Snake venom is a mixture of a large number of biologically active proteins and peptides that can be classified in different categories such as metalloproteinases and disintegrins. Metalloproteinases are zinc-dependent enzymes responsible for degrading proteins of extracellular matrix and also have cytotoxic effect on endothelial cells. Some Bothrops metalloproteinases, like jararhagin and alternagin-C, have been referred to inhibit melanoma cells growth and metastases induced in experimental mice models. Glioblastomas are brain tumours that infiltrate diffusely into regions of the normal brain rendering total surgical extirpation impossible and effective radiotherapy difficult. The effect of Bothrops neuwiedii venom (BN) on glioblastoma cells has not been studied yet. The aim of the present work was to characterise the antitumoral effect of BN on cultured murine glioblastomas cells (RT2).
RT2 cells were treated with varying concentrations of BN and the effects on cells clonogenicity, adhesion and morphology were evaluated. RT2 cells were sensitive to BN venom in a dose-dependent way. Inhibition of cell adhesion, proliferation and morphological disturbs, such as rounded cell shape, could be observed at concentrations higher than 250ng/mL (IC50=372.7ng/mL). In order to shed some light on the mechanisms of these effects we evaluated the BN proteolytic activity, identified by zymography, using gelatin as the substrate (SDS-PAGE-Gelatin). We found that BN presents gelatinolytic activity with optimal pH of 7.2.
Mettaloproteinases with disintegrin domain containing RGD (Arg-Gly-Asp) sequence are very potent inhibitors of platelet aggregation, as well as other cellular interactions with the extracellular matrix. Take together our results and the role of matrix proteins for adhesion and cell survival, we suggest that the anti-tumoral effects of BN on glioblastomas can be ascribed, at least partially, to the involvement of metalloproteinases. Further experiments are in development to identify the specific component of Bothrops neuwiedii venom involved in the anti-tumoral activity and characterise its mechanism of action.