GBEE Inhibits Tumor Angiogenesis In Lewis Lung Cancer And Its Effect On Wnt/?-catenin-VEGF Signaling Pathway

Objective:Study on the activity of Ginkgo biloba exocarp extracts(GBEE)against tumor angiogenesis in vitro and vivo and its influence on Wnt/?-catenin-VEGF(vascular endothelial growth factor)signal pathways and PKB(protein kinase B)by culturing Lewis lung cancer(LLC)cell in vitro and establishing LLC metastasis model of C57BL/6J mice in vivo to reveal partial molecular mechanism of GBEE inhibition of angiogenesis.Methods:The effect of GBEE on proliferation of LLC cells was detected by MTT method in vitro.The mice LLC metastasis model was set up.The C57BL/6J mice were randomly separated into normal,model,positive and GBEE(50,100,200 mg/kg)treatment groups,n=10.Normal group did not receive tumor cell suspension.The mice in normal group and model group were both intragastric gavage(i.g.)normal saline(NS)in a volume of 0.1 mL/10 g(b.w.),positive group was intraperitoneal(i.p.)injection cyclophosphamide(CPA)at a dose of 20 mg/kg(b.w.),the GBEE treatment groups were respectively i.g.GBEE 50,100,and 200 mg/kg(b.w.),once a day for 20 d.After treatment,we calculated the tumor inhibition rate,lung metastasis rate and anti-metastasis rate.HE staining was used to observe the histology of lung and transplanted tumor tissues.The microvessel density(MVD)was measured by immunohistochemistry method in transplanted tumor.The expression levels of VEGF and VEGFR2(Vascular Endothelial Growth Factor Receptor 2)mRNA or Wnt,?-catenin,VEGF,VEGFR2 and p-PKB/PKB protein expression were respectively tested by qRT-PCR or western blot in vitro and vivo.Results:GBEE suppressed the growth of LLC cells in a concentration-dependent way at the concentrations of 5,10,20,40,80 and 160 ?g/mL in vitro.The LLC cells were treated with GBEE(10?20?40 ?g/mL)for 48 h,it can significantly suppress Wnt and ?-catenin protein expression and the content of mRNA of VEGF and VEGFR2 in LLC cells.In vivo,we discovered GBEE(50,100,200 mg/kg)can significantly suppress the growth of C57BL/6J mice LLC transplanted tumor and LLC lung metastasis,and reduce the expression of CD34,which means MVD was inhibited,all of which are in a dose-dependent way.What's more,GBEE can inhibit Wnt,?-catenin,VEGF,VEGFR2 and p-PKB/PKB protein expression and VEGF and VEGFR2 mRNA expression levels in LLC transplanted tumor.Conclusions:The effects of anti-tumor and anti-tumor metastasis by GBEE are in a dose-effect relationship in LLC tumor-bearing mice,depending upon the inhibition of tumor angiogenesis,which may be closely relevant to interfere the expression of Wnt/?-catenin signaling pathway and inhibit the expression of downstream target genes VEGF and VEGFR2,thus affecting the phosphorylation of PKB protein,which belong the downstream signal molecules of VEGF.