Avaliação in vitro do potencial citotóxico de derivados arilaminados nor-β-lapachônicos : estudos de mecanismo de ação

Abstract

The search for new cytotoxic derivatives of nor-β-lapachone is part of a growing and continuous search for new active compounds. The present study aimed to evaluate the cytotoxic potential of four arylamino-nor-β-lapachone derivatives (1-4) and the probable mechanism involved in the cytotoxicity of these compounds. Among the tested derivatives, only three of them (1, 3 and 4) and the precursor molecule elicited a significant antiproliferative effects in all human tumor cell lines used in the study. In experiments with peripheral blood mononuclear cells (PBMC), nor-β-lapachone did not induce cytotoxicity, however its derivatives showed antiproliferative effects whose intensity ranged from moderate to weak, with IC50 values equal to 5.02 µM (1), 12.02 µM (3) and 10.65 µM (4). Moreover, all the compounds showed no hemolytic effects (EC50> 219.29 µM). Nor-β-lapachone and its derivatives (1, 3 and 4) induced apoptosis (mitochondrial pathway) in HL-60 cells, based upon morphological and flow cytometric analysis, and interference on HL-60 cell cycle progression, only at the highest concentration (4 µM). Compounds exposure induced intracellular ROS generation, as well as DNA strand breaks in HL-60 (NQO1−) and DU-145 (NQO1+) cell lines. The co-administration of GSH reduced the cellular sensibility to the toxic effects of the studied compounds. Unlike cells pre-treated with BH (glutathione depletor agent), cells pre-exposed to antioxidants agents (NAC or KI) showed low production of free radicals. On the other hand, dicoumarol (NQO1 inhibitor) prevented the ROS formation and DNA strand breaks only in DU-145 cells, indicating that these compounds can be metabolized by other reductases than NQO1. The DNA damage induced by nor-β-lapachone and its derivatives (1, 3 and 4) in DU-145 cells were partially repaired after 24 h, which agrees with the data of unscheduled DNA synthesis. In addition, all active compounds interfere with DNA repair processes of DNA damage induced by MMS, indicating that this action contributes to compounds cytotoxic effects. All active compounds inhibited the proliferation rate of S. cerevisiae strains defective in the expression of DNA topoisomerases (Top1Δ, Top3Δ and Top1ΔTop3Δ) more pronounced than that observed in wild type strain (BY4741), suggesting that the interference on topoisomerases activities may contributes to the cytotoxicity of active compounds. Genotoxicity and mutagenicity studies with Chinese hamster lung fibroblast cell line (V79), showed that all cytotoxic compounds promoted DNA strand breaks, DNA bases oxidation and micronucleus formation in a concentration (10 µM) far higher than needed to induce the same events in tumor cells. Strengthening the ROS contribution on the mutagenic events, the pre-treatment with NAC prevented the formation of micronucleated cells and reduced the V79 cell sensibility to the cytotoxic effects of the studied compounds. These findings underscore the antitumor properties of nor-β-lapachone and its arylamino derivatives and they can be considered as prototypes for the development of new anticancer agents.