gastric cancer is one of the most frequently diagnosed malignant neoplasms and has the second - highest cancer - related mortality rate in the world . in east asia especially , like china , japan , and korea , more than one million new cases are diagnosed each year .
most patients with gastric cancer are diagnosed at advanced clinical stages with a high ratio of lymph node metastasis . despite a curative operation and postoperative adjuvant therapy ,
currently , the tumor ( t ) node ( n ) metastasis ( m ) stage is still the most important prognostic factor for gastric cancer .
therefore , it is very important to find novel factors for the early diagnostic and prognostic evaluation of gastric cancer .
human dickkopf-1 is a member of the dickkopf gene family , which is composed of dickkopf-1 , dickkopf-2 , dickkopf-3 , and dickkopf-4 , together with a unique dickkopf-3-related protein termed soggy .
several recent studies have also implicated members of the dickkopf family of wnt inhibitors in suppression of human cancer . reduced levels of dickkopf-3
overexpression of dickkopf-1 or dickkopf-3 resulted in inhibition of cell tumorigenicity , cell motility , and invasiveness .
recently it has been demonstrated that expression of dickkopf-1 , -2 , and -3 is reduced in most of melanoma cell lines and most of tumor samples . among the members of the dickkopf family ,
dickkopf-1 is a 35-kda secreted protein involved in embryonic development and known as a potent inhibitor of the wnt signaling pathway , which plays a critical role in cell patterning , proliferation , and fate determination during embryogenesis .
it has been shown that the dickkopf-1 gene has a restricted expression in placenta and mesenchymal stem cells only , but not in other normal tissues .
it has demonstrated that dickkopf-1 was downregulated in human tumors , indicating that it might act as a tumor suppressor .
for example , mikheev et al . showed that dickkopf-1 activates cell death in melanoma cells .
suggested that dickkopf-1 was responsible for the inhibition of cell growth and induction of apoptosis in human mesothelioma cells .
demonstrated that dickkopf-1 suppressed the tumorigenicity of two human breast cancer cell lines that also lack an activated wnt signaling .
dickkopf-1 was found in 26 out of 32 human hepatoblastomas and 5 out of 6 cases of wilms ' tumor .
the expression of dickkopf-1 was also elevated in 21 out of 73 cases of breast cancer , in particular hormoneresistant breast tumors .
additionally , gene expression profiles revealed that dickkopf-1 was overexpressed in prostatic , lung , esophageal , and hepatic carcinoma , serving as a serologic and prognostic biomarker .
wang and zhang also reported that dickkopf-1 is frequently overexpressed in ovarian serous carcinoma and involved in tumor invasion .
these studies suggested that dickkopf-1 is associated with the tumorigenesis and tumor progression . to our knowledge ,
the role of dickkopf-1 expression in gastric cancer was rarely studied . to address this problem , in this study
, we examined the expression of dickkopf-1 in surgical specimens of gastric cancer to explore the possible correlation between dickkopf-1 expression and clinicopathological variables and to determine its prognostic value .
this study was approved by the research ethics committee of huai'an no.1 hospital , china .
gastric cancer tissues were collected from the gastrectomy specimens of 328 patients ( 235 male , 93 female ; median age = 60.0 years ; range = 28~92 years ) treated at the department of surgery , huai'an no.1 hospital , between march 2000 and march 2005 .
tissues were formalin fixed , paraffin embedded , and clinically and histopathologically diagnosed at the departments of gastrointestinal surgery and pathology .
the survival time was counted from the date of surgery to the follow - up deadline , or date of death ( usually the result of cancer recurrence or metastasis ) . according to the who histological classification of gastric cancer formulated in 2002 , there were 245 tubular adenocarcinomas , 12 papillary adenocarcinomas , 22 mucinous adenocarcinomas , and 49 signet - ring cell carcinomas .
nine were highly differentiated adenocarcinomas , 96 were well- or moderately differentiated adenocarcinomas , 220 were poorly differentiated , and 3 were undifferentiated adenocarcinomas .
sixty - eight cases were categorized as stage i , 78 as stage ii , 130 as stage iii , and 52 as stage iv .
three hundred and twenty - eight noncancerous human gastric tissues were obtained from gastrectomy of adjacent gastric cancer margins ( greater than 5  cm ) .
routine chemotherapy was given after surgery to patients with advanced - stage disease , but none of the patients received radiation treatment .
the clinical and pathologic parameters were obtained from the pathological reports and presented in table 1 .
real - time quantitative reverse transcriptase - polymerase chain reaction ( qrt - pcr ) was performed to detect the expression of dikkopf-1 mrna in 20 pairs of human gastric cancer and adjacent noncancerous human gastric tissues .
total rna was isolated from human gastric cancer and adjacent noncancerous human gastric tissues by an rneasy mini kit ( qiagen ) .
random - primed cdna synthesis was performed using quantitect reverse transcription kit ( qiagen ) .
qrt - pcr was performed using a ptc-1000 programmable thermal controller ( mj research , waltham , ma , usa ) with quantitect sybr green pcr kit ( qiagen , valencia , ca , usa ) according to the manufacturer 's instructions . for amplifications , the following primers were designed : dikkopf-1 : forward , 5-aga cca ttg acaact acc agc cgt-3 ; reverse , 5-tct gga ata ccc atc caa ggt gct-3 and gapdh : forward , 5-cct ccg gga aac tgt ggc gtg atg g-3 ; reverse , 5-aga cgg cag gtc agg tcc acc act g-3. each sample was examined in triplicate , and the amounts of the pcr products were nonneoplasticized to gapdh which served as internal control . to confirm the specificity of the anti - dickkopf-1 antibody , western blotting test
human gastric cancer tissues were lysed in 100  l lysis buffer ( 50  mm tris - hcl ph 7.4 , 125  mm nacl , 0.1% ( v / v ) np-40 , 5  mm ethylene diamine tetraacetic acid ( edta ) , 50  mm naf , 50  g / ml phenylmethylsulfonyl fluoride ( pmsf ) , 10  g / ml leupeptin , 10  g / ml soybean trypsin inhibitor , and 1  g / ml aprotinin ] on ice for 10  min ) .
anti - dickkopf-1 polyclonal antibody ( santa cruz biotechnology , santa cruz , ca , usa ) was used in primary reaction .
immunohistochemical study for dickkopf-1 was performed on formalin - fixed , paraffin - embedded , 4-m - thick tissue sections using the avidin - biotin - peroxidase complex method . in brief , the sections were deparaffinized and dehydrated using a graded series of ethanol solutions .
endogenous peroxidase activity was halted through the administration of 0.3% hydrogen peroxidase and methanol for 20  min .
after having been rinsed in phosphate - buffered saline ( pbs ) , the tissue sections were processed in a 0.01  m citrate buffer ( ph 6.0 ) inside a heat - resistant plastic container .
after incubation with rabbit polyclonal primary antibody dickkopf-1 ( santa cruz biotechnology , santa cruz , ca , usa , dilution 1  :  60 ) , which was used in several previous studies [ 16 , 1921 ] , for 2  h at room air temperature , staining was performed by labeled streptavidin - biotin method .
immunostaining was scored by two independent experienced pathologists , who were blinded to the clinicopathological parameters and clinical outcomes of the patients .
the scores of the two pathologists were compared , and any discrepant scores were trained through reexamining the stainings by both pathologists to achieve a consensus score .
the number of positive - staining cells showing immunoreactivity of dickkopf-1 in ten representative microscopic fields was counted , and the percentage of positive cells was calculated .
the percentage scoring of immunoreactive tumor cells was as follows : 0 ( 0% ) , 1 ( 110% ) , 2 ( 1150% ) , and 3 ( > 50% ) .
the staining intensity was visually scored and stratified as follows : 0 ( negative ) , 1 ( weak ) , 2 ( moderate ) , and 3 ( strong ) .
a final score was obtained for each case by multiplying the percentage and the intensity score .
therefore , tumors with a multiplied score exceeding 4 ( i.e. , tumors with a moderate or strong staining intensity of > 10% of the tumor cells ) were deemed to be showing high expression of dickkopf-1 ; all other scores were considered to be low expression . the software of spss version13.0 for windows ( spss inc , il , usa ) was used for statistical analysis .
measurement data were analyzed using the student 's t - test , while categorical data were analyzed using  or fisher 's exact tests .
survival curves were estimated using the kaplan - meier method and the log - rank test was used to compute differences between the curves .
multivariate analysis using the cox proportional hazards regression model was performed to assess the prognostic value of protein expression levels .
correlation coefficients between dickkopf-1 protein expression and clinicopathological findings were estimated using the pearson correlation method .
real - time quantitative rt - pcr was performed to detect the expression of dickkopf-1 mrna in 20 pairs of human gastric cancer and adjacent noncancerous human gastric tissues .
the expression level of dickkopf-1 mrna showed significant difference between gastric cancer tissues and corresponding noncancerous gastric tissues ( figure 1(a ) ) .
the average ratios of dickkopf-1 mrna to gapdh mrna in gastric cancer tissues and noncancerous gastric tissues were 0.68  0.18 and 0.09  0.05 , respectively ( figure 1(b ) ) , which suggested that the expression of dickkopf-1 mrna was significantly higher in gastric cancer tissues than that in corresponding noncancerous gastric tissues ( p < 0.001 ) .
the specificity of anti - dickkopf-1 antibody was confirmed by western blot analysis using lysates of dickkopf-1-expressing gastric cancer tissues . as shown in figure 1(c ) , a single band of dickkopf-1 expression was detected , which was consistent with the results of yamabuki et al . on lung and esophageal carcinoma cells .
a total of 328 pairs of gastric cancer and noncancerous human gastric tissues were evaluated for dickkopf-1 protein expression by immunohistochemical analysis .
high expression was detected in 195/328 ( 59.5% ) human gastric cancer cases and low expression in 133 ( 40.5% ) .
dickkopf-1 was mainly localized in the cytoplasm , and less localized in nuclei of primary cancer cells ( figure 2(a ) ) .
in contrast , none of the noncancerous human gastric tissues showed significant level of immunohistochemical staining ( figure 2(b ) ) .
positive expression of dickkopf-1 correlated with depth of invasion , vessel invasion , lymph node and distant metastasis , and tnm stage ( all p < 0.01 ) , but not with age , gender , tumor size , location , differentiation stage , or histological type ( all p > 0.05 ) ( table 1 ) .
this showed that the depth of invasion ( p = 0.02 ) , lymph node ( p = 0.01 ) and distant metastasis ( p = 0.008 ) , tnm stage ( p = 0.006 ) , and the level of dickkopf-1 expression ( p = 0.002 ) were all independent prognostic factors in patients with gastric cancer .
however , the location of the tumor , tumor size , histological type , differentiation , and vessel invasion had no prognostic value . for patients with stage i , ii , or iii disease , the 5-year survival rate for those with high dickkopf-1 expression were significantly lower than in patients with low expression .
for stage i , the cumulative 5-year survival rate was 90.7% in the low - expression group , but only 71.4% in the high - expression group ( p = 0.001 , figure 3(a ) ) ; for stage ii , the cumulative 5-year survival rate was 69.8% in the low - expression group , but only 54.3% in the high - expression group ( p = 0.001 , figure 3(b ) ) ; for stage iii , the cumulative 5-year survival rate was 55.2% in the low - expression group , but only 21.8% in the high - expression group ( p = 0.0001 , figure 3(c ) ) .
for stage iv the expression of dickkopf-1 did not correlate with the 5-year survival rate ( 14.3% in the low - expression group and 2.2% in the high - expression group ; p = 0.5 ) .
gastric cancer is associated with a much shorter time to recurrence and a shorter survival period after recurrence .
because of the early metastasis and strong invasion , it is very important to estimate the malignant degree and invasion tendency of gastric cancer in order to guide clinical diagnosis and treatment of this disease . in the present study ,
immunohistochemistry was used to analyze the expression levels of dickkopf-1 in 328 clinicopathologically characterized gastric cancer patients .
the results showed that dickkopf-1 protein was significantly upregulated in gastric cancer tissues compared with normal gastric tissues , which was confirmed by real - time qrt - pcr analysis on the expression of dickkopf-1 mrna .
in addition , high levels of dickkopf-1 expression in gastric cancer lesions were associated with depth of invasion , vessel invasion , lymph node and distant metastasis , and tnm stage .
furthermore , the up - regulation of dickkopf-1 was an independent prognostic indicator for gastric cancer . therefore , dickkopf-1 may be a valuable diagnostic marker and therapeutic target for gastric cancer .
several recent studies have demonstrated that dickkopf-1 plays multiple biological roles in a variety of cancers .
gene expression profile analysis revealed that dickkopf-1 was highly transactivated in the great majority of lung cancers and esophageal squamous cell carcinomas .
in contrast , expression of the dickkopf-1 gene , a downstream target of -catenin / tcf , decreases in human colon tumors , indicating its tumor - suppressing role in this neoplasia .
human dickkopf-1 was reported to be responsive to p53 although it has been shown to be induced by dna damage and to sensitize to apoptosis in a p53-independent manner .
however , the involvement of dickkopf-1 in gastric cancers has not been defined . in 2007 , sato et al . reported that dickkopf methylation was frequently observed in gastric cancer cell lines . but shi et al . in 2009 demonstrated that serum concentrations of dickkopf-1 were decreased significantly in groups of patients with gastric cancer compared with healthy controls .
additionally , these previous studies could not clarify the clinical impact of dickkopf-1 expression or the prognostic value for gastric cancer .
therefore , this is the first report to determine the correlation between dickkopf-1 expression and clinical factors in gastric cancer .
our results showed that dickkopf-1 was upregulated in gastric cancer tissues compared with noncancerous tissues and the high or low expression of dickkopf-1 in gastric cancer directly affected clinical factors .
this discrepancy with the previous study of shi et al .   may be due to the small numbers used in their study .
with respect to the results of survival analysis , in patients with stage i , ii , and iii gastric cancer , the 5-year survival rate for those with high dickkopf-1 expression was significantly lower than that of patients with low expression . however
multivariate analysis suggested that the depth of invasion , lymph node and distant metastasis , tnm stage , and dickkopf-1 up - regulation were independent prognostic indicators for gastric cancer .
it is known that dickkopf-1 expression correlates with a reduction in disease - free survival and is an independent predictor of poor prognosis in human esophageal squamous cell carcinoma . in ovarian serous carcinoma
, wang and zhang also showed that the level of dickkopf-1 expression is associated with adverse outcomes .
dickkopf-1 , p - jnk1 , and the coexpression of dickkopf-1 and p - jnk1 were all unfavorable prognosis factors for ovarian serous carcinoma patients .
dickkopf-1 , alone or combined with p - jnk1 , was an independent predictor for the 5-year survival . additionally , dickkopf-1 secreted and stably expressed in cerebral fluids can also be applicable to detect presence of glioblastoma and to develop novel prognostic treatments . in conclusion , our data showed that a subset of patients with gastric cancer had overexpression of dickkopf-1 , which was associated with an aggressive clinical course and poor overall survival .
therefore , dickkopf-1 may play a significant role in the progression of gastric cancer , which was consistent with most other cancers .
in addition , such information may direct us toward novel therapeutic and prognostic possibilities for treating gastric cancer and improving patient outcomes .
this is the first report to suggest a relationship between dickkopf-1 and prognosis in patients with gastric cancer , and further prospective analysis would be worth doing .
 
 aim . to investigate the involvement of dickkopf-1 expression in gastric cancer 
 . methods . 
 dickkopf-1 mrna and protein expression were determined by real - time quantitative reverse transcriptase - polymerase chain reaction ( qrt - pcr ) and immunohistochemistry in specimens of primary cancer and their adjacent noncancerous tissues in gastric cancer patients 
 . results . 
 dickkopf-1 mrna and protein expression levels were both significantly upregulated in gastric cancer lesions compared with adjacent noncancerous tissues . 
 its positive expression was correlated with depth of invasion , vessel invasion , lymph node and distant metastasis , and tnm stage of tumors . 
 additionally , in stages i , ii , and iii gastric cancers , the 5- year survival rate of patients with a high expression of dickkopf-1 was significantly lower than that in patients with low expression . in stage 
 iv , dickkopf-1 expression did not correlate with the 5-year survival rate . 
 further multivariate analysis suggested that the up - regulation of dickkopf-1 was an independent prognostic indicator for gastric cancer . 
 conclusion . 
 a subset of cases with gastric cancer revealed the up - regulation of dickkopf-1 , which was associated with a progressive pathological feature and an aggressive clinical course . 
 therefore , dickkopf-1 expression may be predictor for poor prognosis in patients with gastric cancer . 
 this is the first report describing the involvement of dickkopf-1 in gastric cancer . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion