Abstract
The potential angiogenic effect of roxarsone, a feed additive widely used to promote animal growth worldwide, was demonstrated recently. We explored the mechanism of vascular endothelial growth factor (VEGF) and its receptor (VEGFR) in roxarsone promotion of rat vascular endothelial cells (ECs) and B16F10 mouse xenografts. ECs were treated with 0.1–50 μM roxarsone or with roxarsone plus 10 ng/mL VEGF, VEGFR1 (Flt1), or VEGFR2 (Flk1) antibodies for 12–48 h to examine their role in cell growth promotion. Small interfering RNA (siRNA) targeting Vegf, Flt1, and Flk1 were transfected in the ECs, and we measured the expression level, cell proliferation, migration, and tube formation ability. The siRNA targeting Vegf or Flk1 were injected intratumorally in the B16F10 xenografts of mice that received 25 mg/kg roxarsone orally. Cell viability and VEGF expression following roxarsone treatment were significantly higher than that of the control (P < 0.05), peaking following treatment with 1.0 μM roxarsone. Compared to roxarsone alone, the VEGF antibody decreased cell promotion by roxarsone (P < 0.05), and the Flk1 antibody greatly reduced cell viability compared to the Flt1 antibody (P < 0.01). Roxarsone and Flk1 antibody co-treatment increased supernatant VEGF significantly, while cellular VEGF was obviously decreased (P < 0.01), whereas there was no significant difference following Flt1 antibody blockade. The siRNA against Vegf or Flk1 significantly attenuated the roxarsone promotion effects on EC proliferation, migration, and tube-like formation (P < 0.01), whereas the siRNA against Flt1 effected no obvious differences. Furthermore, the RNA interference significantly weakened the roxarsone-induced increase in xenograft weight and volume, and VEGF and Flk1 expression. Roxarsone promotion of rat EC growth, migration, and tube-like formation in vitro and of B16F10 mouse xenograft model tumor growth and angiogenesis involves a VEGF/Flk1 mechanism.
Highlights
Angiogenesis, which involves endothelial cell (EC) proliferation and migration and the formation of new capillaries, is a critical homeostatic mechanism in organs and tissues[1]
We investigated whether roxarsone promotes rat EC growth via vascular endothelial growth factor (VEGF)/Flk[1] by using targeted small interfering RNA interference of Vegf and its receptor Flt[1] or Flk[1] (VEGFR2) genes, or by antibody blockade of the related signal molecule in cell models of proliferation, migration, and tube formation
Following 12 h, 24 h, 36 h, and 48 h exposure, the ECs treated with roxarsone and 10 ng/mL VEGF had significantly higher relative viability and VEGF expression than the phosphate-buffered saline (PBS)-treated negative control (P < 0.05, Fig. 1a)
Summary
Angiogenesis, which involves endothelial cell (EC) proliferation and migration and the formation of new capillaries, is a critical homeostatic mechanism in organs and tissues[1]. Roxarsone (4-hydroxy-3-nitro-benzenearsonic acid) is widely used in many countries as a feed additive in animal production to enhance weight gain, improve feed efficiency, and control intestinal parasites[13,14] It can be excreted almost as its parent drug in manure[15,16,17]; leaches from poultry litter[18,19,20,21]; and enhances arsenic levels in open water or underground water, soil, or plants in the local area[22,23,24,25]. Our results indicate that VEGF/Flk[1] signaling is involved in roxarsone-induced promotion in rat ECs growth and B16F10 mouse xenografts
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