Avaliação da ativação de apoptose e autofagia como mecanismo de citotoxicidade induzida por um nitrocomposto e uma hidrazona em células tumorais humanas

Abstract

The search for new antitumor drugs is a necessity mainly due to current treatments present high toxicity, low effectiveness and emergence of resistance in some types of cancers. In the process of drug discovery, compounds of different chemical classes are assessed in models predictive and selected for chemical optimization and studies of structure-activity relationship (SAR) to obtain the most promising candidates. We carried out an investigation of mechanism of action and the antitumor potential of two substances: a hydrazone (E6) and nitrocomposto (EBAC). These substances showed low toxicity for normal cells such as human peripheral blood mononuclear cell (PBMC). The evaluation of the mechanism of action was performed on leukemic cell line (HL60) by phase contrast microscopy and LDH release, being able to verify that HL60 cells had features consistent with apoptosis after EBAC and E6 treatment. DNA fragmentation was confirmed after 24 hours of treatment by cell cycle, DNA electrophoresis and fluorescence microscopy after Hoechst 33342-staining. The use of pan-caspase inhibitor (Z-VAD-FMK), Annexin V and PI, quantification of reactive oxygen species and caspase-3 activation confirmed apoptosis. Loss of the mitochondrial membrane potential and caspase -9 detection suggest mitochondrial pathway activation in this cell line. Differences in the mechanism of death were observed for EBAC that induced accumulation of autophagic vacuoles analysis after acridine orange and increasing the amount of viable cells after use of the autophagy inhibitor bafilomycin A1. These results suggest that EBAC induced autophagy and apoptosis simultaneously, which was not observed for E6. The results together show that EBAC and E6 are promising substances with anti-tumor potential and justify the continuation of studies of structure-activity relationship for obtaining more active derivatives.