RESUMO
Entamoeba histolytica is the causative agent of amoebiasis, and Entamoeba dispar is its noninvasive morphological twin. Entamoeba invadens is a reptilian parasite. In the present study, Western blot, phosphatase activity, immunofluorescence, and bioinformatic analyses were used to identify PP2C phosphatases of E. histolytica, E. dispar, and E. invadens. PP2C was identified in trophozoites of all Entamoeba species and cysts of E. invadens. Immunoblotting using a Leishmania mexicana anti-PP2C antibody recognized a 45.2 kDa PP2C in all species. In E. histolytica and E. invadens, a high molecular weight element PP2C at 75 kDa was recognized, mainly in cysts of E. invadens. Immunofluorescence demonstrated the presence of PP2C in membrane and vesicular structures in the cytosol of all species analyzed. The ~75 kDa PP2C of Entamoeba spp. shows the conserved domain characteristic of phosphatase enzymes (according to in silico analysis). Possible PP2C participation in the encystation process was discussed.
Assuntos
Entamoeba/enzimologia , Proteína Fosfatase 2C/metabolismo , Proteínas de Protozoários/metabolismo , Trofozoítos/enzimologia , Sequência de Aminoácidos , Animais , Entamoeba/isolamento & purificação , Entamebíase/parasitologia , Entamebíase/patologia , Humanos , Filogenia , Proteína Fosfatase 2C/química , Proteína Fosfatase 2C/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Homologia de Sequência de Aminoácidos , Trofozoítos/isolamento & purificaçãoRESUMO
Nanomaterials quickly evolve to produce safe and effective biomedical alternatives, mainly silver nanoparticles (AgNPs). The AgNPs' antibacterial, antiviral, and antitumor properties convert them into a recurrent scaffold to produce new treatment options. This work reported the full characterization of a highly biocompatible protein-coated AgNPs formulation and their selective antitumor and amoebicidal activity. The protein-coated AgNPs formulation exhibits a half-inhibitory concentration (IC50) = 19.7 µM (2.3 µg/mL) that is almost 10 times more potent than carboplatin (first-line chemotherapeutic agent) to inhibit the proliferation of the highly aggressive human adenocarcinoma HCT-15. The main death pathway elicited by AgNPs on HCT-15 is apoptosis, which is probably stimulated by reactive oxygen species (ROS) overproduction on mitochondria. A concentration of 111 µM (600 µg/mL) of metallic silver contained in AgNPs produces neither cytotoxic nor genotoxic damage on human peripheral blood lymphocytes. Thus, the AgNPs formulation evaluated in this work improves both the antiproliferative potency on HCT-15 cultures and cytotoxic selectivity ten times more than carboplatin. A similar mechanism is suggested for the antiproliferative activity observed on HM1-IMSS trophozoites (IC50 = 69.2 µM; 7.4 µg/mL). There is no change in cell viability on mice primary cultures of brain, liver, spleen, and kidney exposed to an AgNPs concentration range from 5.5 µM to 5.5 mM (0.6 to 600 µg/mL). The lethal dose was determined following the OECD guideline 420 for Acute Oral Toxicity Assay, obtaining an LD50 = 2618 mg of Ag/Kg body weight. All mice survived the observational period; the histopathology and biochemical analysis show no differences compared with the negative control group. In summary, all results from toxicological evaluation suggest a Category 5 (practically nontoxic) of the Globally Harmonized System of Classification and Labelling of Chemicals for that protein-coated AgNPs after oral administration for a short period and urge the completion of its preclinical toxicological profile. These findings open new opportunities in the development of selective, safe, and effective AgNPs formulations for the treatment of cancer and parasitic diseases with a significant reduction of side effects.
RESUMO
Amoebiasis is a human intestinal disease caused by the parasite Entamoeba histolytica. It has been previously demonstrated that E. histolytica heat shock protein 70 (EhHSP70) plays an important role in amoebic pathogenicity by protecting the parasite from the dangerous effects of oxidative and nitrosative stresses. Despite its relevance, this protein has not yet been characterized. In this study, the EhHSP70 genes were cloned, and the two recombinant EhHSP70 proteins were expressed, purifying and biochemically characterized. Additionally, after being subjected to some host stressors, the intracellular distribution of the proteins in the parasite was documented. Two amoebic HSP70 isoforms, EhHSP70-A and EhHSP70-B, with 637 and 656 amino acids, respectively, were identified. Kinetic parameters of ATP hydrolysis showed low rates, which were in accordance with those of the HSP70 family members. Circular dichroism analysis showed differences in their secondary structures but similarities in their thermal stability. Immunocytochemistry in trophozoites detected EhHSP70 in the nuclei and cytoplasm as well as a slight overexpression when the parasites were subjected to oxidants and heat. The structural differences of amoebic HSP70s with their human counterparts may be used to design specific inhibitors to treat human amoebiasis.
Assuntos
Entamoeba histolytica/genética , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Isoformas de Proteínas/genética , Amebíase/parasitologia , Animais , Núcleo Celular , Dicroísmo Circular , Clonagem Molecular , Citoplasma/metabolismo , Entamoeba histolytica/patogenicidade , Proteínas de Choque Térmico HSP70/classificação , Humanos , Estrutura Secundária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Trofozoítos/metabolismoRESUMO
La amibiasis es un padecimiento que afecta al 10% de la población mundial, y puede tener un comportamiento muy diverso, tanto en el intestino como en diversos órganos (hígado, pulmones, cerebro, piel). Se conoce su ciclo biológico, los síntomas y signos de su penetración al organismo, así como su diagnóstico y tratamiento, pero aún hay controversias sobre los mecanismos moleculares de la patogenicidad de la E. Histolítica, para lo cual se ha utilizado en particular el absceso hepático experimental en Hamsters (AHAH). Durante mucho tiempo se sostuvo que la patogenicidad de E. Histolítica se debía a su capacidad para destruir tejidos, pero encontramos que la E. Histolítica virulenta, per se es incapaz de causar daño al hígado del hámster leucopénico. Este estudio se dedicó a estudiar los mecanismos de virulencia de la amiba mediante la comparación funcional y molecular entre E. Histolítica virulenta y E. Histolítica no virulenta. Encontramos que la virulencia de este parásito no se puede explicar solamente por la actividad de sus moléculas citotóxicas (adhesinas, fosfolipasas y ameboporos) o proteolíticas (proteasas), y los hallazgos sugieren que cuando las amibas virulentas arriban al hígado del hámster y se encuentran una concentración tóxica de oxígeno, éste las sensibiliza a la lisis por el complemento, el peróxido de hidrógeno y el ácido hipocloroso. Las consecuencias de estos hallazgos pueden abrir nuevas perspectivas para el diseño de terapias alternativas para el tratamiento de este padecimiento.
Amoebiasis is a disease that affects 10 % of the world population, and it may have a different behavior when attacks bowels, liver, lungs, brain, etc. Its biological cycle is well known, as well as its symptoms and signs of its penetration into those organs, its diagnosis and treatment, but it is still a controversy on the molecular mechanism of its pathogenesis; to study them it, the experimental hepatic abscess in hamsters has been employed. For years it was considered that the pathogenicity of E. Histolítica was due to its capacity to destroy tissues, but we found that virulent E. Histoliticaperse is unable to produce liver damage in leucopenic hamster; we therefore studied the mechanisms of virulence of the amoeba by functional and molecular comparison between virulent and non virulent E. Histolitica. We found that the parasit virulence cannot be explained only by the activity of citotoxic or proteolytic molecules (adhesines, phospholypases and amebopores, or proteases), and the findings suggest that when amoebas arrives to the hamster liver and find a toxic concentration of oxygen, this sensibilizes them to lysis by complement, hydrogen peroxide and hypoclorose acid. The consequences of those findings may open new perspectives for the design of new therapies for the treatment of this disease.