histone acetylation and deacetylation by histone acetyl transferases and histone deacetylases is involved in the epigenetic regulation in human cells     [ 1 , 2 ] . recently
, this post - translational modification has become a popular molecular target for cancer therapy .
hdac inhibitors ( hdacis ) have demonstrated significant antitumor activity by hyperacetylation of nucleosomal histones resulting in reexpression of repressed genes that produce growth arrest , terminal differentiation , and/or apoptosis in carcinoma cells .
valproic acid ( vpa ) , an hdaci and an antiepileptic agent , causes marked decrease in proliferation of prostate cancer ( pca ) cells in vitro and significant reduction in tumor volume in vivo     [ 4 , 5 ] .
multiple pathways including cell cycle arrest , apoptosis , angiogenesis , and senescence contribute to the antitumor effects of vpa .  
neuroendocrine ( ne ) cells are the third and minor epithelial cell type in prostate , in addition to the more abundant luminal secretory cells and basal cells .
ne cells have dual properties of neurons and endocrine cells and are believed to be involved in the regulation , secretion and differentiation of other prostatic cells .
small cell pca and prostatic carcinoid are relatively rare and are considered pure ne tumors with a poor prognosis .
neuroendocrine differentiation thus has been suggested as a poor prognostic sign by some authors , but the exact role of ne differentiation of the prostate remains unclear , and its prognostic importance in prostate cancer still remains controversial     [ 7 , 9 ] .
the characteristics of ne differentiation in pca are very much similar to those seen in patients who develop this histologic phenotype in non - small - cell lung cancer .
ne cells in prostate express ne markers such as chromogranin a ( cga ) , synaptophysin , b - tubulin , neural cell adhesion molecule ( ncam or cd56 ) , neuron specific enolase ( nse ) , and so forth .
ne cells can be generally identified by electron microscopy or immunohistochemical ( ihc ) staining with antibodies for ne markers .   
recently , some studies have documented increased neuroendocrine markers after in vitro treatment of prostate cancer cell lines with hdacis     [ 9 , 12 ] indicating neuroendocrine transdifferentiation .
in contrast , studies done in neuroendocrine tumors such as carcinoid , pheochromocytoma , and small cell lung cancers have shown vpa and other hdacis to exert antitumor effects     [ 1315 ] .
vpa has been shown to promote apoptosis , reduce ne phenotype and expression of ne markers , and is suggested as a promising therapy for these tumors     [ 16 , 17 ] .
thus the role of hdaci 's in neuroendocrine differentiation still remains unclear and has thus warranted further investigation .
the goal of this study is to carefully determine whether vpa induces ne differentiation in the pca cell lines , in vivo and in   vitro , by studying a variety of markers associated with ne differentiation in numerous pca cell and tumor models .
markers including cga , synaptophysin ,   and ncam were quantified by ihc in a tissue microarray ( tma ) format from several vpa - treated human pca cells grown in vitro and in vivo as tumor xenografts in nude mice .
human prostate cancer cell lines lncap , pc-3 , and du145 were obtained from american type culture collection ( manassas , va ) , and c4 - 2 line was a gift from dr .
all the cells were grown in rpmi 1640 with l - glutamine ( cellgro , herndon , va ) supplemented with 10% heat - inactivated fetal bovine serum ( fbs ; life technologies , inc . ,
carlsbad , ca ) , 5  g / ml ciprofloxacin hydrochloride ( u.s . biological , swampscott , ma ) , and 50  g / ml gentamicin ( quality biological , inc . , gaithersburg , md ) .
cells were allowed to grow until 80% to 90% confluent and harvested with 0.05% trypsin/0.53  mmol
/ l edta ( cellgro , herndon , va ) before each subsequent passage .  
cells were resuspended in 1x phosphate - buffered saline ( ph 7.4 ; biosource , rockville , md ) , mixed 1x with matrigel ( bd biosciences , palo alto , california ) , and injected ( 1  10 per injection ) subcutaneously into the lateral flanks of male athymic nu / nu mice .
vpa ( 1  mol / l ; vpa sodium salt ; sigma , st .
louis , mo ) stock was made in pbs and filters sterilized through a 0.22  m filter .
for in vitro experiments cell lines were treated with 0 , 0.6 , and 1.2  mm vpa for 14 days .
medium was replaced every 48 hours with fresh medium containing vpa . for in vivo experiments , animals received 0.4% w / v vpa in drinking water .
this has been shown to produce blood levels in mouse      comparable to fda approved levels in humans .
animals in treatment arm were treated for 35 days before excision of tumors . in in vivo experiments , chronic treatment implies to long - term treatment with regards to life span .
we considered 35 days of treatment in nude mice ( with life span of 1 year approx . in our experience ) as long - term treatment .
cells treated with different doses of vpa were harvested by trypsinization and resuspended with 5 volumes of cold lysis buffer ( ripa buffer , cat # r0278 , sigma , st .
louis , mo ) and supplemented with protease inhibitor cocktail ( roche , indianapolis , in , usa ) .
equal amounts of proteins were separated by sds - page and the resolved proteins transferred to nitrocellulose membrane .
the membrane was blocked for an hour in blocking buffer [ 100  mm tris - hcl ( ph 7.5 ) , 150  mm nacl , 0.1% tween 20 ] with 5% nonfat dry milk and then incubated with rabbit antiacetylated histone h3 ( upstate , charlottesville , va ) overnight followed by antirabbit igg peroxidase conjugate ( sigma , st .
immunoreactive bands were detected using the enhanced chemiluminescence plus western blotting detection system ( amersham biosciences , piscataway , nj ) according to the manufacturer 's instructions .
anti - cip1/waf1/p21 mouse monoclonal igg ( upstate , charlottesville , va ) , cga ( lk2h10 ) mouse monoclonal antibody ( santa cruz biotechnology , inc .
, santa cruz , ca ) , and antimouse igg peroxidase ( sigma , st .
louis , mo ) were used to normalize protein loading . for in vitro models , cells were harvested and washed in pbs .
resulting cell pellets were incubated for 1 - 2  hr in bouin 's fixative ( 75% saturated picric acid , 20% formalin ( 40% ) , 5% acetic acid , rinsed with 70% ethanol , and dehydrated according to standard procedures with ethanol and xylene .
cells were embedded in paraffin following 90  min of incubation in liquid paraffin at 60c . for in vivo models , tumors were excised and portioned on day 35 .
portions were either immediately frozen in liquid nitrogen and stored at 80c or fixed in buffered formalin and subsequently embedded in paraffin .  
a tissue microarray ( tma ) of the paraffin embedded materials was generated as described previously     .
each array block also contained control normal human prostate tissues and animal xenograft tissues such as bladder , kidney , lung and spleen .
immunohistochemical stains for chromogranin a ( clone lk2h10 , ventana , tucson , az ) , synaptophysin ( polyclonal , cell marque , rocklin , ca ) , and ncam / cd56 ( 123c3.d5 , cell marque , rocklin , ca ) were performed separately on sections cut from the tma .
stained tma slides were scanned ( at 20x magnification setting ) using the aperio imaging system and the images were uploaded and viewed using tmaj     [ 20 , 21 ] .
each array spot was then formed into a composite image for viewing and scoring on a personal computer monitor .
the specimens showed a varying degree of staining intensity and percentage of cells staining . therefore
strong intensity staining was scored as 3 , moderate as 2 , weak as 1 , and negative as 0 . for each intensity score , the percentage of cells with that score was estimated visually .
a combined weighted score consisting of the sum of the percentage of cells staining at each intensity level was calculated for each sample , for example , a case with 50% strong staining , 20% moderate staining , and 10% weak staining would receive a score as follows : ( 50  3 + 20  2 + 10  1 ) = 200 .
one way anova with post - hoc testing was done to evalauate differences in mean staining score between different groups .
the most reliable method to assess ne differentiation in pca is the detection of cga in tumor cells .
the ihc staining of sections constructed of cell lines treated for 14 days with vpa was not able to detect any expression of cga ( figure 1(a ) ) . in order to verify the absence of cga by the ihc in all the cell lines tested , we performed western blot analysis ( figure 1(b ) ) .
results revealed cga expression in these cell lines ; however , cga protein levels were reduced in a dose - dependent manner after vpa treatment .
chronic in vitro treatment of c4 - 2 cells with vpa resulted in increased synaptophysin expression ( mean weighted score 65(4 ) at 1.2  mm versus 34(4 ) at 0.6  mm ( p   =  .002 ) and 18(4 ) at 0  mm ( p  <  .001 ) ) .
significantly increased expression was also found in pc-3 cells treated at 1.2  mm ( mean weighted score 68(2 ) versus 10(2 ) in other two groups , p  <  .001 ) .
however , synaptophysin expression was not altered in lncap and du145 cell lines following vpa treatment ( figure 2 ) .  
chronic in vitro treatment of vpa increased the expression of ncam in lncap ( weighted score 8(3 ) at 0  mm versus 40(3 ) at 0.6  mm , ( p  <  .001 ) ; 40(3 ) at 0.6  mm versus 65(4 ) at 1.2  mm , ( p   =  .002 ) . in pc-3 cells ,
no expression of ncam was seen at 0 and 0.6  mm vpa , but slight expression was seen at 1.2  mm ( mean weighted score 20(5 ) ) .
none or very little ncam staining was seen in c4 - 2 and du145 at either dose ( figure 3 ) .
we have shown previously that administration of 0.4% vpa in mouse drinking water can achieve plasma vpa levels similar to the levels obtained in human patients     .
vpa treatment at these levels was shown to induce acetyl h3k9 , p21 , and reduce tumor volume , thus confirming the pharmacologic activity of vpa     .
animals were sacrificed , and tumors were harvested on day 35 . to investigate the effects of vpa on ne markers of pca tumors in vivo , we evaluated expression of ne markers by ihc on the excised tumors .
ihc did not reveal any cga staining in either treatment or control arms in all cell lines ( figure 4(a ) ) .
tmas from c4 - 2 tumors revealed decreased expression of both synaptophysin ( mean weighted score 47(10 ) versus 15(5 ) , p   <  .001 ) and ncam ( 44(9 ) versus 5(6 ) , p    =  .002 ) in treatment arms ( figure 4(b ) ) .
none of the other arms revealed any significant staining ( weighted scores less than 30 ) for ncam or synaptophysin ( figures 4(b ) and 4(c ) ) .
thus vpa does not induce any ne markers in the physiologically relevant in vivo setting .
ne cells are considered to be derived from local stem cells and are an example of normal , terminally - differentiated cells without proliferative activity .   ne cells in tumor lesions are phenotypically similar to ne cells in normal prostate epithelium in terms of expression of neuropeptides and biogenic amines .
furthermore , dual epithelial characteristics such as prostatic acid phosphatase and/or psa production and ne marker expression , such as cga , are frequently coexpressed in the malignant phenotype of ne cells .
studies evaluating the role of focal ne differentiation in pca prognosis have reported varied results : some reports indicate a negative correlation with prognosis while some show little or no relationship to prognosis     [ 10 , 2328 ] .
histone deacetylase inhibitors are a promising new class of cancer therapy which have antiproliferative and prodifferentiation properties .   for prostate cancer
thus vpa , which is capable of inhibiting hdacs classes i and iia , may be a good option for pca therapy .
in preclinical models , vpa treatment leads to proliferation arrest and differentiation and apoptosis of cancer cells of various tissue origin , while nominal effects were reported in normal cells     [ 2 , 4 , 5 ] .
reported vpa to cause an increase in the secretion of nse in lncap cells ( in vitro ) which may indicate an ne differentiation . in order to better understand the role of vpa in possibly stimulating ne differentiation in pca cell models , we selected the clinically recommended panel of antibodies for the ihc investigation of ne cells in multiple pca cell lines and xenograft tumors .  
chromogranin a or parathyroid secretory protein 1 is a member of the chromogranin - secretogranin family and forms the major constituent in neurosecretory peptide containing dense core granules in ne cells .
cga is highly expressed by cells of neuroendocrine origin , both normal and tumoral , functional and nonfunctional .
while neuron specific enolase ( nse )   has also been used as an ne marker , it is known to be expressed in a variety of non - ne cells and tumors , which has led researchers to question its specificity     [ 30 , 31 ] .
serum cga levels , on the other hand , have been reported to be better predictors of neuroendcocrine differentiation than nse     [ 32 , 33 ] .
thus , cga now is widely regarded as an excellent and more specific marker of ne differentiation . in our study ,
cga expression was not detected by ihc in either control or treated groups in human prostate cell lines of lncap , c4 - 2 , pc-3 , and du145 in vitro or in vivo .
western blotting , being more sensitive , revealed low cga expression in these cell lines ; which reduced further with vpa treatment in a dose - dependent manner .
histone acetylation and p21 induction ( data not shown ) confirmed that active vpa doses were achieved as we have previously demonstrated     [ 5 , 34 ] .
our results are further corroborated by similar reduction in ne markers and ne morphology in ne tumors after treatment with hdacis     [ 13 , 14 , 16 ] .
have previously demonstrated cga to be an important neuropeptide promoting the growth of prostate cancer cells and its suppression leading to programmed cell death in multiple prostate cell lines     .
gong et al . later found antiapoptotic effects of cga to be dependent on a protein kinase b / akt ( an antiapoptotic protein or prosurvival factor ) mediated pathway .
also hdacis have been known to downregulate akt phosphorylation in prostate cancer cells      taken together , it suggests a link between hdaci's - mediated apoptosis and cga inhibition .
further studies will be required to determine the contribution of cga and akt to the vpa therapeutic effect .
synaptophysin and ncam   are other specific and fairly sensitive markers for ne differentiation     [ 38 , 39 ] . in in vitro experiments
, these markers showed varying trends ( increased synaptophysin staining in c4 - 2 and pc-3 cells but unaltered in lncap and du145 ; increased ncam in lncap and pc-3 cells but unaltered in c4 - 2 and du145 ) , and no consistent pattern was seen . in in vivo experiments , the staining did not reveal any significant expression of these markers in any of the xenografts except in c4 - 2 tumors where it revealed a downward trend on treatment .
the findings in our study do not support any neuroendocrine differentiating role of vpa . on the contrary , cga , a very specific marker ,
synaptophysin and ncam showed some inconsistent induction following vpa treatment in some cell lines but , in vivo , vpa treatment did not induce any significant expression of any ne markers .
0.4% vpa in mouse drinking water can achieve plasma vpa levels similar to the therapeutic levels obtained in human patients .
tmas from xenografts of different cell lines either did not stain for ne markers or had very little staining without any induction on vpa treatment .
thus , our data clearly demonstrates that vpa does not induce ne differentiation of pca cells in the physiologically relevant in vivo setting .
 valproic acid ( vpa ) is a histone deacetylase inhibitor that holds promise for cancer therapy . here 
 , we investigate whether vpa treatment induces neuroendocrine differentiation of prostate cancer ( pca ) . 
 a tissue microarray of vpa - treated and untreated tumor xenografts and cell lines of human pca ( lncap , c4 - 2 , du145 , and pc-3 ) were generated   and were analyzed by immunohistochemical analysis ( ihc ) for ne markers chromogranin a ( cga ) , synaptophysin , and ncam ( neural cell adhesion molecule ) . 
 western blot analysis for cga was performed to confirm the results of the tma . 
 ihc analysis did not reveal any induction of cga , synaptophysin , or ncam in any xenograft after vpa treatment in vivo . in vitro , 
 vpa treatment induced little synaptophysin expression in c4 - 2 and pc-3 cells and ncam expression in lncap and pc-3 cells . in the case of cga , 
 vpa treatment decreased its expression in vitro in a dose - dependent manner , as determined by western blot analysis . 
 thus our data demonstrates that vpa does not induce ne differentiation of pca cells in the physiologically relevant in vivo setting . 