The Role Of PI3K/Akt Pathway In Roxarsone Promoting Angiogensis In Vivo And In Vitro

Objective:Roxarsone (Rox),3-nitro-4-hydroxy phenyl arsonic acid, is widely used in the world as an organic arsenic feed additive for livestock and poultry. The absorption of Rox in animal is little, which means the vast majority of Rox is in fecal excretion. The level of arsenic in the environment and the risk of exposure to arsenic in human are subsequently increased with animal manure used as an organic fertilizer. Little information is focused on the angiogenesis roxarsone-induced and its mechanism at present. This paper aims to study the role of PI3K/Akt pathway in roxarsone promoting angiogenesis in vitro and in vivo through the experiments on the rat vascular endothelial cell growth, migration and tube formation and the mouse B16F10 melanoma xenograft model.Methods:MTT assay, BrdU proliferation assay, scratch test and tube formation assay were performed to assess the survival, proliferation, migration and tube formation of rat vascular endothelial cell after different treatments, respectively. Different treatments were as follows: control group (PBS), positive group (10 ng/mL VEGF), roxarsone treatment group (0.1 μmol/L,1.0 μmol/L and 10.0 μmol/L Rox), the inhibitor of PI3K treatment group (20.0 μmol/L LY294002) and co-treatment group (20.0 μmol/L LY294002 plus 1.0μmol/L Rox). The expression of PI3K/Akt signaling molecular and VEGF were detected via Western Blot.In the model of B16F10 melanoma xenograft in vivo, different treatment groups were as follows: control group (PBS), low dose Rox group (1 mg/Kg), middle dose Rox group (5 mg/Kg) and high dose Rox group (25 mg/Kg) at intragastric administration for 7 d once a day after injection of B16F10 cells to mouse. The weight of mice and the volume and weight of tumors were measured. Then the tumors isolated from different groups were stained with H&E or VEGF antibody immunohistochemically. The level of PI3K/Akt signal protein in tumor was detected by Western Blot.Results:The OD rate of endothelial cells treated by 0.1～10.0 μmol/L roxarsone were increased in MTT assay, the number of BrdU-positive cells in proliferation assay, the distances of migration in scratch test and the number of meshes in tube formation assay with higher phosphorylation level of PI3K/Akt signaling and expression of VEGF were also increased,1.0 μmol/L Rox treatment appeared significant differences compared to control group (P<0.05) whereas 0.1 μmol/L and 10.0 μmol/L roxarsone no statistical significance (P>0.05). LY294002, a PI3K inhibitor, could significantly reduce vascular endothelial cell vitality, number of BrdU-positive cells, distances of migration, number of meshes in tube formation assay, activation of PI3K/Akt signaling and expression of VEGF. These parameters of LY294002 plus 1.0 μmol/L Rox co-treatment were significantly decreased compares to 1.0μmol/L Rox (P< 0.01). Roxarsone could promote the growth, migration and tube formation of vascular endothelial cell, in which PI3K/Akt signaling played its regulative role.In the experiment of B16F10 xenografts mice model, the weight and volume of B16F10 xenografts and expression of VEGF and phosphorylation level of PI3K/Akt signaling in tumors were increased at treatments of different dosages of roxarsone (1-25 mg/Kg). Compared with the control group,25 mg/Kg Rox treatment made significant differences (P<0.05), whereas 1 mg/kg and 5 mg/kg roxarsone appeared no statistical significance (P>0.05). Roxarsone may promote the growth of mouse B16F10 xenografts and angiogenesis in tumor, in which PI3K/Akt signaling played an important role.Conclusion:Roxarsone can promote the growth and tube formation of vascular endothelial cell and the growth of mouse B16F10 xenografts. PI3K/Akt signaling was involved in regulation on the angiogenesis induced by roxarsone in vivo and in vitro.