CONICET | Buscador de Institutos y Recursos Humanos

Snake venoms are natural sources of bioactive substances with therapeutic potential. In particular, different types of phospholipases have been shown to possess antitumor and antiangiogenic properties. The venom of the South American Rattlesnake, Crotalus durissus terrificus (C.d.t.), is a complex mixture of proteins. Crotoxin, the main toxin of this venom, contains a basic phospholipase A2 (PLA2) and a non-toxic acidic protein, crotapotin. Thus, the aim of this study was to evaluate the potential effect of a PLA2 isolated from C.d.t. (Cdt-PLA2) on tumoral cell adhesion.Cdt-PLA2 was purified by two chromatographic steps, a gel filtration and a reversed phase HPLC C-18 chromatographs. The purity of the enzyme was verified by SDS-PAGE.Firstly, cytotoxicity of Cdt-PLA2 (5 - 250 μg/mL) was assessed in cultured 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 Cdt-PLA2 (5, 10, 20, 25, and 50 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 PLA2s), considered as 100% adhesion.The obtained results showed a concentration-dependent adhesion inhibition effect of Cdt-PLA2 using non-cytotoxic concentrations. These findings are the beginning of the study of the potential use of this enzyme as an antitumoral.