In contrast to the effect of COX-2 on angiogenesis, the

effects on lymphangiogenesis and lymphatic metastasis remain poorly understood. Recently, some studies have found that COX-2 expression is highly correlated with lymph node metastasis [20, 21]. Several lines of experimental evidence have shown that COX-2 might stimulate VEGFR-3 to promote lymphangiogenesis by up-regulating VEGF-C in breast and lung cancer cells [22, 23]. selleck products However, the role of COX-2 in lymphangiogenesis of gastric carcinoma remains unclear. Using immunohistochemistry, our study aimed to detect the expression of COX-2 and VEGF-C protein and the levels of lymphatic vessel density Combretastatin A4 in vitro (LVD) in human gastric cancer and analyze their correlations with clinicopathological characteristics and prognosis. Methods Patients and specimens Fifty-six patients with

histologically proven gastric adenocarcinoma and who underwent radical gastrectomy JNJ-26481585 order at West China Hospital, Sichuan University, China between January 2001 and October 2002, were included in the present investigation. In this investigation, paracancerous normal mucosal tissues from 25 patients were collected as a control. Patients undergoing neoadjuvant chemotherapy and/or radiotherapy were excluded. TNM staging was carried out according to the American Joint Committee on Cancer (AJCC) classification, and historical grading was performed according to WHO criteria. Paraffin-embedded, formalin-fixed surgical specimens were prepared and collected for immunohistochemical staining. Immunohistochemical staining Specimens were immunostained with the standard labeled streptavidin-biotin protocol. Briefly, after deparaffinization and antigen retrieval, 4-μm tissue sections were incubated with COX-2 antibodies (monoclonal rabbit anti-human, 1:100, Goldenbridge Biotechnology Co, Ltd, Beijing, China) and VEGF-C antibodies (polyclonal rabbit anti-human, 1:100, Goldenbridge Biotechnology Co., Ltd) at 37°C for 1 h then at 4°C overnight. The sections were then incubated with biotinylated goat anti-rabbit immunoglobulin G (1:200, Zymed Laboratories Inc, USA) and subsequently incubated with horseradish

labeled streptavidin Alanine-glyoxylate transaminase (1:200, Zymed Laboratories Inc). 3,3′-Diaminobenzidine was used as a chromogen and hematoxylin as a counterstain. For the staining of lymphatic vessels, a rabbit anti-human D2-40 polyclonal antibody (rabbit polyclonal, Dako Denmark A/S Co., Denmark) was used. The procedure for immunohistochemical staining of D2-40 is similar to that of the COX-2 staining at a dilution of 1:100. Evaluation of immunohistochemical staining The immunohistochemical score (IHS) based on the German immunoreactive score was used for COX-2 and VEGF-C immunohistochemical evaluation [24]. The IHS is calculated by combining the quantity score (percentage of positive stained cells) with the staining intensity score. The quantity score ranges from 0 to 4, i.e.