Expression of Matrix Metalloproteinase-9 mRNA and Vascular Endothelial Growth Factor Protein in Gastric Carcinoma and Its Relationship to Its Pathological Features and Prognosis.код для вставкиСкачать
THE ANATOMICAL RECORD 293:2012–2019 (2010) Expression of Matrix Metalloproteinase-9 mRNA and Vascular Endothelial Growth Factor Protein in Gastric Carcinoma and its Relationship to its Pathological Features and Prognosis QIONG YANG,1 ZAI-YUAN YE,1* JING-XIA ZHANG,1 HOU-QUAN TAO,1 SHU-GUANG LI,1 AND ZHONG-SHENG ZHAO2* 1 Key Laboratory of Gastroenterology, Zhejiang Province, Hangzhou, China 2 Department of Pathology, Zhejiang Provincial People’s Hospital, Hangzhou, China ABSTRACT To investigate matrix metalloproteinase-9 (MMP-9) mRNA and vascular endothelial growth factor (VEGF) protein expression in gastric carcinoma and its correlation with microvascular density, growth-pattern, invasion, metastasis, and prognosis. In situ hybridization of MMP-9 mRNA and immunohistochemistry of VEGF and CD34 proteins were performed on surgical specimens of gastric cancers from 118 patients compared with 20 nonmalignant gastric mucosae. Their relationships to pathological parameters and survival times were determined by statistical analysis. The positive rate of MMP-9 in noncancerous gastric mucosae was signiﬁcantly lower than that of gastric cancer tissue (60.17%, P < 0.01). In patients with cancers of the inﬁltrating type, at stage T3-T4, with vessel invasion, lymphatic metastasis, hepatic, or peritoneal metastasis, the positive expression rates of MMP-9 mRNA, VEGF protein, and CD34 were signiﬁcantly higher than those for patients with tumors of the expanding type (P < 0.01), at stage T1–T2 (P < 0.01), with nonvessel invasion (P < 0.05), without lymphatic metastasis (P < 0.05), and without hepatic (P < 0.001) or peritoneal metastasis (P < 0.001), respectively. Expression of MMP-9 mRNA was positively related to that of VEGF protein (P < 0.001) and microvascular density (P < 0.001). Patients with higher MMP-9 mRNA and VEGF expression demonstrated vivid tumor angiogenesis and poor 5-year survival rate. MMP-9 and VEGF expression is associated with enhanced tumor angiogenesis and may play crucial roles in the invasion and metastasis of gastric carcinoma. Therefore, MMP-9 and VEGF may represent prognostic biomarkers and promising targets for therapeutic intervention. C 2010 Wiley-Liss, Inc. Anat Rec, 293:2012–2019, 2010. V Key words: stomach neoplasms; matrix metalloproteinase-9; vascular endothelial growth factor; metastasis; prognosis Grant sponsor: Zhejiang Province Natural Science Foundation; Grant number: M303843. *Correspondence to: Zai-Yuan Ye, Key Laboratory of Gastroenterology, Zhejiang Province, Hangzhou 310014, Zhejiang, China. E-mail: firstname.lastname@example.org or Z.-S. Zhao, Department of Pathology, Zhejiang Provincial People’s Hospital, Hangzhou 310014, Zhejiang, China. E-mail: email@example.com C 2010 WILEY-LISS, INC. V Received 1 April 2009; Accepted 8 September 2009 DOI 10.1002/ar.21071 Published online 18 November 2010 in Wiley Online Library (wileyonlinelibrary.com). EXPRESSION OF (MMP-9) mRNA AND VEGF PROTEIN INTRODUCTION The incidence and mortality rates of gastric cancer have fallen dramatically in the past 70 years. Despite its decreasing prevalence, gastric carcinoma remains the second leading cause of cancer death worldwide (Crew and Neugut, 2006). Most patients are diagnosed late at the mid-advanced stage, resulting in an overall 5-year survival rate >20%. Tumor cells degrade extracellular matrix (ECM) components to invade surrounding tissues. The process is precisely controlled by ECM-degrading enzymes including matrix metalloproteinases (MMPs). MMP-9 is a zinc-containing enzyme with potent proteolytic activity against a wide range of ECM components such as laminin-5 and type IV collagen, the major constituents of basement membranes. Recent evidence has uncovered multiple functions for MMP-9, in addition to simply degrading ECM, which may partially contribute to tumor angiogenesis and metastasis (Hiratsuka et al., 2002). Aberrantly expressed MMP-9 has been reported in a variety of tumors. However, its clinical role in gastric cancer is still fragmentary. Thus, to explore its malignant inﬂuence and prognostic signiﬁcance, we examined the expression of MMP-9 mRNA and vascular endothelial growth factor (VEGF) and microvascular density (MVD) in 118 tissue samples from gastric cancer patients. The correlation between MMP-9 and tumor angiogenesis and other clinical pathological parameters of gastric carcinomas including growth-pattern, invasion, and metastasis were also analyzed in this study. MATERIALS AND METHODS Patients and Specimens A total of 118 patients (79 male, 39 female; aged 38– 80 years, median age 57.8 years) who underwent gastrectomy for gastric carcinoma at Zhejiang Provincial People’s Hospital from October 1990 to November 1998 were included in this study. Five-year follow-up data was obtained, and the follow-up ended in November 2003. According to the World Health Organization classiﬁcation standard (2002), there were 39 tubular adenocarcinomas, 19 papillary adenocarcinomas, 37 poorly differentiated adenocarcinomas, 12 mucinous adenocarcinomas, and 11 signet ring cell carcinomas. These cancerous tissues of these patients were also classiﬁed into well- and moderately-differentiated (G1 þ G2, 70 patients) and poorly-differentiated (G3 þ G4, 48 patients) types, based on the predominant differentiation mode, and further classiﬁed into expanding (51 patients) and inﬁltrating (67 patients) types. According to the AJCC TNM staging system (Greene et al., 2002), there were 21 cases of stage T1, 26 cases of stage T2, 45 cases of stage T3, and 26 cases of stage T4. There were 89 patients with and 29 without vessel invasion, 84 patients with lymphatic metastasis and 34 without, 55 patients with distant metastasis (35 with peritoneal dissemination, 20 with hepatic metastasis) and 63 without. A control study was carried out on 20 samples obtained from adjacent noninvolved normal gastric mucosa 5 cm away from the primary tumor without hyperplasia or atypical hyperplasia. 2013 Reagents MMP-9 (MK1540) probe: Digoxin-labeled nucleotides for MMP-9 probes were obtained from Boster Biological Technology Limited Company, Wuhan, China. The target gene mRNA sequences were as follows: (1) 50 -TCCCT GCCCG AGACC GGTGA GCTGG ATAGC-30 ; (2) 50 CAACT CGGCG GGAGA CTGT GCGTC TTCCC-30 ; and (3) 50 -CCAGG TGGAC CAAGT GGGCT ACGTG ACCTA30 . Immunohistochemical reagents including primary monoclonal antibodies against VEGF (1:100) and CD34 (1:120) and EnVision kit were obtained from DAKO Denmark. (Produktionsvej 42 DK-2600 Glostrup). The VEGF clone, JH121, and the CD34 clone, QBEnd 10, were used. In Situ Hybridization In situ hybridization was performed on formalin-ﬁxed and parafﬁn-embedded tissue sections according to the manufacturer’s instructions. Samples cut at 4-lm intervals were dewaxed in xylene and rehydrated. The endogenous peroxidase was blocked with 3% hydrogen peroxide. After washing three times with distilled water, the sections were digested at 37 C with Pepsin Reagent for 30 min, ﬁxed with 1% paraformaldehyde plus 1/1,000 DEPC for 10 min, and then incubated in 50-lL prehybridization solution at 40 C for 4 hr. Each slide was then incubated with 20-lL hybridization solution (hybridization probe concentration ¼ 2 ng/lL) overnight in a humidiﬁed chamber at 40 C, washed twice with 2 SSC, once with 0.5 SSC, and ﬁnally three times with 0.2 SSC. After that blocking solution was applied and the superﬂuous liquid was absorbed. Slides were then treated with mouse biotinylated antidigoxigenin for 60 min, washed three times with PBS, and then incubated in SABC for 30 min. The hybridization signal was detected by using the avidin-biotin-peroxidase technique with DAB as the chromogen. The slides were counterstained with Harris hematoxylin, washed, dehydrated in ethanol, cleared in xylene, and enveloped with neutral gum. Negative controls included the following: (1) hybridization solution without probe, and (2) specimens pretreated with RNase. Immunohistochemistry The EnVision two-step method was performed according to the manufacturer’s instructions. Tissue sections were cut at 4-lm intervals, dewaxed with xylene, hydrated by a series of ethanol solutions (100%, 95%, 80%, and 70%). High temperature and high pressure (CD34: 0.01 mol/L sodium citrate buffering solution, pH 6.0; VEGF: 0.01 mol/L EDTA buffering solution, pH 9.0) were applied to facilitate antigen retrieval. The sections were washed once with distilled water, three times with PBS for 5 min, and with inactivated endogenous peroxidase using 3% hydrogen peroxide for 10 min at room temperature. After washing three times with PBS for 5 min, sections were incubated overnight with the primary antibody at 4 C. They were rinsed three times with PBS for 5 min and incubated with goat anti-mouse IgG antibody/HRP polymer for 40 min at 37 C before washing three times with PBS for 5 min. Visualization was achieved with DAB incubation for 3 min. After counterstaining with Harris hematoxylin, the slides 2014 YANG ET AL. were dehydrated in 95% and 100% ethanol, cleared in xylene, and enveloped with neutral gum. The negative control included replacement of the primary antibody with PBS, and the positive slides provided by the reagent kit were used as the positive control. Results Evaluation Cytoplasm stained brown was indicative of the positive expression of MMP-9 mRNA. The staining results ranging from to þþþ were estimated as follows: () (positive cell numbers <10% or no staining); (þ) (positive cell numbers 11%–50%); (þþ) (positive cell numbers 51%–75%); or (þþþ) (positive cell numbers >75%). Signals for VEGF expression was detected as brown in the cytoplasm and/or cell membrane. Based on the positive cell counting, the results were scored as follows: () (no staining); (þ) (positive cell numbers <25%); (þþ) (positive cell numbers 26%–50%); or (þþþ) (positive cell numbers >50%). Calculation of MVD was performed as well. Vessels with a clearly deﬁned lumen or welldeﬁned linear vessel shapes, but not single endothelial cells, were considered for microvascular assessment. Each slide was ﬁrst examined at 100 magniﬁcation to capture ﬁve ‘‘hot spots,’’ deﬁned as areas with highest vascularization. CD34þ vessels were then quantiﬁed in those chosen ﬁelds (200). The average count was taken as MVD and statistically presented as the mean SD. The patients were divided into two groups as high MVD (54.9/mm2) and low MVD (<54.9/mm2). Statistical Analysis Data were analyzed statistically using SPSS 13.0 software. Signiﬁcant differences were evaluated with Student’s t-tests. The v2 test was performed on the numerical data. Survival analysis was carried out using the Kaplan-Meier product-limit method, and survival curves were plotted. The differences were evaluated by the log rank test. A P-value <0.05 was considered statistically signiﬁcant. RESULTS Correlation Between MMP-9 mRNA Expression and Clinical Pathological Parameters Negative results were found in normal gastric mucosae (Fig. 1A), whereas 71 of the 118 (60.7%) gastric cancer samples, including those in early stages, showed positive MMP-9 expression. Signals of positive staining were detected in the cytoplasm of the carcinoma cells, which was usually located near the edge of the carcinoma ﬁltration areas. Most of the greater omentum and peritoneum were also positive for MMP-9 expression (Fig. 1B). In patients with the inﬁltrating type of cancer, at stage T3–T4, with vessel invasion, lymphatic metastasis, hepatic, and peritoneal metastasis, the positive expression rates of MMP-9 mRNA was signiﬁcantly higher than for those patients with the expanding type (v2 ¼ 8.513, P < 0.01), at stage T1–T2 (v2 ¼ 12.706, P < 0.001), without vessel invasion (v2 ¼ 5.664, P < 0.05), without lymphatic metastasis (v2 ¼ 8.446, P < 0.05), or without hepatic and peritoneal metastasis (v2 ¼ 17.378, P < 0.001; v2 ¼ 21.938, P < 0.001). No statistical correlation was revealed between MMP-9 mRNA positive Fig. 1. In situ hybridization expression of MMP-9 mRNA in samples from gastric adenocarcinoma patients: (A) MMP-9 mRNA is negative in adjacent normal gastric mucosa (magniﬁcation 200); (B) Cytoplasm of gastric cancer mucosa demonstrates high positive rates of MMP-9 mRNA (magniﬁcation 200). expression and the carcinoma differentiation (v2 ¼ 1.188, P ¼ 0.276; Table 1). Correlation Between VEGF Expression and Clinical Pathological Features VEGF was rarely expressed in normal gastric mucosae (Fig. 2A). Whereas of 118 gastric carcinoma patients, 64 positively expressed VEGF (54.2%). The staining signal was mainly located in the cytoplasm of tumor cells and usually observed at the invasive edge of tumor cell nests (Fig. 2B). In patients with the inﬁltrating type of tumors, at stage T3–T4, with vessel invasion, lymphatic metastasis, hepatic, and peritoneal metastasis, the VEGF positivity was much more prevalent compared with those for patients with the expanding type (v2 ¼ 10.18, P < 0.01), at stage T1–T2 (v2 ¼ 34.19, P < 0.001), with nonvessel invasion (v2 ¼ 37.29, P < 0.001), without lymphatic metastasis (v2 ¼ 34.71, P < 0.001), and without hepatic or peritoneal metastasis (v2 ¼ 24.53, P < 0.001; v2 ¼ 49.75, P < 0.001). However, no correlation was found between the different histological types (v2 ¼ 3.49, P ¼ 0.062; Table 1). 2015 EXPRESSION OF (MMP-9) mRNA AND VEGF PROTEIN TABLE 1. Correlation of MMP-9 mRNA, VEGF protein, and MVD with clinical pathological characteristics VEGF MMP-9 Growth pattern Expansive Inﬁltrative Histological grade (G) G1 þ G2 G3 þ G4 Invasive depth T1–T2 T3–T4 Vessel invasion No Yes Lymph node metastasis No Yes Distant metastasis No Liver Peritoneal dissemination N þ þþþ 118 51 67 47 28 19 71 23 48 70 48 32 15 38 33 47 71 28 19 19 52 29 89 17 30 12 59 34 84 21 26 13 58 63 20 35 40 2 5 23 18 30 P X2 8.51 <0.01 1.19 >0.05 12.71 5.66 8.45 31.58 17.38a 21.94b <0.001 <0.05 <0.05 <0.001 <0.001a <0.001b þ þþþ 54 32 22 64 19 45 37 17 33 31 37 17 10 54 28 26 1 63 30 24 4 60 50 3 1 13 17 34 X2 P 10.18 <0.01 3.49 >0.05 34.19 <0.001 37.29 <0.001 34.71 <0.001 61.49 <0.001 24.53a 49.75b <0.001a <0.001b MVD (/mm2) 49.45 21.72 64.06 18.76 55.72 21.56 60.70 20.73 44.42 19.96 66.56 17.23 35.68 14.04 64.93 18.07 37.60 15.73 66.24 17.24 42.81 17.23 73.40 8.07 75.79 9.48 t P 3.92 <0.001 1.25 >0.05 6.41 <0.01 7.96 <0.01 8.45 <0.001 7.65a 10.46b <0.01a <0.01b a Compared between patients with liver metastasis and those without distant metastasis. Compared between patients with peritoneal dissemination and those without distant metastasis. b Correlation Between MVD and Clinical Pathological Features Correlation Between MMP-9 mRNA, VEGF Protein, MVD, and Prognosis CD34 staining was positive in vascular endothelial cells. Microvessels in cancerous tissue and surrounding areas were intensely stained from brown to dark brown. In patients with the inﬁltrating type of cancer, at stage T3–T4, with vessel invasion, lymphatic metastasis, hepatic, and peritoneal metastasis (Fig. 3A), the mean MVDs were signiﬁcantly higher than those for patient with the expanding type (t ¼ 3.92, P < 0.001), at stage T1–T2 (t ¼ 6.41, P < 0.01), with nonvessel invasion (t ¼ 7.96, P < 0.01), without lymphatic metastasis (t ¼ 8.45, P < 0.001), and without hepatic or peritoneal metastasis (t ¼ 7.65, P < 0.01; t ¼ 10.46, P < 0.01), respectively (Fig. 3B). No correlation was found within the different histological types (P > 0.05; Table 1). The survival time in patients positive for the expression of MMP-9 mRNA and VEGF protein with an MVD 54.9/mm2 was signiﬁcantly shorter than for patients who were negative for the expression of MMP-9 mRNA (P < 0.001) and VEGF protein (P < 0.01) with an MVD < 54.9/mm2 (P < 0.001). The 5-year survival rate in patients positive for the expression of MMP-9 mRNA and VEGF with an MVD 54.9/mm2 was signiﬁcantly lower than patients negative for expression of MMP-9 mRNA (P < 0.001) and VEGF protein (P < 0.01) with an MVD < 54.9/mm2 (P < 0.001; Figs. 4–6; Table 2). Correlation Between MMP-9 mRNA, VEGF Protein Expression, and MVD The mean MVD (64.28 19.54/mm2) in gastric carcinoma specimens with a positive expression of MMP-9 mRNA was much higher than in the negative expression group (47.88 20.13/mm2; t ¼ 4.410, P < 0.001). Similarly, the mean MVD in gastric carcinoma specimens positive for the expression of VEGF (71.98 13.26/mm2) was higher than in the negative expression group (39.678 14.81/mm2; t ¼12.475, P < 0.001). In addition, the percentage of VEGF expression in carcinoma tissues in conjunction with a positive expression of MMP-9 mRNA was 73.2% (52/71), which was signiﬁcantly higher than the MMP-9 mRNA negative expression group (25.5%, 12/47; P < 0.001). Therefore, there was a positive relationship between the expression of MMP-9 mRNA, VEGF protein, and MVD. DISCUSSION MMP-9 has a wide range of proteolytic activities including the capacity to degrade ECM components. Similar to other members of the family, function of MMP-9 is mainly based on its multidomain structure, which includes a highly conserved catalytic domain, HEXXHXXGXXH. This domain depends on Zn2þ for activation, a C-terminal hemopexin-like domain for collagenase digestion, and three additional repeats of the ﬁbronectin type II motif located in the metalloproteinase domain for the determination of substrate speciﬁcity (Nagase et al., 2006). Imbalanced secretion of MMP-9 and its endogenous inhibitors into the surrounding microenvironment during cancer progression may alter the noncellular compartment, causing tissue remodeling and facilitating tumor cell migration. Aside from its enzymatic activities, MMP-9 also affects cytoskeletal organization through interactions with different families of adhesion receptors and regulates vessel stabilization by controlling pericyte recruitment (Bendeck et al., 2000; Chantrain et al., 2016 YANG ET AL. Fig. 2. Immunohistochemical expression of VEGF in samples from gastric adenocarcinoma patients: (A) VEGF protein is slightly expressed in normal gastric mucosa (magniﬁcation 400); (B) Cytoplasm of gastric cancer mucosa demonstrates a high expression for VEGF protein (magniﬁcation 400). 2004). Associations between MMP-9 expression levels with invasive phenotypes have been increasingly recognized in tumors of different anatomical origins. For example, oncogenes H-ras and v-myc transformed into rat embryo cell lines were proved to be highly metastatic and over-expressed MMP-9 (Himelstein et al., 1997). Cells derived from human lung cancer treated with GCSF, GM-CSF, M-CSF demonstrate an enhanced production of speciﬁc uPA and MMP-9, which was responsible for the subsequent increases in metastatic potential (Pei et al., 1999). Blocking the ERK1/2 or p38-MAPK pathway alone contributes to a decrease in MMP-9 expression and subsequently suppresses the malignancy of renal cancer or squamous cell cancer (Simonc et al., 1998; Hong et al., 2005). Recently, Roelle et al. (2003) have conﬁrmed for the ﬁrst time an expanded role for MMP-9 in the endocrine promotion of pituitary tumors (Roelle et al., 2003). According to their research, the GnRHinduced Src, Ras, and ERK activation and the enhanced crosstalk between GnRHR and EGFR in gonadotropic cells were gelatinase-dependent on the c-Jun NH2-terminal kinase, but not the extracellular signal-regulated kinase1/2 or p38-MAPK. The signaling pathway was sup- Fig. 3. Immunohistochemical expression of CD34 in samples from gastric adenocarcinoma patients: (A) Immunohistochemical expression of CD34 in vascular endothelial cells in gastric carcinoma when MVD 54.90/mm2 (magniﬁcation 200); (B) Immunohistochemical expression of CD34 in vascular endothelial cells in gastric carcinoma when MVD <54.90/mm2 (magniﬁcation 200). posed to be critical for GnRH-mediated upregulation of MMP-9, cell invasion, and motility. On the other hand, the metastasis-association hypothesis was supported by other studies of gene-expression signatures, which suggested MMP-9 was among the 70 genes comprising a gene signature that are capable of predicting distant metastasis in lymph node-negative breast cancer patients (van’t et al., 2002). Likewise, gastric carcinomas coincide with a notably upregulated MMP-9 expression. In fact, poorer survival rates were found in patients with elevated plasma MMP-9 levels compared with patients with normal MMP-9 plasma levels. However, MMP-9 in serum did not correlate well with the invasiveness or survival time of gastric cancer (Wu et al., 2007). As uncontrolled ECM remodeling of basement membranes is a common feature of metastasis, we compared MMP-9 mRNA expression in human gastric cancer tissues with nontumor mucosae using in situ hybridization. A signiﬁcantly enhanced production of MMP-9 was observed in cancer tissues, especially those tumors characteristic of the inﬁltrating type, at stage T3–T4, with vessel or lymphatic invasion, and with hepatic or EXPRESSION OF (MMP-9) mRNA AND VEGF PROTEIN Fig. 4. Survival curves with positive and negative MMP-9 mRNA expression in gastric adenocarcinoma (P < 0.001). Fig. 5. Survival curves with positive and negative VEGF expression in gastric adenocarcinoma (P < 0.001). peritoneal metastasis. This aberrant proteinase upregulation may represent an important biological mechanism responsible for the increasing malignant behavior of gastric cancer. Tumors require angiogenesis to continue to grow. One critical event implicated in the migration and proliferation of vascular endothelial cells is the proteolytic degradation of basement membranes and ECM components by matrix metalloproteinases (MMPs). In this study, MMP9 mRNA in the gastric cancer tissues was upregulated and coincided with VEGF expression and microvascular density. These ﬁndings support an emerging notion that MMP-9 is a positive regulator of tumoral angiogenesis (Cox et al., 2000). Hawinkels et al. (2008) showed that 2017 Fig. 6. Survival curves with MVD < 54.90/mm2 and 54.90/mm2 in gastric adenocarcinoma (P < 0.001). neutrophil-derived MMP-9 was able to release the biologically active VEGF165 from the ECM of colon cancer once liberated from heparan sulfates (Hawinkels et al., 2008). Heissig et al. (2002) found MMP-9/ mice given 5-FU treatment showed a delayed hematopoietic recovery compared with the MMP-9þ/þ mice. MMP-9 secreted within the bone marrow altered the stem cell-stromal cell interaction in the microenvironment. These effects of MMP-9 have been shown to be enhanced by SDF-1, VEGF, and G-CSF (Lévesque et al., 2003; Xu et al., 2005). The cleavage of vascular cell adhesion molecule-1, expressed by BM stromal cells, and the release of the soluble c-kit ligand from a membrane bound state, has been largely implicated in promoting stem cell differentiation, accelerating hematopoietic reconstitution, as well as enhancing their mobilization into peripheral circulation. Consequently, these properties suggest a key role for the proteolytic cleavage of some cytokines in vasculogenesis (Jeon et al., 2007). In conclusion, our study identiﬁed that gastric cancer mucosae produced a signiﬁcantly higher level of MMP-9 mRNA compared with healthy ones. The positive rates of MMP-9 expression were remarkably elevated in tumor features associated with the inﬁltrating type, at stage T3–T4, with vessel invasion, lymphatic metastasis, and hepatic or peritoneal metastasis when compared with the expanding type of gastric cancer, at stage T1–T2, with nonvessel invasion, without lymphatic metastasis, without hepatic and peritoneal metastasis. Tumors with different differentiations did not generate such a distinction. On the other hand, evidence from immunohistochemistry revealed a positive correlation between the expression proﬁles of MMP-9, VEGF, and CD34, representing a vivid tumor vascular growth pattern. To explore the survival inﬂuence of those molecules, Kaplan–Meier survival analysis was then performed. The curves indicated that gastric tumors expressing high levels of MMP-9 mRNA, and VEGF and CD34 protein were more inclined to become invasive. 2018 YANG ET AL. TABLE 2. Correlation of MMP-9 mRNA, VEGF protein, and MDV with survival period N MMP-9 mRNA VEGF MVD þþþþ þþþþ <54.90 54.90 47 71 54 64 47 71 Mean survival period (months) 88.38 40.24 114.53 32.13 122.61 30.58 6.37 5.95 6.66 4.25 6.63 3.75 Consequently, patients with these types of tumors had a poorer 5-year survival rate. In light of the earlier observations, we believe MMP-9 and VEGF may serve as useful molecular biomarkers to predict the aggressiveness of gastric carcinoma and to determine the prognosis of tumors which express it. Therapeutic interventions aimed at this two promising targets are worth exploring in future longitudinal studies. However, most clinical trials of anti-MMPs compounds to date have failed to obtain satisfactory effects. Even so, we can still draw some inspirations from the updated attempts. For example, In the study of Kunigal, antisense Ad-MMP-9 were successfully applied to decrease the levels of transcription factors nuclear factor—kappa B and activator protein, both of which participated in the triggering of the Fas-Fas ligand apoptotic cascade, and ﬁnally overcame the radiotherapy resistance of breast cancer (Kunigal et al., 2008). Thus, to solve this problem of poor response, multiple transcription factor consensus binding motifs in the regulatory regions, including those for SP-1, Ets, AP-1, and REB, might be the logical candidates for targeting (Himelstein et al., 1997; Ma et al., 2001). Taken together, MMPs is such a large family that the functions of which are so complex that far beyond our current understanding. Considering that expression of MMPs is mainly controlled at the level of transcription, and a number of transcriptional sites have already been characterized now (Ahn et al., 2008; Kim et al., 2008). Novel strategies to target this type of proteinase more selectively or to design drugs against critical transduction signals or key gene expression during the progression of gastric cancer may provide some advantages for the survival of patients with gastric carcinoma. LITERATURE CITED Ahn JS, Kim MK, Hahn JH, Park JH, Park KH, Cho BR, Park SB, Kim DJ. 2008. Tissue transglutaminase-induced down-regulation of matrix metalloproteinase -9. Biochem Biophys Res Commun 376:743–747. Bendeck MP, Irvin C, Reidy M, Smith L, Mulholland D, Horton M, Giachelli CM. 2000. Smooth muscle cell matrix metalloproteinase production is stimulated via alpha(v)beta(3) integrin. Arterioscler Thromb Vasc Biol 20:1467–1472. Chantrain CF, Shimada H, Jodele S, Groshen S, Ye W, Shalinsky DR, Werb Z, Coussens LM, DeClerck YA. 2004. Stromal matrix metalloproteinase-9 regulates the vascular architecture in neuroblastoma by promoting pericyte recruitment. Cancer Res 64: 1675–1686. v2 P 32.71 <0.001 44.44 <0.001 57.49 <0.001 5-year survival rate (%) 83.0 28.2 70.4 10.9 95.7 19.7 (39/8) (20/51) (38/16) (17/47) (45/2) (12/59) v2 P 33.98 <0.001 43.85 <0.001 63.38 <0.001 Cox G, Jones JL, O’Byrne KJ. 2000. Matrix metalloproteinase 9 and the epidermal growth factor signal pathway in operable non-small cell lung cancer. Clin Cancer Res 6:2349–2355. Crew KD, Neugut AI. 2006. Epidemiology of gastric cancer. World J Gastroenterol 12:354–362. Greene FL, Page DL, Fleming ID, Fritz A, Balch CM, Haller DG, Morrow M. 2002. Stomach. In: American Joint Committee on Cancer. AJCC cancer staging manual. 6th ed. New York, NY: Springer. p 99–106. Hawinkels LJ, Zuidwijk K, Verspaget HW, de Jonge-Muller ES, van Duijn W, Ferreira V, Fontijn RD, David G, Hommes DW, Lamers CB, Sier CF. 2008. VEGF release by MMP-9 mediated heparan sulphate cleavage induces colorectal cancer angiogenesis. Eur J Cancer 44:1904–1913. Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett NR, Crystal RG, Besmer P, Lyden D, Moore MA, Werb Z, Raﬁi S. 2002. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell 109:625–637. Himelstein BP, Lee EJ, Sato H, Seiki M, Muschel RJ. 1997. Transcriptional activation of the matrix metalloproteinase-9 gene in an H-ras and v-myc transformed rat embryo cell line. Oncogene 14:1995–1998. Hiratsuka S, Nakamura K, Iwai S, Murakami M, Itoh T, Kijima H, Shipley JM, Senior RM, Shibuya M. 2002. MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lungspeciﬁc metastasis. Cancer Cell 2:289–300. Hong S, Park KK, Magae J, Ando K, Lee TS, Kwon TK, Kwak JY, Kim CH, Chang YC. 2005. Ascochlorin inhibits matrix metalloproteinase-9 expression by suppressing activator protein-1-mediated gene expression through the ERK1/2 signaling pathway: inhibitory effects of ascochlorin on the invasion of renal carcinoma cells. J Biol chem 280:25202–25209. Jeon SH, Chae BC, Kim HA, Seo GY, Seo DW, Chun GT, Kim NS, Yie SW, Byeon WH, Eom SH, Ha KS, Kim YM, Kim PH. 2007. Mechanisms underlying TGF-beta1-induced expression of VEGF and Flk-1 in mouse macrophages and their implications for angiogenesis. J Leukoc Biol 81:557–566. Kim DS, Jeon OH, Lee HD, Yoo KH, Kim DS. 2008. Integrin alphavbeta3-mediated transcriptional regulation of TIMP-1 in a human ovarian cancer cell line. Biochem Biophys Res Commun 377:479–483. Kunigal S, Lakka SS, Joseph P, Estes N, Rao JS. 2008. Matrix metalloproteinase-9 inhibition down-regulates radiation-induced nuclear factor-kappa B activity leading to apoptosis in breast tumors. Clin Cancer Res 14:3617–3626. Lévesque JP, Hendy J, Winkler IG, Takamatsu Y, Simmons PJ. 2003. Granulocyte colony-stimulating factor induces the release in the bone marrow of proteases that cleave c-KIT receptor (CD117) from the surface of hematopoietic progenitor cells. Exp Hematol 31:109–117. Ma Z, Qin H, Benveniste EN. 2001. Transcriptional suppression of matrix metalloproteinase-9 gene expression by IFN-c and IFNbeta: critical role of STATS-l alpha. J Immunol 167:5150–5159. Nagase H, Visse R, Murphy G. 2006. Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc Res 69:562–573. Pei XH, Nakanishi Y, Takayama K, Bai F, Hara N. 1999. Granulocyte, granulocyte-macrophage, and macrophage colony- EXPRESSION OF (MMP-9) mRNA AND VEGF PROTEIN stimulating factors can stimulate the invasive capacity of human lung cancer cells. Br J Cancer 79:40–46. Roelle S, Grosse R, Aigner A, Krell HW, Czubayko F, Gudermann T. 2003. Matrix metalloproteinases 2 and 9 mediate epidermal growth factor receptor transactivation by gonadotropin-releasing hormone. J Biol Chem 278:47307–47318. Simon C, Goepfert H, Boyd D. 1998. Inhibition of the p38 mitogenactivated protein kinase by SB 203580 blocks PMA-induced Mr 92,000 type IV collagenase secretion and in vitro invasion. Cancer Res 58:1135–1139. van ’t Veer LJ, Dai H, van de Vijver MJ, He YD, Hart AA, Mao M, Peterse HL, van der Kooy K, Marton MJ, Witteveen AT, Schreiber 2019 GJ, Kerkhoven RM, Roberts C, Linsley PS, Bernards R, Friend SH. 2002. Gene expression proﬁling predicts clinical outcome of breast cancer. Nature 415:530–536. Wu CY, Wu MS, Chiang EP, Chen YJ, Chen CJ, Chi NH, Shih YT, Chen GH, Lin JT. 2007. Plasma matrix metalloproteinase-9 level is better than serum matrix metalloproteinase-9 level to predict gastric cancer evolution. Clin Cancer Res 13:2054–2060. Xu M, Bruno E, Chao J, Huang S, Finazzi G, Fruchtman SM, Popat U, Prchal JT, Barosi G, Hoffman R; MPD Research Consortium. 2005. Constitutive mobilization of CD34þ cells into the peripheral blood in idiopathic myeloﬁbrosis may be due to the action of a number of proteases. Blood 105:4508–4515.