prostate cancer is the most diagnosed male malignancy and the second leading cause of cancer - related death among american men .
fortunately , mortality rates have increased less than incidence rates in many countries . in brazil , according to the national cancer institute , prostate carcinoma ( pca ) is the second most common cancer , in incidence , after skin tumors ( non - melanoma ) , and , in terms of mortality , it is exceeded only by lung cancer .
the etiologic factors associated with prostate cancer are varied , encompassing both host genetic and environmental influences .  
glycosylation is one of the most common co- or posttranslational modifications . inside a cell ,
complex glycosylation pathways assemble these oligosaccharides and attach them to proteins and lipids as they travel to the cell surface .  
furthermore , by virtue of their peripheral location , particular oligosaccharide epitopes on proteins or lipids exert key functions in important intercellular communication processes such as fertilization , immune response , pathogen anchoring , or metastasis .
these particular carbohydrate epitopes are recognized by membrane - anchored carbohydrate - recognition domains of different molecules such as receptors , enzymes , antibodies , or lectins .
the latter class comprises proteins of nonimmune origin that display specificity for terminal or subterminal carbohydrates , forming non - covalent bonds . on account of these specific affinities ,
lectins have been extensively used as histochemical probes to characterize various cell types at various stages of differentiation and maturation of cancer [ 4 , 6 , 7 ] .
thus , modern lectin histochemistry has become a valuable instrument to analyze patterns of glucide composition in glycoconjugates and their modifications in the cell during malignant transformation in tumors . 

cratylia mollis ( camaratu bean ) is a native forage from the semiarid region of the northeast of brazil , and its seeds are considered an important lectin source ( cramoll ) , supplying multiple molecular forms ( cramoll 1 to 4 ) with different carbohydrate specificities [ 9 , 10 ] .  
the aim of the present investigation was to evaluate the binding pattern of two preparations containing isoforms from c. mollis seed lectin ( cramoll 1,4 , a preparation containing cramoll 1 and cramoll 4 glucose / mannose - specific and cramoll 3 galactose - specific ) in human prostate tissues .
con a and pna were used for binding comparison since they possess the same carbohydrate specificity as cramoll 1,4 and cramoll 3 , respectively .
formalin - fixed , paraffin - embedded tissue blocks of eight cases of normal human prostate were obtained from the obit identification service at the federal university of pernambuco , brazil ; 61 cases of bph and 82 cases of pca were obtained from the tissue bank of the university hospital of the federal university of pernambuco , brazil .
patient ages varied between 31 and 69 years ( mean 44 ) at the time of diagnosis for normal tissues , 46 and 92 years ( mean 69 ) for bph , and 52 and 88 years ( mean 66 ) for pca .
cramoll 1,4 and cramoll 3 were extracted , isolated , and purified from seeds of c. mollis according to correia and coelho   and paiva and coelho   at the glycoprotein laboratory at the biochemistry department of the federal university of pernambuco , brazil .
cramoll 1,4 and cramoll 3 hrp conjugation was performed according to beltro et al . .
canavalia ensiformis agglutinin ( concanavalin a ( con - a ) ) and arachis hypogea ( peanut ) agglutinin ( pna ) conjugated to hrp ( con a - hrp and pna - hrp ) were purchased from sigma ( st .
basic and native polyacrylamide gel electrophoresis ( 10%  w / v ) was carried out with conjugated and nonconjugated c. mollis isoforms .
tissue slices ( 4  m ) were cut , adhered to albumin - treated slides , deparaffinized in xylol , and hydrated in graded ethanol ( 100%70% ) .
/ v ) trypsin solution for 2 minutes at 37c and afterwards with 0.3%  ( v / v ) methanolic hydrogen peroxide solution for 15 minutes at 25c .
sections were incubated with hrp - conjugated lectins ( con - a - hrp , cramoll 1,4-hrp , pna - hrp , and cramoll 3-hrp , at various concentrations 8 , 15 , 25 , 30 and 60  g / ml ) for 2 hours at 4c .
slices were washed ( twice for 5 minutes ) with 10  mm phosphate buffer saline solution ( pbs ) ph 7.2 , containing 0.15  m nacl , after each step .
lectin staining was visualized with 3,3-diaminobenzidine- ( dab- ) hydrogen peroxide in pbs for 4 minutes at 25c .
thereafter , tissues were rinsed in distilled water , counterstained with haematoxylin , dehydrated in graded ethanol , cleared in xylol , and mounted .
inhibition lectin - carbohydrate binding controls were performed using the specific sugars at a final concentration of 0.3  m for each lectin ( methyl--d - mannopyranoside for cramoll 1,4 and con - a ; d - galactose for cramoll 3 and pna ) .
tissue sections were examined using an optical microscope ( nikon eclipse 50i , usa ) .
staining intensity was determined as the pattern observed in at least 20% of cells with cytoplasm or membrane staining , in luminal secretion , corpora amylacea , and stroma and scored in four categories : 0no staining , 1weak staining , 2moderate staining , and 3intense staining . for image acquisition , an image analyses system ( software nis 
was analyzed by nonparametric tests ( mann - whitney test and kruskal - wallis , followed by multiple comparison posttest of dunn ) with a significance level of 95% ( p < .05 ) using graphpad prism version 5.00 .
conjugation efficiency of cramoll 3 and cramoll 1,4 to hrp was evaluated using gel electrophoresis for basic and native proteins .
the results showed that conjugated lectins migrated more slowly than their respective nonconjugated counterparts ( figure 1 ) .
corpora amylacea were observed in 50.8% of the 61 cases of bph . the gleason score for pca ( n = 82 ) was of 2 + 2 = 4   in 3 samples , 2 + 3 = 5 in 1 , 3 + 2 = 5 in 1 , 3 + 3 = 6 in 24 , 3 + 4 = 7 in 36 , 4 + 3 = 7 in 3 , 3 + 5 = 8 in 3 , 4 + 4 = 8 in 2 , 4 + 5 = 9 in 3 , 5 + 4 = 9 in 3 , and 5 + 5 = 10 in 3 .
corpora amylacea were visualized in 40.2% of pca in which the gleason score was usually 3 .
cytoplasm of normal prostate cells was weakly and moderately stained by con - a and cramoll 1,4 , both at 15 
normal tissue stroma was moderately stained by con - a but no staining was observed with cramoll 1,4 ( figures 2(a ) and 2(b ) ) .
hyperplastic epithelial cells were stained by lectins , con - a , and cramoll 1,4 , mainly in the apical cytoplasm and cell borders .
cramoll 1,4 staining pattern in bph was intense in most cases ( figures 2(e ) and 2(f ) ) . in pca samples , characterized by heterogeneous gleason scores
, con - a and cramoll 1,4 staining intensity decreased while the malignancy degree increased , that is , in well differentiated tissues both lectins showed moderate staining while in undifferentiated tissues no staining was observed .
pca cell staining was more evident using cramoll 1,4 ( figures 2(i ) to 2(l ) ) .
figure 3(a ) presents the statistical comparison of the staining patterns of cramoll 1,4 and con - a in normal and transformed prostate tissues .
pna and cramoll 3 ( 25  g / ml  galactose specific ) weakly stained the normal tissue and did not present a different staining pattern between stroma and glandular tissue ( figures 2(c ) and 2(d ) ) .
bph and pca neoplastic cells were not differentially stained by these two lectins , rending a non - staining or a weakly heterogeneous pattern ( figures 2(g ) and 2(h ) ) . in pca pna and cramoll 3 presented a focal and intense staining pattern in luminal secretion and apical membrane of glandular tumor cells ( figures 2(m ) and 2(n ) ) .
pca stroma was moderately stained by pna and cramoll 3 . statistical comparison between staining patterns of cramoll 3 and
the staining pattern for cramoll 1,4 and cramoll 3 was more intense than that observed when commercial lectins were used ( figures 4(a ) to   4(d ) ) . in pca
lectin staining was completely abolished in all tissues by inhibition of the lectin carbohydrate - binding site with solutions of methyl--d - mannopyranoside ( 0.3  m ) for con - a and cramoll 1,4 and of d - galactose ( 0.3  m ) for pna and cramoll 3 .
cramoll 3 and cramoll 1,4 were properly conjugated to hrp . enzyme conjugation efficiency of in - house purified proteins is very important to assure the quality of histochemical assays .
con - a and cramoll 1,4 ( glucose / mannose specific ) data were similar to those reported by morales et al .   using ( galanthus nivalis agglutinin gna10  g / ml ) and con - a ( 20  g / ml ) for normal and hyperplastic prostate cells .
they observed that normal prostatic glands showed low content or nonaccessibility to glucose / mannose residues when compared to the hyperplastic glands in which the high staining pattern indicates a high content or accessibility to these saccharides in secretory cells ( apical cytoplasm of epithelium and luminal content ) .
the lectin staining of secreted glycoconjugates with mannose / glucose residues is observed in human hyperplastic prostatic glands .  
the increase in glucose / mannose glycocalyx content can be related to high production of glycoconjugates bearing these residues as observed by lectin histochemistry . in prostatic hyperplastic glands
the presence of acid phosphatase which contains n - linked high - mannose chains in its structure was observed by jakob et al . .
the present study also suggests that the increased lectin staining in bhp could be related to an increased secretor activity of prostatic cells in this pathology .  
con a staining of normal prostate stroma is in disagreement with the results of arenas et al .   that observed a weak stroma staining of normal , bph , and pca tissues .
this occurred probably because they used different enzymatic treatments that may have cleaved expressed stroma glycoconjugates bearing glucose / mannose residues in contrast to our trypsin treatment .  
con - a and cramoll 1,4 different staining patterns indicate that the latter has greater diagnostic value for prostate diseases than the commercially available lectin .
such differences in staining can be explained by their similar but not equal quaternary structure which influences the stabilization of the lectin - carbohydrate complex .
thus , the glycoconjugates expressed in pathological prostatic tissues seem to be more likely or prone to form more stable complexes with cramoll 1,4 . in the present study ,
pna and cramoll 3 increased staining patterns from normal to pca tissues are in agreement with the results of janssen et al .   who observed that the binding of pna increases from bph to cancers .
these authors also established a significant positive correlation between the number of pna acceptors and those of prostatic - specific antigen .
arenas et al . , using pna , found an increasing staining pattern of epithelial cells in normal and bph to pca .
we observed that using pna as well as cramoll 3 in pca , galactose residues content increased only in luminal secretion and the apical membrane of malignant tumor glands and not in the cytoplasm of epithelium cells .
the absence and/or nonaccessibility of galactose residues in epithelial cell glycoconjugates can be evaluated using a neuraminidase pretreatment instead of or together with trypsin ( as used in our work ) in order to expose galactose nonaccessible residues due to the presence of sialic acid residues .
pna staining in pca stroma suggests that the development of pca is associated with an increase in galactose residues in glycoconjugates at luminal border of prostate cells and stroma .
the low incidence of corpora amylacea in pca can not be used to exclude malignancy as also observed by christian and collaborators .
corpora amylacea contain an amyloid substance , 2-microglobulin , a double content of sugars ( glucosamine and galactose ) in relation to proteins and sulfur atoms that suggest the presence of glycosaminoglycans [ 12 , 18 ] .
our labeling for bph glands and normal prostate are similar to those observed by morales et al . .
the intense lectin staining in corpora amylacea of secretory cells of bph is probably due to the accumulation of keratan sulfate after luminal secretion , a component which has been identified in both prostatic secretor cells and corpora amylacea .
the origin and function of corpora amylacea have been debated but its function continues to be unknown .
research indicates that corpora amylacea have a secretory origin [ 12 , 20 ] . beyond structural roles in increasing the protein stability , protecting from proteolysis , and improving the protein solubility
, glycosylation promotes also general functional diversity reflected on the high structural possibilities of glycans   which can be acting as key events in apoptosis , immunomodulation , and antiproliferative activity in tumor cells . and
the lectins remain in focus to decipher these diverse glycan chains , being intensively used as cyto- and histochemistry tool for diagnosis and prognosis of cancer .
our results showed that c. mollis lectin isoforms are able to recognize cells and an extracellular structure ( corpora amylacea ) of prostatic tissues . proving that lectins bind
differently the glycocode of normal and tumor cells and are able to detect subtle neoplastic changes of histologically related or similar pathologies ; these two isoforms of a brazilian northeast native forage plant can be used as auxiliary tools in prostate cancer diagnosis compared to commercial lectins such as con - a and pna .
the knowledge of the saccharide identity of cell glycoconjugates enables them to be used as targets for drug delivery and immunomodulation .
 lectins , proteins which selectively recognize carbohydrates , have been used in histochemistry for the evaluation of changes in glycosylation in processes of cellular differentiation and/or dedifferentiation . 
 cratylia mollis seed lectins ( cramoll 1,4 and cramoll 3 ) , conjugated to horseradish peroxidase , were used as histochemical probes in human prostate tissues : normal ( np ) , hyperplasia ( bph ) , and prostate carcinoma ( pca ) . the staining pattern of con - a and cramoll 1,4 in bph was more intense than in np . 
 these lectins also showed staining differences between bph and pca ; the latter showing decreased staining intensity with an increased degree of malignancy . 
 pna and cramoll 3 stained epithelial cells similarly in all diagnoses although they did present intense staining of pca glands lumen . 
 corpora amylacea were not differentially recognized by any of the lectins . 
 cramoll 1,4 and cramoll 3 seed lectins present themselves as candidates for histochemical probes for prostate pathologies when compared to commercial lectins such as con - a and pna . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions