RESUMO
The lipopolysaccharide (LPS) of Gram-negative bacteria is recognized on human monocytes and macrophages by TLR4 and MD2 and induces the production of inflammatory cytokines; the LPSâ¯+â¯IgG complexes co-stimulation increases the cytokine production, mediated by the Fc-γRIIa (CD32a). We stimulated human CD14â¯+â¯monocytes or THP-1 cells with LPS or LPSâ¯+â¯soluble human IgG (sIgG) and TNF-α transcription and production, assessed RT-qPCR, ELISA, or flow cytometry, was enhanced by 30% upon LPSâ¯+â¯sIgG compared to LPS stimulation. LPSâ¯+â¯sIgG co-stimulation affected the NF-κB pathway (p65 phosphorylation and nucleus translocation, and IkB- α degradation). The biochemical inhibition of IRAK 1/4 and Syk kinases suppressed the enhancer effect of LPSâ¯+â¯sIgG on TNF- α production, suggesting the involvement of both MyD88 dependent and independent pathways. Our results suggest that during LPS activation, sIgG may participate in a TLR4 - Fc-γR crosstalk.
Assuntos
Imunoglobulina G/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Receptores de IgG/metabolismo , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Células Cultivadas , Citocinas/metabolismo , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-2/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Antígeno 96 de Linfócito/imunologia , Antígeno 96 de Linfócito/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , NF-kappa B/metabolismo , Receptor Cross-Talk/fisiologia , Receptores de IgG/imunologia , Transdução de Sinais/efeitos dos fármacos , Células THP-1 , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Canonical Wnt signaling is altered in most cases of colorectal cancer. Experimental evidence indicates that protein phosphatase 2A (PP2A) may play either positive or negative roles in Wnt signaling but its precise in vivo functions remain elusive. In this work, using colon cultured cell lines we showed that basal PP2A activity is markedly reduced in malignant cells compared to non-malignant counterparts. We found that whereas normal or cancer cells displaying not altered Wnt signaling express mRNAs coding for PP2A-A scaffold α and ß isoforms, cancer cells which have altered Wnt signaling do not express the Aß isoform mRNA. Remarkably, we found that the Aß protein levels are lost in all colon cancer cells, and in patients' tumor biopsies. In addition, all cancer cells exhibit higher levels of RalA activity, compared to non-malignant cells. Rescue experiments to restore Aß expression in malignant RKO cells, diminished the RalGTPase activation and cell proliferation, indicating that the Aß isoform acts as tumor suppressor in colon cancer cells. Reciprocal co-immunoprecipitation and immunofluorescence studies showed that the PP2A-C and -Aα subunits, expressed in all colon cells, interact in vivo with ß-catenin only in malignant cells. Selective inhibition of PP2A did not significantly affect cellular apoptosis but induced dose-dependent negative effects in ß-catenin-mediated transcriptional activity and in cell proliferation of malignant cells, indicating that the residual PP2A activity found in malignant cells, mediated by -C and Aα core subunits, is essential to maintain active Wnt signaling and cell proliferation in colon cancer cells.
Assuntos
Neoplasias do Colo/genética , Proteína Fosfatase 2/genética , Subunidades Proteicas/genética , Via de Sinalização Wnt/genética , Linhagem Celular Tumoral , GTP Fosfo-Hidrolases/genética , Células HT29 , Humanos , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Transcrição Gênica/genética , beta Catenina/genéticaRESUMO
Senescence phenotype can be achieved by multiple pathways. Most of them involve the activation of negative cell cycle regulators as well as a shift to an oxidative status. However, the exact participation of these events in senescence establishment and maintenance is not completely understood. In this study we investigated the content of three final cell cycle regulators, as well as the redox state in some critical points during the pre-senescent and the full-senescent states. Our results highlight the existence of a critical pre-phase in senescent phenotype establishment, in which cell proliferation stops with the participation of the cell cycle inhibitors, and a second maintenance stage where the exacerbated pro-oxidant state inside the cell induces the physiological decline characteristic in senescent cells.
Assuntos
Senescência Celular/fisiologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Animais , Antioxidantes/farmacologia , Pontos de Checagem do Ciclo Celular/fisiologia , Proliferação de Células , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Cromanos/farmacologia , DNA/biossíntese , Fibroblastos/efeitos dos fármacos , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Camundongos , Oxirredução , Fenótipo , Carbonilação ProteicaRESUMO
Breast cancer (BC) is the most frequent malignant neoplasia and leading cause of cancer mortality for women. A timely diagnosis of BC is crucial to ensure the best chances of survival. Among the various screening tools for BC, antibodies directed towards self-antigens or tumor-associated antigens (autoantibodies) have emerged as an alternative to image-based screening modalities. However, little attention has been paid to the global diversity of autoantibodies. This work aimed to analyze the diversity of autoantibodies reactive to antigens expressed by the BC cell line T47D in the sera of Mexican women with BC, benign breast pathology (BBP), or without breast pathology (WBP). We found that the diversity of antibodies in the sera was higher in the BC and BBP groups than in the WBP group. Likewise, the diversity changed with the progression of BC. Our results show and measure the complexity of the antibody response in breast health and disease.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Neoplasias da Mama/patologia , Autoanticorpos , Antígenos de NeoplasiasRESUMO
Heat shock protein 90 subfamily is composed by two cytosolic isoforms known as Hsp90α and Hsp90ß. Endothelial nitric oxide synthase (eNOS) is regulated by Hsp90, however the specific role of each Hsp90 isoform on NO production has not been established. This study was designed to evaluate the effect of Hsp90α and Hsp90ß over-expression on eNOS/NO pathway. Rat Hsp90α and Hsp90ß were cloned into pcDNA3.1(+) and transfected in human embryonic kidney cells (HEK-293). Hsp90α and Hsp90ß transfection was corroborated by Western blot analysis and their effect on NO production (NO(2)/NO(3)), eNOS protein and its phosphorylation at Ser1177 and Thr495, as well as Akt/PKB Ser473 phosphorylation was determined. The interaction of Hsp90α and Hsp90ß with eNOS and the dimer/monomer ratio of Hsp90, as well as O(2)(-) generation were also assessed. After transfection, Hsp90α and Hsp90ß levels were significantly increased in HEK-293 cells. The Hsp90α over-expression induced a significant increase in NO(2)/NO(3) levels, an effect that was associated with increased phosphorylation of eNOS Ser 1177 and Akt/PKB Ser473, as well as with a greater Hsp90α dimerization. Noteworthy, pcHsp90ß transfection reduced significantly NO(2)/NO(3) and increased O(2)(-) generation. These effects were associated with a reduction of eNOS dimeric conformation, increased eNOS Thr495 phosphorylation, reduced Akt/PKB phosphorylation, and by a greater amount of monomeric Hsp90ß conformation. These data show for first time that Hsp90α and Hsp90ß differentially modulate NO and O(2)(-) generation by eNOS through promoting changes in eNOS conformation and phosphorylation state.
Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Superóxidos/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Dimerização , Células HEK293 , Proteínas de Choque Térmico HSP90/genética , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno , RatosRESUMO
Nutritional status, in particular overweight and obesity, as well as sedentarism and high-fat diet consumption, are important risk factors to develop chronic diseases, which have a higher impact on the elderly's health. Therefore, these nutritional problems have become a concern to human healthspan and longevity. The fatty acids obtained thru the diet or due to fatty acid synthesis during obesity accumulate within the body generating toxicity and cell death. Fat is not only stored in adipose tissue, but it can also be stored in skeletal muscle. Palmitic acid (PA) has been reported as one of the most important saturated free fatty acids; it is associated to chronic oxidative stress and increased mitochondrial ROS production causing cell death by apoptosis. In skeletal muscle, palmitate has been associated with various pathophysiological consequences, which lead to muscle deterioration during aging and obesity. Since molecules that modify redox state have been proven to prevent cellular damage by inducing a hormetic response, the aim of this study was to evaluate if tert-butylhydroquinone (tBHQ) could activate an antioxidant hormetic response that would be able to protect L6 myoblasts from palmitate toxic effect. Our results provide evidence that tBHQ is able to protect L6 myoblasts against the toxicity induced by sodium palmitate due to a synergistic activation of different signaling pathways such as Nrf2 and NF-κB.
Assuntos
Hidroquinonas/farmacologia , Mitocôndrias/metabolismo , Mioblastos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Idoso , Animais , Apoptose , Linhagem Celular , Hormese , Humanos , Mioblastos/fisiologia , Fator 2 Relacionado a NF-E2/metabolismo , Palmitatos/toxicidade , Ratos , Transdução de SinaisRESUMO
Dendritic cells (DC) and macrophages (Mphi) are well known as important effectors of the innate immune system and their ability to produce IL-12 indicates that they possess the potential of directing acquired immunity toward a Th1-biased response. Interestingly, the intracellular parasite Leishmania has been shown to selectively suppress Mphi IL-12 production and are DC the principal source of this cytokine. The molecular details of this phenomenon remain enigmatic. In the present study we examined the effect of Leishmania mexicana lipophosphoglycan (LPG) on the production of IL-12, TNF-alpha, and IL-10 and nuclear translocation of NF-kappaB. The results show that LPG induced more IL-12 in human DC than in monocytes. This difference was due in part to nuclear translocation of NF-kappaB, since LPG induced more translocation in DC than in monocytes. These results suggest that Leishmania LPG impairs nuclear translocation of NF-kappaB in monocytes with the subsequent decrease in IL-12 production.
Assuntos
Células Dendríticas/imunologia , Glicoesfingolipídeos/farmacologia , Interleucina-12/biossíntese , Leishmania mexicana/fisiologia , Monócitos/imunologia , NF-kappa B/metabolismo , Animais , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/parasitologia , Humanos , Interleucina-10/biossíntese , Interleucina-12/análise , Leishmania mexicana/química , Leishmania mexicana/imunologia , Monócitos/efeitos dos fármacos , Monócitos/parasitologia , NF-kappa B/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossínteseRESUMO
The Trypanosoma cruzi rRNA gene promoter was characterized by deletion and point mutation analyses. A core of 89 bp was identified as the minimal region with full promoter activity. This core region is flanked upstream by a control element that stimulates its activity, and downstream by a novel down regulating region of about 200 bp. A point mutation analysis of the transcription start region evidenced 7 contiguous nucleotides where individual substitutions produced in all cases a defective promoter. It is generally accepted that the anciently speciated trypanosomatids lack strict promoters for protein coding genes transcribed by RNA polymerase II. The occurrence of a well structured rRNA gene promoter in these species suggests an early appearance of the RNA polymerase I promoters in the evolution of eukaryotic cells.
Assuntos
Regiões Promotoras Genéticas , RNA de Protozoário/genética , RNA Ribossômico/genética , Sequências Reguladoras de Ácido Nucleico , Trypanosoma cruzi/genética , Animais , Sequência de Bases , Mutação PuntualRESUMO
BACKGROUND: The biochemical bases for hormone dependence in breast cancer have been recognized as an important element in tumor resistance, proliferation and metastasis. On this respect, dexamethasone (Dex) dependent protection against TNF-alpha-mediated cell death in the MCF-7 cell line has been demonstrated to be a useful model for the study of this type of cancer. Recently, cytoplasmic signaling induced by steroid receptors has been described, such as the activation of the PI3K/Akt and NF-kappaB pathways. We evaluated their possible participation in the Dex-dependent protection against TNF-alpha-mediated cell death. RESULTS: Cellular cultures of the MCF-7 cell line were exposed to either, TNF-alpha or TNF-alpha and Dex, and cell viability was evaluated. Next, negative dominants of PI3K and IkappaB-alpha, designed to block the PI3K/Akt and NF-kappaB pathways, respectively, were transfected and selection and evaluation of several clones overexpressing the mutants were examined. Also, correlation with inhibitor of apoptosis proteins (IAPs) expression was examined. Independent inhibition of these two pathways allowed us to test their participation in Dex-dependent protection against TNF-alpha-cytotoxicity in MCF-7 cells. Expression of the PI3K dominant negative mutant did not alter the protection conferred by Dex against TNF-alpha mediated cell death. Contrariwise, clones expressing the IkappaB-alpha dominant negative mutant lost the Dex-conferred protection against TNF-alpha. In these clones degradation of c-IAP was accelerated, while that of XIAP was remained unaffected. CONCLUSION: NF-kappaB, but not PI3K/Akt activation, is required for the Dex protective effect against TNF-alpha-mediated cell death, and correlates with lack of degradation of the anti-apoptotic protein c-IAP1.
Assuntos
Apoptose/efeitos dos fármacos , Dexametasona/farmacologia , NF-kappa B/fisiologia , Proteína Oncogênica v-akt/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/fisiopatologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Fosfatase 1 de Especificidade Dupla , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/fisiologia , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/fisiologia , NF-kappa B/genética , Proteína Oncogênica v-akt/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/fisiologia , Proteína Fosfatase 1 , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/fisiologia , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/fisiologia , Transfecção , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/fisiologiaRESUMO
Replicative senescence is a cellular response to stress that has been postulated to serve as a tumor suppression mechanism and a contributor to aging. Numerous experimental studies have demonstrated that an apparently identical senescent state can also be prematurely induced in vitro in different cell types following sublethal oxidative stress stimuli. The former suggests a molecular link between cell cycle regulation and cell survival that could involve regulatory proteins such as Bcl-2. There is strong evidence that, in addition to its well-known effects on apoptosis, Bcl-2 is involved in antioxidant protection and regulation of cell cycle progression. The aim of this work was to determine if the protection against oxidative stress mediated by Bcl-2 could prevent or delay oxidative stress-induced senescence. Using a retroviral infection system, Bcl-2 was overexpressed in primary, nonembryonic mice fibroblasts obtained from lungs derived from 2-month-old CD1 mice. Fibroblasts overexpressing Bcl-2 were exposed to 75 microM H2O2 for 2 h to induce SIPS. The rate of proliferation and the increment of senescent cells were then determined. Our results indicate that overexpression of Bcl-2 protected primary fibroblasts against oxidative stress-mediated reduction in cell proliferation, but did not prevent premature senescence.
Assuntos
Senescência Celular/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Células Cultivadas , Feminino , Fibroblastos/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Espécies Reativas de Oxigênio/metabolismoRESUMO
The changes that accompany aging may be a result of oxidative damage to DNA that accumulates as a result of aging and age-related illnesses. Furthermore, a higher susceptibility is thought to be more common among elderly than young individuals. In the present study, we examined the severity of DNA damage caused by carbon tetrachloride (CCl4) and H2O2 in cells from young (2 month old) and older (14 month old) mice using both in vivo and in vitro exposures. CCl(4) is known to generate radical oxidative species (ROS) throughout its biotransformation in the liver. Therefore, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxdGuo) was quantified in liver DNA obtained from young and older mice treated with CCl4. In addition, DNA single-strand breaks were measured by the Comet assay in primary lung fibroblasts cultured from young and older mice and treated in vitro with H2O2. Intracellular ROS production and mitochondrial enzyme activity were determined in parallel. 8-oxodGuo levels were significantly higher in older mouse liver DNA than younger, and increased significantly with CCl4 treatment. When the basal DNA damage was subtracted, the net damage was almost equal for both. In addition, untreated cells cultured from older mice had significantly greater levels of strand breaks than cells derived from young mice. H2O2 increased the level of damage in both cell cultures. Our findings indicate that the DNA damage observed in older animals probably results from the accumulation of endogenous damage with age, perhaps due to insufficient repair, which enhances the injury caused by exposure to the toxic agents.
Assuntos
Envelhecimento/efeitos dos fármacos , Tetracloreto de Carbono/toxicidade , Dano ao DNA , Peróxido de Hidrogênio/toxicidade , Estresse Oxidativo/efeitos dos fármacos , 8-Hidroxi-2'-Desoxiguanosina , Animais , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Ensaio Cometa , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Feminino , Fígado/metabolismo , Camundongos , Camundongos Mutantes , Espécies Reativas de Oxigênio/metabolismo , Espectrofotometria , Sais de Tetrazólio , TiazóisRESUMO
Prior exposure to lipopolysaccharides (LPS) induces a state of cell resistance to subsequent LPS restimulation, known as endotoxin tolerance, mainly by repressing the expression of pro-inflammatory cytokines. We established an endotoxin tolerance model in human monocytes Endotoxin-tolerant cells showed a decrease in IκBα degradation and diminished expression of Tumor necrosis factor (TNF) (both messenger RNA [mRNA] and protein content). The myeloid differentiation factor 88 (MyD88)/MyD88 splice variant (MyD88s) ratio, an indirect way to test the Toll-like receptor 4 (TLR4) MyD88-dependent signaling cascade, did not change in endotoxin-tolerant cells when compared to LPS-stimulated or -unstimulated ones. Remarkably, cell population analysis indicated a significant increase of the CD14+ CD16+ subset only under the endotoxin-tolerant condition. Furthermore, endotoxin-tolerant cells produced higher amounts of C-X-C motif chemokine 10 (CXCL10), a typical MyD88-independent cytokine.
Assuntos
Endotoxinas/imunologia , Tolerância Imunológica , Monócitos/imunologia , Quimiocina CXCL10/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/imunologia , Masculino , Monócitos/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Fenótipo , Receptores de Superfície Celular/metabolismo , Receptores de IgG/metabolismo , Transdução de SinaisRESUMO
The Immunoglobulin G (IgG) antibody response to different protein antigens of the mammary ductal carcinoma by adult women affected by Breast Cancer (BC) distinguishes at least 103 proteins that differ in their molecular weights (MW). The IgG producing cell clones (nodes) coexist with each other in each individual organism and share energy resources among themselves, as well as factors that control the level of expression and Specificity of their IgG antibodies. So, it can be proposed that among them there is a Network of interconnections (links) unveiled by the antigens, which specifically react with the IgG antibodies produced by the clones. This Network possibly regulates IgG antibodies' activity and effectiveness. We describe the Network of nodes and links that exists between the different antigens and their respective IgG producing cell clones against the extracted protein antigens from the cells of the T47D Cell-Line, in 50 women with BC, 50 women with Benign Breast Pathology (BBP) and 50 women without breast pathology (H). We have found that women with BBP have the highest number of Links, followed by the H group and, lastly, the women with BC, a finding which suggests that cancer interferes with the Connectivity between the IgG producing cell clones and blocks the expression of 322 links in women with BBP and 32 links in women with H. It is also plausible that the largest number of links in the women with BBP indicates the Network's state of arousal that provides protection against BC. On the other hand, there were many missing links in the BC group of women; the clone which lost more links in the BC group was the hub 24, which point to some of the antigens of T47D as potentially useful as vaccines, as the immune system of women with BBP is well aware of them.
Assuntos
Reações Antígeno-Anticorpo/imunologia , Antígenos de Neoplasias/imunologia , Neoplasias da Mama/imunologia , Mama/imunologia , Redes Reguladoras de Genes , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/análise , Mama/patologia , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/imunologia , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/imunologia , Carcinoma Lobular/patologia , Estudos de Casos e Controles , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Adulto JovemRESUMO
Cells can respond to damage and stress by activating various repair and survival pathways. One of these responses can be induced by preconditioning the cells with sublethal stress to provoke a prosurvival response that will prevent damage and death, and which is known as hormesis. Bcl-2, an antiapoptotic protein recognized by its antioxidant and prosurvival functions, has been documented to play an important role during oxidative-conditioning hormesis. Using an oxidative-hormetic model, which was previously established in the L929 cell line by subjecting the cells to a mild oxidative stress of 50 µM H2O2 for 9 h, we identified two different transductional mechanisms that participate in the regulation of Bcl-2 expression during the hormetic response. These mechanisms converge in activating the nuclear transcription factor NF-κB. Interestingly, the noncanonical p50 subunit of the NF-κB family is apparently the subunit that participates during the oxidative-hormetic response.
Assuntos
Peróxido de Hidrogênio/farmacologia , Subunidade p50 de NF-kappa B/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Hormese , Humanos , Camundongos , NF-kappa B/genética , Subunidade p50 de NF-kappa B/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Transdução de Sinais/efeitos dos fármacosRESUMO
Leishmania mexicana (Lm) causes localized (LCL) and diffuse (DCL) cutaneous leishmaniasis. DCL patients have a poor cellular immune response leading to chronicity. It has been proposed that CD8 T lymphocytes (CD8) play a crucial role in infection clearance, although the role of CD8 cytotoxicity in disease control has not been elucidated. Lesions of DCL patients have been shown to harbor low numbers of CD8, as compared to patients with LCL, and leishmanicidal treatment restores CD8 numbers. The marked response of CD8 towards Leishmania parasites led us to analyze possible functional differences between CD8 from patients with LCL and DCL. We compared IFNγ production, antigen-specific proliferation, and cytotoxicity of CD8 purified from PBMC against autologous macrophages (MO) infected with Leishmania mexicana (MOi). Additionally, we analyzed tissue biopsies from both groups of patients for evidence of cytotoxicity associated with apoptotic cells in the lesions. We found that CD8 cell of DCL patients exhibited low cytotoxicity, low antigen-specific proliferation and low IFNγ production when stimulated with MOi, as compared to LCL patients. Additionally, DCL patients had significantly less TUNEL+ cells in their lesions. These characteristics are similar to cellular "exhaustion" described in chronic infections. We intended to restore the functional capacity of CD8 cells of DCL patients by preincubating them with TLR2 agonists: Lm lipophosphoglycan (LPG) or Pam3Cys. Cytotoxicity against MOi, antigen-specific proliferation and IFNγ production were restored with both stimuli, whereas PD-1 (a molecule associated with cellular exhaustion) expression, was reduced. Our work suggests that CD8 response is associated with control of Lm infection in LCL patients and that chronic infection in DCL patients leads to a state of CD8 functional exhaustion, which could facilitate disease spread. This is the first report that shows the presence of functionally exhausted CD8 T lymphocytes in DCL patients and, additionally, that pre-stimulation with TLR2 ligands can restore the effector mechanisms of CD8 T lymphocytes from DCL patients against Leishmania mexicana-infected macrophages.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Leishmania/imunologia , Leishmaniose Tegumentar Difusa/imunologia , Receptor 2 Toll-Like/imunologia , Adulto , Linfócitos T CD8-Positivos/parasitologia , Células Cultivadas , Feminino , Glicoesfingolipídeos/imunologia , Humanos , Leishmania/fisiologia , Leishmaniose Tegumentar Difusa/parasitologia , Masculino , Pessoa de Meia-Idade , Receptor 2 Toll-Like/agonistas , Adulto JovemRESUMO
A series of 6-substituted 2-(N-trifluoroacetylamino)imidazopyridines have been synthesized and their bioactivities were evaluated. Compounds 6a, 6c, and 11a were the most active compounds with modest cytotoxic activity against six human cancer cell lines U251 (glioma), PC-3 (prostate), K-562 (leukemia), HCT-15 (colon), MCF7 (breast) and SK-LU-1 (lung). The cell cycle analysis showed that compounds 6a, 6c, and 11a induce a G2/M phase cell cycle arrest on SK-LU-1 cell line where inhibition of CDK-1 and CDK-2 may be implicated.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Flúor/química , Imidazóis/farmacologia , Piridinas/farmacologia , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Humanos , Imidazóis/síntese química , Imidazóis/química , Neoplasias Pulmonares/tratamento farmacológico , Estrutura Molecular , Piridinas/síntese química , Piridinas/químicaRESUMO
A series of 2-methylimidazo[1,2-a]pyridine- and quinoline-substituted 2-aminopyrimidines derivatives were synthesized using a convenient synthetic route. We evaluate the isosteric replacement of methyl groups in 4-(2-methylimidazo[1,2-a]pyridin-3-yl)-N-p-tolylpyrimidin-2-amine (compound 1) by trifluoromethyl groups and the isosteric substitution of the 2-methylimidazo[1,2-a]pyridin-3-yl scaffold by quinolin-4-yl or quinolin-3-yl moieties. The replacement of hydrogen by fluorine does not affect notably the cytotoxic activity and CDK inhibitor activity in this series. Quinolin-4-yl-substituted compound, 8, presents cytotoxic activity and is most effective and selective against CDK1/CycA than against CDK2/CycB. Compound 11, which has a quinolin-3-yl moiety is CDK inhibitor but presents null cytotoxic activity. Quinolin-4-yl-substituted compounds constitute a new lead of cytotoxic and CDK inhibitor compounds from which more compelling and selective inhibitors can be designed.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Piridinas/química , Pirimidinas/química , Pirimidinas/farmacologia , Quinolinas/química , Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Quinases Ciclina-Dependentes/antagonistas & inibidores , Humanos , Concentração Inibidora 50 , Pirimidinas/síntese químicaRESUMO
Hormesis is the process whereby exposure to a low dose of a chemical agent induces an adaptive effect on the cell or organism. This response evokes the expression of cytoprotective and antioxidant proteins, allowing pro-oxidants to emerge as important hormetic agents. The antiapoptotic protein Bcl-2 is known to protect cells against death induced by oxidants; it has been suggested that Bcl-2 might also modulate steady-state reactive oxygen species levels. The aim of this work was to find out if Bcl-2 might play a role during the hormetic response and in Nrf-2 activation. We have established a model to study the oxidative conditioning hormesis response (OCH) by conditioning the cell line L929 with 50muM H(2)O(2) for 9h. This condition did not induce oxidative damage nor oxidative imbalance, and OCH cells maintained a 70-80% survival rate after severe H(2)O(2) treatment compared to nonconditioned cells. When cells were pretreated with the Bcl-2 inhibitor HA14-1 or were silenced with Bcl-2-siRNA, both the hormetic effect and the Nrf-2 nuclear translocation previously observed were abrogated. Our results suggest a sequence of causal events related to increase in Bcl-2 expression, induction of Nrf-2 activation, and sustained expression of cytoprotective proteins such as GST and gammaGCS.
Assuntos
Núcleo Celular/metabolismo , Fibroblastos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Animais , Benzopiranos/farmacologia , Linhagem Celular , Citoproteção/efeitos dos fármacos , Citoproteção/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Peróxido de Hidrogênio/farmacologia , Camundongos , Fator 2 Relacionado a NF-E2/genética , Nitrilas/farmacologia , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Interferente Pequeno/genética , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
BACKGROUND: Several common aspects of endothelial phenotype, such as the expression of cell adhesion molecules, are shared between metastasis and inflammation. Here, we analyzed VCAM-1 variants as biological markers of these two types of endothelial cell activation. With the combination of 2-DE and western blot techniques and the aid of tunicamycin, we analyzed N-glycosylation variants of VCAM-1 in primary human endothelial cells stimulated with either TNF or tumoral soluble factors (TSF's) derived from the human breast cancer cell line ZR75.30. RESULTS: Treatments induced a pro-adhesive endothelial phenotype. 2D western blots analysis of cells subjected to both treatments revealed the expression of the two known VCAM-1 isoforms and of previously unknown isoforms. In particular TSFZR75.30 induced an isoform with a relative molecular mass (Mr) and isoelectric point (pI) of 75-77 kDa and 5.0, respectively. CONCLUSION: The unknown isoforms of VCAM-1 that were found to be overexpressed after treatment with TSF's compared with TNF, could serve as biomarkers to discriminate between inflammation and metastasis. 2D western blots revealed three new VCAM-1 isoforms expressed in primary human endothelial cells in response to TSF stimulation. Each of these isoforms varies in Mr and pI and could be the result of differential glycosylation states.
RESUMO
Aging is a process where individuals decrease the performance of their physiological systems and cellular stress response, making them more susceptible to disease and death. The increase in DNA damage associated with age might be recognized as the accumulation of physiological and environmentally induced mutations accompanied with a decline in DNA repair. DNA mismatch repair (MMR) is the main postreplicative correction pathway, which is known to decrease with age. However, since infrequent occurrence of direct DNA damage contrasts with the extensive cell and tissue dysfunction seen in older individuals, the withdrawing of DNA-repairing systems might be also related to epigenetic changes, such as DNA methylation. It has been reported that the physiological stress related to breeding might accelerate the acquisition of aging-related markers; therefore, the aim of this work was to link age with epigenetic modifications in this animal population. Hence, the correlation of Msh2 gene silencing with the deterioration of breeding female mice associated to aging was determined. Combined bisulfite restriction analysis assay was used to compare methylation on DNA isolated from twelve-month-old retired breeders against nulliparous female mice aged-matched, and two-month-old young adults. Our experiments clearly reveal Msh2 promoter hypermethylation associated to the aging process. A higher degree methylation was additionally observed in breeding females DNA. Nevertheless, this additional methylation did not correlate with a further decrease Msh2 mRNA, suggesting that the increase in methylation in old retired breeder might account for further epigenetic changes that could additionally promote the aging process.