Expression and Biological Relationship of Vascular Endothelial Growth Factor-A and Matrix Metalloproteinase-9 in Gastric Carcinoma

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1 The Journal of International Medical Research 2011; 39: [first published online as 39(6) 1] Expression and Biological Relationship of Vascular Endothelial Growth Factor-A and Matrix Metalloproteinase-9 in Gastric Carcinoma S YANG a, Z ZHAO a, R WU a, H LU, X ZHANG, C HUAN, C WANG, X WU AND G GUAN Department of Oncology, The Union Hospital of Fujian Medical University, Fuzhou, China Protein expression of vascular endothelial growth factor A (VEGF-A) and matrix metalloproteinase 9 (MMP-9) was studied in gastric carcinoma patients in relation to clinicopathological characteristics and prognosis. Fifty-four samples of gastric carcinoma tissue and 15 samples of adjacent normal gastric mucosal tissue were examined immunohistochemically. Expression rates of VEGF-A (66.7%) and MMP-9 (63.0%) in carcinoma tissue were significantly higher than in normal tissue (6.7% for both proteins). VEGF-A and MMP- 9 expression was associated with tumour size, invasion depth, lymph node metastasis, degree of histological differentiation and pathological stage, but not age or sex. VEGF-A expression was positively correlated with that of MMP-9. Expression of VEGF-A and MMP-9 were each inversely correlated with 5-year survival. VEGF-A and MMP-9 were overexpressed in tumours compared with normal tissue; they may act together to increase carcinogenesis and the progression, invasion and metastasis of gastric carcinoma, and could be used as biomarkers for the prognosis of gastric carcinoma. KEY WORDS: GASTRIC CARCINOMA; STOMACH NEOPLASMS; VASCULAR ENDOTHELIAL GROWTH FACTOR A; MATRIX METALLOPROTEINASE 9; IMMUNOHISTOCHEMISTRY; PROGNOSIS Introduction Gastric carcinoma is one of the most common cancers in China. Surgery (including lymph node dissection) accompanied by preoperative chemotherapy is still the most important management strategy for this cancer; 1,2 however, this comprehensive regimen has a poor outcome in advanced-stage gastric carcinoma. 3,4 a These authors contributed equally to this study. Research has shown that the proliferation, invasion and metastasis of tumour cells, which are highly associated with angiogenic and other cell factors, are the main factors reducing the effectiveness of cancer treatment; moreover, they can lead directly to death. 5 The angiogenic factor, vascular endothelial growth factor A (VEGF-A), can induce endothelial cell division, stimulate cell growth, promote vascularization and increase microvascular permeability, thus 2076

2 favouring the proliferation of tumour cells and the formation of a vascular net. However, the multistep process of tumour invasion and metastasis is a complex one in which tumour cells interact continuously with host cells. 6,7 Primary tumour cells may escape from the primary lesion, start to synthesize proteins actively and invade the extracellar matrix (ECM) and basement membrane. Matrix metalloproteinase 9 (MMP-9) can increase the invasiveness and metastasis of tumour cells through processes such as degradation of the ECM and basement membrane and the induction and intensification of angiogenesis. 8,9 The present study used immunohistochemistry to investigate how expression of VEGF-A and MMP-9 protein in gastric carcinoma tissue is related to clinicopathological characteristics. Patients and methods PATIENTS AND SAMPLE COLLECTION Tissue samples that had been fixed in 4% formaldehyde and then embedded in paraffin prior to storage were used. Stored paraffin blocks of gastric carcinoma tissue samples were selected randomly from a total of 150 blocks that originated from patients who had attended the Union Hospital of Fujian Medical University between July 2001 and July 2002 for radical subtotal gastrectomy or total gastrectomy. Patients selected for inclusion all had full clinical data available (n = 54). Control tissue samples (n = 15) consisted of normal gastric mucosal tissue from areas surrounding the tumours that had been collected during the same period as the gastric carcinoma tissue samples. The distance between the normal tissue sampling site and the tumour was > 5 cm and control tissues were verified pathologically. Clinical staging of the tumours was according to the 1997 Union for International Cancer Control tumour, node, metastasis (TNM) staging scheme and pathological classification was according to the World Health Organization 1990 scheme. 10,11 Ethical approval for the study was provided by the Ethics Committee of The Union Hospital of Fujian Medical University (Ref. No ). Written consent for the use of tissue samples in the study was provided by the patients and healthy subjects, or by a relative if the patient was too ill to provide consent themselves. SAMPLE PREPARATION AND IMMUNOHISTOCHEMICAL STAINING Consecutive 5 µm slices of the paraffin block embedded tissue were cut and the slices were floated onto microslides using poly-l-lysine and then dried at 70 C in an oven for 4 h. Expression of VEGF-A and MMP-9 proteins was detected immunohistochemically using methods described by Kase et al. 12 and Kallakury et al., 13 respectively. A streptavidin peroxidase immuno histo - chemical kit with primary monoclonal antibodies specific to VEGF-A and MMP-9, and 3,3 -diaminobenzidine chromogen (all supplied by Fuzhou Maxim, Fuzhou, China) was used according to the manufacturer s instructions. The primary antibody was replaced with 0.01 M phosphate-buffered saline (PBS), ph 7.4, to provide a negative control. The positive control was provided by Fuzhou Maxim. All chemicals used in the study were of analytical purity. SEMIQUANTITATIVE ASSESSMENT OF VEGF-A AND MMP-9 EXPRESSION The presence of brown yellow stained particles (claybank expression) in the cytoplasm indicated positivity for VEGF-A 2077

3 and MMP-9 protein. For each slice, 10 randomly selected microscope fields at 200 magnification were examined and 100 tumour cells were counted in each field. The percentage of cells with positively staining cytoplasm was calculated and the mean value determined. A semiquantitative integration method was used to obtain a final score for positive expression; this method combined data for the percentage of positive cells and staining intensity. Cells that were unstained, lightly stained, moderately stained and strongly stained were assigned a score of 0, 1, 2 and 3, respectively; similarly, slices with 0%, 1 25%, 26 50% and > 50% stained cells were scored 0, 1, 2 and 3, respectively. The scores were summed and a total score 3 was classified as positive expression and a total score of 0 2 as negative expression. STATISTICAL ANALYSES The data were analysed using the Statistical Package for Social Sciences (SPSS version 10.0; SPSS Inc., Chicago, IL, USA). The χ 2 test was used to analyse categorical data, the κ statistic for correlation analysis, the Kaplan Meier method to analyse survival rate and the log-rank test to analyse intergroup differences in survival rate. A P-value < 0.05 was considered to be statistically significant. Results DEMOGRAPHIC AND CLINICAL DATA A total of 54 gastric carcinoma tissue samples and 15 control tissue samples from normal gastric mucosal tissue surrounding the tumours were used. The demographic and clinicopathological data of the patients from whom these samples originated are given in Table 1. The mean age of the patients was 53.3 years (range years) and 26 patients were aged > 60 years. The male : female ratio was 2.2 : 1 (37 males, 17 females). Ten tumours were classified pathologically as high grade, nine as moderate grade, 33 as low grade and two as undifferentiated. Forty-four of the 54 patients had lymph node metastasis. The depth of tumour invasion was pt1 or pt2 in 12 cases and pt3 or pt4 in 42 cases. The TNM stage was I or II in 15 cases and III or IV in 39 cases. None of the patients underwent preoperative radiotherapy or chemotherapy. The clinical follow-up period was months and median overall survival time was 33 months (range 5 74 months). EXPRESSION OF VEGF-A AND MMP-9 The presence of claybank staining in cytoplasm, indicating expression of VEGF-A (Fig. 1) and MMP-9 (Fig. 2), was mainly observed in tumour cells and, occasionally, in normal gastric mucosal cells surrounding tumours. The rates of expression (percentage of cells showing positive staining) were significantly higher in the gastric carcinoma tissues than in normal gastric mucosal tissues for both VEGF-A (66.7 and 6.7%, respectively; P < 0.05) and MMP-9 (63.0 and 6.7%, respectively; P < 0.05). Expression rates for VEGF-A and MMP-9 were significantly related to tumour size (P = and P < 0.001, respectively), depth of invasion (P < for both), degree of differentiation (P < and P = 0.019, respectively), lymph node metastasis (P < and P = 0.006, respectively) and pathological stage (P < for both), but not to patient age or sex (Table 1). CORRELATION BETWEEN VEGF-A AND MMP-9 EXPRESSION Out of a total of 36 VEGF-A-positive gastric carcinoma tissue samples, 28 (77.8%) also expressed MMP-9 and, of the 18 VEGF-A- 2078

4 TABLE 1: Demographic and clinicopathological characteristics of patients with gastric carcinoma in relation to tumour cell expression of vascular endothelial growth factor A (VEGF-A) and matrix metalloproteinase 9 (MMP-9) detected by immunohistochemistry VEGF-A expression MMP-9 expression Statistical Statistical Characteristic Cases (%) Positive Negative χ 2 significance Positive Negative χ 2 significance Age 0.59 NS 3.61 NS > 60 years 26 (48.1) years 28 (51.9) Sex 0.17 NS 0.03 NS Male 37 (68.5) Female 17 (31.5) Tumour size (diameter) 6.29 P = P < > 5 cm 25 (46.3) cm 29 (53.7) Depth of invasion P < P < pt1 or pt2 12 (22.2) pt3 or pt4 42 (77.8) Degree of differentiation P < P = Moderate or high 19 (35.2) Undifferentiated or 35 (64.8) low Lymph node metastasis P < P = No 10 (18.5) Yes 44 (81.5) TNM stage P < P < I or II 15 (27.8) III or IV 39 (72.2) TNM, tumour, node, metastasis; NS, not statistically significant (P > 0.05). 2079

5 100 µm FIGURE 1: Immunohistochemical detection of vascular endothelial growth factor A (brown yellow particles in the cytoplasm) in gastric carcinoma tissue 100 µm FIGURE 2: Immunohistochemical detection of matrix metalloproteinase 9 (brown yellow particles in the cytoplasm) in gastric carcinoma tissue 2080

6 negative gastric carcinoma tissue samples, six (33.3%) expressed MMP-9. The correlation between VEGF-A and MMP-9 expression in gastric carcinoma tissue was statistically significant (κ = 0.432; P < 0.05). VEGF-A AND MMP-9 EXPRESSION AND PROGNOSIS Kaplan Meier survival curve analysis indicated that expression of VEGF-A and expression of MMP-9 were inversely correlated with survival rate. The 5-year survival rates for patients whose carcinoma tissues expressed and did not express VEGF-A were 16.7% (6/36 patients) and 38.9% (7/18 patients), respectively (χ 2 = 4.63; P < 0.05; Fig. 3) and the corresponding values for MMP-9 were 20.6% (7/34 patients) and 30.0% (6/20 patients), respectively (χ 2 = 6.57; P < 0.05; Fig. 4). Discussion The proliferation of tumour cells is closely related to tumour invasiveness and metastasis, since tumour cell proliferation is a precondition for both these characteristics. Angiogenesis is required for tumour growth, haematogenous and lymphatic invasion, and metastasis. Tumours, therefore, cannot grow, invade or metastasize without the formation of a vascular phenotype. The angiogenic factor VEGF-A can specifically induce endothelial cell division and promote vascularization, and can also increase microvascular permeability and exosmosis of plasma fibrin. VEGF-A is, therefore, able to nurture tumour cells before the formation of blood vessels and provide a fibrous net along which different types of angiogenic cells can migrate. Research has shown that VEGF-A is expressed at a high level in gastric carcinoma tissue and that its expression is correlated with clinicopathological characteristics: raised VEGF-A expression is usually accompanied by greater aggressiveness of the tumour. 14,15 The Negative VEGF-A expression Cumulative survival Positive VEGF-A expression Duration of survival (months) FIGURE 3: Five-year survival curves of gastric carcinoma patients whose tumours expressed or did not express vascular endothelial growth factor A (VEGF-A) protein (expression versus non-expression: χ 2 = 4.63; P < 0.05) 2081

7 Negative MMP-9 expression Cumulative survival Positive MMP-9 expression Duration of survival (months) FIGURE 4: Five-year survival curves of gastric carcinoma patients whose tumour cells expressed or did not express matrix metalloproteinase 9 (MMP-9) (expression versus non-expression: χ 2 = 6.57; P < 0.05) present study showed that the rate of expression of VEGF-A (percentage of cells showing positive staining) was significantly higher in gastric carcinoma tissue than in normal gastric mucosal tissue. In addition, VEGF-A expression was significantly higher in tumours > 5 cm in diameter, in tumours that had invaded to a depth of pt3 or pt4, when lymph node metastases were present, or when the TNM stage was III or IV compared with other tumours (i.e. expression of VEGF-A was higher when the pathological stage was worse). Moreover, the 5-year survival rate was significantly lower for patients with VEGF-A-positive tumours than for those with VEGF-A-negative tumours. These results suggest that VEGF-A is important for the growth, invasiveness and metastasis of gastric carcinoma, and may be of use as a biomarker for evaluating both the biological behaviour of tumours and the patient s prognosis. Degradation of the ECM and basement membrane is a key component of the processes of tumour invasion and metastasis. MMPs, which are proteinases related to invasion and metastasis, may be able to degrade the molecules forming the fibrin net of the ECM and basement membrane. MMP- 9 has the highest molecular weight among the proteins of the MMP family. It is produced first as a zymogen, which is then activated by conversion into type IV collagenase that is able to degrade type IV collagen, type V collagen and gelatin in the ECM adjacent to the surface of a tumour. Tumour cells can then invade the surrounding tissue because of the consequent deficit in the basement membrane, through structures such as blood vessels and lymphatic vessels, thus leading to metastasis. The role of MMP-9 in gastric carcinoma has attracted considerable attention. Sier et al. 16 found a low survival 2082

8 rate in postoperative patients with gastric carcinoma and high expression of MMP-9, regardless of the clinical stage. Kabashima et al. 17 showed a correlation between high expression of MMP-9 mrna and metastasis of gastric carcinoma; MMP-9 expression therefore appears to reflect the metastatic potency and prognosis of gastric carcinoma. Using reverse transcription polymerase chain reaction analysis and immunohistochemical methods, other workers have found a relationship between MMP-9, VEGF-A and invasion and metastasis of gastric carcinoma. 18,19 Their results showed that expression of MMP-9 protein and its mrna was higher in the metastatic group than in other patients. In the present study, expression of MMP-9 in gastric carcinoma tissue was significantly higher than that in normal gastric mucosal tissue. Moreover, MMP-9 expression was significantly higher in patients with larger tumour size, deeper invasion, lower differentiation, the presence of lymph node metastases and more advanced TNM stage. The 5-year survival rate of patients with tumours expressing MMP-9 was significantly lower than that of patients with tumours not expressing MMP-9. These results suggest that MMP-9 promotes the development of gastric carcinoma and that a high level of expression may favour the growth, invasion and metastasis of gastric carcinoma. MMP-9 could potentially be used as a biomarker to evaluate the biological behaviour of tumours and patient prognosis. There is evidence for interaction between VEGF-A and MMP-9 with regard to angiogenesis and the proliferation, invasion and metastasis of tumour cells. Bergers et al. 20 and Lubbe et al. 21 showed that MMP-9 could not only degrade the ECM, because of its powerful barrier-breaking capacity, but could also promote the formation of new vessels. Shou et al. 22 concluded that MMPs may induce tumour cells to excrete VEGF-A. Zucker et al. 23 found that VEGF-A could induce the expression of endothelial urokinase-type plasminogen activator and its receptor, and then indirectly promote the expression of MMP-9 and increase its activity. These workers considered that MMP- 9 and VEGF-A may act in a synergistic manner in the genesis and development of tumours. Riedel et al. 24 reported that MMP-9 could participate directly in angiogenesis as a regulating factor and could also co-operate with VEGF-A. Deryugina et al. 25 reported that overexpression of MMP-9 was associated with increased production of VEGF-A, and that a high level of expression of VEGF-A could promote MMP-9 transcription. Other researchers have found activator protein 1 (AP-1) binding sites in the transcriptionregulating zone or promoter of MMP-9. AP-1, as a third nuclear messenger, may transfer signals for angiogenesis and matrix degradation into the nucleus. 26 It may, therefore, regulate MMP-9 and VEGF-A expression at the gene level and induce a malignant phenotype in which the matrix degrades, blood vessels are formed and invasion and metastasis occur easily. 26 In the present study, VEGF-A and MMP-9 coexisted in gastric carcinoma tissue in 28 cases and expression of these two proteins was significantly positively correlated. This suggests that VEFG-A may increase the expression of MMP-9, and vice versa, such that they may jointly promote local angiogenesis, tumour invasion and metastasis in patients with gastric carcinoma. Expression of VEGF-A and MMP-9 proteins in the present study was also detected in normal gastric mucosal tissue surrounding the tumours, though the percentage of positive cells was lower than in tumour 2083

9 tissue. It may be that tumour cells were present in this tissue, although no such cells were detected immunohistochemically. We suggest, therefore, that tumour growth may activate some form of regulatory process and, thereby, increase VEGF-A and MMP-9 expression in tumour cells and adjacent normal cells. Büchler et al. 27 showed that anoxia could stimulate VEGF-A mrna expression by between 2- and 50-fold in tumour cells and normal cells and, thus, might promote angiogenesis. Several reports have described key roles for VEGF-A and MMP-9 in the growth, invasion and metastasis of tumours, but the conclusions drawn have not always been in agreement and some researchers have detected no relationship between prognosis and the expression of VEGF-A and MMP ,29 Further research is, therefore, needed in order to deepen our understanding of the mechanisms of growth, invasion and metastasis of gastric carcinoma, and to provide a basis for the development of techniques for diagnosis, treatment and prognosis determination. 30,31 Conflicts of interest The authors had no conflicts of interest to declare in relation to this article. Received for publication 13 February 2011 Accepted subject to revision 12 April 2011 Revised accepted 11 September 2011 Copyright 2011 Field House Publishing LLP References 1 Takayama S, Wakasugi T, Funahashi H, et al: Strategies for gastric cancer in the modern era. World J Gastrointest Oncol 2010; 2: Shigeoka H, Imamoto H, Nishimura Y, et al: Complete response to preoperative chemoradiotherapy in highly advanced gastric adenocarcinoma. World J Gastrointest Oncol 2010; 2: Zhang D, Fan D: Multidrug resistance in gastric cancer: recent research advances and ongoing therapeutic challenges. Expert Rev Anticancer Ther 2007; 7: Afridi SP, Bano F, Shafiq-ur-Rahman: Pattern and presentation of carcinoma stomach. J Coll Physicians Surg Pak 2011; 21: Rak J, Milsom C, Yu J: Vascular determinants of cancer stem cell dormancy do age and coagulation system play a role? APMIS : Sato Y: Anti-angiogenic drugs. Nippon Rinsho 2010; 68: [in Japanese, English abstract]. 7 Groblewska M, Mroczko B, Szmitkowski M: The role of selected matrix metalloproteinases and their inhibitors in colorectal cancer development. Postepy Hig Med Dosw (Online) 2010; 64: [in Polish, English abstract]. 8 Tuder RM, Yun JH: Vascular endothelial growth factor of the lung: friend or foe. Curr Opin Pharmacol 2008; 8: Mroczko B, Łukaszewicz-Zajac M, Guzińska- Ustymowicz K, et al: Expression of matrix metalloproteinase-9 in the neoplastic and interstitial inflammatory infiltrate cells in gastric cancer. Folia Histochem Cytobiol 2009; 47: Borchard F: Classification of gastric carcinoma. Hepatogastroenterology 1990; 37: von Rahden BH, Feith M, Stein HJ: Carcinoma of the cardia: classification as esophageal or gastric cancer? Int J Colorectal Dis 2005; 20: Kase S, Osaki M, Honjo S, et al: Expression of cyclo-oxygenase-2 is correlated with high intratumoral microvessel density and low apoptotic index in human esophageal squamous cell carcinomas. Virchows Arch 2003; 442: Kallakury BV, Karikehalli S, Haholu A, et al: Increased expression of matrix metalloproteinases 2 and 9 and tissue inhibitors of metalloproteinases 1 and 2 correlate with poor prognostic variables in renal cell carcinoma. Clin Cancer Res 2001; 7: Vidal O, Metges JP, Elizalde I, et al: High preoperative serum vascular endothelial growth factor levels predict poor clinical outcome after curative resection of gastric cancer. Br J Surg 2009; 96: Cabuk D, Basaran G, Celikel C, et al: Vascular endothelial growth factor, hypoxia-inducible factor 1 alpha and CD34 expressions in earlystage gastric tumors: relationship with pathological factors and prognostic impact on survival. Oncology 2007; 72: Sier CF, Kubben FJ, Ganesh S, et al: Tissue levels of matrix metalloproteinases MMP-2 and MMP-9 are related to the overall survival of 2084

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