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1.
Cell Biol Int ; 45(2): 411-421, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33140880

RESUMO

Breast cancer is one of the most common cancers in the female population worldwide, and its development is thought to be associated with genetic mutations that lead to uncontrolled and accelerated growth of breast cells. This abnormal behavior requires extra energy, and indeed, tumor cells display a rewired energy metabolism compared to normal breast cells. Inorganic phosphate (Pi) is a glycolytic substrate of glyceraldehyde-3-phosphate dehydrogenase and has an important role in cancer cell proliferation. For cells to obtain Pi, ectoenzymes in the plasma membrane with their catalytic site facing the extracellular environment can hydrolyze phosphorylated molecules, and this is an initial and possibly limiting step for the uptake of Pi by carriers that behave as adjuvants in the process of energy harvesting and thus partially contributes to tumor energy requirements. In this study, the activity of an ectophosphatase in MDA-MB-231 cells was biochemically characterized, and the results showed that the activity of this enzyme was higher in the acidic pH range and that the enzyme had a Km = 4.5 ± 0.5 mM para-nitrophenylphosphate and a Vmax = 2280 ± 158 nM × h-1 × mg protein-1 . In addition, classical acid phosphatase inhibitors, including sodium orthovanadate, decreased enzymatic activity. Sodium orthovanadate was able to inhibit ectophosphatase activity while also inhibiting cell proliferation, adhesion, and migration, which are important processes in tumor progression, especially in metastatic breast cancer MDA-MB-231 cells that have higher ectophosphatase activity than MCF-7 and MCF-10 breast cells.


Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Fosfatos/metabolismo , Neoplasias de Mama Triplo Negativas , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Neoplasias de Mama Triplo Negativas/enzimologia , Neoplasias de Mama Triplo Negativas/patologia
2.
Front Oncol ; 8: 441, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30460192

RESUMO

[This corrects the article DOI: 10.3389/fonc.2018.00090.].

3.
Front Oncol ; 8: 353, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30234016

RESUMO

In spite of a great deal of work, the biochemical mechanisms underlying tumorigenesis and metastasis are not yet fully understood. Specifically regarding metastasis many authors consider that malignancy is caused by the accumulation of mutations. However, evidence is gathering to show that tumors are composed of heterogeneous cell populations subjected to selective pressures. In this micro evolutionary scenario, intra- and extra-cellular selective pressures will determine which subpopulations of tumor cells will thrive and be able to dissociate from the tumor as autonomous metastatic cells. We propose here that alteration of conformations of transcription factors confer novel non-canonical functions that may induce oncogenesis and metastasis in a mutation independent manner. We argue that the functional plasticity of transcription factors is due to intrinsically disordered domains (IDRs) of proteins. IDRs prevent spontaneous folding of proteins into well-defined three-dimensional structures. Because most transcription factors contain IDRs, each could potentially interact with many ligands. This high degree of functional pleiotropy would then be ultimately responsible for the metastatic phenotype. The conformations of proteins can be altered by chemical chaperones collectively known as osmolytes. Osmolytes are small organic molecules permeable through biological membranes that can accumulate in cells, increase the thermodynamic stability of proteins, modulate enzyme activity and prevent protein aggregation. Thus, by modifying IDRs, osmolytes could subvert the homeostatic regulatory network of cells. Untargeted metabolomic analysis of oral cancer cells showed that those with the greatest metastatic potential contained several osmolytes that were absent in the non-metastatic cells. We hypothesize that high concentrations of osmolytes might promote conformational alterations of transcription factors that favor metastatic behavior. This hypothesis is eminently testable by investigating whether: (a) the intracellular microenvironment of metastatic cells differs from non-metastatic cells and whether osmolytes are responsible for this change and (b) high intracellular concentrations of osmolytes are sufficient to induce structural modifications in regulatory protein so as to establish novel interactive networks that will constitute the metastatic phenotype. Synthetic cell penetrating peptides mimicking IDRs could act as sensitive probes. By exposing the peptides to the microenvironments of living tumor and metastatic tumor cells one should be able to compare the chemical shifts as revealed by spectra obtained by nuclear magnetic resonance (NMR).

4.
Front Oncol ; 8: 90, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29675398

RESUMO

Cancer outcome has improved since introduction of target therapy. However, treatment success is still impaired by the same drug resistance mechanism of classical chemotherapy, known as multidrug resistance (MDR) phenotype. This phenotype promotes resistance to drugs with different structures and mechanism of action. Recent reports have shown that resistance acquisition is coupled to metabolic reprogramming. High-gene expression, increase of active transport, and conservation of redox status are one of the few examples that increase energy and substrate demands. It is not clear if the role of this metabolic shift in the MDR phenotype is related to its maintenance or to its induction. Apart from the nature of this relation, the metabolism may represent a new target to avoid or to block the mechanism that has been impairing treatment success. In this mini-review, we discuss the relation between metabolism and MDR resistance focusing on the multiple non-metabolic functions that enzymes of the glycolytic pathway are known to display, with emphasis with the diverse activities of glyceraldehyde-3-phosphate dehydrogenase.

5.
Biochem Biophys Rep ; 10: 267-275, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28955754

RESUMO

MAGE-A10 is a member of the MAGE protein family (melanoma associated antigen) which is overexpressed in cancer cells. Although MAGE-A10 has been characterized for some time and is generally associated to metastasis its function remains unknown. Here we describe experiments using as models oral squamous cell carcinoma (OSCC) cell lines displaying increasing metastatic potential (LN1 and LN2). These cell lines were transduced with lentivirus particles coding for short hairpin against MAGE-A10 mRNA. Repression of MAGE-A10 expression in LN2 cells altered their morphology and impaired growth of LN1 and LN2 cell lines. Furthermore, repression of MAGE-A10 expression increased cell-cell and cell matrix adhesion. Furthermore shMAGEA10 cells were shown to assemble aberrantly on a 3D culture system (microspheroids) when compared to cells transduced with the control scrambled construct. Cell migration was inhibited in knocked down cells as revealed by two different migration assays, wound healing and a phagokinetic track motility assay. In vitro invasion assay using a leiomyoma tissue derived matrix (myogel) showed that shMAGEA10 LN1 and shMAGEA10 LN2 cells displayed a significantly diminished ability to penetrate the matrices. Concomitantly, the expression of E-cadherin, N-cadherin and vimentin genes was analyzed. shMAGEA10 activated the expression of E-cadherin and repression N-cadherin and vimentin transcription. Taken together the results indicate that MAGE-A10 exerts its effects at the level of the epithelial-mesenchymal transition (EMT) presumably by regulating the expression of adhesion molecules.

6.
FEBS J ; 283(1): 54-73, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26417966

RESUMO

Efforts to understand the mechanistic principles driving cancer metabolism and proliferation have been lately governed by genomic, transcriptomic and proteomic studies. This paper analyzes the caveats of these approaches. As molecular biology's central dogma proposes a unidirectional flux of information from genes to mRNA to proteins, it has frequently been assumed that monitoring the changes in the gene sequences and in mRNA and protein contents is sufficient to explain complex cellular processes. Such a stance commonly disregards that post-translational modifications can alter the protein function/activity and also that regulatory mechanisms enter into action, to coordinate the protein activities of pathways/cellular processes, in order to keep the cellular homeostasis. Hence, the actual protein activities (as enzymes/transporters/receptors) and their regulatory mechanisms ultimately dictate the final outcomes of a pathway/cellular process. In this regard, it is here documented that the mRNA levels of many metabolic enzymes and transcriptional factors have no correlation with the respective protein contents and activities. The validity of current clinical mRNA-based tests and proposed metabolite biomarkers for cancer detection/prognosis is also discussed. Therefore, it is proposed that, to achieve a thorough understanding of the modifications undergone by proliferating cancer cells, it is mandatory to experimentally analyze the cellular processes at the functional level. This could be achieved (a) locally, by examining the actual protein activities in the cell and their kinetic properties (or at least kinetically characterize the most controlling steps of the pathway/cellular process); (b) systemically, by analyzing the main fluxes of the pathway/cellular process, and how they are modulated by metabolites, all which should contribute to comprehending the regulatory mechanisms that have been altered in cancer cells. By adopting a more holistic approach it may become possible to improve the design of therapeutic strategies that would target cancer cells more specifically.


Assuntos
Genômica , Neoplasias/metabolismo , Neoplasias/patologia , Fenótipo , Animais , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/diagnóstico , Neoplasias/genética , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Biochem J ; 473(6): 703-15, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26699902

RESUMO

Tumours display different cell populations with distinct metabolic phenotypes. Thus, subpopulations can adjust to different environments, particularly with regard to oxygen and nutrient availability. Our results indicate that progression to metastasis requires mitochondrial function. Our research, centered on cell lines that display increasing degrees of malignancy, focused on metabolic events, especially those involving mitochondria, which could reveal which stages are mechanistically associated with metastasis. Melanocytes were subjected to several cycles of adhesion impairment, producing stable cell lines exhibiting phenotypes representing a progression from non-tumorigenic to metastatic cells. Metastatic cells (4C11+) released the highest amounts of lactate, part of which was derived from glutamine catabolism. The 4C11+ cells also displayed an increased oxidative metabolism, accompanied by enhanced rates of oxygen consumption coupled to ATP synthesis. Enhanced mitochondrial function could not be explained by an increase in mitochondrial content or mitochondrial biogenesis. Furthermore, 4C11+ cells had a higher ATP content, and increased succinate oxidation (complex II activity) and fatty acid oxidation. In addition, 4C11+ cells exhibited a 2-fold increase in mitochondrial membrane potential (ΔΨmit). Consistently, functional assays showed that the migration of cells depended on glutaminase activity. Metabolomic analysis revealed that 4C11+ cells could be grouped as a subpopulation with a profile that was quite distinct from the other cells investigated in the present study. The results presented here have centred on how the multiple metabolic inputs of tumour cells may converge to compose the so-called metastatic phenotype.


Assuntos
Glutamina/metabolismo , Melanócitos/fisiologia , Melanoma/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio/fisiologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Glucose/metabolismo , Glutaminase/metabolismo , Glutamina/genética , Lactatos/metabolismo , Melanócitos/patologia , Melanoma/patologia , Potenciais da Membrana/fisiologia , Metabolismo , Camundongos , Oxirredução , Fenótipo
8.
Arch Toxicol ; 88(7): 1327-50, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24792321

RESUMO

Significant efforts have been made for the development of new anticancer drugs (protein kinase or proteasome inhibitors, monoclonal humanized antibodies) with presumably low or negligible side effects and high specificity. However, an in-depth analysis of the side effects of several currently used canonical (platin-based drugs, taxanes, anthracyclines, etoposides, antimetabolites) and new generation anticancer drugs as the first line of clinical treatment reveals significant perturbation of glycolysis and oxidative phosphorylation. Canonical and new generation drug side effects include decreased (1) intracellular ATP levels, (2) glycolytic/mitochondrial enzyme/transporter activities and/or (3) mitochondrial electrical membrane potentials. Furthermore, the anti-proliferative effects of these drugs are markedly attenuated in tumor rho (0) cells, in which functional mitochondria are absent; in addition, several anticancer drugs directly interact with isolated mitochondria affecting their functions. Therefore, several anticancer drugs also target the energy metabolism, and hence, the documented inhibitory effect of anticancer drugs on cancer growth should also be linked to the blocking of ATP supply pathways. These often overlooked effects of canonical and new generation anticancer drugs emphasize the role of energy metabolism in maintaining cancer cells viable and its targeting as a complementary and successful strategy for cancer treatment.


Assuntos
Antineoplásicos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Animais , Antineoplásicos/efeitos adversos , Desenho de Fármacos , Glicólise/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Terapia de Alvo Molecular , Neoplasias/patologia
9.
PLoS One ; 8(12): e82484, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24358189

RESUMO

Transthyretin (TTR) is a tetrameric beta-sheet-rich protein. Its deposits have been implicated in four different amyloid diseases. Although aggregation of the wild-type sequence is responsible for the senile form of the disease, more than one hundred variants have been described thus far, most of which confer a more amyloidogenic character to TTR, mainly because they compromise the stability of the protein in relation to monomer formation, which upon misfolding is intrinsically aggregation-prone. We report the case of a Brazilian patient suffering from a severe cardiomyopathy who carries a rare mutation in exon 2 of the TTR gene that results in an Ala to Asp substitution at position 19 (A19D). The putative pathogenic mechanisms of this variant were analyzed in silico. We constructed a structural model for the A19D tetramer from which its thermodynamic stability was compared to that displayed by the V30M (more amyloidogenic than WT-TTR) and T119M (non-amyloidogenic) variants. The FoldX force field predicted that A19D and V30M are 10.88 and 8.07 kCal/mol less stable than the WT-TTR, while T119M is 5.15 kCal/mol more stable, which is consistent with the aggregation propensities exhibited by these variants. We analyzed the step in which the tetramer-dimer-monomer-unfolded monomer equilibrium might contribute the most to the increased or decreased amyloidogenicity in each variant. Our results suggest that the concentration of four non-native negative charges occur inside thyroxine-binding channels, and the loss of contacts at both the tetrameric and dimeric interfaces would account for an overall decreased stability of the tetramer and the consequent enhanced amyloidogenicity of the A19D variant. As far as we know, this is the first description of a non-V30M mutation in Brazil.


Assuntos
Amiloidose/metabolismo , Cardiomiopatias/metabolismo , Pré-Albumina/metabolismo , Amiloidose/genética , Cardiomiopatias/genética , Humanos , Masculino , Mutação , Pré-Albumina/genética , Desnaturação Proteica , Estrutura Quaternária de Proteína
10.
FEBS J ; 280(22): 5737-49, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24034837

RESUMO

To determine the extent to which the supply of the precursor 2-oxoglutarate (2-OG) controls the synthesis of lysine in Saccharomyces cerevisiae growing exponentially in high glucose, top-down elasticity analysis was used. Three groups of reactions linked by 2-OG were defined. The 2-OG supply group comprised all metabolic steps leading to its formation, and the two 2-OG consumer groups comprised the enzymes and transporters involved in 2-OG transformation into lysine and glutamate and their further utilization for protein synthesis and storage. Various 2-OG steady-state concentrations that produced different fluxes to lysine and glutamate were attained using yeast mutants with increasing activities of Krebs cycle enzymes and decreased activities of Lys synthesis enzymes. The elasticity coefficients of the three enzyme groups were determined from the dependence of the amino acid fluxes on the 2-OG concentration. The respective degrees of control on the flux towards lysine (flux control coefficients) were determined from their elasticities, and were 1.1, 0.41 and -0.52 for the 2-OG producer group and the Lys and Glu branches, respectively. Thus, the predominant control exerted by the 2-OG supply on the rate of lysine synthesis suggests that over-expression of 2-OG producer enzymes may be a highly effective strategy to enhance Lys production.


Assuntos
Ácidos Cetoglutáricos/metabolismo , Lisina/biossíntese , Saccharomyces cerevisiae/metabolismo , Ciclo do Ácido Cítrico/genética , Enzimas/genética , Enzimas/metabolismo , Cinética , Redes e Vias Metabólicas , Modelos Biológicos , Mutação , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
Parasitology ; 140(9): 1085-95, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23673212

RESUMO

SMYB1 is a Schistosoma mansoni protein highly similar to members of the Y-box binding protein family. Similar to other homologues, SMYB1 is able to bind double- and single-stranded DNA, as well as RNA molecules. The characterization of proteins involved in the regulation of gene expression in S. mansoni is of great importance for the understanding of molecular events that control morphological and physiological changes in this parasite. Here we demonstrate that SMYB1 is located in the cytoplasm of cells from different life-cycle stages of S. mansoni, suggesting that this protein is probably acting in mRNA metabolism in the cytoplasm and corroborating previous findings from our group that showed its ability to bind RNA. Protein-protein interactions are important events in all biological processes, since most proteins execute their functions through large supramolecular structures. Yeast two-hybrid screenings using SMYB1 as bait identified a partner in S. mansoni similar to the SmD3 protein of Drosophila melanogaster (SmRNP), which is important in the assembly of small nuclear ribonucleoprotein complexes. Also, pull-down assays were conducted using immobilized GST-SMYB1 proteins and confirmed the SMYB1-SmRNP interaction. The interaction of SMYB1 with a protein involved in mRNA processing suggests that it may act in processes such as turnover, transport and stabilization of RNA molecules.


Assuntos
Proteínas de Helminto/metabolismo , RNA de Helmintos/metabolismo , RNA Mensageiro/metabolismo , Schistosoma mansoni/metabolismo , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Transporte Biológico , Citoplasma/metabolismo , Feminino , Biblioteca Gênica , Proteínas de Helminto/genética , Imuno-Histoquímica , Masculino , RNA de Helmintos/genética , RNA Mensageiro/genética , Coelhos , Schistosoma mansoni/genética , Técnicas do Sistema de Duplo-Híbrido
12.
Tumour Biol ; 33(3): 739-48, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22407532

RESUMO

Previously, we demonstrated that A549, a human lung cancer cell line, could be adapted to the free radical nitric oxide (NO●). NO● is known to be over expressed in human tumors. The original cell line, A549 (parent), and the newly adapted A549-HNO (which has a more aggressive phenotype) serve as a useful model system to study the biology of NO●. To see if tumor cells can similarly be adapted to any free radical with the same outcome, herein we successfully adapted A549 cells to high levels of hydrogen peroxide (HHP). A549-HHP, the resulting cell line, was more resistant and grew better then the parent cell line, and showed the following characteristics: (1) resistance to hydrogen peroxide, (2) resistance to NO●, (3) growth with and without hydrogen peroxide, and (4) resistance to doxorubicin. Gene chip analysis was used to determine the global gene expression changes between A549-parent and A549-HHP and revealed significant changes in the expression of over 1,700 genes. This gene profile was markedly different from that obtained from the A549-HNO cell line. The mitochondrial DNA content of the A549-HHP line determined by quantitative PCR favored a change for a more anaerobic metabolic profile. Our findings suggest that any free radical can induce resistance to other free radicals; this is especially important given that radiation therapy and many chemotherapeutic agents exert their effect via free radicals. Utilizing this model system to better understand the role of free radicals in tumor biology will help to develop new therapeutic approaches to treat lung cancer.


Assuntos
Adaptação Fisiológica , Adenocarcinoma/metabolismo , Peróxido de Hidrogênio/farmacologia , Neoplasias Pulmonares/metabolismo , Adenocarcinoma/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA Mitocondrial , Doxorrubicina/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Resistência a Medicamentos/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética
13.
Immunobiology ; 216(3): 275-84, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20851496

RESUMO

Dendritic cells (DCs) are professional antigen-presenting cells with attributes for priming/activating T cells and mediating immune responses. Considering the importance of DCs in the initiation of immune responses, it will be of interest to study their mechanisms of regulation. Histone-modifying enzymes, such as histone deacetylases (HDACs), are critical in controlling chromatin organization. The aim of our study was to investigate DC differentiation under the influence of sodium butyrate (NaB), a short chain fatty acid that is a histone deacetylase inhibitor. Monocytes from healthy individuals were differentiated into immature DCs with IL-4 and GM-CSF in the presence or absence of NaB. DC differentiation was evaluated by CD14 and CD1a expression by flow cytometry. We observed that monocytes stimulated to differentiate in the presence of NaB displayed colony formation and dendritic cell morphology, lost CD14 and showed decreased secretion of IL-1ß. The acquisition of CD1a, however, was impaired. Being a natural short chain fatty acid, NaB may regulate CD1a acquisition independently of its HDAC inhibitory activity. We observed that the addition of peroxisome proliferator-activated receptor γ (PPAR-γ) antagonist (GW9662) did not reverse NaB effect, suggesting this was not the pathway involved. On the other hand, CD1a can also be induced by toll like receptors 2 (TLR 2) agonists, such as Pam3Cys, and NaB inhibited this effect. Our data suggest that the histone deacetylase inhibitor NaB instead of impairing DC differentiation inhibits the acquisition of CD1a induced both by cytokines and by TLR 2 agonist stimulus. Furthermore, this occurs at the transcriptional level as NaB led to a decrease in mRNA levels of CD1a and upregulation of CD1d.


Assuntos
Antígenos CD1/genética , Butiratos/farmacologia , Células Dendríticas/metabolismo , Receptor 2 Toll-Like/metabolismo , Anilidas/farmacologia , Antígenos CD1d/genética , Diferenciação Celular , Células Cultivadas , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Histona Desacetilases/metabolismo , Humanos , Interleucina-1beta/metabolismo , Interleucina-4/farmacologia , Receptores de Lipopolissacarídeos/genética , Monócitos/efeitos dos fármacos , PPAR gama/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor 2 Toll-Like/agonistas
14.
Recent Pat Anticancer Drug Discov ; 6(1): 15-25, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21110821

RESUMO

Recent results obtained from research on the intermediary metabolism of tumor cells have uncovered the biochemical reprogramming that takes place upon malignant transformation. Many features have been highlighted that are currently being exploited for specific chemotherapy. Many more will become available shortly as a consequence of the recognition of potentially useful targets for treatment. General interest in this area can be gauged by the number of recent patents that have been deposited, or are in the process of application. Because the metabolic subversion that is a hallmark of cancer cells involves a disruption of its homeostasis, the regulatory pathways dealt with in this review were broadly divided into those that encompass the main stages of the cell cycle and its various regulatory mechanisms and those that involve the aerobic glycolysis typical of cancer cells. It becomes apparent that both, the cell cycle and the intermediary metabolism are interconnected and rely on reactions many of which are dependent on kinases and phosphatases. Kinases and phosphatases are responsive to cellular redox signaling and may have a key role in determining whether cells progress towards malignant transformation as a result of continuous oxidative stress. The results discussed here underline aspects of the signaling pathways that lend themselves to specific inhibition by natural and synthetic compounds. The mitochondria and its role in programmed cell death are briefly commented, but special emphasis is placed on biochemical regulation at the level of chromatin structure, particularly the reactions that involve acetylation and deacetylation of histones. Within this context, inhibitors that act on histone deacetylases are discussed as promising alternatives to available treatments.


Assuntos
Antineoplásicos/uso terapêutico , Ciclo Celular/fisiologia , Metabolismo Energético/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ciclo Celular/genética , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/fisiologia , Metabolismo Energético/genética , Inibidores Enzimáticos/uso terapêutico , Humanos , Terapia de Alvo Molecular/métodos , Terapia de Alvo Molecular/tendências , Neoplasias/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/genética
15.
Oncol Rep ; 21(6): 1599-604, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19424642

RESUMO

It has been suggested that the blood clotting initiator protein, tissue factor (TF), participates in tumor growth, metastasis and angiogenesis. In addition, a family of G protein-coupled-receptors known as protease-activated receptors (PARs) has also been implicated in tumor biology. These receptors might be activated by blood coagulation proteases thus eliciting a number of pro-tumoral responses, including the expression of interleukin-8 (IL-8). Therefore, in this study we analyzed the expression of TF, PAR-1, PAR-2 and IL-8 genes in patients with esophageal cancer, one of the most aggressive neoplastic diseases. Total RNA was extracted from tissue samples (tumor and the corresponding normal mucosa) obtained from patients submitted to esophagectomy or endoscopy and further analyzed by semi-quantitative reverse transcriptase-polymerase (RT-PCR) and/or real-time quantitative PCR (qPCR). Expression of full-length transmembrane TF was significantly higher in tumor samples whereas no differences were observed in alternatively spliced TF transcripts. Tumor tissue showed increased mRNA levels for PAR-1 but not PAR-2. Remarkably, IL-8 expression was not detected in most normal tissues but showed very high expression in tumor samples. As expected, qPCR revealed greater differences in the expression pattern of all transcripts analyzed but the general profile was very similar to that observed by RT-PCR. Altogether our data suggest a possible role for blood clotting proteins in the biology of human esophageal cancer.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Receptor PAR-1/genética , Tromboplastina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Brasil , Neoplasias Esofágicas/cirurgia , Esofagectomia , Esofagoscopia , Feminino , Humanos , Interleucina-8/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , Receptor PAR-2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
16.
Ann N Y Acad Sci ; 1153: 153-63, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19236338

RESUMO

Ouabain, a known inhibitor of the Na,K-ATPase, has been shown to regulate a number of lymphocyte functions in vitro and in vivo. Lymphocyte proliferation, apoptosis, cytokine production, and monocyte function are all affected by ouabain. The ouabain-binding site occurs at the alpha subunit of the enzyme. The alpha subunit plays a critical role in the transport process, and four different alpha-subunit isoforms have been described with different sensitivities to ouabain. Analysis by RT-PCR indicates that alpha1, alpha2, and alpha3 isoforms are all present in murine lymphoid cells obtained from thymus, lymph nodes, and spleen. In these cells ouabain exerts an effect at concentrations that do not induce plasma membrane depolarization, suggesting a mechanism independent of the classical inhibition of the pump. In other systems, the Na,K-ATPase acts as a signal transducer in addition to being an ion pump, and ouabain is capable of inducing the activation of various signal transduction cascades. Neither resting nor concanavalin A (Con A)-activated thymocytes had their levels of phosphorylated-extracellular signal-regulated kinase (P-ERK) modified by ouabain. However, ouabain decreased p38 phosphorylation induced by Con A in these cells. The pathway induced by ouabain in lymphoid cells is still unclear but might vary with the type and state of activation of the cell.


Assuntos
Sistema Imunitário/metabolismo , Fatores Imunológicos/metabolismo , Ouabaína/metabolismo , Animais , Humanos , Linfócitos/metabolismo , Potenciais da Membrana , ATPase Trocadora de Sódio-Potássio/metabolismo
17.
Mol Biochem Parasitol ; 146(2): 180-91, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16427147

RESUMO

Metazoan species diversification in general and the adaptation of parasites to their life-style in particular are due, not only to the evolution of different structural or metabolic proteins, but also to changes in the expression patterns of the corresponding genes. In order to explore the conservation/divergence of transcriptional regulation in the platyhelminth parasite Schistosoma mansoni, we are studying the structures and functions of transcriptional mediators. CREB-binding protein (CBP) and p300 are closely related transcriptional coactivators that possess histone acetyltransferase (HAT) activity that can modify chromatin to an active relaxed state. They are also thought to link transcription factors to the basic transcriptional machinery and to act as integrators for different regulatory pathways. Here we describe the cloning and functional characterization of S. mansoni CBP. SmCBP1 comprises 2093 amino acids and displays a conserved modular domain structure. The HAT domain was shown to acetylate histones with a marked activity toward H4. Functional studies showed that SmCBP1 could interact physically with the nuclear receptor SmFtz-F1 and also potentiated its transcriptional activity in the CV-1 cell line. Screening of the EST and genomic sequence databases with the SmCBP1 sequence allowed us to characterize a second CBP gene in S. mansoni. SmCBP2 shows a high degree of sequence identity to SmCBP1, particularly in the HAT domain. Phylogenetic studies show that these peptides are more closely related to each other than to either mammalian CBP or p300, suggesting that they derive from a platyhelminth-specific duplication event. Both genes are expressed at all life-cycle stages, but differences in their relative expression and structural variations suggest that they play distinct roles in schistosome gene regulation.


Assuntos
Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Schistosoma mansoni/química , Schistosoma mansoni/genética , Fatores de Transcrição de p300-CBP/química , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Animais , Linhagem Celular , Clonagem Molecular , DNA de Helmintos/química , DNA de Helmintos/genética , Regulação da Expressão Gênica , Genes de Helmintos , Proteínas de Helminto/genética , Histonas/metabolismo , Dados de Sequência Molecular , Morfogênese , Filogenia , Ligação Proteica , Estrutura Terciária de Proteína , RNA de Helmintos/análise , RNA Mensageiro/análise , Schistosoma mansoni/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Fatores de Transcrição de p300-CBP/genética
18.
J Infect Dis ; 190(4): 843-52, 2004 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-15272414

RESUMO

Adult Schistosoma mansoni digest large amounts of host hemoglobin and release potentially toxic heme inside their guts. We have previously demonstrated that free heme in S. mansoni is detoxified through aggregation, forming hemozoin (Hz). Possible mechanisms of heme aggregation and the effects of chloroquine (CLQ) on formation of Hz and on the viability of this parasite have now been investigated. Different fractions isolated from S. mansoni, such as crude whole-worm homogenates, total lipid extracts, and Hz itself promoted heme aggregation in vitro in a CLQ-sensitive manner. Treatment of S. mansoni-infected mice with CLQ led to remarkable decreases in total protein, Hz content, and viability of the worms, as well as in parasitemia and deposition of eggs in mouse livers. These results indicate that inhibition of formation of Hz in S. mansoni, by CLQ, led to an important decrease in the overall severity of experimental murine schistosomiasis. Taken together, the results presented here suggest that formation of Hz is a major mechanism of heme detoxification and a potential target for chemotherapy in S. mansoni.


Assuntos
Cloroquina/uso terapêutico , Heme/antagonistas & inibidores , Schistosoma mansoni/efeitos dos fármacos , Esquistossomose/tratamento farmacológico , Animais , Fracionamento Celular , Cloroquina/farmacologia , Estudos de Coortes , Modelos Animais de Doenças , Desenho de Fármacos , Feminino , Heme/metabolismo , Hemeproteínas/antagonistas & inibidores , Hemeproteínas/biossíntese , Injeções Intraperitoneais , Fígado/parasitologia , Camundongos , Contagem de Ovos de Parasitas , Parasitemia , Schistosoma mansoni/isolamento & purificação , Schistosoma mansoni/metabolismo
20.
Mem. Inst. Oswaldo Cruz ; 87(supl.4): 67-70, 1992. ilus
Artigo em Inglês | LILACS | ID: lil-125628

RESUMO

Protein extracts obtained from male and female shistosomes were incubated with a gender-specific gene, F-10, transcribed only in adult females and encoding a major egg-shell protein. The protein/DNA interaction was measured using the band shift, DNase-I-footprinting and UV cross-linking techniques. The results showed a clear band shift when a 302 bp restriction fragment containing the 3'end of the gene was incubated with either female or male proteins. This fragment also contained a putative steroid hormone regulatory element (HRE). In contrast, only the male proteins produced a shift with the 495 bp fragment corresponding to the middle region of the gene. DNase I footprinting showed that proteins from males and females interacted with the F-10 gene by binding to multiple adjacent sites along the DNA, thus generatingrelatively long protected fragments of approximately 100 bp. This result suggested that the adjacent binding of several moles of proteins occured at the 5'end of the gene. UV cross-linking between schistosome proteins and a 21 bp synthetic oligonucleotide the F-10 HRE, evidence proteins having MWS of 30,45 and 65 kDNA. These proteins are presumably involved in the regulation of transcription of the F-10 gene


Assuntos
Proteínas de Bactérias , Schistosoma mansoni/análise , Maturidade Sexual
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