CONICET | Buscador de Institutos y Recursos Humanos

The vast majority of snakebite envenomings in northeastern Argentina are caused by Bothrops diporus (yarará chica). In addition to its systemic effects, B. diporus venom induces prominent local tissue damage. Phospholipase A2 (PLA2) enzymes from viperid venoms are often key players in the pathogenesis of these local effects.. In the present study, we isolated a basic PLA2 from B. diporus venom, and examined its potential adhesion inhibition effect on a tumoral cell line.Purification was made by a two-step procedure utilizing ion exchange (HiTrap SP XL-AKTAprime) and gel filtration chromatography (Sephadex G-75). SDS-PAGE of the isolated enzyme showed a single typical band of 14 kDa and PLA2 activity was evidenced by the formation of hemolytic halos in agarose-erythrocyte egg yolk gels. Cytotoxic activity of the PLA2 (1.5-500 µg/mL-3h incubation) was determined on a murine tumoral epithelial cell line (LM3) grown in DMEM-5% FBS at 37°C-5% CO2.Non-cytotoxic concentrations were selected for adhesion inhibition assay. Briefly, LM3 cells (3×104/well) were preincubated for 30 min at 37°C with PLA2 (1.2, 1.5, 2.5, 3, 5 and 10 μg/mL) or culture medium (control) and then added to 96-well plates. After 1.5 h, non-adherent cells were removed by careful washing and aspiration with PBS. Adherent cells were fixed and stained with crystal violet. The percentage of cell adhesion was determined by comparison of the absorbance readings (620nm) with the mean absorbance of control cells (not exposed to the PLA2), considered as 100% adhesion. Results indicate that the isolated enzyme PLA2 induces a dose-dependent inhibition of cell adhesion. Even with the lowest dose assayed (1.25 μg/mL) a 20% of this effect was observed and with 10 μg/mL, 60% of cells didn´t adhere to the substrate. Although more studies are needed, these findings demonstrate the therapeutic potential of this basic PLA2 isolated from B. diporus venom.