Biotechnological application of protein Leuc-B isolated from Bothrops leucurus venom as a prototype for antitumoral radiopharmaceutical

According to the report of the International Agency for Research on Cancer, the growth of this disease implies the death of 17 million people a year by 2030. Although the knowledge on development of cancer is growing considerably, just a few advances in the diagnosis and therapy has been achieved. Faced with this scenario, it is clear the need for new substances more specifics with low toxicity to the patient, which can be used for diagnosis and treatment of cancer. Membrane receptors over expressed in tumor cells are promising target candidates for development of diagnostic and therapeutical tools. Integrins are a family of hetero dimeric cell surface adhesion receptors able to recognize and bind to proteins in the extracellular matrix (ECM). This recognition is mainly through the RGD domains presents in both the cell surface as in the protein from the ECM. Various integrins have been identified as regulators of tumor progression. The RGD domain is also found in some snake venoms named disintegrins. Disintegrins inhibit cell-matrix and a cell-cell interactions mediated by integrins and it has been shown that these proteins are able to inhibit metastasis in processes dependent on integrin. The disintegrin-like (ECD), as well as RGD-disintegrin are also able to bind to cell surface integrins and inhibit their adhesion to the natural ligands. In this work it was purified from Bothrops leucurus venom (VBL), a metalloproteinase-class P-III with disintegrin-like domain (ECD), Leucurolisina B (Leuc-B). This metalloproteinase and the crude venom were used to evaluate their applicability in the differential detection of tumors. In vitro results demonstrated that both VBL and Leuc-B have potent antitumoral effect on several cancer cell lines: U87, T98, RT2 (glioblastoma), MCF7 (breast), Ehrlich and UACC (melanoma) with IC50 values of approximately 0.6 μM. The morphological changes observed in these strains when treated with Leuc-B, and data from the DAPI staining solution and acridine/ethidium bromide indicate that the antitumoral effect of these substances occurs via apoptosis. Radioactive probes of Leuc-B (131/125I-Leuc-B) were synthesized with high specific activity and high radiochemical purity. Biodistribution studies, performed by intravenous injection and intratumoral flow in mice bearing Ehrlich tumor showed a significant tumor uptake Leuc-B ( p <0.05). These results show the potential of Leuc-B as a template for the development of drugs and radiopharmaceuticals for diagnosis and therapy of tumors. (author