The Role Of PLCλ/PKC Pathway In Angiogensis Promotion Of Roxarsone In Vivo And In Vitro

[Objective] Roxarsone (3-nitro-4-hydroxy phenyl arsenic acid, Rox), has been widely used in the world as a feed additive because of its antibacterial anticoccidial, feed efficiency improvation, and animal growth promotion. Roxarsone can promote endothelial cells growth in vitro in previous studies, but little studies are focused on the angiogenesis and its mechanism of roxarsone induced. Based on the culture of rat endothelial cells in vitro, tube formation and mouse B16 xenograft model, the role of PLCy/PKC pathway in angiogensis promotion of roxarsone in vivo and in vitro were inverstigated by MTT assay, BrdU assay, cell proliferation assay, immunohistochemistry and Western Blot methods.[Methods] In the cuture of rat vascular endothelial cells in vitro, the different treatment groups were designed as follows:PBS,0.1 μM Rox,1.0 μM Rox,10 μM Rox,10 ng/mL VEGF,40 μM U73122, and 40 μM U73122+1.0 μM Rox. The level of cell viability, proliferation, cell migration and vessel-like structures forming ability were determinated by MTT asaay, BrdU assay, scratch test and tube formation assay. The protein expression of PLCy/PKC signaling was detected by Western-blot technique. In the mouse B16 melanoma xenograft experiments, different treatment as follows:PBS,1 mg/kg Rox,5 mg/kg Rox,25 mg/kg Rox,10 mg/kg SU5416 and 10 mg/kg SU5416 +5 mg/kg Rox. Each treatment group was orally administrated for continuous 7 d after injection of B16 cells. The body weight of mice, the volume and weight of tumor striped were measured, HE staining and CD31 immunohistochemistry of tumor tissue, the protein expression of PLCy/PKC signaling by Western Blot were mead.[Results] The MTT data of endothelial cells at 24 h incubation showed that the OD values of 1.0μM Rox group was significantly increased compared to PBS control (P<0.05), there was no significant difference between 0.1 μM and 10 μM Rox. The OD values of 1.0 μM Rox+U73122 group was significantly decreased compared to 1.0 μM Rox (P<0.01).In the BrdU test for detection proliferation of endothelial cells at 24h, BrdU-positive cells in 1.0 μM Rox group was significantly increased compared with PBS control group, (P<0.05); those in 0.1 μM Rox group and 10 μM Rox group were also increased, but no significant difference. BrdU-positive cells in 1.0 μM Rox+U73122 group was significantly decreased compared with 1.0 μMRox group (P<0.01).Scratch test results showed that cell migration in 1.0 vM Rox group was significantly increased compared with PBS control group (P<0.05); 0.1μM Rox group and 10μM Rox group compared with the PBS control group were also increased, but no significant difference; that of 1.0 μM Rox+U73122 was significantly reduced compared with 1.0 μM Rox migration (P<0.01).Tube formation assay showed that the number of tubes in 1.0 μM Rox group was significantly increased compared with PBS control group (P<0.05); number of tubes in 0.1μM Rox group and 10μM Rox were also increased but there was no significant difference. The number of tubes in 1.0 μM Rox+U73122 group was significantly reduced compared with 1.0 μM Rox group (P<0.01).Western Blot analysis showed that different concentrations Rox significantly promote the expression of PLCy, PKC phosphorylated protein (P<0.05),1.0μM Rox group with PBS control group was significant differences (P<0.01). The level of PLCy, PKC phosphorylated protein in 1.0 μMRox+U73122 group was significantly reduced compared with 1.0μMRox group (P<0.01).In model of mice B16 xenograft, showed that tumor weight and volume in different concentrations Rox groups were all increased, but that in 5,25mg/kg Rox appeared significant differences compared with PBS control (P<0.05). The tumor weight and volume in 10 mg/kg SU5416+5 mg/kg Rox group were significantly decreased compare with 5 mg/kg Rox (P<0.01). HE staining and CD31 immunohistochemistry for tumor tissue showed that angiogenesis of tumors in different concentrations Rox treated groups were appeared to promotion; whereas the necrotic area and poor vessel formation in 10 mg/kg SU5416+5 mg/kg Rox group were appeared compared with 5 mg/kg Rox group. The level of PLCy, PKC phosphorylated protein in different concentrations of Rox by Western Blot analysis were significantly promoted (P<0.05). The PLCy, PKC phosphorylation in 10 mg/kg SU5416+5 mg/kg Rox group was significant decreased compared with 5 mg/kg Rox group (P<0.01).[Conclusion] Roxarsone can promote the growth of vascular endothe lial cell and angiogenesis, as well as promote the growth of mouse B16 xenograft and its blood vessel. PLCy/PKC signaling pathways play an important role in the angiogenesis promotion of roxarsone.