Lack of any Prognostic Relationship between Adiponectin Receptor ( Adipo R 1 / R 2 ) Expression for Early / Advanced Stage Gastric Cancer

Adiponectin (ApN, 30 kDa) is a complement C1qrelated protein that is mainly secreted from adipose tissue (Maeda et al., 1996). It is present in two forms, which include full length ApN (fAdipo) and biologically active globular ApN (gApN) (Kadowaki and Yamauchi 2005). It has been shown that ApN levels decrease in some conditions, such as insulin resistance and hyperinsulinemia (Diez and Iglesias 2003). ApN exhibits antiatherogenic effects by inhibiting vascular smooth muscle and endothelial cells (Yokota et al., 2000; Arita et al., 2002). In addition, ApN induces antiangiogenesis and has antitumoral effects (Brakenhielm et al., 2004; Ishikawa et al., 2007; Ogunwobi and Beales 2008). It has been reported that the concentration of ApN is low in obese patients (Cnop et al., 2009). Therefore, there may be a relationship between low ApN concentration and obesityrelated malignancies (Joshi and Lee, 2014). It has also been reported that there are reduced serum concentrations of ApN in gastric, esophageal, colorectal


Introduction
Adiponectin (ApN, 30 kDa) is a complement C1qrelated protein that is mainly secreted from adipose tissue (Maeda et al., 1996).It is present in two forms, which include full length ApN (fAdipo) and biologically active globular ApN (gApN) (Kadowaki and Yamauchi 2005).
It has been shown that ApN levels decrease in some conditions, such as insulin resistance and hyperinsulinemia (Diez and Iglesias 2003).ApN exhibits antiatherogenic effects by inhibiting vascular smooth muscle and endothelial cells (Yokota et al., 2000;Arita et al., 2002).In addition, ApN induces antiangiogenesis and has antitumoral effects (Brakenhielm et al., 2004;Ishikawa et al., 2007;Ogunwobi and Beales 2008).It has been reported that the concentration of ApN is low in obese patients (Cnop et al., 2009).Therefore, there may be a relationship between low ApN concentration and obesityrelated malignancies (Joshi and Lee, 2014).
It has also been reported that there are reduced serum concentrations of ApN in gastric, esophageal, colorectal
As the grade of renal cell cancer increases, the serum ApN levels decrease, and metastatic tumors tend to express lower Adipo-R2 (Pinthus et al., 2008).Serum ApN levels are lower in patients with advanced stage lung cancer than in those with local lung cancer (Petridou et al., 2007).Otani and colleagues hypothesized that ApN receptors would be downregulated in the early stages of cancer development in cancer cells that are protected from the antiproliferative effects of ApN (Otani et al., 2010).Based on these data, it has been hypothesized that Adipo-R1 and Adipo-R2 may have prognostic significance in early stage and advanced stage gastric cancer.In this study, we investigate the clinicopathologic characteristics, survival, and the relationship between Adipo-R1 and Adipo-R2 expression (immunohistochemically) in cases with early stage and advanced stage gastric cancer and in those who underwent surgical resection.The significance of Adipo-R1 and Adipo-R2 expression in tumor aggression is evaluated.

Materials and Methods
The patients who underwent surgical gastric resection were enrolled from the archives of a local university hospital.Eighteen patients (8 female, 10 male, median age 58.33±15.83years) with early stage gastric cancer and 39 patients (10 female, 29 male, median age 63.38±10.27years) with advanced stage gastric cancer were included in this study.
Tumors confined to the mucosa and submucosa were considered to be early stage gastric cancer, while any infiltration beyond the submucosa was considered to be advanced stage gastric cancer.All patients were graded and staged histologically according to the World Health Organization (WHO) and TNM system.For statistical analyses, the patients with mucinous and signet ring cell were grouped and compared to those with tubular ones.A statistical analysis comparing pTNM1 and pTNM2 with pTNM3 and pTNM4 was conducted.Follow-up length ranged from 1 month to 84 months (mean for early stage gastric carcinoma: 38.28±31.90months, mean for advanced stage gastric cancer: 21.03±15.77months).This study was approved by the local ethics committee.

Immunohistochemistry
A d i p o n e c t i n r e c e p t o r 1 a n d a d i p o n e c t i n receptor 2 expression in tissues was determined by immunohistochemical staining.Adipo-R1 and Adipo-R2 (ab126611 and ab77612, respectively; Abcam, Inc., Cambridge, MA, USA; working dilution 1/250) were used as the primary antibodies.A streptavidin-avidin-biotin method was used for immunohistochemical staining as follows.Four-micron thick sections were deparaffinized in an oven overnight at 60°C.The sections were dipped three times in Xylene for 5 minutes each and three times in 96% citrate buffer (pH 6.0) for 5 minutes each.Then, they were boiled in a 750 Watt microwave oven with distilled water at 5 minutes intervals for a total of 20 minutes.After incubation for 20 minutes at room temperature, they were washed 2 times with PBS.The sections were dried, and then were incubated for 15 minutes in a 25 o C humid chamber with 3% hydrogen peroxide.They were again washed with PBS and kept 10 minutes in protein blocking solution.The sections were incubated for 1 hour with primary antibody, and then washed twice with PBS for 3   minutes each.After 15 minutes in the secondary antibody (Biotinylated Link), they were washed with PBS and then put in DAB chromogen for 10 minutes.
The sections were washed with distilled water and evaluated by light microscopy according to the prevalence and severity of staining.The preparations having significant staining in the tissue were considered to be positive (Figure1, 2, and 3).

Statistical analysis
Descriptive statistics were calculated for all of the study data.The Kolmogorov -Smirnov test was used to determine whether the continuous variables satisfied the normality assumption.
The Mann Whitney U test was used to compare all variables between groups.A Pearson's chi-square test was used for the comparison of categorical variables.A correlation test was used to examine the relationships between variables, and a value of p<0.05 was considered statistically significant.A Kaplan-Meier analysis and a log rank test were used to compare survival time (stage age etc.) distributions.All analyses were performed with the SPSS 13.0 package program.

Results
There was no significant difference between the expression of adiponectin receptor 1 and adiponectin receptor 2 in patients with early stage and advanced stage gastric cancer.The patients' demographic data are presented in Table 1.Eighteen (8 female, 10 male) of the cases had early stage gastric carcinoma, and 39 (10 female, 29 male) had advanced stage gastric carcinoma.There were significant differences between those with early stage and advanced stage gastric cancer in terms of T stage, N stage, TNM stage, histological differentiation, and perineural and lymphatic invasion.The expression of Adipo-R1 and Adipo-R2 was poor in both groups (p=0.510,p=0.167, respectively).In addition, there were no significant relationships between Adipo-R1 and Adipo-R2 expression and age, sex, tumor location, tumor size, lymph node involvement, TNM stage, early and advanced stage gastric cancer, histologic subtypes, differentiation, Helicobacter pylori infection, or perineural, lymphatic, and venovascular invasion.There was no significant relationship between Adipo-R1 and Adipo-R2 expression in terms of overall survival and  present in 16 of 39 patients (41%).The relationship between TNM stage and Adipo-R2 expression was nearly significant (p=0.054)(Table 2).In terms of histologic grade, Adipo-R2 expression was significantly higher in moderately differentiated tumors when compared to welldifferentiated tumors (p=0.011).
Tumor stage (pT1+pT2/pT3+pT4) was identified as the determining factor (p=0.004) for both overall survival and disease-free survival.Only venovascular invasion was statistically significant (p=0.018) in overall survival analysis (Table 3).Although none of the variables were significant in progression free survival analysis, perineural invasion came close (p=0.052).

Discussion
Adiponectin is an adipokine that is abundant in the circulation and has contradictory functions and multiple features in tumorigenesis.These multiple and complex roles include metabolic regulation, changes in the tumor microenvironment, and direct effects on cancer cells (Hebbard and Ranscht 2014).Current evidence supports that adiponectin is a new risk factor for cancer and has a potential role as a diagnostic and prognostic biomarker (Dalamaga et al., 2012).
The ideal strategy for treating cancer cells is the down-regulation of adiponectin receptors in the early stages of cancer development, which would prevent  progression free survival (Figure 4 and 5).
The clinicopathologic characteristics of the patients are shown in Table 1.In early stage gastric cancer, Adipo-R1 expression was present in 2 of 18 patients (11.1%), and Adipo-R2 expression was present in 4 of 18 (22.2%)patients.In those with advanced stage gastric cancer, Adipo-R1 expression was present in 7 of 39 patients (17.9%), and Adipo-R2 expression was DOI:http://dx.doi.org/10.7314/APJCP.2014.15.11.4711Lack of Prognostic Influence of Adiponectin Receptor Expression in Gastric Cancer the antiproliferative effects of adiponectin.It has been proposed that AdipoR1/R2 expression is downregulated by gastric epithelial malignant transformation (Otani et al., 2010).Ishikawa et al. proposed that low plasma adiponectin levels increase the risk for gastric cancer and play a role in its progression.They found that plasma adiponectin levels in gastric cancer were lower than those of a healthy control group.The plasma adiponectin level was also very low in upper gastric cancer (Ishikawa et al., 2005).
Moreover, adiponectin inhibits the proliferation of gastric cancer cell lines and peritoneal dissemination.It also has antineoplastic effects in gastric cancer.Adiponectin signals through Adipo-R1/R2 receptors (Ishikawa et al., 2007).
Seker et al. found that plasma adiponectin levels were higher in undifferentiated gastric tumors than in well-differentiated grade gastric tumors.However, they found no relationship between the patients' adiponectin levels and histopathological variables or demographic characteristics (Seker et al., 2010).
However, adiponectin receptor expression was lower in prostate cancer (Mistry et al., 2006;Michalakis et al., 2007).Baressi et al. reported that expression of adiponectin receptors 1 and 2 was significantly different in intestinal type gastric cancer patients and those with diffuse-type.Moreover, they found a statistically significant relationship between overall survival and Adipo-R1/R2 expression (Barresi et al., 2009;Tsukada et al., 2011).
Tsukada et al. found an inverse relationship between Adipo-R1 expression and tumor growth in gastric cancer.They proposed that Adipo-R1 expression can contribute significantly to the improvement of prognosis.However, they stated that Adipo-R2 expression has no effect on prognosis and has no relationship with clinicopathological factors (Tsukada et al., 2011).
Herein we investigate the relationship between the clinicopathological characteristics of cases with early and advanced stage gastric cancer with immunohistochemicallydetected Adipo-R1 and Adipo-R2 expression based on pTNM stage.In addition, we conducted disease-free and overall survival analysis.In terms of histological grade, Adipo-R2 expression was significantly higher (p=0.011) in moderately differentiated tumors than in well-differentiated tumors.There was nearly a statistically significant relationship between TNM stage and Adipo-R2 expression (p=0.054).Adipo-R2 expression tends to be a little higher in cases with advanced stage gastric cancer.There is a strong relationship between H. pylori infection and gastric cancer (Karami et al., 2013).However, there was no significant relationship between H. pylori infection and Adipo-R1/R2 expression according to tumor stage.Further studies considering the many other factors involved in the pathogenesis of gastric cancer with larger sample sizes are necessary.
In conclusion, Adipo-R1 and Adipo-R2 are present in early and advanced stage gastric cancer.Unfortunately, there is no statistical difference with regards to survival.