Hong-Xia Li, Xin-Ming Chang, Zheng-Jun Song, Shui-Xiang He, Department of gastroenterology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, Shaanxi Province, China
Correspondence to: Dr.Hong-Xia Li, Department of Gastroenterology, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, Shaanxi Province, China. hx1105sina.com
Telephone: +86-29-5324101
Received: 2002-10-09 Accepted: 2002-11-09

Abstract
AIM: To evaluate the expression of cyclooxygenase (COX-2) and the relationship with tumor angiogenesis and advancement in gastric adenocarcinoma.
METHODS: Immunohistochemical stain was used for detecting the expression of COX-2 in 45 resected specimens of gastric adenocarcinoma; the monoclonal antibody against CD34 was used for displaying vascular endothelial cells, and microvascular density (MVD) was detected by counting of CD34-positive vascular endothelial cells. Paracancerous tissues were examined as control.

RESULTS: Immunohistological staining with COX-2-specific polyclonal antibody showed cytoplasmic staining in the cancer cells, some atypical hyperplasia and intestinal metaplasia,as well as angiogenic vasculature present within the tumors and prexisting vasculature adjacent to cancer lesions. The rate of expression of COX-2 and MVD index in gastric cancers were significantly increased, compared with those in the paracancerous tissues (77.78 vs 33.33 %, 58.13±19.99 vs 24.02±10.28, P<0.01, P<0.05, respectively). In 36 gastric carcinoma specimens with lymph node metastasis, the rate of COX-2 expression and MVD were higher than those in the specimens without metostasis (86.11 vs 44.44 %, 58.60±18.24 vs 43.54±15.05, P<0.05, P<0.05, respectively).The rate of COX-2 expression and MVD in the specimens with invasive serosa were significantly higher than those in the specimens without invasion to serosa (87.88 vs 50.0 %, 57.01±18.79 vs 42.35±14.65, P<0.05, P<0.05). Moreover, MVD in COX-2-positive specimens was higher than that in COX-2-negative specimens (61.29±14.31 vs 45.38±12.42,P<0.05). COX-2 expression was positively correlated with MVD (r=0.63, P<0.05).

CONCLUSION: COX-2 expression might correlate with the occurance and advancement of gastric carcinoma and is involved in tumor angiogenesis in gastric carcinoma. It is likely that COX-2 by inducing angiogenesis can be one of mechanisms which promotes invasion and metastasis of gastric carcinoma. It may become a new therapeutic target for anti-angiogenesis.

Li HX, Chang XM, Song ZJ, He SX. Correlation between expression of cyclooxygenase-2 and angiogenesis in human gastric adenocarcinoma. World J Gastroenterol 2003; 9(4): 674-677
http://www.wjgnet.com/1007-9327/9/674.htm


INTRODUCTION
COX is a key enzyme in the conversion of arachidonic acid to prostaglandin, and two isoforms of COX, namely COX-1 and COX-2, have been identified^[1,2]. COX-1 is constitutively expressed in many tissues and is considered to be involved in various physiological functions, whereas COX-2 is induced by pathological stimuli, such as inflammation, various growth factors and cytokines produced by tumor cells^[1-3].
   Epidemiologic studies showed that nonsteroidal anti-inflammatory drugs (NSAIDs), known to inhibit COX, could reduce the incidence rate and mortality from digestive tract carcinomas^[4-10]. In rodent models of FAP, a genetic disease leading to colonic carcinoma, blockade of COX-2, suppresses intestinal polyp formation^[11]. Increased COX-2 expression has been reported in colorectal, pancreatic, hepatocellular and other cancers^[12-20]. Taken together, these data provide strong evidence for the importance of COX-2 in oncogenesis.
   It has been reported that tumor angiogenesis play an important role in tumor growth, invasion and metastasis^[21-25].We investigated the expression of COX-2, MVD in human gastric cancer. The aim of this study was to determine the relationship between COX-2 and tumor angiogenesis,and the development, progression of gastric cancer. The further understanding of oncogenesis might provide a new approach to tumor therapy.

MATERIALS AND METHODS
Materials
45 patients with gastric adenocarcinomas confirmed pathologically underwent gastrectomy in our hospital from January 2000 to October 2001. From these subjects, gastric tumor and paracancerous tissues (more than 5 cm away from the lesion) were obtained from resected specimen. Among them, 35 were male, and 10 female, with a mean age of 57.51±10.73 (33 to 78). Patients who had received radiotherapy or chemotherapy before gastrectomy were excluded.Histologically, they were classified by the WHO criteria, 5 were highly differentiated adenocarcinoma, 10 moderately-differentiated, 27 poorly-differentiated, 3 undifferentiated. As regards to the size of cancer, 20 were <5 cm, 25≥5 cm. 33 tumors invaded to the serosa and 12 tumors not.36 cases had local lymph node metastasis.

COX-2, MVD expression and distribution
77.78 % (35/45) cases of gastric carcinomas showed COX-2 positive expression while high-expression was detected in 22 cases, low-expression in 13. 33.33 % (15/45) cases of paracancerous tissues showed COX-2 positive expression while high-expression was only detected in 3 cases. The rate and density of COX-2 expression in cancerous tissues were significantly higher that in paracancerous tissues (x²=18, x²=6.09, P<0.005, P<0.05, respectively). The positive COX-2 staining was mainly diffusely located as brownish yellow stained granules in the cytoplasm. Immunohistological analysis revealed cytoplasmic staining in the neoplastic cells (Figure1),atypical hyperplasia and intestinal metaplasia. In addition,COX-2 was also detected in the angiogenic vasculature present within the tumors and preexisting vasculature adjacent to cancer lesions (Figure 2). In contrast, normal epithelium or stroma occasionally showed weak staining pattern or didn't.
   The mean MVD in gastric carcinoma was significantly higher than that in para-cancerous tissues (58.13±19.99, 24.02±10.28, t=10.18, P<0.001). The positive expression of CD34 was mainly presented as brownish yellow or brownish granules in the cytoplasm of vascular endothelial cell (Figure 3). New blood vessles in the cancerous lesions had no regular contour and were not evenly distributed.

The relationship between the rate of COX-2 expression and MVD
The result showed that MVD (61.29±14.31) in the COX-2-positive gastric cancerous tissues was higher than that (45.38±12.43) in the COX-2-negative one (t=5.64, P<0.001). The expression of COX-2 was positively correlated with MVD (r=0.63, P<0.05).

The relationship between the expression of COX-2, MVD and pathological features of gastric carcinoma
In Table 1, the associations between COX-2, MVD expression and the pathological features were shown. Both COX-2 and MVD were not correlated with tumor size, tumor histological type. However, there was correlation between COX-2, MVD and depth of invasion and lymph-node metastasis of gastric carcinoma respectively.

Figure 1 COX-2 expression in gastric adenocarcinoma.
Figure 2 COX-2 expression was also detected in the vasculature within gastric adenocarcinoma.
Figure 3 CD34 expression in the cytoplasm of vascular endothelial cell within gastric adenocarcinoma.

Table 1 The relationship between COX-2, MVD and pathological features of gastric carcinoma

DISCUSSION
Human gastric mucosa normally expresses no detectable levels of COX-2 protein^[27,28]. In the current study, we found that the rate of COX-2 expression in gastric cancer was significantly increased, compared with that in the paracancerous tissues, the expression of COX-2 showed cytoplasmic staining, not only in cancerous cells but also in precancerous lesion such as atypical hyperplasia and intestinal metaplasia. A similar pattern of COX-2 expression has previously been found in human gastric cancer^[29-34]. The above data demonstrated that COX-2 was up-regulated in human gastric cancer, suggesting COX-2 may play an important role in occurrence of gastric cancer,being a relatively early event in the carcinogenesis of stomach.
   Here, we also analyzed the relationship between COX-2 expression and clinical pathological features in gastric carcinoma. It was shown that the rate of COX-2 expression was correlated closely with the depth of tumor invasion, indicating COX-2 may contribute to invasive growth of gastric carcinoma. The rate of COX-2 expression of gastric carcinoma with lymph-node metastasis was higher than that without suggesting the increase of its expression in gastric cancer tissue can promote lymph-node metastasis. It seemed more likely that COX-2 probably heightened viability and increased infiltrative potential of gastric cancer. The mechanism was not clear. Tsujii concluded that overexpression of the COX-2 gene as a result of transfection promoted invasiveness in wild type human colon carcinoma cell lines through the induction of metalloproteinase-2 and membrane-type metalloproteinase^[35]. Rat intestinal epithelial cells that overexpressed COX-2 protein were found to be resistant to butyrate-induced apoptosis and had elevated bcl-2 protein expression and decrease expression of both E-cadherin and the transforming growth factor-b receptor^[36]. Each of these changes has been linked to enhanced tumorigenic potential and increased tumor invasiveness. Therefore, the above data further indicated that COX-2 might play an important role in gastric tumorigenesis and tumor progression.
   Recently the relation of COX-2 and tumor angiogenesis is emphasized. One of the mechanisms by which PGE₂ supports tumor growth is by inducing the angiogenesis necessary to supply oxygen and nutrients to tumors >2 mm in diameter^[37,38]. Masferrer^[39] reported that SC-236, a COX-2-selective inhibitor, was effective in reducing angiogenesis driven by bFGF in the matriel rat model, whereas SC-560, a COX-1-selective inhibitor was ineffective. He also observed COX-2 expression in newly formed blood vessels within tumors grown in animals, whereas under normal physiological condibions the quiescent vasculature expressed only the COX-1 enzyme, indicating COX-2-derived prostaglandins contributed to tumor angiogenesis^[40]. In our study, COX-2 expression was also detected in the angiogenic vasculature present within the tumors and preexisting vasculature adjacent to cancer lesions, suggesting COX-2 may induce newly formed blood vessels to sustain tumor cell viability and growth. COX-2 was also expressed within atypical hyperplasia, intestinal metaplasia and neovasculature in the paracancerous tissue, indicating COX-2 may promote precancerous lesion to cancer by new blood vessel formation.
   MVD is a reliable index of tumor angiogenesis^[41]. We found that the MVD in COX-2 positive tumors was significantly higher than that in COX-2 negative tumors, MVD in gastric carcinoma was higher than that in paracancerous tissues, suggesting its distribution was similar to the pattern of COX-2 in gastric carcinoma. A close correlation was present between MVD and COX-2 (P<0.01), indicating COX-2 was closely related to tumor angiogenesis further, and may be one of important factors involved in gastric carcinoma angiogenesis. In addition, MVD in the specimens with lymph node metastasis was significantly higher than that without and it was also correlated closely with the depth of tumor invasion,suggesting that tumor angiogenesis in gastric carcinomas might result in cancer cells entering blood circulation, and the lymph node metastasis could be promoted when the gastric cancer cells invade lymphatic vessels. Both COX-2 and MVD were associated with the depth of invasion and lymph-node metastasis, suggesting the effect of COX-2 on angiogenesis can promote metastatic protential as well as tumor invasiveness. Therefore, inducing tumor angiogenesis may be one of mechanisms which COX-2 promotes the development and metastasis of gastric cancer.
   In conclusion, COX-2 expression in gastric adenocarcinoma was higher than that in the paracancerous tissues, and was related to lymph node metastasis and the depth of invasion, suggesting COX-2 might correlate with the occurance and advancement of gastric carcinoma; COX-2 expression in gastric carcinoma was closely related to MVD, suggesting COX-2 might be involved in tumor angiogenesis in gastric carcinoma, it is likely that COX-2 inducing angiogenesis may be one of mechanisms which COX-2 promotes the invasion, metastasis of tumor in gastric carcinoma. These findings suggest that COX-2 may be a new therapeutic target for anti-angiogenesis.

REFERENCES
1    Williams CS, DuBois RN. Prostaglandin endoperoxide synthase: why two isoforms? Am J Physiol 1996; 270: G393-G400
2    Eberhart CE, DuboisRN. Eicosanoids and the gastrointestinal tract. Gastroenterology 1995; 109: 285-301
3    DuBois RN, Awad J, Morrow J, Roberts LJ 2nd, Bishop P. Regulation of eicosanoid production and mitogenesis in rat
      intestinal epithelial cells by tranasforming growth factor-alpha and phorbol ester. J Clin Inves1994; 93: 493-498
4    Farrow DC, Vaughan TL, Hansten PD, Stanford JL, Risch HA, Gammon MD, Chow WH, Dubrow R, Ahsan H, Mayne
      ST, Schoenberg JB, West AB, Rotterdam H, Fraumeni JF Jr, Blot WJ.Use of aspirin and other nonsteroidal
      antiinflammatory drugs and risk of esophageal and gastric cancer. Cancer Epidemiol Biomarkers Prev 1998; 7: 97-102
5    Garcia-Rodriguez LA, Huerta-Alvarez C. Reduced risk of colorectal cancer among long-term users of aspirin and
      nonsteroidal antiinflammatory drugs. Epidemiology 2001; 12: 88-93
6    Morgan G. Non-steroidal anti-inflammatory drugs and the chemoprevention of colorectal and nonaspirin oesophageal
      cancers. Gut 1996; 38: 646-648
7    Reeves MJ, Newcomb PA, Trentham-Dietz A, Storer BE, Remington PL. Nonsteroidal anti-inflammatory drug use
      and protection against colorectal cancer in women. Cancer Epidemiol Biomarkers Prev 1996; 5: 955-960
8    Thun MJ. NSAID use and decreased risk of gastrointestinal cancers. Gastroenterol Clin North Amer1996; 25: 333-348
9    Funkhouser EM, Sharp GB. Aspirin and reduced risk of esophageal carcinoma. Cancer 1995; 76: 1116-1119
10  Zhuang ZH, Wang LD. NSAID and digestive tract carcinomas.Shijie Huaren Xiaohua Zazhi 2001; 9: 1050-1053
11  Oshima M, Dinchuk JE, Kargman SL, Oshima H, Hancock B, Kwong E, Trzaskos JM, Evans JF, Taketo MM. Suppression
      of intestinal polyposis in Apc deta716 knockout mice by inhibition of cyclooxygenase 2(COX-2). Cell 1996; 87: 803-809
12  Tsujii M, Kawano S, DuBois RN. Cyclooxygenase-2 expression in human colon cancer cells increase metastatic potential.
      Proc Natl Acad Sci USA 1997; 94: 3336-3340
13  Chan G, Boyle JO, Yang EK, Zhang F, Sacks PG, Shah JP, Edelstein D, Soslow RA, Koki AT, Woerner BM, Masferrer
      JL, Dannenberg AJ. Cyclooxygenase-2 expression is up-regulated in squamous cell carcinome of the head and neck. Cancer
      Res 1999; 59: 991-994
14  Tucker ON, Dannenberg AJ, Yang EK, Zhang F, Teng L, Daly JM, Soslow RA, Masferrer JL, Woerner BM, Koki AT, Fahey
      TJ. Cyclooxygenase-2 expression is up-regulated in human pancreatic cancer.Cancer Res 1999; 59: 987-990
15  Zimmermann KC, Sarbia M, Weber AA, Borchard F, Gabbert HE, Schror K. Cyclooxygenase-2 expression in human
      esophageal carcinoma. Cancer Res 1999; 59: 198-204
16  Shiota G, Okubo M, Noumi T, Noguchi N, Oyama K, Takano Y, Yashima K, Kishimoto Y, Kawasaki H. Cyclooxygenase-
      2 expression in hepatocellular carcinoma. Hepatogastroenterology 1999; 46: 407-412
17  Shirahama T, Sakakura C. Overexpression of cyclooxygenase-2 in squamous cell carcinoma of the urinary bladder. Clin
      Cancer Res 2001; 7: 558-561
18  Soslow RA, Dannenberg AJ, Rush D, Woerner BM, Khan KN, Masferrer J, Koki AT. COX-2 is expressed in
      human pulmonary,colonic,and mammary tumors. Cancer 2000; 89: 2637-2645
19  Wu QM, Li SB, Wang Q, Wang DH, Li XB, Liu CZ. The expression of COX-2 in esophageal carcinoma and its relation
      to clinicoptahologic characteristics. Shijie Huaren Xiaohua Zazhi 2001; 9: 11-14
20  Qiu DK, Ma X, Peng YS, Chen XY. Significance of cyclooxygenase-2 expression in human primary hepatocellular
      carcinoma. World J Gastroenterol 2002; 8: 815-817
21  Che X, Hokita S, Natsugoe S, Tanabe G, Baba M, Takao S, Aikou T. Tumor angiogenesis related to growth pattern and
      lymph node metastasis in early gastric cancer. Chin Med J 1998; 111: 1090-1093
22  Shimoyama S, Kaminishi M. Increased angiogenin expression in gastric cancer correlated with cancer progression. J
      Cancer Res Clin Oncol 2000; 126: 468-474
23  Yoshikawa T, Yanoma S, Tsuburaya A, Kobayashi O, Sairenji M, Motohashi H, Noguchi Y. Angiogenesis inhibitor,
      TNP-470,suppresses growth of peritoneal disseminating foci.Hepatogastroenterology 2000; 47: 298-302
24  Xiangming C, Hokita S, Natsugoe S, Tanabe G, Baba M, Takao S, Kuroshima K, Aikou T. Angiogenesis as an unfavorable
      factor related to lymph node metastasis in early gastric cancer. Ann Surg Oncol 1998; 5: 585-589
25  Maehara Y, Hasuda S, Abe T, Oki E, Kakeji Y, Ohno S, Sugimachi K. Tumor angiogenesis and micrometastasis in
      bone marrow of patients with early gastric cancer.Clin Cancer Res 1998; 4: 2129-2134
26  Weidner N, Folkman J, Pozza F, Bevilacqua P, Allred EN, Moore DH, Meli Gasparini G. Tumor anginogenesis:a new
      significant and independent prognostic indicator in early state breast carcinoma. J Natl Cancer Inst 1992; 84: 1875-1887
27  Mizuno H, Sakamoto C, Matsuda K, Wada K, Uchida T, Noguchi H, Akamatsu T, Kasuga M. Induction of cyclooxygenase 2
      in gastric mucosal lesions and its inhibition by the specific antagonigt delays healing in mice. Gastroenterology
      1997; 112: 389-397
28  Kargman S, Charleson S, Cartwright M, Frank J, Riendeau D, Mancini J, Evans J, O扤eill G. Characterization of
      prostaglandin G/H synthase 1 and 2 in rat, dog, monkey and human gastrointestinal tracts. Gastroenterology
      1996; 111: 445-454
29  Uefujk K, Ichikura T, Mochizuki H, Shinomiya N. Expression of cyclooxygenase-2 protein in gastric adenocarcinoma. J
      Surg Oncol 1998; 69: 168-172
30  Ratnasinghe D, Tangrea JA, Roth MJ, Dawsey SM, Anver M, Kasprzak BA, Hu N, Wang QH, Taylor PR. Expression
      of cyclooxygenase-2 in human adenocarcinomas of the gastric cardia and corpus. Oncel Rep 1999; 6: 965-968
31  Lim HY, Joo HJ, Choi JH, Yi JW, Yang MS, Cho DY, Kim H, Nam DK, Lee KB, Kim HC. Increased expression of cyclooxygenase-
      2 protein human gastric carcinoma. Chin Cancer Res 2000; 6: 519-525
32  van Rees BP, Saukkonen K, Ristimaki A, Polkowski W, Tytgat GN, Drillenburg P, Offerhaus GJ. Cyclooxygenase-2
      expression during carcinogenesis in the human stomach. J Pathol 2002; 196:171-179
33  Ohon R, Yoshinaga K, Fujita T, Hasegawa K, Iseki H, Tsunozaki H, Ichikawa W, Nihei Z, Sugihara K. Depth of invasion
      parallels increased cyclooxygenase-2 levels in patents with gastric carcinoma. Cancer 2001; 91: 1876-1881
34  Xue YW, Zhang QF, Zhu ZB, Wang Q, Fu SB. Expression of cyclooxygenase-2 and clinicopathologic features in human
      gastric adenocarcinoma. World J Gastroenterol 2003; 9: 250-253
35  Murata H, Kawano S, Tsuji S, Tsuji M, Sawaoka H, Kimura Y, Shiozaki H, Hori M. Cyclooxgenase-2 overexpressing
      enhances lymphatic invasion and metastasis in human gastric carcinoma. Am J Gastroenterology 1999; 94: 451-455
36  Tsujii M, DuBois RN. Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin
      endoperoxide synthase-2. Cell 1995; 83: 493-501
37  Form DM, Auerbach R. PGE2 and angiogenesis. Proc Soc Exp Biol Med 1983; 172: 214-218
38  Hanahan D, Folkman J. Patterns and emerging mechenisms of the angiogenic swithch during tumorigenesis. Cell
      1996; 86: 353-364
39  Masferrer JL, Koki A, Seibert K. COX-2 inhibitors.A new class of antiangiogentic agents. Ann N Y Acad Sci
      1999; 889: 84-86
40  Masferrer JL, Leahy KM, Koki AT, Zweifel BS, Settle SL, Woerner BM, Edwards DA, Flickinger AG, Moore RJ, Seibert
      K. Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors. Cancer Res 2000; 60: 1306-1311
41  Fukumura D, Jain RK. Role of nitric oxide in angiogenesis and microcirculation in tumors. Cancer Metastasis Rev
      1998; 17: 77-89