A basic phospholipase A2 from Bothrops diporus venom inhibits cancer cell adhesion and migration

Abstract

The vast majority of snakebite envenomings in northeastern Argentina are caused by Bothrops diporus. Thevenom of this species causes local tissue damage characterized by myonecrosis, hemorrhage, blistering, andedema. In the present study, we isolated a basic PLA 2 from B. diporus venom, and examined its potentialadhesion and migration inhibition effects on a tumoral cell line.Purification was made by a two-step procedure: ion exchange (HiTrapSP XL-AKTAprime) and gel filtrationchromatography (Sephadex G-75). Enzyme non-cytotoxic concentrations on a murine tumoral epithelial cellline (LM3) were selected for adhesion and migration inhibition assays. In order to evaluate cell adhesion, LM3cells (3×10 4 /well) were preincubated for 30 min at 37°C with PLA 2 (1.25-10 μg/mL) or culture medium (DMEM5% FBS-100% adhesion) and then added to 96-well plates. After 1.5 h, adherent cells were fixed and stainedwith crystal violet and the percentage of cell adhesion was determined. Cell migration was measured bywound-healing assay. Cells were grown in a 6-well plate and a wound was created by a sterile pipette tip.Culture medium alone or PLA 2 (0.125-0.25 μg/mL) were added to the cells and incubated for 24 h. Woundwidths were measured and percentage of cell migration was calculated.Results indicate that the isolated enzyme PLA 2 induces a dose-dependent inhibition of cell adhesion andmigration. The lowest dose assayed (1.25 μg/mL) evidenced a 20% of adhesion inhibition effect and with 10μg/mL, 60% of cells didn´t adhere to the substrate. On the other hand, the wound scratch in control cells wascompletely closed after 24 h of incubation. However, treatment with 0.125 and 0.25 μg/mL of PLA 2 resulted inthe suppression of cell migration in a concentration-dependent manner, decreasing respect to controls by 8and 55% respectively.Although more studies are needed, these findings demonstrate the therapeutic potential of this basic PLA 2isolated from B. diporus venom.