Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 29
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Int J Mol Sci ; 21(2)2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31936346

RESUMO

The aim of the manuscript is to discuss the influence of plant polyphenols in overcoming multidrug resistance in four types of solid cancers (breast, colorectal, lung and prostate cancer). Effective treatment requires the use of multiple toxic chemotherapeutic drugs with different properties and targets. However, a major cause of cancer treatment failure and metastasis is the development of multidrug resistance. Potential mechanisms of multidrug resistance include increase of drug efflux, drug inactivation, detoxification mechanisms, modification of drug target, inhibition of cell death, involvement of cancer stem cells, dysregulation of miRNAs activity, epigenetic variations, imbalance of DNA damage/repair processes, tumor heterogeneity, tumor microenvironment, epithelial to mesenchymal transition and modulation of reactive oxygen species. Taking into consideration that synthetic multidrug resistance agents have failed to demonstrate significant survival benefits in patients with different types of cancer, recent research have focused on beneficial effects of natural compounds. Several phenolic compounds (flavones, phenolcarboxylic acids, ellagitannins, stilbens, lignans, curcumin, etc.) act as chemopreventive agents due to their antioxidant capacity, inhibition of proliferation, survival, angiogenesis, and metastasis, modulation of immune and inflammatory responses or inactivation of pro-carcinogens. Moreover, preclinical and clinical studies revealed that these compounds prevent multidrug resistance in cancer by modulating different pathways. Additional research is needed regarding the role of phenolic compounds in the prevention of multidrug resistance in different types of cancer.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Flavonoides/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Flavonoides/efeitos adversos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia
2.
Cell Cycle ; 17(6): 766-779, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29417873

RESUMO

Human leukemia Jurkat T cells were analyzed for apoptosis and cell cycle by flow cytometry, using the Annexin V/propidium iodide (PI) standard assay, and a simple PI staining in Triton X-100/digitonin-enriched PI/RNase buffer, respectively. Cells treated with doxorubicin or menadione displayed a very strong correlation between the apoptotic cell fraction measured by the Annexin V/PI assay, and the weight of a secondary cell population that emerged on the forward scatter (FS)/PI plot, as well as on the side scatter (SS)/PI and FL1/PI plots generated from parallel cell cycle recordings. In both cases, the Pearson correlation coefficients were >0.99. In cell cycle determinations, PI fluorescence was detected on FL3 (620/30 nm), and control samples exhibited the expected linear dependence of FL3 on FL1 (525/40 nm) signals. However, increasing doses of doxorubicin or menadione generated a growing subpopulation of cells displaying a definite right-shift on the FS/FL3, SS/FL3 and FL1/FL3 plots, as well as decreased PI fluorescence, indicative of ongoing fragmentation and loss of nuclear DNA. By gating on these events, the resulting fraction of presumably sub-cycling cells (i.e. cells with cleaved DNA, counting sub-G0/G1, sub-S and sub-G2/M cells altogether) was closely similar to the apoptotic rate assessed by Annexin V/PI labeling. Taken together, these findings suggest a possible way to recognize the entire population of cells undergoing apoptotic DNA cleavage and simultaneously determine the cell cycle distribution of non-apoptotic cells in PI-labeled cell samples with various degrees of DNA fragmentation, using a simple and reproducible multiparametric analysis of flow cytometric recordings.


Assuntos
Fragmentação do DNA , Citometria de Fluxo/métodos , Propídio/química , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Doxorrubicina/toxicidade , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Células Jurkat , Vitamina K 3/toxicidade
3.
Int J Food Sci Nutr ; 69(5): 584-597, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29157036

RESUMO

In this study, we evaluated the effects of epigallocatechin-3-O-gallate (EGCG) in two cancer cell lines, A-431 overexpressing ErbB1 and SK-BR-3, overexpressing ErbB2. EGCG treatment showed dose-dependent collapse of mitochondrial membrane potential (Δψm), increase in reactive oxygen species (ROS) production, changes in nuclear morphology and reduced viability. Flow cytometry data indicated that EGCG partially decreases the phosphorylation of several proteins involved in cell proliferation and survival: pErbB1(Y1173, Y1068), pAkt(S473) and pERK(Y204). EGCG affected the clonogenic growth in both cell lines with an EC50 of 2.5 and 5.4 µM for A-431 and SK-BR-3, respectively. Wound scratch assay demonstrated that EGCG inhibited the healing in dose-dependent manner and the effect was correlated with partial reduction in phosphorylation of pFAK(S910). Our data suggest that EGCG administration might reduce the unfavourable traits, particularly associated with ErbB1/EGFR overexpression.


Assuntos
Neoplasias da Mama/classificação , Neoplasias da Mama/tratamento farmacológico , Catequina/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Catequina/química , Catequina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Estrutura Molecular , Espécies Reativas de Oxigênio
5.
Pharmacol Res ; 103: 300-17, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26687095

RESUMO

We have investigated the growth-suppressive action of epigallocatechin-3-gallate (EGCG) on human leukemia Jurkat T cells. Results show a strong correlation between the dose-dependent reduction of clonogenic survival following acute EGCG treatments and the EGCG-induced decline of the mitochondrial level of Ca(2+). The cell killing ability of EGCG was synergistically enhanced by menadione. In addition, the cytotoxic effect of EGCG applied alone or in combination with menadione was accompanied by apoptosis induction. We also observed that in acute treatments EGCG displays strong antioxidant properties in the intracellular milieu, but concurrently triggers some oxidative stress generating mechanisms that can fully develop on a longer timescale. In parallel, EGCG dose-dependently induced mitochondrial depolarization during exposure, but this condition was subsequently reversed to a persistent hyperpolarized mitochondrial state that was dependent on the activity of respiratory Complex I. Fluorimetric measurements suggest that EGCG is a mitochondrial Complex III inhibitor and indicate that EGCG evokes a specific cellular fluorescence with emission at 400nm and two main excitation bands (at 330nm and 350nm) that may originate from a mitochondrial supercomplex containing dimeric Complex III and dimeric ATP-synthase, and therefore could provide a valuable means to characterize the functional properties of the respiratory chain.


Assuntos
Catequina/análogos & derivados , Células Jurkat/efeitos dos fármacos , Vitamina K 3/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Catequina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células Jurkat/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
6.
PLoS One ; 9(7): e101575, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25010413

RESUMO

Na+/H+ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na+-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na+/H+ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respective physiological functions. Here we present the characterization of the Na+/H+ antiporter NhaA from Salmonella enterica serovar Thyphimurium LT2, the causing agent of food-born human gastroenteritis and typhoid like infections. The recombinant antiporter was functional in vivo and in vitro. Expression of its gene complemented the Na+-sensitive phenotype of an E. coli strain that lacks the main Na+/H+ antiporters. Purified to homogeneity, the antiporter was a dimer in solution as accurately determined by size-exclusion chromatography combined with multi-angle laser-light scattering and refractive index monitoring. The purified antiporter was fully capable of electrogenic Na+(Li+)/H+-antiport when reconstituted in proteoliposomes and assayed by solid-supported membrane-based electrophysiological measurements. Transport activity was inhibited by 2-aminoperimidine. The recorded negative currents were in agreement with a 1Na+(Li+)/2H+ stoichiometry. Transport activity was low at pH 7 and up-regulation above this pH value was accompanied by a nearly 10-fold decrease of KmNa (16 mM at pH 8.5) supporting a competitive substrate binding mechanism. K+ does not affect Na+ affinity or transport of substrate cations, indicating that selectivity of the antiport arises from the substrate binding step. In contrast to homologous E. coli NhaA, transport activity remains high at pH values above 8.5. The antiporter from S. Typhimurium is a promising candidate for combined structural and functional studies to contribute to the elucidation of the mechanism of pH-dependent Na+/H+ antiporters and to provide insights in the molecular basis of species-specific growth and survival strategies.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Salmonella typhimurium/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Fenômenos Eletrofisiológicos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Salmonella typhimurium/fisiologia , Sódio/metabolismo , Especificidade da Espécie , Especificidade por Substrato
7.
J Photochem Photobiol B ; 139: 76-84, 2014 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-24912405

RESUMO

The study of the photoinduced ultraweak photon emission in the optical wavelength range, namely the Delayed Luminescence, from human cells and tissues has an increasingly growing interest in view of its possible application in optical biopsy. Due to the low level, dedicated experimental set-up are necessary to reveal such photoluminescence signal. The paper reviews the results obtained in the field of cancer research, by using the experimental equipment for fast ultraweak luminescence analysis ARETUSA developed at the National Southern Laboratories of the National Nuclear Physics Institute (LNS-INFN), in Catania, Italy. Delayed Luminescence signals from normal and cancer cells are compared and the relationship between Delayed Luminescence and apoptosis is investigated.


Assuntos
Medições Luminescentes/instrumentação , Neoplasias/patologia , Apoptose , Humanos , Modelos Biológicos
8.
Leuk Res ; 38(7): 836-49, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24862793

RESUMO

The flavonoid quercetin and menadione (vitamin K3) are known as potent apoptogens in human leukemia Jurkat T cells. We explored some underlying mechanisms and the potential relevance of the combination quercetin-menadione for clinical applications. In acute treatments, quercetin manifested a strong antioxidant character, but induced a transient loss of Δψm, likely mediated by opening of the mitochondrial permeability transition pore. After removal of quercetin, persistent mitochondrial hyperpolarization was generated via stimulation of respiratory Complex I. In contrast, menadione-induced Δψm dissipation was only partially and transiently reversed after menadione removal. Results indicate that Ca(2+) release is a necessary event in quercetin-induced cell death and that the survival response to quercetin is delineated within 1h from exposure. Depending on dose, the two agents exhibited either antagonistic or synergistic effects in reducing clonogenicity of Jurkat cells. 24-h combinatorial regimens at equimolar concentrations of 10-15 µM, which are compatible with a clinically achievable (and safe) scheme, reduced cell viability at efficient rates. Altogether, these findings support the idea that the combination quercetin-menadione could improve the outcome of conventional leukemia therapies, and warrant the utility of additional studies to investigate the therapeutic effects of this combination in different cellular or animal models for leukemia.


Assuntos
Leucemia/tratamento farmacológico , Quercetina/farmacologia , Vitamina K 3/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Células Jurkat , Leucemia/patologia , Potenciais da Membrana/efeitos dos fármacos , NAD/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Rotenona/farmacologia
9.
Int J Biol Macromol ; 65: 176-87, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24444882

RESUMO

The polymorphism of amyloid fibrils is potentially crucial as it may underlie the natural variability of amyloid diseases and could be important in developing a fuller understanding of the molecular basis of protein deposition disorders. This study examines morphological differences in lysozyme fibrils and the implications of these differences in terms of cytotoxicity. The structural characteristics of amyloid fibrils formed under two different experimental conditions (acidic and neutral) were evaluated using spectroscopic methods, atomic force microscopy and image analysis. Growth curves and apoptotic/necrotic assays were used to determine the cytotoxic effect of fibrils on the LLC-PK1 renal cells. The results reveal that both types of mature lysozyme amyloid fibrils are actively involved in the cytotoxic process, however each exhibit different levels of cytotoxicity. Fibrils formed at acidic pH affect cell growth in a dose-dependent manner, but a threshold-dependent inhibition of cell growth was observed in the case of lysozyme fibrils prepared at neutral pH. Experiments examining the mechanism of the cell death suggest that both types of mature lysozyme fibrils trigger late apoptosis/necrosis at different fibril concentrations. Our findings clearly indicate that the intrinsic differences between amyloid fibrils due to their polymorphism result in different degrees of cytotoxicity.


Assuntos
Amiloide/química , Amiloide/toxicidade , Células Epiteliais/efeitos dos fármacos , Rim/citologia , Muramidase/química , Muramidase/toxicidade , Multimerização Proteica , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citotoxinas/química , Citotoxinas/toxicidade , Células Epiteliais/citologia , Concentração de Íons de Hidrogênio , Necrose/induzido quimicamente , Estrutura Secundária de Proteína
10.
J Nat Prod ; 77(2): 250-7, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24456004

RESUMO

Since the administration of synthetic medicines is associated with drug resistance and undesired side effects, utilization of natural compounds could be an alternative and complementary modality to inhibit or prevent the development of tumors. Epigallocatechin 3-O-gallate (EGCG, 1), the major flavan component of green tea, and genistein (2), a soy isoflavonoid, are known to have chemopreventive and chemotherapeutic effects against cancer. This study demonstrated that both flavonoids inhibit cell proliferation, an effect enhanced under serum-free conditions. Compound 1, but not 2, induced downregulation of ErbB1 and ErbB2 in mammary and epidermoid carcinoma cells, and its inhibitory effect on cell viability was mediated by the 67 kDa laminin receptor (67LR). While 1 was superior in inducing cell death, 2 was more efficient in arresting the tumor cells in the G2/M phase. Furthermore, number and brightness analysis revealed that 1 decreased the homoclustering of a lipid raft marker, glycosylphosphatidylinositol-anchored GFP, and it also reduced the co-localization between lipid rafts and 67LR. The main conclusion made is that the primary target of 1 may be the lipid raft component of the plasma membrane followed by secondary changes in the expression of ErbB proteins. Compound 2, on the other hand, must have other unidentified targets.


Assuntos
Anticarcinógenos/química , Anticarcinógenos/farmacologia , Catequina/análogos & derivados , Flavonoides/farmacologia , Genisteína/farmacologia , Receptores de Laminina/efeitos dos fármacos , Proteínas Ribossômicas/efeitos dos fármacos , Chá/química , Algoritmos , Anticarcinógenos/isolamento & purificação , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/metabolismo , Catequina/química , Catequina/isolamento & purificação , Catequina/farmacologia , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Flavonoides/química , Flavonoides/isolamento & purificação , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Genes erbB-1/genética , Genes erbB-1/fisiologia , Genes erbB-2/genética , Genes erbB-2/fisiologia , Genisteína/química , Genisteína/isolamento & purificação , Humanos , Estrutura Molecular , Polifenóis/farmacologia , Receptores de Laminina/genética , Receptores de Laminina/metabolismo , Receptores de Laminina/fisiologia , Glycine max/química
11.
J Biomed Opt ; 18(12): 127006, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24365956

RESUMO

The role of mitochondrial complex I in ultraweak photon-induced delayed photon emission [delayed luminescence (DL)] of human leukemia Jurkat T cells was probed by using complex I targeting agents like rotenone, menadione, and quercetin. Rotenone, a complex I-specific inhibitor, dose-dependently increased the mitochondrial level of reduced nicotinamide adenine dinucleotide (NADH), decreased clonogenic survival, and induced apoptosis. A strong correlation was found between the mitochondrial levels of NADH and oxidized flavin mononucleotide (FMNox) in rotenone-, menadione- and quercetin-treated cells. Rotenone enhanced DL dose-dependently, whereas quercetin and menadione inhibited DL as well as NADH or FMNox. Collectively, the data suggest that DL of Jurkat cells originates mainly from mitochondrial complex I, which functions predominantly as a dimer and less frequently as a tetramer. In individual monomers, both pairs of pyridine nucleotide (NADH/reduced nicotinamide adenine dinucleotide phosphate) sites and flavin (FMN-a/FMN-b) sites appear to bind cooperatively their specific ligands. Enhancement of delayed red-light emission by rotenone suggests that the mean time for one-electron reduction of ubiquinone or FMN-a by the terminal Fe/S center (N2) is 20 or 284 µs, respectively. All these findings suggest that DL spectroscopy could be used as a reliable, sensitive, and robust technique to probe electron flow within complex I in situ.


Assuntos
Complexo I de Transporte de Elétrons , Espectrometria de Fluorescência/métodos , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/efeitos dos fármacos , Complexo I de Transporte de Elétrons/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Células Jurkat , Cinética , NAD/química , NAD/metabolismo , NADP/química , NADP/metabolismo , Rotenona/farmacologia , Desacopladores/farmacologia
12.
Arch Biochem Biophys ; 540(1-2): 145-59, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24211435

RESUMO

Ryanodine receptors are generally thought to possess a high-affinity activating cytosolic Ca(2+) site and a low-affinity inhibitory cytosolic Ca(2+) site. By performing conformation selective measurements in which quercetin was used as a fluorescent marker for RyR3 (ryanodine receptor type 3) in Jurkat cells, we now find that the rectified RyR3 channel in open conformation may be regulated in situ by two cytosolic activating Ca(2+) sites, of low and high affinity, respectively, whereas no inhibitory Ca(2+) effect could be delineated. In the closed rectified channel, as well as in the open hindered channel, only the high affinity activating Ca(2+) site and the inhibitory Ca(2+) site were functional, whereas in the closed hindered channel all three regulatory Ca(2+) sites appeared to be operational. RyR3 also seems to possess one activating and two inhibitory quercetin sites. Corresponding Hill coefficients and affinities of these regulatory sites were estimated. Quercetin cellular uptake exhibited an initial rapid phase (~1.04 min), followed by slow accumulation of free quercetin inside the cytosol (~34.5 min). The RyR3-mediated Ca(2+) release current increased from a baseline of 247 to 287 pA in 1 min. after addition of 50 µM quercetin and then declined slowly to a plateau of 265 pA.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Corantes Fluorescentes/metabolismo , Quercetina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Citosol/metabolismo , Humanos , Ativação do Canal Iônico , Células Jurkat , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Espectrometria de Fluorescência
13.
Pflugers Arch ; 465(8): 1101-19, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23475436

RESUMO

Because channels of intracellular organelles are not directly accessible to the patch-clamp technique, the activity (open probability) of intracellular ion channels in intact cells has so far eluded direct examination. Here, we present strong evidence that the ratio F380/F440 of the quercetin-specific cellular fluorescence emitted at 540 nm upon excitation at 380/440 nm reflects the open probability of an endoplasmic reticulum Ca(2+) release channel, the ryanodine receptor (RyR), in both intact and permeabilized Jurkat cells. The time course of the Ca(2+) release signal induced by high levels of quercetin in intact cells and that of F380/F440 were strongly correlated. The RyR specific inhibitor, ryanodine, the RyR type 3 and 1 but not type 2 specific inhibitor, dantrolene, as well as the non-specific RyR inhibitor, ruthenium red, depressed consistently the quercetin-induced Ca(2+) transient. Confocal microscopy confirmed that the dual fluorescent signal emitted by quercetin colocalizes with the endoplasmic reticulum, not the mitochondria. A novel regulatory mechanism was identified whereby RyR activity under physiological conditions is partially suppressed (hindered channel), whereas the channel becomes nearly fully activated after exposure to millimolar concentrations of bulk cytosolic Ca(2+) and subsequent chelation of Ca(2+) (rectified channel). Upon rectification, the dependence of F380/F440 on the cytosolic Ca(2+) concentration was remarkably similar to that of the open probability of the RyR type 3, not 1 or 2, reported from bilayer experiments. So, quercetin appears to be a semi-specific fluorescent probe for the activity of ryanodine receptors, which in our Jurkat (clone E6.1) cell preparations probably reports the type 3 RyR activity.


Assuntos
Corantes Fluorescentes/metabolismo , Células Jurkat/metabolismo , Quercetina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Linhagem Celular Tumoral , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Humanos
14.
Food Chem Toxicol ; 61: 94-100, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23523830

RESUMO

Cell membrane fluidity, which can be altered by oxidative stress, plays an important role in the cell physiology. Flavonoids are among the most studied food substances that prevent and/or reduce oxidative stress, but their action mechanisms are far from being understood. We performed a study on the effect of quercetin and epigallocatechin-3-gallate on 2-Dimyristoyl-sn-glycero-3-phosphocholine small unilamellar vesicles (SUVs) with different amounts of cholesterol, using Laurdan as a fluorescent probe, to put into evidence the perturbations of the phospholipid membrane fluidity and local lipid order in an attempt to decipher the action mechanism of the flavonoids at the cell membrane level. Results indicate that polyphenols modulate the transition from the gel phase to the liquid crystalline phase of SUVs in all studied membranes. SUVs with cholesterol have by themselves higher phase transition temperature and the presence of polyphenols stabilizes further the membrane. Quercetin has a dose-dependent effect on the fluidity and local order of the lipid membranes, whilst epigallocatechin-3-gallate action is not dose-dependent, the differences being attributable to the hydrophobic/hydrophilic character of the substances. The findings are discussed within the frame of earlier reports on the effect of polyphenols on artificial membranes.


Assuntos
Catequina/análogos & derivados , Membrana Celular/efeitos dos fármacos , Colesterol/química , Lipossomos/química , Quercetina/farmacologia , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Catequina/farmacologia , Membrana Celular/química , Membrana Celular/metabolismo , Dimiristoilfosfatidilcolina/química , Relação Dose-Resposta a Droga , Corantes Fluorescentes/química , Lauratos/química , Fosfolipídeos/química , Temperatura
15.
Oxid Med Cell Longev ; 2012: 498914, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22829956

RESUMO

Following previous work, we investigated in more detail the relationship between apoptosis and delayed luminescence (DL) in human leukemia Jurkat T cells under a wide variety of treatments. We used menadione and hydrogen peroxide to induce oxidative stress and two flavonoids, quercetin, and epigallocatechin gallate, applied alone or in combination with menadione or H(2)O(2). 62 MeV proton beams were used to irradiate cells under a uniform dose of 2 or 10 Gy, respectively. We assessed apoptosis, cell cycle distributions, and DL. Menadione, H(2)O(2) and quercetin were potent inducers of apoptosis and DL inhibitors. Quercetin decreased clonogenic survival and the NAD(P)H level in a dose-dependent manner. Proton irradiation with 2 Gy but not 10 Gy increased the apoptotic rate. However, both doses induced a substantial G(2)/M arrest. Quercetin reduced apoptosis and prolonged the G(2)/M arrest induced by radiation. DL spectroscopy indicated that proton irradiation disrupted the electron flow within Complex I of the mitochondrial respiratory chain, thus explaining the massive necrosis induced by 10 Gy of protons and also suggested an equivalent action of menadione and quercetin at the level of the Fe/S center N2, which may be mediated by their binding to a common site within Complex I, probably the rotenone-binding site.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Flavonoides/farmacologia , Leucemia/patologia , Luminescência , Oxidantes/toxicidade , Prótons , Catequina/análogos & derivados , Catequina/farmacologia , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Humanos , Peróxido de Hidrogênio/toxicidade , Células Jurkat , Cinética , NADP/metabolismo , Teoria Quântica , Quercetina/farmacologia , Fatores de Tempo , Vitamina K 3/toxicidade
16.
Gen Physiol Biophys ; 31(1): 47-55, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22447830

RESUMO

Quercetin and epigallocatechin gallate are two of the most abundant polyphenols in dietary plants, including apples, onions, red wine and green tea. The bioactivity of polyphenols is linked to their ability to interact with cell membranes without being internalized. The aim of the present study was to assess the short-time effect of these polyphenols on membrane anisotropy and transmembrane potential of U937 monocytes and Jurkat T lymphoblasts. Results showed that quercetin and epigallocatechin gallate induced, after 20 minutes cell exposure, a dose-dependent increase of membrane anisotropy and polarization. Anisotropy increase was correlated with the reduction of lipid peroxidation. Our results could indicate that the antioxidant capacity of the tested polyphenols is due to their stabilizing effect on the cell membranes, thus contributing to cell protection in various pathologies and as adjuvant therapy in highly toxic treatment regimens.


Assuntos
Catequina/análogos & derivados , Membrana Celular/fisiologia , Fluidez de Membrana/fisiologia , Potenciais da Membrana/fisiologia , Quercetina/farmacologia , Antioxidantes/farmacologia , Catequina/farmacologia , Membrana Celular/efeitos dos fármacos , Humanos , Células Jurkat , Fluidez de Membrana/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Estatística como Assunto , Células U937
17.
Eur Biophys J ; 2012 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-22302013

RESUMO

Quercetin (QCT) is an important bioactive natural compound found in numerous edible plants. Since the lipid bilayer represents an essential compound of the cell membrane, QCT's direct interaction with this structure is of great interest. Therefore, we proposed to study the effects of QCT on DMPC liposomes containing cholesterol (Chol), and for this purpose Laurdan fluorescence was used. As a fluorescent probe, Laurdan is able to detect changes in membrane phase properties. When incorporated in lipid bilayers, Laurdan emits from two different excited states, a non-relaxed one when the bilayer packing is tight and a relaxed state when the bilayer packing is loose. The main tool for quantifying QCT's effects on phospholipid membranes containing Chol has been the analysis, the decomposition of Laurdan emission spectra in sums of two Gaussian functions on energy. This kind of approach has allowed good analysis of the balance between the two emitting states of Laurdan. Our results show that both Laurdan emission states are present to different extents in a wide temperature range for DMPC liposomes with Chol. QCT is decreasing the phase transition temperature in pure DMPC liposomes as proved by generalized polarization (GP) values. QCT also quenches Laurdan fluorescence, depending on the temperature and the presence of Chol in the membrane. Stern-Volmer constants were calculated for different lipid membrane compositions, and the conclusion was that the relaxed state favors the nonradiative transitions of the fluorophore.

18.
J Mol Model ; 18(2): 721-36, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21562824

RESUMO

We use a detailed modeling formalism based on numerical simulations of local calcium release events where the blurring of the image, the presence of diffusional barriers provided by large organelles situated close to the release site, as well as the variable position of the scan line with respect to the release site are taken into consideration. We have investigated the effect of the fluorescence noise fluctuations on the accuracy in computing the signal mass from linescan recordings and obtained a quantitative description of both the signal mass and the local increase in the free Ca(2+) level as a function of the release current, the release duration and the orientation of the scan line, for three different levels of noise magnitudes. The model could provide a very good fit to a wide set of available experimental data regarding the signal mass of puffs visualized by fluorescence microscopy in the Xenopus oocyte loaded with 40 µM Oregon Green-1 in the absence of the calcium chelator EGTA. Numerical simulations also predict the amplitude and the kinetics of calcium signals evolving in the absence of the indicator, and indicate that sub-maximal activation of IP(3) receptors could produce in average levels of about 2 µM and 0.4 µM free Ca(2+) close to a release site located in the animal or in the vegetal hemisphere, respectively, whereas the maximal levels reached in more rare events could be 11 µM and 4 µM, respectively.


Assuntos
Cálcio/química , Simulação por Computador , Modelos Teóricos , Animais , Cálcio/metabolismo , Corantes Fluorescentes , Cinética , Microscopia de Fluorescência , Oócitos/metabolismo , Fatores de Tempo
19.
Cell Biochem Biophys ; 58(3): 169-79, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20697976

RESUMO

Menadione (MD) is an effective cytotoxic drug able to produce intracellularly large amounts of superoxide anion. Quercetin (QC), a widely distributed bioflavonoid, can exert both antioxidant and pro-oxidant effects and is known to specifically inhibit cell proliferation and induce apoptosis in different cancer cell types. We have investigated the relation between delayed luminescence (DL) induced by UV-laser excitation and the effects of MD, hydrogen peroxide, and QC on apoptosis and cell cycle in human leukemia Jurkat T-cells. Treatments with 500 µM H2O2 and 250 µM MD for 20 min produced 66.0 ± 4.9 and 46.4 ± 8.6% apoptotic cell fractions, respectively. Long-term (24 h) pre-exposure to 5 µM, but not 0.5 µM QC enhanced apoptosis induced by MD, whereas short-term (1 h) pre-incubation with 10 µM QC offered 50% protection against H2O2-induced apoptosis, but potentiated apoptosis induced by MD. Since physiological levels of QC in the blood are normally less than 10 µM, these data can provide relevant information regarding the benefits of flavonoid-combined treatments of leukemia. All the three drugs exerted significant effects on DL. Our data are consistent with (1) the involvement of Complex I of the mitochondrial respiratory chain as an important source of delayed light emission on the 10 µs-10 ms scale, (2) the ability of superoxide anions to quench DL on the 100 µs-10 ms scale, probably via inhibition of reverse electron transfer at the Fe/S centers in Complex I, and (3) the relative insensitivity of DL to intracellular OH• and H2O2 levels.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Quercetina/farmacologia , Vitamina K 3/farmacologia , Proteínas de Ciclo Celular/metabolismo , Citometria de Fluxo , Humanos , Células Jurkat , Cinética , Leucemia/tratamento farmacológico , Leucemia/patologia , Espectrometria de Fluorescência
20.
Gen Physiol Biophys ; 29(1): 41-9, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20371879

RESUMO

The unilamellar liposomes of dimyristoylphosphatidylcholine (DMPC), marked with chlorophyll a (Chla), have been chosen as suitable models in studies aimed at determining the effects of natural antioxidant quercetin (QCT) and Hofmeister series anions on lipid bilayers. The variations of steady state fluorescence emission intensity of Chla have been recorded and the values of Chla fluorescence anisotropy, under constant temperature and viscosity, at pH = 7.3-7.4 have been measured. Two types of experiments have been performed. In the first type of experiment, the concentration of anions was maintained constant and the concentration of QCT was varied (from 0 to 100 micromol/l). In the second type of experiment, the concentration of QCT was constant (30 micromol/l) and the concentration of anions was varied (from 0 to 152 mmol/l). The quenching of Chla fluorescence by QCT pleads in favor of QCT insertion at interface water-lipid, in the vicinity of the polar heads of lipids from liposomal bilayer at physiological pH and temperature. Fluorescence anisotropy of Chla in liposomes brought more evidences for QCT localizations at lipid/water interface. Chla is sensing a more rigid microenvironment when QCT is added to the lipid bilayer and specific effects of the Hofmeister series anions.


Assuntos
Antioxidantes/química , Clorofila/química , Bicamadas Lipídicas/química , Lipossomos/química , Quercetina/química , Ânions , Clorofila A
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...