Utilizaçăo dos aminoácidos L-arginina e L-glutamina e produçăo de mediadores inflamatórios pelas células Walker 256 /

Orientadora : Maria Benigna Martinelli de OliveiraCo-orientadora : Maria Eliane Merlin RochaTese (doutorado) - Universidade Federal do Paraná, Setor de Cięncias Biológicas, Programa de Pós-Graduaçăo em Bioquímica. Defesa: Curitiba, 2008Inclui bibliografia e anexo

Utilizaçăo dos aminoácidos L-arginina e L-glutamina e produçăo de mediadores inflamatórios pelas células Walker 256 /

Orientadora : Maria Benigna Martinelli de OliveiraCo-orientadora : Maria Eliane Merlin RochaTese (doutorado) - Universidade Federal do Paraná, Setor de Cięncias Biológicas, Programa de Pós-Graduaçăo em Bioquímica. Defesa: Curitiba, 2008Inclui bibliografia e anexo

Increased cellular uptake of lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles due to surface modification with folic acid

Lauryl gallate loaded in superparamagnetic poly (methyl methacrylate) nanoparticles surface modified with folic acid were synthesized by miniemulsion polymerization in just one step. In vitro biocompatibility and cytotoxicity assays on L929 (murine fibroblast), human red blood, and HeLa (uterine colon cancer) cells were performed. The effect of folic acid at the nanoparticles surface was evaluated through cellular uptake assays in HeLa cells. Resultsshowed that the presence of folic acid did not affect substantially the polymer particle size (~120 nm), the superparamagnetic behavior, the encapsulation efficiency of lauryl gallate (~87 %), the Zeta potential (~38 mV) of thepolymeric nanoparticles or the release profile of lauryl gallate. The release profile of lauryl gallate from superparamagnetic poly(methyl methacrylate) nanoparticles presented an initial burst effect (0?1 h) followed by a slow andsustained release, indicating a biphasic release system.Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles with folic acid did not present cytotoxicity effects on L929 and human red blood cells. However, free lauryl gallate presented significant cytotoxic effects on L929 and human red blood cells at all tested concentrations. The presence of folic acid increased the cytotoxicity of lauryl gallate loaded in nanoparticles on HeLa cells due to a higher cellular uptake when HeLa cells were incubated at 37 °C. On the other hand, when the nanoparticles were incubated at low temperature (4 °C) cellular uptake was not observed, suggesting that the uptake occurred by folate receptor mediated energy-dependent endocytosis. Based on presented results our work suggeststhat this carrier system can be an excellent alternative in targeted drug delivery by folate receptor.Fil: Feuser, Paulo Emilio. Universidade Federal de Santa Catarina; BrasilFil: Carpio Arévalo, Juan Marcelo. Universidade Federal do Paraná; BrasilFil: Lima, Enio Junior. Comisión Nacional de Energía Atómica. Gerencia del Área de Investigación y Aplicaciones No Nucleares. Gerencia de Física. Laboratorio de Resonancias Magnéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; ArgentinaFil: Rodrigues Rossi, Gustavo. Universidade Federal do Paraná; BrasilFil: Trindade, Edvaldo da Silva. Universidade Federal do Paraná; BrasilFil: Merlin Rocha, Maria Eliane. Universidade Federal do Paraná; BrasilFil: Virtuoso Jacques, Amanda. Universidade Federal de Santa Catarina; BrasilFil: Ricci Júnior, Eduardo. Universidade Federal do Rio de Janeiro; BrasilFil: Santos Silva, Maria Claudia. Universidade Federal de Santa Catarina; BrasilFil: Sayer, Claudia. Universidade Federal de Santa Catarina; BrasilFil: Hermes de Araújo, Pedro H.. Universidade Federal de Santa Catarina; Brasi

Novel properties of melanins include promotion of DNA strand breaks, impairment of repair, and reduced ability to damage DNA after quenching of singlet oxygen

Melanins have been associated with the development of melanoma and its resistance to photodynamic therapy (PDT). Singlet molecular oxygen (102), which is produced by ultraviolet A solar radiation and the PDT system, is also involved. Here, we investigated the effects that these factors have on DNA damage and repair. Our results show that both types of melanin (eumelanin and pheomelanin) lead to DNA breakage in the absence of light irradiation and that eumelanin is more harmful than pheomelanin. Interestingly, melanins were found to bind to the minor grooves of DNA, guaranteeing close proximity to DNA and potentially causing the observed high levels of strand breaks. We also show that the interaction of melanins with DNA can impair the access of repair enzymes to lesions, contributing to the perpetuation of DNA damage. Moreover, we found that after melanins interact with 102, they exhibit a lower ability to induce DNA breakage; we propose that these effects are due to modifications of their structure. Together, our data highlight the different modes of action of the two types of melanin. Our results may have profound implications for cellular redox homeostasis, under conditions of induced melanin synthesis and irradiation with solar light. These results may also be applied to the development of protocols to sensitize melanoma cells to PDT. (c) 2012 Elsevier Inc. All rights reserved.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (Brasilia, DF, Brazil)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (Brasilia, DF, Brazil)CAPESCAPESCTINFRA/FINEPCTINFRA/FINEPFundacao AraucariaFundacao AraucariaInstituto Nacional de Ciencia e Tecnologia de Processos Redox em Biomedicina (Redoxoma)Instituto Nacional de Ciencia e Tecnologia de Processos REDOX em Biomedicina-Redoxom

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2).

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO2, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma

Preparation and characterization of 4-nitrochalcone-folic acid-poly(methyl methacrylate) nanocapsules and cytotoxic activity on HeLa and NIH3T3 cells

Chalcones of natural origin are plant metabolites which have been explored because of the cytotoxic effects towards tumor cells. In this study, the synthetic chalcone 4-nitrochalcone (4NC) and its encapsulated form in folic acid-poly(methyl methacrylate) (PMMA) nanocapsules (4NC-FA-PMMA) were investigated towards the cytotoxic effects on erytrocytes, mouse embryonic fibroblasts cells (NIH3T3), and tumor cells (HeLa cells). Characterization of 4NC-FA-PMMA presented spherical morphology with nanocapsule-type structure, a mean size of 170 ± 6 nm, a negative zeta potential of (−40 ± 4 mV), and an entrapment efficiency of ~80%. In HeLa cells, 4NC induced a dose-dependent reduction in cell viability, with an IC value of 46.7 μM. The cytotoxicity was confirmed by morphological alterations, cell death, and an increase in the population of hypodiploid cells. When 4NC-FA-PMMA nanocapsules were employed at concentrations of 15 and 30 μM the reduction in cell viability was higher than that of 4NC. In addition, 4NC and 4NC-FA-PMMA nanocapsules did not present any cytotoxic effect on the NIH3T3 cells and human erythrocytes up to 50 μM. These results demonstrated that the 4NC encapsulation in PMMA nanocapsules with folic acid-modified surface is a better system to promote selective cytotoxic effects to HeLa cells. Therefore, this formulation could be considered a promising preparation with potential chemotherapeutic action

Annexin V-FITC and propidium iodide staining of HepG2 treated with 1,3,4-thiadiazolium derivatives (the experimental conditions are described in the Materials and Methods section 2.5.3).

<p>The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 25 μM for 18–24 h. Then, the cells were collected with trypsin and 10.000 events were analyzed by flow cytometry by FL2 and FL1 filters. (A) control, (B) MI-D, (C) MI-J, (D) MI-4F and (E) MI-2,4diF. The figures show representative dot-plot with the different cell populations: left-bottom = labeled cells; left-top = PI labeled; right-top = doubly labeled; right-bottom = annexin V labeled. The results were expressed as mean ± SD of three independents experiments.</p

Effects of 1,3,4-thiadiazolium derivatives on hepatocytes morphology (the experimental conditions are described in the Materials and Methods section 2.6).

<p>Hepatocytes were incubated with the derivatives at 25 μM for 24 h. The images were obtained using inverted microscope. A: control (untreated cells); B-E: treatments by MI-D, MI-J, MI-4F and MI-2,4diF, respectively. The scale is indicated by black bars representing 50 μm. The photographs represent three different experiments in triplicate.</p

Effects of 1,3,4-thiadiazolium derivatives on HepG2 cell morphology (the experimental conditions are described in the Materials and Methods section 2.6).

<p>The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 5 μM for 3 h. The images were captured with a 100X magnification; they correspond to control (A), MI-D (B), MI-J (C), MI-4F (D) and MI-2,4diF (E). The scale is indicated by black bars representing 0.02 mm. The arrows show morphological modifications as blebs, increased volume and vacuolization. The photographs represent three different experiments in triplicate.</p

Annexin V-FITC and propidium iodide staining of hepatocytes treated by 1,3,4-thiadizolium derivatives (the experimental conditions are described in the Materials and Methods section 2.5.3).

<p>Hepatocytes were incubated with derivatives at 25 μM for 20 h. The images (10X magnification) were captured with an AXIOVERT 40CSFL fluorescence microscope. The scale is indicated by white bars representing 100 μm. The annexin V-FITC-positive cells are stained in green, and the PI-positive cells in red. The images represent (A) control (untreated cells), (B) ASA positive control at 20 mM, (C) MI-D, (D) MI-J, (E) MI-4F and (F) MI-2,4diF. The figures represent three different experiments in triplicate.</p