RESUMO
Streptomyces hygroscopicus UFPEDA 3370 was fermented in submerged cultivation and the biomass extract was partitioned, obtaining a fraction purified named EB1. After purification of EB1 fraction, nigericin free acid was obtained and identified. Nigericin presented cytotoxic activity against several cancer cell lines, being most active against HL-60 (human leukemia) and HCT-116 (human colon carcinoma) cell lines, presenting IC50 and (IS) values: 0.0014 µM, (30.0) and 0.0138 µM (3.0), respectively. On HCT-116, nigericin caused apoptosis and autophagy. In this study, nigericin was also screened both in vitro and in silico against a panel of cancer-related kinases. Nigericin was able to inhibit both JAK3 and GSK-3ß kinases in vitro and its binding affinities were mapped through the intermolecular interactions with each target in silico.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/patologia , Nigericina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Streptomyces/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Domínio Catalítico , Linhagem Celular Tumoral , Humanos , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/química , Janus Quinase 3/metabolismo , Simulação de Acoplamento Molecular , Nigericina/química , Nigericina/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismoRESUMO
A new Ca(2+) intracellular store, the acidocalcisome, has been reported in trypanosomatids. It has been characterized physiologically as a Ca(2+) store sensitive to nigericin. The Ca(2+)/H(+)-ATPase is the system responsible for Ca(2+) accumulation, which depends on a pH gradient formed by ATP- and PPi-dependent proton pumps. In this work we present physiological and morphological evidences for the presence of acidocalcisomes in Trypanosoma evansi. The parasites were purified and loaded with the fluorescent dye Fura 2-AM in order to detect the intracellular changes of Ca(2+) levels in individual cells. The simultaneous incubation of T. evansi cells with ionomycin and nigericin led to large release of Ca(2+) (ca. 200 nM) from intracellular stores, which was not observed with either agent alone. On the other hand, no enhancement of the nigericin-induced Ca(2+) release was observed in the presence of oligomycin. Additionally, the pretreatment with bafilomycin decreases the nigericin-induced Ca(2+) release. These results confirm the presence of an intracellular non-mitochondrial acidic Ca(2+) storage compartment. These results suggest that H(+)-ATPase is involved in the process of Ca(2+) accumulation into the acidocalcisomes. Furthermore, the cells loaded with acridine orange exhibited abundant fluorescent vacuoles, which were sensitive to nigericin or bafilomycin A(1). Electronic transmission microscopy observations demonstrated the presence of electron dense particles in the parasites. High levels of inorganic pyrophosphate and triphosphate were detected in perchloric acid extracts of T. evansi by high resolution 31P NMR. Taken together, these results present the first evidence for the presence of acidocalcisomes in T. evansi.