RESUMO
Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium that presents resistance to several antibiotics, thus, representing a major threat to human and animal health. Phage-derived products, namely lysins, or peptidoglycan-hydrolyzing enzymes, can be an effective weapon against antibiotic-resistant bacteria. Whereas in Gram-positive bacteria, lysis from without is facilitated by the exposed peptidoglycan layer, this is not possible in the outer membrane-protected peptidoglycan of Gram-negative bacteria. Here, we suggest the encapsulation of lysins in liposomes as a delivery system against Gram-negative bacteria, using the model of P. aeruginosa. Bioinformatic analysis allowed for the identification of 38 distinct complete prophages within 66 P. aeruginosa genomes (16 of which newly sequenced) and led to the identification of 19 lysins of diverse sequence and function, 5 of which proceeded to wet lab analysis. The four purifiable lysins showed hydrolytic activity against Gram-positive bacterial lawns and, on zymogram assays, constituted of autoclaved P. aeruginosa cells. Additionally, lysins Pa7 and Pa119 combined with an outer membrane permeabilizer showed activity against P. aeruginosa cells. These two lysins were successfully encapsulated in DPPC:DOPE:CHEMS (molar ratio 4:4:2) liposomes with an average encapsulation efficiency of 33.33% and 32.30%, respectively. The application of the encapsulated lysins to the model P. aeruginosa led to a reduction in cell viability and resulted in cell lysis as observed in MTT cell viability assays and electron microscopy. In sum, we report here that prophages may be important sources of new enzybiotics, with prophage lysins showing high diversity and activity. In addition, these enzybiotics following their incorporation in liposomes were able to potentiate their antibacterial effect against the Gram-negative bacteria P. aeruginosa, used as the model.
Assuntos
Prófagos , Pseudomonas aeruginosa , Animais , Antibacterianos/farmacologia , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Humanos , Lipossomos , Peptidoglicano/metabolismo , Prófagos/metabolismo , Pseudomonas aeruginosa/metabolismoRESUMO
The bacterial enzyme asparaginase is the main treatment option for acute lymphoblastic leukemia. However, it causes side effects, such as immunological reactions, and presents undesirable glutaminase activity. As an alternative, we have been studying asparaginase II from Saccharomyces cerevisiae, coded by ASP3 gene, which was cloned and expressed in Pichia pastoris. The recombinant asparaginase (ASP) presented antileukemic activity and a glutaminase activity 100 times lower in comparison to its asparaginase activity. In this work, we describe the development of a delivery system for ASP via its covalent attachment to functionalized polyethylene glycol (PEG) polymer chains in the outer surface of liposomes (ASP-enzymosomes). This new delivery system demonstrated antiproliferative activity against K562 (chronic myeloid leukemia) and Jurkat (acute lymphocytic leukemia) cell lines similar to that of ASP. The antiproliferative response of the ASP-enzymosomes against the Jurkat cells suggests equivalence to that of the free Escherichia coli commercial asparaginase (Aginasa®). Moreover, the ASP-enzymosomes were stable at 4 °C with no significant loss of activity within 4 days and retained 82% activity up to 37 days. Therefore, ASP-enzymosomes are a promising antileukemic drug.
Assuntos
Antineoplásicos/química , Asparaginase/química , Leucemia/tratamento farmacológico , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Asparaginase/genética , Asparaginase/metabolismo , Asparaginase/farmacologia , Composição de Medicamentos/métodos , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células Jurkat , Células K562 , Leucemia/patologia , Lipossomos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Células Tumorais CultivadasRESUMO
Resistance to chemotherapy is a major problem facing current cancer therapy, which is continuously aiming at the development of new compounds that are capable of tackling tumors that developed resistance toward common chemotherapeutic agents, such as doxorubicin (DOX). Alongside the development of new generations of compounds, nanotechnology-based delivery strategies can significantly improve the in vivo drug stability and target specificity for overcoming drug resistance. In this study, multifunctional gold nanoparticles (AuNP) have been used as a nanoplatform for the targeted delivery of an original anticancer agent, a Zn(II) coordination compound [Zn(DION)2]Cl2 (ZnD), toward better efficacy against DOX-resistant colorectal carcinoma cells (HCT116 DR). Selective delivery of the ZnD nanosystem to cancer cells was achieved by active targeting via cetuximab, NanoZnD, which significantly inhibited cell proliferation and triggered the death of resistant tumor cells, thus improving efficacy. In vivo studies in a colorectal DOX-resistant model corroborated the capability of NanoZnD for the selective targeting of cancer cells, leading to a reduction of tumor growth without systemic toxicity. This approach highlights the potential of gold nanoformulations for the targeting of drug-resistant cancer cells.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ouro/química , Nanopartículas Metálicas/química , Zinco/administração & dosagem , Animais , Antineoplásicos/farmacologia , Cetuximab/administração & dosagem , Cetuximab/farmacologia , Complexos de Coordenação/administração & dosagem , Complexos de Coordenação/farmacologia , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Zinco/farmacologiaRESUMO
Milk thistle (Silybum marianum) is well-known for its antioxidant activity due to the presence of silymarin. Albeit some studies show a potential for skin inflammation, its activity against dermal MMP-9 and MMP-2 remains to be studied. Silymarin isolated from an S. marianum herbal extract was tested for gelatinase inhibition in the presence of isolated MMP-9 and in dermal adenocarcinome HaCaT cells. Silymarin was then further tested in vivo, using a cutaneous inflammation mice model mediated by reactive oxygen species. Silymarin was able to significantly inhibit gelatinolytic activity in vitro without impairing cell growth and viability. Furthermore, inhibition was more pronounced in cells than with the isolated gelatinase, suggesting an additional effect upon metabolic pathways. In vivo, silymarin was able to reduce ear edema up to 74% and attenuated histological lesions. Results highlight silymarin potential for application in skin inflammatory disorders via gelatinase inhibition.
RESUMO
Breast cancer (BC) brain metastases (BCBM) is a severe condition frequently occurring in the triple-negative subtype. The study of BCBM pathogenesis and treatment has been hampered by the difficulty in establishing a reliable animal model that faithfully recapitulates the preferential formation of brain metastases. The injection of BC cells in the carotid artery of mice has been proposed but the procedure is challenging, with the metastatic pattern being scarcely characterized. In this work, we thoroughly describe an improved procedure, highlighting the tricks and challenges of the process, and providing a characterization of the brain and peripheral metastatic pattern at the cellular and molecular level. Triple-negative BC (4T1) cells were inoculated in the common carotid artery of BALB/c mice. Brains and peripheral organs were harvested at 7-14 days for the histological characterization of the metastases' pattern and the immunofluorescence analysis of specific markers. With our surgical procedure, both mouse death and procedure-associated weight loss were negligible. Brain metastases mostly occurred in the hippocampus, while sparse peripheral lesions were only detected in the lungs. Brain-colonizing BC cells presented proliferative (Ki-67) and epithelial (pan-cytokeratin and tomato lectin) features, which account for metastases' establishment. The presented surgical approach constitutes an important and reliable tool for BCBM studies.
Assuntos
Neoplasias Encefálicas , Neoplasias de Mama Triplo Negativas , Animais , Camundongos , Humanos , Artéria Carótida Primitiva , Modelos Animais de Doenças , Camundongos Endogâmicos BALB CRESUMO
Discovery of new selective anticancer, anti-inflammatory, and anti-microbial agents is a crucial and necessary step to ensure a pipeline for innovative products to improve disease management. Several new bioactive agents derived from plants have been investigated and an example is the steroidal glycoalkaloid (SGA) class of natural products found in plants, investigated for their health-beneficial biological activities. Among them, α-tomatine is a SGA derived from the plant parts of unripe green tomatoes. In this review we aimed at searching for two different perspectives to study α-tomatine from green tomatoes, namely from its dual action point of view: as an anti-nutrient and as a health promoter. The aspects associated to its synthesis and degradation were considered. Finally, the current strategies for its extraction from natural sources and the methodologies commonly used for its identification and quantification were discussed.
Assuntos
Anti-Infecciosos , Solanum lycopersicum , Anti-Infecciosos/metabolismo , Anti-Inflamatórios/metabolismo , Humanos , Solanum lycopersicum/metabolismo , Tomatina/análogos & derivados , Tomatina/metabolismoRESUMO
Leishmaniasis is a widespread tropical infection; cutaneous leishmaniasis (CL) is the most common form of this disease known to cause significant morbidity. Trace metals, including selenium, zinc, iron, and copper, are required for the activity of several enzymes involved in immune system responses. The aim of this research was to measure the serum levels of selenium (Se), zinc (Zn), copper (Cu), iron (Fe), and Zn/Cu ratio in patients with CL. In this case-control study, 80 patients with CL and 80 healthy volunteers (not exposed to CL) from a CL endemic region in southwest Iran agreed to participate. Both clinical and parasitological verifications were made to include each subject as a CL-positive case. A questionnaire was completed for each participant which included the following criteria: age (year), height (cm), weight (kg), body mass index (kg/m2), and duration of disease (day). The biochemical assays were performed according to the standard protocols, and the values of Zn, Cu, Se, and Fe were expressed in micrograms per deciliter (µg/dl). All results were expressed as mean ± standard deviation (SD), and the statistical significance level was defined to be less than 0.05 (P < 0.05). There were no statistically significant differences in terms of mean values of age, body weight, height, and body mass index between CL patients and the control group (P > 0.05). The mean ± SD concentrations of Zn, Fe, and Se in the control group were found to be 118.87 ± 6.35 µg/dl, 123.00 ± 8.40 µg/dl, and 11.26 ± 1.88 µg/dl, respectively. These trace elements (TEs) were statistically lower (P < 0.001) in patients with CL (case group) with values of 83.05 ± 7.32 µg/dl for Zn, 86.51 ± 10.09 µg/dl for Fe, and 3.83 ± 1.20 µg/dl for Se. We have also observed that serum levels of Cu in CL-positive group were significantly higher than in the controls (P < 0.001). Furthermore, CL patients had significantly lower Zn/Cu ratio than controls (0.63 ± 0.05 µg/dl vs. 1.11 ± 0.10; P < 0.001). The alternation in serum levels of TEs may be a part of the defense strategy of the organism. Based on these results, it can be suggested that serum levels of these TEs can be a useful marker to estimate the prognosis of CL infection.
Assuntos
Cobre , Ferro , Leishmaniose Cutânea , Selênio , Oligoelementos , Zinco , Estudos de Casos e Controles , Cobre/análise , Cobre/sangue , Humanos , Irã (Geográfico)/epidemiologia , Ferro/análise , Ferro/sangue , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/epidemiologia , Selênio/sangue , Zinco/análise , Zinco/sangueRESUMO
The aim of this work was to prepare and characterize inclusion complexes between a synthetic curcumin analog (dibenzalacetone, DBA) and beta-cyclodextrin (ß-CD); and to evaluate their in vitro antileishmanial activity. DBA was synthetized and characterized by spectroscopic methods and the inclusion complexes were obtained by kneading and lyophilization (LIO) in 1:1 and 1:2 stoichiometries. Phase solubility and dissolution assays showed a 40-fold increase in the aqueous solubility of DBA and its complete dissolution from LIO 1:1 formulation after 120 min respectively. Solid-state characterization by differential scanning calorimetry and near infrared spectroscopy demonstrated the inclusion of DBA in the ß-CD cavity at the molar ratios tested, with LIO 1:1 formulation being the most stable. Using nuclear magnetic resonance experiments, the protons inside the cavity of ß-CD were the most affected after the inclusion of DBA molecule. The cellular viability of THP-1 macrophage cells treated with plain DBA, ß-CD and DBA/CD inclusion complexes showed that the plain DBA and DBA/CD at 1:2 stoichiometry presented toxicity, while ß-CD alone and DBA/CD at 1:1 stoichiometry showed no toxicity up to 640 µg mL-1. The in vitro assay with free-living promastigotes demonstrated that plain DBA and ß-CD had IC50 of < 10 and > 320 µg mL-1 respectively, while only inclusion complexes with 1:1 stoichiometry showed antiproliferative activity with IC50 = 51.3 µg mL-1. Using the amastigote intracellular forms, there was also a difference between the plain and ß-CD complexed DBA with complexes of 1:1 and 1:2 stoichiometry presenting EC50 = 66.3 µg mL-1 and 58.9 µg mL-1 respectively. The study concluded that DBA/CD at 1:1 molar ratio has the potential to decrease the intrinsic toxicity of plain DBA towards Leishmania host cells, which may be a therapeutic advantage in the application of these compounds.