animal experiments. The biological functions closely depend on the activity of BDKRB2 gene. BDKRB2 (bradykinin receptor B2, NM-000623.3) is also termed B2R or BK-2 receptor. This gene encodes a receptor for bradykinin, which elicits many responses including vaso- dilation, edema, smooth muscle spasm and pain ﬁber stimulation. Some study revealed that bradykinin regulates the secretion and biosynthesis of endothelin-1 through kinin B2 receptor in melanoma cells (Andoh et al., 2010). Although BDKRB2 expression has been identiﬁed on some cancer tissues (Wu et al., 2002), little is known about its biological functions on gastric carcinoma, especially on IRX1-related anti-angiogenic mechanism. Our new ﬁnd- ings will facilitate the understanding of IRX1-mediated anti-cancer mechanisms on gastric cancer. Results Enforcing IRX1 on gastric cancer cells suppresses peritoneal spreading and lung metastasis in nude mice A total of 75 nude mice were enrolled in these experiments. Among them, 30 mice were used for IRX1 inﬂuences peritoneal spreading and metastasis J Jiang et al peritoneal spreading study and 45 for pulmonary metastatic study. The experimental results were summar- ized in Table 1. The peritoneal nodules were markedly suppressed in IRX1 overexpression group, compared with mock or controls (1.0±0.70 vs 4.4±1.14, Po0.001; Figure 1a). The tumorigenicity of pulmonary metastasis was also observed. In IRX1 overexpression group, 2 out of 15 mice show tumorigenicity in lung, which is signiﬁcantly lower than that in mock (11/15) and controls (10/15) (P ¼ 0.002; Figure 1b). Meanwhile, we assayed the microvessel density by CD34 immunohisto- chemical stain as well as VM density by periodic acid- schiff (PAS) histochemical stain. We found a signiﬁcant reduction of microvessel density and VM density in tumor tissues of SCG-7901/IRX1 group, compared with mock or controls (Figure 2). Enforcing IRX1 on gastric cancer cells suppresses tubular formation of HUVECs Human umbilical vein endothelial cells (HUVECs) (2 104 cells/well) were suspended in collected super- natants from SGC-7901, SGC-7901/vector and SCG- 7901/IRX1, respectively. After 18 h incubation at 37 1C 4499 Table 1 Animal experiments for peritoneal spreading and pulmonary metastasis Animal models Peritoneal spreading Pulmonary metastasis Cells lines SGC-7901 SGC-7901/vector SGC-7901/IRX1 SGC-7901 SGC-7901/vector SGC-7901/IRX1 Cell number 2 106 2 106 2 106 2 106 2 106 2 106 Time 4 weeks 4 weeks 4 weeks 2 months 2 months 2 months Tumorigenicity 4.4±1.14 4.2±0.83 1.0±0.70 11/15 10/15 2/15 P-value o0.001 0.002 Figure 1 Effect of enforcing IRX1 on peritoneal spreading and metastasis. A 0.1-ml suspension (2 106 cells/ml) per mouse was injected into peritoneal cavity or caudal vena. (a) The peritoneal nodules were markedly suppressed in IRX1 overexpression group, compared with mock or controls (1.0±0.70 vs 4.2±0.83, 4.4±1.14, *Po0.001). (b) Metastatic cancer mass is easily observed microscopically in mock or controls, compared with experimental animal (the tumor is indicated with arrow, the bar indicates 50 mm). In IRX1 overexpression group, 2 out of 15 mice show tumorigenicity in lung, which is signiﬁcantly lower than that in mock (11/15) and controls (10/15) (P ¼ 0.002). Oncogene