Xiao-jie Li ,Hai-long Xie *,San-ju Lei,Hui-qiu Cao ,Tian-Yun MengYu-lin Hu

1Department of Pathology, 1st People’s Hospital of Chenzhou, Chenzhou 423000, China

2Institute of Cancer Research, University of South China, Hengyang 421001, China

3Department of Otorhinolaryngology, 1st People’s Hospital of Chenzhou, Chenzhou 423000, China

INTRODUCTION

Gastric cancer (GC)is the most common cancer in Eastern Asia and South America.Although the incidence in western countries is much lower than that in Asia,it is still a significant worldwide health burden,only second to lung tumors as a leading cause of cancer death[1].The occurrence and development of gastric carcinoma is a complex multi-step process controlled by multiple factors.The activation of oncogenes (such as myc,K-ras,c-erbB-2 and K-sam)and deactivation of antioncogenes [such as p53,adenomatous polyposis coli(APC)and p16]play important roles.Therefore,it is important to identify new oncogenes involved in occurrence and development of gastric carcinoma for prognosis and clinical treatment of GC[2].

Carbonic anhydrase (CA)is a type of zinccontaining metalloenzyme.At least 16 types of CA isozymes have been reported in mammals with different tissue distribution and catalytic properties[3].CAI,CAII,CAIX and CAXII are highly tumorrelated[4,5],and among them,CAIX and CAXII are related to soft tissue sarcoma,breast cancer and nonsmall cell lung cancer[6-8].Human CAII gene,which locates at chromosome 8q22,codes a cytoplasmic protein that has the highest turnover rate and widest tissue distribution among the known human CA isozymes[3].CAII is correlated with several types of tumors[9-12].In our previous study,the expression of CAII was down-regulated in GC[2].However,the relationship between CAII expression and tumor invasion as well as metastasis in gastric carcinoma has not been examined.In the present study,we aimed to investigate the expression of CAII in GC and explore its correlation with some clinicopathologic characteristics of GC.

MATERIALS AND METHODS

Clinical Specimens

Retrospective analysis was performed on gastric carcinoma patients who had undergone gastrectomy between August 2004 and March 2007 in the pathological department of the 1st Hospital of Chenzhou.The mean age of the patients was 49 years,ranging from 23 to 79 years.A total of 78 males and 34 females (2.29∶1)were included in our study,of which 65 patients were under 55 years old and 47 patients were over 55 years old.In terms of tumor size,78 specimens were larger than 5 cm,and 36 specimens were smaller than 5 cm.According to tumor differentiation,we investigated 20 highly-differentiated tumors (17.86%),29 moderately-differentiated tumors (25.89%)and 63 poorly-differentiated tumors (56.25%).In terms of the depth of tumor invasion,20 (17.86%)and 92 (82.14%)patients were classified as early type and advanced type,respectively.Seventy-five (66.96%)patients were lymph node metastasis negative,whereas 37 (33.04%)were lymph node metastasis positive.No adjuvant radiotherapy or chemotherapy was administered on those patients before surgery.Multiple sections were microscopically examined to confirm the degree of differentiation,the depth of invasion and lymph node metastasis.One representative block was then selected for immunohistochemical study.Twenty normal mucosa and 38 intraepithelial neoplasia paraffin blocks derived from subtotal gastrectomy specimens because of duodenal ulcer.The study was approved by the Ethical Committee for Clinical Research of the Hospital.

Immunohistochemistry

Immunoperoxidase staining of formalin-fixed and paraffin-embedded tissue sections was performed by an ordinary biotin-streptavidin method.Briefly,sections were deparaffinized in xylene,heated with 10 mmol/L citrate buffer (pH 6.0)in a pressure cooker for 5 min and washed with phosphate-buffered saline(PBS,pH 7.2).In order to block endogenous peroxidase activity,sections were immersed in methanol containing 0.3% hydrogen peroxide (H2O2)at room temperature for 20 min.Then the sections were blocked with 10% normal calf serum in PBS for 10 min.Subsequently,the sections were incubated with anti-CAII mouse monoclonal antibody (1∶200)(Santa Cruz Biotechnology Inc.,USA)for 1 h in a humidified chamber.After incubating with secondary antibody and avidin-biotin complex reagent,color reaction was developed in 0.02% H2O2in Tris buffer (pH 8.0).Hematoxylin was used for counterstaining.The positive control was positive slides bought from Santa Cruz Biotechnology Inc.,USA.In addition,a parallel negative control without primary antibody was established.

Evaluation of Immunostaining

Yellow particles in the cytoplasm or cytomembrane meant positive staining.The scores were evaluated in terms of staining intensity as follows∶ 0,no reaction; +,weak reaction; ++,moderate reaction; and +++,strong reaction.In the statistical analyses,the specimens were grouped into two categories based on the staining intensity and positive cells∶ CAII(+)tumors,including more than 10% of neoplastic cells exhibiting moderate or strong reaction; and CAII(-)tumors,including less than 10% of neoplastic cells with moderate or strong reaction,or weak or negative immunostaining results.

Analysis of Survival Rate

We collected 112 patients’ postal address and telephone number from the medical record department of the 1st Hospital of Chenzhou.Then,we contacted patients or their family members,and collected 50 cases until July 2010.Finally,we drew the five-year survival curves.

Statistical Analysis

The correlation between CAII expression of neoplastic cells and clinicopathological factors was evaluated by chi-squared test or Fisher’s exact test.P＜0.05 was considered statistically significant.The logrank test was used in the survival analysis.All statistical analyses were carried out by using SPSS 13.0(SPSS Inc.,Chicago,IL,USA)

RESULTS

Immunostaining Analysis of CAII Expression in Normal Mucosa,Intraepithelial Neoplasia and GC

Table 1 shows that the positive rate of CAII protein was 28.57% (32/112)in 112 tumor specimens,63.15%(24/38)in intraepithelial neoplasia,and 100% (20/20)in normal mucosa.Figure 1 shows that CAII protein was expressed in cytoplasm,and CAII expression at the protein level in normal mucosa was stronger than that in intraepithelial neoplasia and GC.

Correlation between CAII Expression and Age or Sex

The expression rates of CAII was 29.48% and 26.47% in male and female GC specimens,respectively,and there was no correlation between CAII expression and age.

Table 1.Expression of CAII in normal mucosa,intraepithelial neoplasia and GC

Table 2.Correlations between CAII expression and clinicopathologic characteristics of GC

Correlation between CAII Expression and Tumor Size

We showed that 28.95% of the cases whose tumor sizes were smaller than 5 cm expressed CAII and 27.78% of the cases whose tumor sizes were larger than 5 cm expressed CAII.There was no correlation between CAII expression and tumor size.

Correlation between CAII Expression and Differentiation,Stage as well as Metastasis

Table 2 shows that differentiation,stage and lymph node metastasis were positively correlated with CAII expression among the 112 specimens with gastric carcinoma.The positive rate of CAII protein was significantly lower in poorly-differentiated gastric carcinoma than that of moderately- or welldifferentiated gastric carcinoma (15.94% vs.31.03% or 60.00%,P＜0.05).The positive rate of CAII protein was significantly higher in gastric carcinoma at early stages than that at advanced stages (70.00% vs.19.57%,P＜0.001).Furthermore,the positive rate of CAII protein was significantly lower in gastric carcinoma with lymph node metastasis than that without lymph node metastasis (10.81% vs.37.33%,P＜0.05).

Analysis of Survival Rate

We collected 50 GC patients until July 2010.Fourteen patients were CAII(+),and 36 patients were CAII(-)tumors.Three CAII(+)patients are alive.The CAII(+)patients had longer survival time.Figure 2 shows that the patients with CAII(+)tumors had a slightly higher survival rate than those with CAII(-)tumors,and the difference was statistically significant(P=0.024,log-rank test).

Figure 1.Immunohistochemical staining of CAII protein was located in cytoplasm.A,B: normal gastric mucosa; C:intraepithelial neoplasia; D: GC.

Figure 2.Survival of GC with CAII expression.The patients with CAII(+)tumors showed a better survival rate than those with CAII(-)tumors (P=0.024,log-rank test).

DISCUSSION

Tumorigenesis is a multi-step process accompanied with genetic alterations of precancerous cells and the simultaneous microenvironment building up,which promotes transformation[13].CA is a type of zinccontaining metalloenzyme.CAII is a cytoplasmic protein that has the highest turnover rate and widest tissue distribution among the known human CA isozymes.In tumor,CAII is involved in acidifying the extracellular microenvironment,and tumors show much stronger invasiveness in acidified microenvironment[14-16].

A previous study showed that the extracellular pH of solid tumor was more acidic than that of adjacent normal tissue[17].In contrast,the intracellular pH estimated by31P-magnetic resonance spectroscopy is identical or slightly higher in tumor compared with normal tissue[18].In order to establish the pH gradient between the extracellular and intracellular compartments,tumor cells express ion transport proteins,including vacuolar H+-ATPase,Cl-/HCO3-exchanger and Na+/H+exchanger[19,20].Many tumors also express CAs,which catalyze the production of H+and HCO3-ions in a reversible reaction∶ H2O+CO2H++HCO3-[21-23].Recently,Ivanov,et al.hypothesized that tumorassociated transmembrane isozymes CAII and XII may be involved in acidifying the extracellular microenvironment surrounding cancer cells.Consequently,a microenvironment,which is suitable for tumor growth and spread,is created[24].CAIV,CAIX or CAXII may produce bicarbonate ions outside of the cells.Then the bicarbonate ions are transported inside the cells by bicarbonate/chloride exchangers and catalyzed by CAII to titrate protons.On the other hand,CAII binds to the carboxyl terminus of human bands 3 protein,which catalyzes the exchange of bicarbonate for chloride[25].Buffering protons facilitate proton secretion and protect the cell from intracellular acidification[26-28].CAII is interrelated to antigen of vascular endothelium,and its expression is related to amplification of epidermal growth factor receptor(EGFR).The anti-tumor mechanism of CAII is similar to that of EGFR.Yoshiura,et al.[29]suggested that endothelial CAII can be a potential target antigen for dendritic cell therapy.

Recently,Supuran,et al.[30]showed that the pH value of tumor environment can be changed through modulating the CA activity.This effect may favorably influence the anti-tumor effect of sulfonamide CA inhibitor or other concomitantly used anti-tumor agents.Thus,it is very important to examine the potential of CA inhibitor/activator in treating tumor and inhibiting tumor progression[31,32].However,its relevance to GC remains unclear.Based on these considerations,our study showed that there was a significant reduction of cytosolic CAII expression in GC.The positive rate of CAII protein was 28.57%(32/112),63.60% (24/38)and 100% (20/20)in gastric carcinoma,intraepithelial neoplasia and normal mucosa,respectively.This is consistent with the previous study[33].In 112 gastric carcinoma specimens,the differentiation,stage and lymph node metastasis were positively correlated with CAII expression.Because of less metastatic gastric carcinoma specimens,we did not compare difference between primary and metastatic gastric carcinoma.In order to know whether CAII is an independent prognosis index,we analyzed the survival rate of patients with GC.In the present study,the patients with CAII(+)tumors had a slightly higher survival rate than those with CAII(-)tumors,and the difference was statistically significant (P=0.024,log-rank test).Therefore,we think that CAII may be a good prognosis index.

Since CAII affects the tumor development by changing tumor’s microenvironment and energy metabolism,the reduction of CAII expression may promote GC cell motility and contribute to tumor growth and metastasis.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

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