world 
 health organization ( who ) and the international federation of 
 gynecology and obstetrics ( figo ) [ 17 , 18 ] .
patients 
 were divided into 3 groups according to the classification of 
 tumor type : nonneoplastic ( n = 15 ) , benign ( n = 28 ) , or malignant ( n = 20 ) ( which included cystadenomas of 
 borderline malignancy ) . characterization of patient groups is presented in 
 table 1 .
cystic 
 fluid samples were aseptically collected by punction immediately following 
 resection of tissue .
the collected fluids were immediately stored on ice until centrifugation ( 180 g , 15 minutes ) was performed .
cell 
 supernatants were transferred to fresh tubes , and the cell pellets were 
 resuspended in phosphate - buffered saline ( pbs ) .
the 
 levels of no metabolites ( nitrite plus nitrate ) in cystic samples were 
 determined by enzymatically reducing the nitrate present with nitrate reductase 
 as previously described .
briefly , 50   l of nondiluted sample was incubated 
 with an equal volume of reductase buffer ( 0.1 m potassium phosphate ( ph 7.5 ) , 
 1 mm nadph , 10 mm fad , 4u of nitrate 
 reductase / ml ) for 20 hours at 37c . 

a standard nitrate curve was obtained by incubating sodium nitrate ( 10 to 200   m ) with reductase buffer .
briefly , the samples were incubated with an equal volume of 
 freshly prepared griess reagent ( 1% sulfanilamide , 0.1% naphthylenediamine 
 dihydrochloride in 5% phosphoric acid ) .
absorbance at 550 nm was determined 
 using a multi - well plate reader ( multiskan mcc/340mkii , flow laboratories ) and 
 the results were reported as micromoles of no3 + no2 .
specimens 
 obtained from surgical resection were fixed in 10% formalin before being 
 processed in paraffin .
sections stained with hematoxylin - eosin were reviewed by 
 a pathologist and a representative section for each case was selected for 
 immunohistochemical analysis . selected sections 
 were deparaffinized , rehydrated , and heated in a microwave oven in 0.01 m citrate buffer ( ph 
 6.0 ) for 30 minutes .
endogenous peroxidase activity was blocked by 3% hydrogen 
 peroxide for 10 minutes , followed by a wash with pbs .
the sections were 
 incubated overnight at 4c with an anti - inos rabbit polyclonal antibody ( santa cruz biotechnology , 1:200 ) . 

the conjugate used was the avidin - biotin peroxidase detection solution ( dako 
 cytomation lsab and system - hrp ) .
the signal was visualized using 
 diaminobenzidine ( dako cytomation liquid dab and substrate chromogen system , dako ) . 

slides were counterstained with harris 's haematoxylin , dehydrated , cleared , and 
 mounted .
a skin sample with chronic granulomatous inflammation known to be 
 positive for inos was used as a positive control .
two independent observers 
 evaluated the sections and the intensity of staining was evaluated subjectively 
 using the following designations : 0 ( no signal ) , 1 ( weak ) , 2 ( medium ) , 3 
 ( strong ) .
when scores of multiple tissue stainings were combined , scores 
 that were 1 were labeled  weak intensity  , and scores 2 were labeled 
  strong intensity  .
for immunohistochemical staining , the 
 concordance between staining intensity scores was calculated according to the 
 following classifications : kappa < 0.4 : slight concordance ; kappa 0.4 and < 0.8 : moderate concordance ; kappa 0.8 and < 1 : 
 strong concordance ; kappa = 1 : perfect concordance .
the fisher 's exact test was used to compare inos immunohistochemistry results and to assess the 
 relationship between inos expression and no levels with the stage and grade of 
 the ovarian cancer samples analyzed .
data for nitrate levels were 
 expressed as the mean + /   standard deviation ( sd ) and values were compared by 
 anova followed by the tukey test for individual comparisons .
the correlation 
 between intracystic nitrate levels and inos immunolabeling was 
 tested using spearman 's rank correlation coefficient .
sixty - three 
 randomly selected women receiving pelvic mass outpatient services from the discipline 
 of gynecology and obstetrics of the federal university of tringulo mineiro 
 ( uftm ) were enrolled in this study . for those diagnosed with nonneoplastic 
 tumors ( n = 15 ) , 10 ( 66.7% ) had serous ovarian cysts and 5 ( 33.3% ) had functional 
 cysts ( corpus luteum , follicular , and theca lutein cysts ) .
the 28 patients 
 diagnosed with benign neoplasias included 11 ( 39.3% ) with serous cystadenoma , 6 
 ( 21.4% ) with mucinous cystadenoma , 6 ( 21.4% ) with mature teratoma , 3 ( 10.7% ) 
 with cystadenofibroma , as well as 1 ( 3.6% ) with serous- and 1 ( 3.6% ) with 
 mucinous - cystadenoma associated with brenner 's tumor .
for the 20 patients 
 diagnosed with malignant neoplasias , the cases included 7 ( 35% ) serous 
 adenocarcinoma , 3 ( 15% ) granulosa cell tumor , 3 ( 15% ) mucinous cystadenoma of 
 borderline malignancy , 2 ( 10% ) mucinous cystadenocarcinoma , 1 ( 5% ) endometrioid 
 adenocarcinoma , 1 ( 5% ) granulosa cell tumor associated with brenner 's tumor , 1 ( 5% ) anaplasic 
 adenocarcinoma , 1 ( 5% ) immature teratoma with epidermoid carcinoma , and 1 ( 5% ) 
 serous cystadenoma of borderline malignancy . in the ovarian cancer patient group , the number of 
 each tumor stage resected included 5 ( 25% ) of i - a , 1 ( 5% ) of i - b , 2 ( 10% ) of i - c , 1 ( 5% ) of 
 iii - a , 10 ( 50% ) of iii - c , and 1 ( 5% ) of iv . in the iii - c and iv stages , five 
 complete surgeries , four satisfactory citorreductions and only 2 unsatisfactory 
 citorreductions were performed .
patients receiving adjuvant first - line 
 chemotherapy received either a combination of cisplatin , epirubicin , and 
 cyclophosphamide for epithelial tumors , or cisplatin , etoposide , and bleomycin for 
 granulosa cell tumors of stages i - b .
of the 12 carcinomas , 5 ( 41.7% ) were 
 well differentiated , 4 ( 33.3% ) were moderately differentiated , and 3 ( 25% ) were 
 poorly differentiated .
five patients were diagnosed with malignant ovarian 
 tumors had died before their follow - up .
there were 
 sufficient samples to perform inos immunohistochemistry on 15 nonneoplastic 
 tumors , 21 benign tumors , and 18 malignant tumors ( figures 1 and 2 ) .
the 
 results are summarized in table 2 . samples with strong inos staining ( scored 2 ) were more frequently found in ovarian cancer samples than in nonneoplastic ( p = .0014 ) or benign neoplasia samples ( p = .0003 ) .
samples of malignant ovarian cancer 
 tissue were further classified into well - differentiated ( n = 11 ) or moderately / poorly 
 differentiated ( n = 7 ) tumors . for the well - differentiated tumors , 8 ( 72.7% ) presented 
 strong staining of inos while the other 3 presented weak or medium staining of 
 inos .
for the moderately / poorly differentiated tumor samples , all showed 
 weak / medium intensity of inos .
overall , well - differentiated tumors presented a 
 higher frequency of strong inos expression compared to moderately / poorly 
 differentiated carcinomas ( p = .004 ; fisher 's exact test ) .
no 
 statistically significant correlation was found between the intensity of inos staining 
 and tumor stage .
of the 5 ( 27.8% ) 
 patients diagnosed with malignant ovarian tumors that had died by the time of 
 follow - up , only one of the samples previously collected from those five 
 individuals showed strong expression for inos .
cystic   fluid 
 samples were collected at the time of surgical resection and were subsequently 
 tested for no metabolite levels .
cystic fluids from 1 nonneoplastic tumor , 1 benign neoplasia , and 2 malignant ovarian 
 tumors were not tested due to the viscous consistency of those fluids .
the mean 
 levels of no metabolites detected in the malignant tumor samples ( 75.7 m , n = 18 ) were significantly higher ( p = .045 ) 
 than the no metabolite levels for benign ovarian tumors ( 38.5 m , n = 27 ) .
however , statistically significant 
 differences were not detected between no levels of malignant neoplasia samples versus 
 nonneoplastic samples ( 40.9 m , n = 14 ) ( figure 3 ) . to examine 
 whether intracystic no metabolite levels could be predictive for tumor stage ,
patient 
 samples were divided into two groups : those with no metabolite levels < 80 m and 
 those with no metabolite levels > 80 m .
the value of 
 80 m was derived from the median value of no detected 
 in malignant tumor samples ( 81.6 m ) .
no stage ii 
 samples had detectable levels of no metabolites . for stage i 
 samples , 6 ( 85.7% ) had no 
 metabolite levels > 80 m , and only 1 ( 14.3% ) had no 
 metabolite 
 levels < 80 m .
in contrast , 3 of stage 
 iii / iv tumor samples ( 27.3% ) had no metabolite levels > 80 m 
 and 8 ( 72.7% ) had no metabolite levels < 80 m . 

these results indicate that intracystic no 
 metabolite levels > 80 m were significantly more 
 frequent in stage i samples than in stage iii / iv samples 
 ( p = .0498 ; 
 fisher exact test ) .
however , there was no significant correlation between no 
 metabolite levels and the grade of tumor differentiation .
correlations between no production and inos expression in the collected tumor tissues are 
 summarized in table 3 .
differences in inos expression between 
 nonneoplastic , benign and malignant ovarian neoplasias suggest a role for no in 
 ovarian carcinogenesis .
our experiments revealed greater expression of inos in 
 malignant ovarian neoplasias than in benign or nonneoplastic tumors .
there were 
 two cases of ovarian cancer that did not show inos immunoreactivity , while 
 approximately one - third of benign and nonneoplastic tumor samples were positive 
 for inos expression .
our results are consistent with other studies that have 
 shown that a majority of ovarian malignant neoplasias present nos activity , while 
 inos is detected at lower levels in patients without cancer .
although we 
 hypothesize that inos may be a marker to detect malignant disease , it is not 
 considered by others to be a marker exclusive to malignant disease .
expression 
 of nos in malignant tissue derived from gynecological , breast , central nervous 
 system , gastric , and colorectal tumors has been reported , suggesting its role 
 in cancer progression [ 2529 ] .
a positive correlation between inos expression 
 and an increased density of tumor microvessels in human colorectal cancer was 
 shown by cianchi et al .
, 
 and inos expression has been associated with increased vascularization and 
 tumor invasion in endometrial malignant neoplasia .
correspondingly , 
 patients with lung cancer , prostate cancer , or cervical cancer treated with no 
 inhibitors showed antivascular activity .
these data support the hypothesis 
 that the inhibition of inos may provide a new therapeutic option for the treatment 
 of ovarian cancer .
recently , it was demonstrated that inos 
 expression in serous and low - grade carcinomas was significantly higher than in nonserous 
 and high - grade carcinomas .
advanced stage tumors expressed low levels of 
 inos and were associated with a shorter mean survival time although this was 
 not determined to be a statistically significant correlation . in our study , 
 patients with malignant tumors had significantly higher levels of intracystic 
 no and inos expression in well - differentiated carcinomas compared to moderately / poorly 
 differentiated carcinomas .
in addition , positive inos expression in ovarian 
 carcinoma had been identified as a positive disease - related survival indicator 
 , and was found to be consistent with the high levels of inos activity and 
 no production of nonmetastatic cells versus metastatic cells . in 
 contrast to our findings , a separate study found that patients with advanced ovarian serous 
 tumors express inos and cox-2 and experience a shorter disease - free interval 
 and survival rate .
in addition , patients negative for inos expression 
 presented a complete clinical response to a first - line treatment of 
 chemotherapy .
a separate study found a positive correlation of no synthesis 
 with tumor progression in a breast cancer model . in 
 a study by taveres - murta et al . , 
 increased levels of no metabolites in the tumor microenvironment were found in 
 patients with ovarian cancer , but not in patients with benign neoplasia . 

similarly , supernatants of cell cultures obtained from well - differentiated , 
 malignant ovarian tumors were found to contain higher levels of no metabolites 
 compared to cell cultures from patients with poorly differentiated tumors . 
 however , to our knowledge , this is the first study to evaluate both intracystic 
 no production and inos expression from the same tumor tissue .
our results 
 suggest that intracystic no is not produced by tumor cells since no significant 
 correlation was found between the levels of no metabolites detected and the 
 intensity of inos expression detected by immunohistochemical staining of the 
 tumor tissue .
instead , an analysis of no production from effusions ( ascitic , 
 cystic , or pleural fluids ) of ovarian malignant tumors showed a significant 
 correlation between the percentage of macrophages present and detectable levels 
 of no metabolites , suggesting that macrophages play a significant role in the 
 production of no in the tumor microenvironment .
an 
 association between increased intracystic leukocyte infiltrates and no 
 production has previously been associated with ovarian cancer .
no produced 
 by ovarian carcinoma cell lines has also been shown to correlate with the 
 extent of tumor cell apoptosis observed .
these results , in combination 
 with our data , suggest that the level of inos expressed by tumor cells and 
 infiltrating leukocytes accounts for the intracystic no production detected 
 although it can not be ruled out that the inos associated with ovarian tumors 
 could also be expressed by immune cells [ 36 , 37 ] .
studies using activated 
 macrophages treated with nos - specific inhibitors have shown an inhibition of no 
 production and induced cytotoxicity , suggesting that activated macrophages may 
 mediate no - dependent cytotoxicity .
high 
 concentrations of no can mediate cytotoxic activity against tumor cells , while 
 low concentrations have been associated with angiogenesis .
correspondingly , high concentrations of no 
 have not been found to be maintained in many malignant neoplasias .
these 
 opposing actions of no have been attributed to factors such as differences in 
 the isoform of nos expressed , the level of nos expression , and the type of cell 
 involved in either in vitro or in vivo 
 systems . in ovarian cancer 
 cell lines ,
high levels of no donors or strong expression of the inos suppressed 
 survivin levels ( a human gene that is part of the inhibitor of apoptosis family ) , and have been shown to induce apoptosis .
it is 
 hypothesized that no signaling could contribute to therapy resistance in 
 epithelial ovarian cancer by modulating survivin expression since low levels of 
 no are associated with resistance to carboplatin- and paclitaxel - induced 
 apoptosis .
the ability to modulate nos gene expression may 
 represent an opportunity to control the growth and metastasis of tumors in vivo .
in this study , increased expression of 
 inos and increased production of no metabolites were detected in the tumor 
 microenvironment of ovarian cancer samples compared to benign and nonneoplastic 
 ovarian tissue samples . for ovarian cancer samples , inos expression correlated with 
 the extent of tumor differentiation and intracystic no metabolite levels 
 correlated with the tumor stage .
we 
 hypothesize that the absence of a correlation between no production and inos 
 expression indicates that different cell types are involved in inos expression , 
 and that controlling no production and inducing nos may represent valuable strategies 
 in the prevention of benign tissue transitioning into well - differentiated 
 malignant ovarian tumors .
 tumor sections from nonneoplastic ( n = 15 ) , benign ( n = 28 ) , and malignant ovarian tumors ( n = 20 ) were obtained from 63 women . 
 immunohistochemistry of the tumor sections demonstrated that inducible nitric oxide synthase ( inos ) expression was increased in ovarian cancer samples compared to nonneoplastic or benign tumor samples . using the griess method , nitric oxide ( no ) metabolite levels 
 were also found to be elevated in malignant tumor samples compared to benign tumor samples ( p < .05 ) . 
 for stage i ovarian cancer , intracystic no levels > 80 m were more frequent than no levels < 80 m , and inos expression in well - differentiated carcinomas was greater than in moderately / poorly differentiated carcinomas ( p < .05 ) . 
 these data suggest an important role for no in ovarian carcinogenesis . 