Basic phospholipases A2 isolated from Bothrops diporus snake venom inhibits endothelial tubulogenesis in vitro

Abstract

Angiogenesis is a regulated process of growth and remodeling of new blood vessels from preexisting vasculature, which is correlated with a variety of physiological processes. However, when this process is deregulated contributes to the pathogenesis of numerous disorders such as the survival and progression of solid tumors. Snake venoms are a complex mixture that contains biologically active molecules, being phospholipases A2 (PLA2s) one of the most abundant components with pharmacological potential. In the present study, two basic PLA2s (PLA2-I and PLA2-II) were isolated from the Argentine northeastern snake Bothrops diporus venom and their potential inhibitory effect on in vitro tubulogenesis was evaluated. Toxins were purified by reverse phase high-performance liquid chromatography (RP-HPLC) on a C18 column. Venom (2 mg) was dissolved in 200 µL of 0.1% trifluoroacetic acid (TFA). Elution was performed at 1 mL/min using a gradient of acetonitrile with 0.1% TFA. Protein concentrations were estimated by measuring the absorbance at 280 nm. Phospholipase activity was assessed and purity and apparent molecular mass were determined by SDS-PAGE. Cytotoxic activity of PLA2-I and PLA2-II (1.25-30 μg/mL-24h of incubation) was determined using the tEnd murine endothelial cell line (CVCL_6272) cultured in DMEM-5% FBS at 37°C-5% CO2. For tubulogenesis assay, 96-well plates were coated with 50 μL of Geltrex at 4 °C and incubated for 30 min at 37 °C. Then tEnd cells were seeded (1 x 104 cells/well) with non-cytotoxic concentrations (25 μg/mL) of PLA2s or culture medium (control group). The morphological changes induced at 8h were evaluated using a phase contrast microscopy. Photographs were taken and angiogenic parameters were quantified using ImageJ software. Results showed an inhibitory effect on tubulogenesis process with both toxins assayed in comparison to controls. There was a decrease in the nodes, segments and meshes when cells were incubated in the presence of PLA2-I or PLA2-II, the former having a greater inhibitory effect. Although more studies are needed to elucidate the triggered mechanisms of action, these results suggest a potential antimetastatic effect of these snake toxins.