Expression of Annexin A 3 in Gastric Cancer and its Correlation with Proliferation and Apoptosis

Gastric cancer is one of the most frequent digestive malignancies in the world (Vogelaar et al., 2012). In China, gastric cancer is the second cause of mortality related with cancer and constitutes approximately 30% of all worldwide gastric cancer new cases. Lots of gastric cancer patients are diagnosed at advanced stage and have high rates of lymph node metastasis. Despite some improvements are acquired in treatment of gastric cancer, prognosis is still unfavourable for patients. Thus there is an urgent need for finding effective molecular markers to improve the survival time of gastric cancer patients. Annexin A3 is a member of a calcium-binding protein family that includes five groups (A-E) (Gerke et al., 2002; Gerke et al., 2005). As Ca2+-dependent phospholipid-binding proteins, Annexin A-E possesses a variety of biological activities, such as regulating ion channels, mediating inflammatory response, participating in membrane trafficking (Raynal et al., 1994; Caohuy et al., 1996; Perretti et al., 2003). Recently, Annexin A3 was indentified as a novel biomarker in different types of cancers, including prostate cancer (Wozny et al., 2007; Kollermann et al., 2008; Schostak et al., 2009), lung cancer (Liu et al., 2009) and colorectal cancer (Xie et al., 2013). However little is known about the effects of Annexin A3 in gastric cancer. The aim of our study was to explore Annexin A3 expression in gastric cancer samples and its


Introduction
Gastric cancer is one of the most frequent digestive malignancies in the world (Vogelaar et al., 2012).In China, gastric cancer is the second cause of mortality related with cancer and constitutes approximately 30% of all worldwide gastric cancer new cases.Lots of gastric cancer patients are diagnosed at advanced stage and have high rates of lymph node metastasis.Despite some improvements are acquired in treatment of gastric cancer, prognosis is still unfavourable for patients.Thus there is an urgent need for finding effective molecular markers to improve the survival time of gastric cancer patients.
Annexin A3 is a member of a calcium-binding protein family that includes five groups (A-E) (Gerke et al., 2002;Gerke et al., 2005).As Ca 2+ -dependent phospholipid-binding proteins, Annexin A-E possesses a variety of biological activities, such as regulating ion channels, mediating inflammatory response, participating in membrane trafficking (Raynal et al., 1994;Caohuy et al., 1996;Perretti et al., 2003).Recently, Annexin A3 was indentified as a novel biomarker in different types of cancers, including prostate cancer (Wozny et al., 2007;Kollermann et al., 2008;Schostak et al., 2009), lung cancer (Liu et al., 2009) and colorectal cancer (Xie et al., 2013).However little is known about the effects of Annexin A3 in gastric cancer.The aim of our study was to explore Annexin A3 expression in gastric cancer samples and its

Expression of Annexin A3 in Gastric Cancer and its Correlation with Proliferation and Apoptosis
Jing-Ming Zhai 1& , Shi-Jun Sun 3& , Wei Wang 1 , Chao Zeng 2 * potential value as a prognostic marker for gastric cancer.
Interestingly, Annexin A3 could inhibit apoptosis of ovarian cancer cells (Yan et al., 2010).Moreover, downregulated Annexin A3 expression promoted apoptosis and restrain proliferation of gallbladder cancer cells (Tan et al., 2010).Taking into account the few data concerning role of Annexin A3 in proliferation and apoptosis of gastric cancer, we initiated study the correlation of Annexin A3 with Ki-67, Bcl-2 and Bax.Therefore, Annexin A3 expression patterns and its roles in tumorigenesis of gastric cancer was thoroughly analyzed in the present study.

Patients and tissue samples
Gastric cancer specimens were collected from the Department of Pathology, the First Affiliated Hospital Henan University of Science and Technology between 2007 and 2008.For immunohistochemistry assay, gastric cancer cases included 80 paraffin-embedded samples, with mean age of 56 years (range, 45-70 years).Clinicopathologic features including age, gender, differentiation degree, tumor volume, invasion depth, TNM stage and lymph node metastasis were detailed in Table 1.The informed consent on collection of samples was obtained by each patient.The study was approved by the Medical Ethics Committee of the first affiliated hospital of Henan University of Science and Technology.

Tissue microarray and immunohistochemistry
Tissue microarrays (TMAs) were constructed as the method described previously (Xie et al., 2003).Briefly, paraffin-embedded tissue blocks and the corresponding H&E-stained slides were overlaid for TMA sampling.Triplicate cylindrical tissues with 0.6-mm diameter were punched from selected tumor areas of individual donor tissue and re-embedded into a recipient paraffin block at the designated place.4-μm sections were cut from the TMAs deparaffinized by routine techniques.The slides were microwaved.The slides were microwaved in citrate buffer for 5 min for antigen retrieval.

Evaluation of Annexin A3, Ki-67, Bcl-2 and Bax expression
Annexin A3, Bcl-2 and Bax were scored according to the intensity (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and the percentage(extent staining) of tumor cells that were stained (0, no positive cells; 1, >10% of cancer cells stained; 2, 10%-50% of cancer cells stained; 3, >50% of cancer cells stained; 4, >75% of cancer cells staining positive).If the product of multiplication between staining intensity and the percentage of positive cells is ≥2, it is thought as immunoreaction positive (+).Ki-67 was scored according the positive number of 100 gastric cancer cells at high magnification.Serum was performed as negative control for the primary antibody.

Statistical analysis
All statistical analyses were performed by using SPSS 13.0 for Windows (SPSS, Inc, Chicago, IL).The correlation between Annexin A3 and clinicopathologic factors was evaluated by Fisher's exact test.Multivariate analysis was done by using Cox proportional hazards regression.Overall survival curves were generated according to the Kaplan-Meier method.A result was considered significant when the p value is <0.05.

Annexin A3 expression in gastric cancer tissues
Annexin A3 was detected by immunohistochemistry in 80 gastric cancer cases.Immunohistochemical results were confirmed by two sophisticate pathologists.Annexin A3 immunreactivity was found in 49 of the 80 (61.3%) cases.However, Annexin A3 staining in gastric tissues adjacent to cancer was negative.More importantly, as shown in Figure 1, Annexin A3 expression was only present in the cytoplasm of gastric cancer cells.In all 80 gastric cancer cases, Annexin A3 expression was scored positively in 49 cases and 18 of them had strong immunoexpression.

Correlation between Annexin A3 and clinicopathological factors in gastric cancers
To better elucidate the clinical significance of Annexin A3 in gastric cancer, the association between Annexin A3 expression and the clinicopathologic factors was evaluated.1, positive expression of Annexin A3 was significantly correlated with tumor size (p=0.022)and TNM stage (p=0.001).Moreover, there was no association between Annexin A3 expression and the other clinicopathologic parameters, such as gender, age, differentiation degree, depth of invasion and lymph node metastasis (p>0.05).

Annexin A3 is an independent prognostic biomarker for poor outcome of gastric cancer patients
To better clarify the role of Annexin A3 in prognosis of gastric cancer, Kaplan-Meier survival curves was employed to evaluate overall survival time of patients.Patients with positive Annexin A3 expression had worse prognosis compared with those with negative expression (Figure 2) (p<0.05).Univariate and multivariate analyses were used to assess the influence of Annexin A3 expression and other clinicopathological factors on the prognosis of gastric cancer patients.Univariate Cox regression analysis indicated high Annexin A3 expression, lymph node metastasis, TNM stage, invasion depth was significantly correlated with overall survival of patients (Table 2).By multivariate analyses, TNM stage, lymph node metastasis and Annexin A3 expression were associated with poor overall survival.Hence, Annexin A3 expression is an indicator of a poor prognosis for gastric cancer patients.In all clinicopathologic parameters, lymph node metastasis was the most independent factor as a prognostic biomarker (p=0.011)(Table 2).In accordance with our results, previous study found TNM and lymph node metastasis are all independent prognostic factors affecting the survival of patients with gastric cancer (Cao et al., 2013).

Association between Annexin A3 and proliferation index (pI)
To investigate the effect of Annexin A3 on proliferation of gastric cancer, we study the association between Annexin A3 and proliferation index.In the present study, proliferation index was reflected by means of Ki-67.As shown in Figure 3, PI in the positive Annexin A3 expression group was significantly higher (59.2±7.8)compared with that in the negative Annexin A3 expression group (20.9±6.3)(Table 3; p<0.01).These results indicated Annexin A3 might be involved in proliferation of gastric cancer cells.

Correlation between Annexin A3 and Bcl-2, Bax
In all gastric cancer specimens, 35 among 49 Annexin A3-stained cases showed Bcl-2 immunoreactivity.As shown in Figure 3, Bcl-2 expression was located in the cytoplasm of gastric cancer cells.Moreover, among 31 cases with Annexin A3 negative expression, 21 cases had not detected Bcl-2 staining.However, apart from 7 positive expression cases, the other samples showed no Bax immunoreactivity (Figure 3).

Discussion
Recently, more attention has been focused on Annexin A3 as its key roles in tumorigenesis of different tumors.Some studies reported that Annexin A3 expression was   up-regulated in lung (Wu et al., 2013), breast (Zeng et al., 2013) and colorectal cancer (Xie et al., 2013).Furthermore, increased expression of Annexin A3 was correlated with lymph node metastasis and poor survival rate in lung cancer.However, Annexin A3 protein expression was essentially reduced and it could be an independent adverse prognostic factor in renal-cell (Bianchi et al., 2010) and papillary thyroid cancer (Jung et al., 2010).Faced with two apparently contradictory statements, it raised our interest to investigate the functions of Annexin A3 protein in conjunction with clinical outcome of the patients with gastric cancer.This study is the first one that systematically explored the Annexin A3 expression in gastric cancers.In our study, Annexin A3 expression was significantly stronger in gastric cancer tissues compared with their normal counterpart.Immunohistochemistry assay demonstrated that cases with positive Annexin A3 expression had bigger tumor volume, which might indicated Annexin A3, to some extent, involved in proliferation of gastric cancer cells.Moreover, Annexin A3 expression was associated with the TNM stage.Consequently, Annexin A3 overexpression may suggest that patients have a worse prognosis.The following results confirmed that Annexin A3 was an independent prognostic marker for gastric cancer by Kaplan-Meier log rank analysis and multivariate analyses.
Previous study found Annexin A3 expression was associated with tumor volume.Hence, the correlation between Annexin A3 and proliferation, apoptosis of gastric cancer was investigated.Experimental results demonstrated Annexin A3 was associated with Ki-67, indicating Annexin A3 might be participated in proliferation of gastric cancer.In the Bcl-2 protein family, proapoptotic member Bax and antiapoptotic member Bcl-2 are the active effectors and regulators (Li et al., 2013).Taking into account the important role of Bcl-2 and Bax in determining apoptosis of cancer cells, the association between Annexin A3 and Bcl-2, Bax was needs to be explored.Further study found the positive rate of Bcl-2 in Annexin A3-positive cases was significantly higher compared with that in Annexin A3negative cases.It is worthwhile to note that only 7 cases have Bax immunoreactivity, suggesting Bax expression is irrelevant to Annexin A3.Therefore, Annexin A3 might be involved in tumorigenesis of gastric cancer by regulating the proliferation and apoptosis of gastric cancer cells.
In conclusion, annexin A3 is an independent prognostic factor for gastric cancer and it will be a potentially new target in gastric cancer therapy.More importantly, Annexin A3 has vital effects on proliferation and apoptosis of gastric cancer cells.The precise molecular mechanisms of Annexin A3 participate in tumorigenesis of gastric cancer need further investigation.

Figure 1 .
Figure 1.Expression Pattern of Annexin A3 in Gastric Cancer Tissues.(A) Normal gastric tissues showed no immunoreactivity of Annexin A3. (B) High Annexin A3 expression was detected in the well-differentiated gastric cancer (×200).(C) Annexin A3 expression was shown in moderatelydifferentiated gastric cancer.(D) Annexin A3 expression was shown in poor-differentiated gastric cancer (×200)

Figure
Figure 2. The Gastric Cancer Patients with Positive Annexin A3 Expression had Worse Prognosis than Those with Negative Expression of Annexin A3 by Using Kaplan-Meier Survival Analysis

Table 1 . Correlation between Annexin A3 Expression and Clinicopathological Factors of Gastric Cancer Patients
Positive Annexin A3 expression was found in 31 of 42 (73.8%)cases with larger tumor volume, whereas only 18 of 38 (47.4%) smaller cases showed positive Annexin A3 staining.Therefore, our data indicated enhanced Annexin A3 expression was associated with tumor size.Additionly, 30 of 37 Ⅲ and Ⅳ stage cases showed high Annexin A3 expression, however, 19 of 43 tumors positive Annexin A3 expression was only present inⅠand Ⅱstage cases, *p<0.05 doi.org/10.7314/APJCP.2014.15.7.3001Expression of Annexin A3 in Gastric Cancer andits Correlation with Proliferation and Apoptosis indicating Annexin A3 might participate in advancement of gastric cancer.As shown in Table