Involvement Of HIF-1?/Glycolysis In Growth Promotion Of Rat Vascular Endothelial Cells Induced By Roxarsone

Objective:Roxarsone(Rox),a kind of feed additive,is little absorbed by livestock and poultry,which most of it is excreted in faeces and enter into the environment increasing the risk of arsenic exposure in humans.In our previous studies,Rox could promote vascular endothelial cells growth in vitro and tumor angiogenesis in vivo.The up-regulated expression of hypoxia inducible factor-1?(HIF-1?)and mitochondrial dysfunction in vascular endothelial cells were found.HIF-1? regulated the expression of many related proteins in glycolysis.However,little information was focused on the involvement of glycolysis in the growth promotion of vascular endothelial cells and the angiogenesis induced by Rox at present.In the research,the cell viability,ATP content,lactic acid(LD)release and aldolase(ALD)level were measured based on the culture of rat vascular endothelial cells treated with Rox or HIF-1? related inhibitors in vitro and mouse B16F10 melanoma xenograft models in vivo.Western Blot and immunohistochemistry were used to detect the expressions of HIF-1? and glycolysis related proteins ALD A and glucose transporter 1(GLUT1).This paper aims to study the effects of glycolysis and HIF-1? in the growth promotion of vascular endothelial cells induced by Rox.Methods:In the experiments of rat vascular endothelial cells in vitro:?The treatment groups were as follows:control group(0?M Rox),groups of Rox treatment(0.1,1.0 and 10.0 ?M Rox).The proliferation activity,ATP content and LD release of rat vascular endothelial cells were measured.The activity and content of ALD were detected by ELISA and the expressions of ALD A and GLUT1 proteins were detected by Western Blot.?With HIF-1? related inhibitors,the treatment groups were as follows:control group(0?M Rox),1.0 ?M Rox group(1.0 ?M Rox),HIF-1? inhibitor group(50.0 ?M YC-1),co-treatment group 1(50.0 ?M YC-1+1.0 ?M Rox),PI3K inhibitor group(20.0 ?M LY294002)and co-treatment group 2(20.0 ?M LY294002+1.0?M Rox).The activity of rat vascular endothelial cells was measured by MTT assay,and the expressions of ALD A,GLUT1 and HIF-1?proteins were detected by Western Blot.In the experiments of mouse B16 melanoma xenograft models in vivo,the groups of different treatments were as follows:PBS group(negative control group),low dose group(1 mg/kg),medium dose group(5 mg/kg)and high dose group(25 mg/kg)of Rox,YC-1 group(15 mg/kg),YC-1(15 mg/kg)+medium dose group(5 mg/kg)of Rox.When the volume of the tumors was approximately 100-150mm~3,Rox was administered intragastrically and YC-1 was administered intraperitoneally once a day for 7 days.The body weight of mice and the volume/weight of tumors were measured.Then the tumors were stained with H&E or HIF-1?antibody immunohistochemically and the expressions of ALD A,GLUT1 and HIF-1?proteins in tumor were detected by Western Blot.Results:In the culture of rat vascular endothelial cells in vitro:?The proliferation of rat vascular endothelial cells were promoted by 0.1-10.0?M Rox.The production of ATP and the release of LD,the activity and content of ALD were increase significantly and the expressions of ALD A and GLUT1 proteins were significantly up-regulated(P<0.05).The activity of ALD in 0.1,1.0 and 10.0 ?M Rox groups were 1.08,1.24 and 1.09 times compared with that in control group.The expression of ALD A was 1.56,1.83 and 1.70 times,the GLUT1 was 1.50,1.66 and 1.56 times compared with that in control group.?With the inhibitors YC-1 and LY294002,the activity of rat vascular endothelial cells and the expressions of ALD A,GLUT1 and HIF-1?proteins were inhibited significantly(P<0.05).Compared with 1.0 ?M Rox group,the expressions of ALD A and GLUT1 proteins was significantly decreased in YC-1+1.0 ?M Rox group(P<0.05),the expressions of ALD A,GLUT1 and HIF-1? proteins were extremely significantly decreased in LY294002+1.0 ?M Rox group(P<0.01).In vivo,the volume and weight of transplanted tumors were increased significantly in 5-25 mg/kg Rox groups,the expressions of ALD A,GLUT1 and HIF-1? proteins of tumor tissue were significantly up-regulate(P<0.05).The growth and angiogenesis of tumor tissue were be found increased by HE staing analysis.However,YC-1 significantly inhibited the growth of tumor tissue and the expressions of ALD A,GLUT1 and HIF-1? proteins(P<0.05).The growth of tumor tissue and the expressions of ALD A,GLUT1 and HIF-1? proteins were significantly decreased in YC-1+medium dose group compared with medium dose group(P<0.01).Conclusion:The growth and glycolysis of rat vascular endothelial cells,as well as the growth and glycolysis of mouse B16F10 melanoma xenografts were be promoted by roxarsone.HIF-1?-glycolysis were involved in regulation on the growth promotion of rat vascular endothelial cells and tumor tissue induced by roxarsone in vivo and in vitro.