Effect Of Non-anticoagulant N-desulfated Heparin On Expression Of Basic Fibroblast Growth Factor In Vivo And Vitro, Angiogenesis And Metastasis Of Orthotopic Implantation Of Human Gastric Carcinoma

Gastric cancer is the common alimentary tract cancer in China in terms of incidence. It is one of the malignancies that do serious harm to people's health with a high mortality and is short of effective therapeutic methods. Heparin, a highly sulfated proteoglan, has been extensively used as an anticoagulant drug for a long time. Aside from its anticoagulant action, heparin binds to various growth factors, cytokines, and extracellular proteins and consequently is able to affect migration of cancer cells and angiogenesis in tumors. However, clinical use of heparin in treatment of tumor is limited by its strong anticoagulant activity, which may cause severe bleeding complications.Chemically modified heparin shows a significantly reduced anticoagulant activity and enhanced ability to interact with FGF, VEGF and hepatocyte growth factor, which are known to stimulate angiogenesis. In this study, we investigated the effect of N-desulfated heparin on expression of bFGF in vitro and in vivo, using ELISA and RT-PCR method.PART I Inhibition of N-deslfated heparin on bFGF gene expression and tumor angiogenesis of orthotopic implantation of human gastric carcinomaObjective To investigate the effect of N-desulfated heparin on tumor metastasis,tumor angiogenesis and basic fibroblast growth factor(bFGF) gene expression of orthotopically implatated human gastric carcinoma in NODSCID mice. Methods Human gastric cancer SGC-7901 tissues were orthotopically implanted into the stomach of the NODSCID mice. Twenty mice were randomly divided into two groups which received either intravenous injection of 0.9% NaCl solution(0.9%NaCl solution group) or 10mg/kg N-desulfated heparin(N-desulfated heparin group ) twice weekly for three weeks. Mice were sacrificed six weeks after implantation. Tissues from stomach and other organs were obtained for hisopathological evaluation .The intratumoral microvessel density(MVD) in tumor was evaluated immunochemically. Real time PCR technique was used to detect bFGF mRNA expression.Results:The tumor metastasis rates were 9/10 in 0.9% NaCl solution group and 2/10 in N-desulfated heparin group(P<0.05).MVD was 9.1±3.4 in 0.9% NaCl solution group and 4.7±1.8 in N-desulfated heparin group (t=3.617,P<0.05).bFGF mRNA expression was lower in N-desulfated heparin group (2.60±0.56% ) than that in 0.9% NaCl solution group(30.65±6.84%).Conclusions: N-desulfated heparin can inhibit the metastasis of gastric cancer through inhibiting tumor bFGF gene expression and tumor angiogenesis with no obvious anticoagulant activity.PART II Inhibition of N-desulfated heparin on bFGF expression of SGC-7901 gastric carcinoma cellAIM: To investigate the effect of N-desulfated heparin on bFGF expression of human gastric carcinoma SGC-7901 cells in vitro. METHODS: In vitro human gastric carcinoma SGC-7901 cells were treated with N-desulfated heparin in different concentration (0.1,1mg/ml, N-desulfated heparin group), and treated with medium (control group), 3 samples each of the parallel. ELISA and real time PCR method were used to detect the bFGF(basic fibroblast growth factor) expression of human gastric tumor cells at 12h and 24 h, respectively. RESULTS: ELISA method results:In the control group, the bFGF expression of human gastric cells was significantly increased with the extension of time in vitro (12h vs 24h,P<0.05 ) .Compared with control group, bFGF expression was decreased significantly in different concentration (0.1,1mg/ml, N-desulfated heparin group) . Treated with 0.1 1mg/ml N-desulfated heparin, bFGF expression was decreased significantly(12h vs 24h , P<0.05). Moreover, treated with 1mg/ml N-desulfated heparin for 12,24h, bFGF expression was decreased significantly(P<0.05), compared with 0.1 mg/ml N-desulfated heparin. Real time PCR method: in each of N-desulfated heparin groups, bFGF mRNA expression was decreased compared with control group (P<0.05). The inhibitory effect of N-desulfated heparin on bFGF mRNA expression of human gastric carcinoma cells in vitro was with dose 1mg/ml>0.1mg/ml, suggesting that it had dose-dependent effects. In the same N-desulfated heparin concentration, the expression of bFGF mRNA at 24h was lower than that at 12h (P<0.05, 1mg/ml, N-desulfated heparin group), suggesting that the inhibition of N-desulfated heparin on bFGF mRNA expression of human gastric carcinoma cells had time-dependent effects. CONCLUSION: N-desulfated heparin can inhibit bFGF expression of human gastric carcinoma cells in vitro, and with dose, time-dependent.