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Analysis of the cytotoxic effects of purified components from Phoneutria nigriventer spider venom on tumor cells

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Author(s):
Marcus Vinícius Campoy Chichizola Caballero Alves
Total Authors: 1
Document type: Master's Dissertation
Press: Campinas, SP.
Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
Defense date:
Examining board members:
Catarina Raposo Dias Carneiro; Laura de Oliveira Nascimento; Maria Helena Rodrigues Mesquita Britto
Advisor: Catarina Raposo Dias Carneiro
Abstract

Gliomas comprise the type of primary tumor most commonly found in the brain, originating from neuronal progenitor cells or transformed glial cells. Grade IV gliomas are called glioblastomas (GBs), which correspond to more than 50% of gliomas and are characterized by their high mortality rate. As a multifactorial and complex disease, cancer treatment is a major challenge and GBs represent a separate challenge in terms of therapy and prognosis; therefore, the development of new therapeutic approaches has great social relevance. Animal venoms contain biologically active molecules with specific targets in cells and tissues. Peptide-based therapy, one of the main classes of molecules present in spider venoms, is a vast and promising field for agents with emerging antineoplastic actions. Recently, our research group demonstrated that the venom of the Spider Phoneutria nigriventer (PnV) has cytotoxic effects in GB strains. The present study aims to select purified components of the venom with effect on viability / death, cell cycle and proliferation of the human GB NG97 lineage, as well as to contribute to clarify the mechanism of action behind these effects, investigating mTOR and related proteins control of apoptosis. The cells were first treated with the PnV fractions: F1 (less than 3kDa), F2 (between 3 and 10 kDa) and F3 (greater than 10 kDa). From these results, the fractions F1 + F2 (low weight) were repurified, obtaining 11 subfractions (SF1 - SF11). The treatments were performed during 1, 5, 24 and / or 72 h and then viability (MTT), proliferation (CFSE), death (Annexin V / propidium iodide - IP) and cell cycle (IP) tests were used ; an mTOR inhibitor (rapamycin, a drug already used to treat various neoplasms) was used in the experiments with SFs and western blotting was done for BCL-2 and BAX. The F1 and F2 fractions showed more relevant effects on the observed parameters and were repurified in subfractions (SF1 - SF11); of these, SFs3 and 4 were the ones that showed the most significant antitumor effects. The previous inhibition of mTOR by rapamycin had a synergistic effect on SFs, reducing cell viability even more significantly compared to untreated control. The BAX protein, related to the induction of apoptosis, was increased by SFs3 and 4 and, even more expressed when mTOR was inhibited, corroborating the synergistic effect of the drugs. BCL-2, related to anti-apoptotic mechanisms, was reduced by SFs and there was no difference when rapamycin was previously used. Therefore, taken together, the results point to components present in SFs3 and 4 as potential prototypes for the development of new drugs for the treatment of GB and indicate that these compounds can be used in combination therapy associated with rapamycin. Future studies will be conducted to characterize and synthesize the molecules and to evaluate the efficacy and safety in pre-clinical models (AU)

FAPESP's process: 17/24331-7 - Analysis of the effect and mechanism of action of P. nigriventer venom and its isolated toxins on the development of brain tumor implanted in mice
Grantee:Marcus Vinícius Campoy Chichizola Caballero Alves
Support Opportunities: Scholarships in Brazil - Master