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1.
Int J Mol Sci ; 25(7)2024 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-38612439

RESUMO

Prostate cancer (PCa) is the most prevalent non-cutaneous cancer in men. Early PCa detection has been made possible by the adoption of screening methods based on the serum prostate-specific antigen and Gleason score (GS). The aim of this study was to correlate gene expression with the differentiation level of prostate adenocarcinomas, as indicated by GS. We used data from The Cancer Genome Atlas (TCGA) and included 497 prostate cancer patients, 52 of which also had normal tissue sample sequencing data. Gene ontology analysis revealed that higher GSs were associated with greater responses to DNA damage, telomere lengthening, and cell division. Positive correlation was found with transcription factor activator of the adenovirus gene E2 (E2F) and avian myelocytomatosis viral homolog (MYC) targets, G2M checkpoints, DNA repair, and mitotic spindles. Immune cell deconvolution revealed high M0 macrophage counts and an increase in M2 macrophages dependent on the GS. The molecular pathways most correlated with GSs were cell cycle, RNA transport, and calcium signaling (depleted). A combinatorial approach identified a set of eight genes able to differentiate by k-Nearest Neighbors (kNN) between normal tissues, low-Gleason tissues, and high-Gleason tissues with high accuracy. In conclusion, our study could be a step forward to better understanding the link between gene expression and PCa progression and aggressiveness.


Assuntos
Redes Reguladoras de Genes , Neoplasias da Próstata , Masculino , Humanos , Neoplasias da Próstata/genética , Ciclo Celular , Divisão Celular , Adenoviridae
2.
Int J Mol Sci ; 24(2)2023 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-36675137

RESUMO

The aim of the study was to use transcriptomics data to identify genes associated with advanced/aggressive breast cancer and their effect on survival outcomes. We used the publicly available The Cancer Genome Atlas (TCGA) database to obtain RNA sequence data from patients with less than five years survival (Poor Prognosis, n = 101), patients with greater than five years survival (Good Prognosis, n = 200), as well as unpaired normal tissue data (normal, n = 105). The data analyses performed included differential expression between groups and selection of subsets of genes, gene ontology, cell enrichment analysis, and survival analyses. Gene ontology results showed significantly reduced enrichment in gene sets related to tumor immune microenvironment in Poor Prognosis and cell enrichment analysis confirmed significantly reduced numbers of macrophages M1, CD8 T cells, plasma cells and dendritic cells in samples in the Poor Prognosis samples compared with Good Prognosis. A subset of 742 genes derived from differential expression analysis as well as genes coding for immune checkpoint molecules was evaluated for their effect on overall survival. In conclusion, this study may contribute to the better understanding of breast cancer transcriptomics and provide possible targets for further research and eventual therapeutic interventions.


Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/genética , Prognóstico , Agressão , Linfócitos T CD8-Positivos , Análise de Dados , Microambiente Tumoral/genética
3.
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
4.
Int J Mol Sci ; 20(5)2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823649

RESUMO

Prostate cancer is the one of the most frequently diagnosed cancers among men over the age of 50. Several lines of evidence support the observation that polyphenols have preventive and therapeutic effects in prostate cancer. Moreover, prostate cancer is ideal for chemoprevention due to its long latency. We propose here an equilibrated lifestyle with a diet rich in polyphenols as prophylactic attempts to slow down the progression of localized prostate cancer or prevent the occurrence of the disease. In this review, we will first summarize the molecular mechanisms of polyphenols in prostate cancer with a focus on the antioxidant and pro-oxidant effects, androgen receptors (AR), key molecules involved in AR signaling and their transactivation pathways, cell cycle, apoptosis, angiogenesis, metastasis, genetic aspects, and epigenetic mechanisms. The relevance of the molecular mechanisms is discussed in light of current bioavailability data regarding the activity of polyphenols in prostate cancer. We also highlight strategies for improving the bioavailability of polyphenols. We hope that this review will lead to further research regarding the bioavailability and the role of polyphenols in prostate cancer prevention and treatment.


Assuntos
Antineoplásicos/farmacocinética , Polifenóis/farmacocinética , Neoplasias da Próstata/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Disponibilidade Biológica , Humanos , Masculino , Polifenóis/farmacologia , Polifenóis/uso terapêutico
5.
Int J Mol Sci ; 19(12)2018 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-30487390

RESUMO

Colorectal cancer is one of the leading causes of death, and the third most diagnosed type of cancer, worldwide. It is most common amongst men and women over 50 years old. Risk factors include smoking, alcohol, diet, physical inactivity, genetics, alterations in gut microbiota, and associated pathologies (diabetes, obesity, chronic inflammatory bowel diseases). This review will discuss, in detail, the chemopreventive properties of some dietary compounds (phenolic compounds, carotenoids, iridoids, nitrogen compounds, organosulfur compounds, phytosterols, essential oil compounds, polyunsaturated fatty acids and dietary fiber) against colorectal cancer. We present recent data, focusing on in vitro, laboratory animals and clinical trials with the previously mentioned compounds. The chemopreventive properties of the dietary compounds involve multiple molecular and biochemical mechanisms of action, such as inhibition of cell growth, inhibition of tumor initiation, inhibition of adhesion, migration and angiogenesis, apoptosis, interaction with gut microbiota, regulation of cellular signal transduction pathways and xenobiotic metabolizing enzymes, etc. Moreover, this review will also focus on the natural dietary compounds' bioavailability, their synergistic protective effect, as well as the association with conventional therapy. Dietary natural compounds play a major role in colorectal chemoprevention and continuous research in this field is needed.


Assuntos
Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/prevenção & controle , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/prevenção & controle , Animais , Anticarcinógenos/uso terapêutico , Quimioprevenção/métodos , Humanos
6.
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
8.
J Oncol ; 2017: 1532534, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28286519

RESUMO

ErbB proteins overexpression, in both normal and mutated forms, is associated with invasive forms of cancer prone to metastasis and with stronger antiapoptotic mechanisms and therefore more challenging to treat. Downstream effectors of ErbB receptors mediating these phenotypic traits include MAPK, STAT, and PI3K/AKT/mTOR pathways. Various phytochemical compounds were studied for their large number of biological effects including anticancer activity. Among these compounds, epigallocatechin-3-gallate (EGCG), the main catechin from green tea leaves, and curcumin, component of the curry powder, constituted the object of numerous studies. Both compounds were shown to act directly either on ErbB expression, or on their downstream signaling molecules. In this paper we aim to review the involvement of ErbB proteins in cancer as well as the biologic activity of EGCG and curcumin in ErbB expressing and overexpressing malignancies. The problems arising in the administration of the two compounds due to their reduced bioavailability when orally administered, as well as the progress made in this field, from using novel formulations to improved dosing regimens or improved synthetic analogs, are also discussed.

9.
Phys Chem Chem Phys ; 18(28): 18855-67, 2016 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-27350395

RESUMO

The generation of Aß amyloid aggregates in the form of senile plaques in the brain is one of the pathological hallmarks of Alzheimer's disease (AD). There is no cure for AD and one of the recent treatment strategies is focused on the inhibition of amyloid fibrillization of Aß peptide. Fullerene C60 has been proposed as a candidate for destroying Aß aggregates but it is not soluble in water and its toxicity to cells remains largely ambiguous. To overcome these drawbacks, we synthesized and studied the effect of water-soluble fullerenol C60(OH)16 (fullerene C60 carrying 16 hydroxyl groups) on the amyloid fibrillization of Aß40 peptide in vitro. Using a Thioflavin T fluorescent assay and atomic force microscopy it was found that C60(OH)16 effectively reduces the formation of amyloid fibrils. The IC50 value is in the low range (µg ml(-1)) suggesting that fullerenol interferes with Aß40 aggregation at stoichiometric concentrations. The in silico calculations supported the experimental data. It was revealed that fullerenol tightly binds to monomer Aß40 and polar, negatively charged amino acids play a key role. Electrostatic interactions dominantly contribute to the binding propensity via interaction of the oxygen atoms from the COO(-) groups of side chains of polar, negatively charged amino acids with the OH groups of fullerenol. This stabilizes contact with either the D23 or K28 of the salt bridge. Due to the lack of a well-defined binding pocket fullerenol is also inclined to locate near the central hydrophobic region of Aß40 and can bind to the hydrophobic C-terminal of the peptide. Upon fullerenol binding the salt bridge becomes flexible, inhibiting Aß aggregation. In order to assess the toxicity of fullerenol, we found that exposure of neuroblastoma SH-SY5Y cells to fullerenol caused no significant changes in viability after 24 h of treatment. These results suggest that fullerenol C60(OH)16 represents a promising candidate as a therapeutic for Alzheimer's disease.


Assuntos
Doença de Alzheimer/metabolismo , Amiloide/química , Proteínas Amiloidogênicas/química , Fulerenos/química , Tiazóis/química , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Benzotiazóis , Simulação por Computador , Tiazóis/metabolismo
10.
Molecules ; 20(12): 22578-620, 2015 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-26694341

RESUMO

The review will discuss in detail the effects of polyphenols on breast cancer, including both the advantages and disadvantages of the applications of these natural compounds. First, we focus on the characterization of the main classes of polyphenols and then on in vitro and in vivo experiments carried out in breast cancer models. Since the therapeutic effects of the administration of a single type of polyphenol might be limited because of the reduced bioavailability of these drugs, investigations on combination of several polyphenols or polyphenols with conventional therapy will also be discussed. In addition, we present recent data focusing on clinical trials with polyphenols and new approaches with nanoparticles in breast cancer. Besides the clinical and translational findings this review systematically summarizes our current knowledge about the molecular mechanisms of anti-cancer effects of polyphenols, which are related to apoptosis, cell cycle regulation, plasma membrane receptors, signaling pathways and epigenetic mechanisms. At the same time the effects of polyphenols on primary tumor, metastasis and angiogenesis in breast cancer are discussed. The increasing enthusiasm regarding the combination of polyphenols and conventional therapy in breast cancer might lead to additional efforts to motivate further research in this field.


Assuntos
Anticarcinógenos/farmacologia , Neoplasias da Mama/prevenção & controle , Polifenóis/farmacologia , Animais , Anticarcinógenos/administração & dosagem , Inibidores da Aromatase/administração & dosagem , Inibidores da Aromatase/farmacologia , Quimioprevenção , Dieta , Glicólise/efeitos dos fármacos , Humanos , Polifenóis/administração & dosagem
11.
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
12.
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
13.
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
14.
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
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.
Proteomics ; 11(10): 2063-70, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21480528

RESUMO

Both fluorescence resonance energy transfer (FRET) and proximity ligation assay (PLA) are techniques used in the investigation of protein interactions but the latter has not been evaluated in a systematic way, prompting us to compare their performance quantitatively. Proteins were labeled with oligonucleotide- or fluorophore-conjugated antibodies and their proximity was analyzed by flow cytometry in order to obtain statistically robust data. Both intermolecular and intramolecular PLA signals reached saturation at high expression levels. At the same time, the FRET efficiency was independent of, while the FRET signal exhibited a strict linear correlation with the expression levels of proteins. When the density of oligonucleotide- and fluorophore-conjugated antibodies was systematically changed by competition with unlabeled antibodies the FRET signal was linearly proportional to the amount of bound fluorophore-tagged antibodies, whereas the PLA signal was again saturated. The saturation phenomenon in PLA could not be eliminated by decreasing the duration of the rolling circle amplification reaction. Our data imply that PLA is a semiquantitative measure of protein colocalizations due to non-linear effects in the reaction and that caution should be exercised when interpreting PLA data in a quantitative way.


Assuntos
Citometria de Fluxo/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Mapeamento de Interação de Proteínas/métodos , Marcadores de Afinidade/análise , Marcadores de Afinidade/química , Marcadores de Afinidade/metabolismo , Anticorpos/química , Anticorpos Monoclonais/química , Anticorpos Monoclonais Humanizados , Carbocianinas/química , Linhagem Celular Tumoral , Humanos , Modelos Lineares , Dinâmica não Linear , Trastuzumab
17.
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
18.
J Neurosci ; 28(3): 737-48, 2008 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-18199773

RESUMO

We describe two new transgenic mouse lines for studying pathological changes of Tau protein related to Alzheimer's disease. They are based on the regulatable expression of the four-repeat domain of human Tau carrying the FTDP17 (frontotemporal dementia and parkinsonism linked to chromosome 17) mutation deltaK280 (Tau(RD)/deltaK280), or the deltaK280 plus two proline mutations in the hexapeptide motifs (Tau(RD)/deltaK280/I277P/I308P). The deltaK280 mutation accelerates aggregation ("proaggregation mutant"), whereas the proline mutations inhibit Tau aggregation in vitro and in cell models ("antiaggregation mutant"). The inducible transgene expression was driven by the forebrain-specific CaMKIIalpha (calcium/calmodulin-dependent protein kinase IIalpha) promoter. The proaggregation mutant leads to Tau aggregates and tangles as early as 2-3 months after gene expression, even at low expression (70% of endogenous mouse Tau). The antiaggregation mutant does not aggregate even after 22 months of gene expression. Both mutants show missorting of Tau in the somatodendritic compartment and hyperphosphorylation in the repeat domain [KXGS motifs, targets of the kinase MARK (microtubule affinity regulating kinase)]. This indicates that these changes are related to Tau expression rather than aggregation. The proaggregation mutant causes astrogliosis, loss of synapses and neurons from 5 months of gene expression onward, arguing that Tau toxicity is related to aggregation. Remarkably, the human proaggregation mutant Tau(RD) coaggregates with mouse Tau, coupled with missorting and hyperphosphorylation at multiple sites. When expression of proaggregation Tau(RD) is switched off, soluble and aggregated exogenous Tau(RD) disappears within 1.5 months. However, tangles of mouse Tau, hyperphosphorylation, and missorting remain, suggesting an extended lifetime of aggregated wild-type Tau once a pathological conformation and aggregation is induced by a proaggregation Tau species.


Assuntos
Neurônios/patologia , Sinapses/patologia , Tauopatias/metabolismo , Tauopatias/patologia , Proteínas tau/química , Fatores Etários , Animais , Morte Celular/fisiologia , Detergentes/farmacologia , Modelos Animais de Doenças , Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão/métodos , Mutação/fisiologia , Emaranhados Neurofibrilares/metabolismo , Emaranhados Neurofibrilares/patologia , Neurônios/ultraestrutura , Fosforilação , Estrutura Terciária de Proteína , Sarcosina/análogos & derivados , Sarcosina/farmacologia , Coloração pela Prata/métodos , Sinapses/metabolismo , Sinapses/ultraestrutura , Proteínas tau/efeitos dos fármacos , Proteínas tau/genética
19.
J Biol Chem ; 282(43): 31755-65, 2007 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-17716969

RESUMO

Neurofibrillary lesions are characteristic for a group of human diseases, named tauopathies, which are characterized by prominent intracellular accumulations of abnormal filaments formed by the microtubule-associated protein Tau. The tauopathies are accompanied by abnormal changes in Tau protein, including pathological conformation, somatodendritic mislocalization, hyperphosphorylation, and aggregation, whose interdependence is not well understood. To address these issues we have created transgenic mouse lines in which different variants of full-length Tau are expressed in a regulatable fashion, allowing one to switch the expression on and off at defined time points. The Tau variants differ by small mutations in the hexapeptide motifs that control the ability of Tau to adopt a beta-structure conformation and hence to aggregate. The "pro-aggregation" mutant DeltaK280, derived from one of the mutations observed in frontotemporal dementias, aggregates avidly in vitro, whereas the "anti-aggregation" mutant DeltaK280/PP cannot aggregate because of two beta-breaking prolines. In the transgenic mice, the pro-aggregation Tau induces a pathological conformation and pre-tangle aggregation, even at low expression levels, the anti-aggregation mutant does not. This illustrates that abnormal aggregation is primarily controlled by the molecular structure of Tau in vitro and in the organism. Both variants of Tau become mislocalized and hyperphosphorylated independently of aggregation, suggesting that localization and phosphorylation are mainly a consequence of increased concentration. These pathological changes are reversible when the expression of Tau is switched off. The pro-aggregation Tau causes a strong reduction in spine synapses.


Assuntos
Modelos Animais de Doenças , Sinapses/patologia , Tauopatias/patologia , Proteínas tau/química , Proteínas tau/metabolismo , Motivos de Aminoácidos , Animais , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Mutação , Fosforilação , Conformação Proteica , Isoformas de Proteínas , Tauopatias/genética , Tauopatias/metabolismo , Proteínas tau/genética
20.
Traffic ; 7(7): 873-88, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16734669

RESUMO

Amyloid-beta, a peptide derived from the precursor protein APP, accumulates in the brain and contributes to the neuropathology of Alzheimer's disease. Increased generation of amyloid-beta might be caused by axonal transport inhibition, via increased dwell time of APP vesicles and thereby higher probability of APP cleavage by secretase enzymes residing on the same vesicles. We tested this hypothesis using a neuronal cell culture model of inhibited axonal transport and by imaging vesicular transport of fluorescently tagged APP and beta-secretase (BACE1). Microtubule-associated tau protein blocks vesicle traffic by inhibiting the access of motor proteins to the microtubule tracks. In neurons co-transfected with CFP-tau, APP-YFP traffic into distal neurites was strongly reduced. However, this did not increase amyloid-beta levels. In singly transfected axons, APP-YFP was transported in large tubules and vesicles moving very fast (on average 3 microm/s) and with high fluxes in the anterograde direction (on average 8.4 vesicles/min). By contrast, BACE1-CFP movement was in smaller tubules and vesicles that were almost 2x slower (on average 1.6 microm/s) with approximately 18x lower fluxes (on average 0.5 vesicles/min). Two-colour microscopy of co-transfected axons confirmed that the two proteins were sorted into distinct carriers. The results do not support the above hypothesis. Instead, they indicate that APP is transported on vesicles distinct from the secretase components and that amyloid-beta is not generated in transit when transport is blocked by tau.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/biossíntese , Proteínas tau/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Animais , Células Cultivadas , Córtex Cerebral/metabolismo , Chlorocebus aethiops , Regulação da Expressão Gênica , Humanos , Neurônios/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Transporte Proteico , Ratos , Ratos Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Ganglionares da Retina/metabolismo , Proteínas tau/genética
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