RESUMO
Breast cancer (BC) is a heterogeneous disease composed of multiple subtypes with different molecular characteristics and clinical outcomes. The metastatic process in BC depends on the transcription factors (TFs) related to epithelial-mesenchymal transition (EMT), including the master regulator Twist1. However, its role beyond EMT in BC subtypes remains unclear. Our study aimed to investigate the role of Twist1, beyond EMT, in the molecular subtypes of BC. In patients, we observed the overexpression of TWIST1 in the HER2+ group. The silencing of TWIST1 in HER2+ BC cells resulted in the upregulation of 138 genes and the downregulation of 174 genes compared to control cells in a microarray assay. In silico analysis revealed correlations between Twist1 and important biological processes such as the Th17-mediated immune response, suggesting that Twist1 could be relevant for IL-17 signaling in HER2+ BC. IL-17 signaling was then examined, and it was shown that TWIST1 knockdown caused the downregulation of leading members of IL-17 signaling pathway. Taken together, our findings suggest that Twist1 plays a role on IL-17 signaling in HER2+ BC.
Assuntos
Neoplasias da Mama/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Interleucina-17/imunologia , Proteínas Nucleares/imunologia , Receptor ErbB-2/imunologia , Transdução de Sinais/imunologia , Proteína 1 Relacionada a Twist/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Interleucina-17/genética , Proteínas Nucleares/genética , Receptor ErbB-2/genética , Transdução de Sinais/genética , Proteína 1 Relacionada a Twist/genéticaRESUMO
Breast cancer (BC) has been extensively studied, as it is one of the more commonly diagnosed cancer types worldwide. The study of miRNAs has increased what is known about the complexity of pathways and signaling and has identified potential biomarkers and therapeutic targets. Thus, miRNome profiling could provide important information regarding the molecular mechanisms involved in BC. On average, more than 430 miRNAs were identified as differentially expressed between BC cell lines and normal breast HMEC cells. From these, 110 miRNAs were common to BC subtypes. The miRNome enrichment analysis and interaction maps highlighted epigenetic-related pathways shared by all BC cell lines and revealed potential miRNA targets. Quantitative evaluation of BC patient samples and GETx/TCGA-BRCA datasets confirmed MYB and EZH2 as potential targets from BC miRNome. Moreover, overall survival was impacted by EZH2 expression. The expression of 15 miRNAs, selected according to aggressiveness of BC subtypes, was confirmed in TCGA-BRCA dataset. Of these miRNAs, miRNA-mRNA interaction prediction revealed 7 novel or underexplored miRNAs in BC: miR-1271-5p, miR-130a-5p, and miR-134 as MYB regulators and miR-138-5p, miR-455-3p, miR-487a, and miR-487b as EZH2 regulators. Herein, we report a novel molecular miRNA signature for BC and identify potential miRNA/mRNAs involved in disease subtypes.
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Using chip array assays, we identified differentially expressed genes via a comparison between luminal A breast cancer subtype and normal mammary ductal cells from healthy donors. In silico analysis confirmed by western blot and immunohistochemistry revealed that C-JUN and C-FOS transcription factors are activated in luminal A patients as potential upstream regulators of these differentially expressed genes. Using a chip-on-chip assay, we identified potential C-JUN and C-FOS targets. Among these genes, the NRIP1 gene was revealed to be targeted by C-JUN and C-FOS. This was confirmed after identification and validation with transfection assays specific binding of C-JUN and C-FOS at consensus binding sites. NRIP1 is not only upregulated in luminal A patients and cell lines but also regulates breast cancer-related genes, including PR, ESR1 and CCND1. These results were confirmed by NRIP1 siRNA knockdown and chip array assays, thus highlighting the putative role of NRIP1 in PGR, ESR1 and CCND1 transcriptional regulation and suggesting that NRIP1 could play an important role in breast cancer ductal cell initiation.
Assuntos
Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína 1 de Interação com Receptor Nuclear/metabolismo , Adulto , Idoso , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Ciclina D1/genética , Ciclina D1/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células MCF-7 , Pessoa de Meia-Idade , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína 1 de Interação com Receptor Nuclear/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , TranscriptomaRESUMO
Among the childhood diseases, B-cell acute lymphocytic leukemia (B-ALL) is the most frequent type of cancer. Despite recent advances concerning disease treatment, cytotoxic chemotherapy remains the first line of treatment in several countries, and the modifications induced by such drugs in the organism are still poorly understood. In this context, the present study provided a comparative high-throughput proteomic analysis of the cumulative changes induced by chemotherapeutic drugs used in the induction phase of B-ALL treatment in both peripheral blood (PB) and bone marrow compartment (BM) samples. To reach this goal, PB and BM plasma samples were comparatively analyzed by using label-free proteomics at two endpoints: at diagnosis (D0) and the end of the cumulative induction phase treatment (D28). Proteomic data was available via ProteomeXchange with identifier PXD021584. The resulting differentially expressed proteins were explored by bioinformatics approaches aiming to identify the main gene ontology processes, pathways, and transcription factors altered by chemotherapy, as well as to understand B-ALL biology in each compartment at D0. At D0, PB was characterized as a pro-inflammatory environment, with the involvement of several downregulated coagulation proteins as KNG, plasmin, and plasminogen. D28 was characterized predominantly by immune response-related processes and the super expression of the transcription factor IRF3 and transthyretin. RUNX1 was pointed out as a common transcription factor found in both D0 and D28. We chose to validate the proteins transthyretin and interferon-gamma (IFN-γ) by commercial kits and expressed the results as PB/BM ratios. Transthyretin ratio was augmented after induction chemotherapy, while IFN-γ was reduced at the end of the treatment. Considering that most of these proteins were not yet described in B-ALL literature, these findings added to understanding disease biology at diagnosis and highlighted a possible role for transthyretin and IFN-γ as mechanisms related to disease resolution.
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Mesenchymal stromal cells (MSCs) were first used as a source for cell therapy in 1995; however, despite their versatility and unambiguous demonstration of efficacy and safety in preclinical/phase I studies, the positive effect of MSCs in human phase III studies did not resemble the success obtained in mouse models of disease. This dissonance highlights the need to more thoroughly study the immunobiology of MSCs to make better use of these cells. Thus, we aimed to study the immunobiology of MSCs by using chip array analysis as a method for general screening to obtain a global picture in our model study and found IFNy and IL-17 signaling as the first two "top canonical pathways" involved in MSCs immunomodulation. The role of IFNy in triggering the immunosuppressive properties of MSCs is well recognized by many groups; however, the role of IL-17 in this process remains uncertain. Interestingly, in contrast to IFNy, which actively improved the MSCs-mediated immunosuppression, IL-17 did not improve directly the MSCs-mediated immunosuppression. Instead, IL-17 signaling induced the migration of MSCs and inflammatory cells, bringing these cell types together and increasing the likelihood of the lymphocytes sensing the immunosuppressive molecules produced by the MSCs. These effects also correlated with high levels of cytokine/chemokine production and metalloprotease activation by MSCs. Importantly, this treatment maintained the MSCs safety profile by not inducing the expression of molecules related to antigen presentation. In this way, our findings highlight the possibility of using IL-17, in combination with IFNy, to prime MSCs for cell therapy to improve their biological properties and thus their therapeutic efficacy. Finally, the use of preactivated MSCs may also minimize variations among MSCs to produce more uniform therapeutic products. In the not-so-distant future, we envisage a portfolio of MSCs activated by different cocktails specifically designed to target and treat specific diseases. Graphical abstract.
Assuntos
Movimento Celular , Terapia de Imunossupressão , Interferon gama/metabolismo , Interleucina-17/metabolismo , Células-Tronco Mesenquimais/metabolismo , Movimento Celular/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Teste de Cultura Mista de Linfócitos , Células-Tronco Mesenquimais/imunologia , Fenótipo , Transdução de SinaisRESUMO
CAPN3 is a muscle-specific and an intrinsically disordered protein. Thus, as a scaffolding protein CAPN3 could play a role during early stages of myogenesis. To test this hypothesis, we studied the distribution and function of CAPN3 during myogenesis using embryonic chick muscle cells grown in vitro. Super-resolution microscopy showed CAPN3 distribution in (i) amorphous patches in myoblasts, (ii) a region near the nuclei of myotubes; (iii) adhesion plaques in myotubes, (iv) stress fiber-like structures in myotubes, and (v) filaments in fibroblasts. Downregulation of CAPN3 induced a decrease in the number of muscle cells and in the size of myotubes formed. These data show a diverse intracellular distribution of CAPN3, compatible with a scaffolding protein, and suggest a multitude of different interactions of CAPN3 with other partners during muscle formation.
Assuntos
Calpaína/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/metabolismo , Animais , Embrião de Galinha , Fibroblastos/metabolismo , Espaço Intracelular/metabolismo , Modelos Biológicos , Desenvolvimento MuscularRESUMO
Breast cancer treatment has advanced enormously in the last decade. Most of this is due to advances reached in the knowledge regarding tumor biology, mainly in the field of diagnosis and treatment. This review brings information about how the genomics-based information contributed to advances in breast cancer diagnosis and prognosis perspective, as well as presents how tumor biology discoveries fostered the main therapeutic approaches available to treat such patients, based on a personalized point of view.
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Biomarcadores Tumorais/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Ensaios Clínicos como Assunto , Detecção Precoce de Câncer , Feminino , Genômica , Humanos , Terapia de Alvo Molecular , Mutação , Medicina de Precisão , PrognósticoRESUMO
Lysosomes and acidic compartments are involved in breaking down of macromolecules, membrane recycling, and regulation of signaling pathways. Here, we analyzed the role of acidic compartments during muscle differentiation and the involvement of the Wnt/beta-catenin pathway in lysosomal function during myogenesis. Acridine orange was used to localize and quantify acidic cellular compartments in primary cultures of embryonic muscle cells from Gallus gallus. Our results show an increase in acidic compartment size and area, as well as changes in their positioning during the initial steps of myogenesis. The inhibition of lysosomal function by either the chloroquine Lys05 or the downregulation of LAMP-2 with siRNA impaired chick myogenesis, by inhibiting myoblast fusion. Two activators of the Wnt/beta-catenin pathway, BIO and Wnt3a, were able to rescue the inhibitory effects of Lys05 in myogenesis. These results suggest a new role for the Wnt/beta-catenin pathway in the regulation of acidic compartment size, positioning, and function in muscle cells.
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Proteínas Aviárias/metabolismo , Desenvolvimento Muscular , Mioblastos Esqueléticos/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Embrião de GalinhaRESUMO
BACKGROUND: Witches' broom disease (WBD) of cacao (Theobroma cacao L.), caused by Moniliophthora perniciosa, is the most important limiting factor for the cacao production in Brazil. Hence, the development of cacao genotypes with durable resistance is the key challenge for control the disease. Proteomic methods are often used to study the interactions between hosts and pathogens, therefore helping classical plant breeding projects on the development of resistant genotypes. The present study compared the proteomic alterations between two cacao genotypes standard for WBD resistance and susceptibility, in response to M. perniciosa infection at 72 h and 45 days post-inoculation; respectively the very early stages of the biotrophic and necrotrophic stages of the cacao x M. perniciosa interaction. RESULTS: A total of 554 proteins were identified, being 246 in the susceptible Catongo and 308 in the resistant TSH1188 genotypes. The identified proteins were involved mainly in metabolism, energy, defense and oxidative stress. The resistant genotype showed more expressed proteins with more variability associated with stress and defense, while the susceptible genotype exhibited more repressed proteins. Among these proteins, stand out pathogenesis related proteins (PRs), oxidative stress regulation related proteins, and trypsin inhibitors. Interaction networks were predicted, and a complex protein-protein interaction was observed. Some proteins showed a high number of interactions, suggesting that those proteins may function as cross-talkers between these biological functions. CONCLUSIONS: We present the first study reporting the proteomic alterations of resistant and susceptible genotypes in the T. cacao x M. perniciosa pathosystem. The important altered proteins identified in the present study are related to key biologic functions in resistance, such as oxidative stress, especially in the resistant genotype TSH1188, that showed a strong mechanism of detoxification. Also, the positive regulation of defense and stress proteins were more evident in this genotype. Proteins with significant roles against fungal plant pathogens, such as chitinases, trypsin inhibitors and PR 5 were also identified, and they may be good resistance markers. Finally, important biological functions, such as stress and defense, photosynthesis, oxidative stress and carbohydrate metabolism were differentially impacted with M. perniciosa infection in each genotype.
Assuntos
Agaricales/imunologia , Cacau/microbiologia , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas/imunologia , Doenças das Plantas , Agaricales/fisiologia , Biomarcadores , Brasil , Cacau/genética , Quitinases/genética , Quitinases/metabolismo , Perfilação da Expressão Gênica , Genótipo , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Domínios Proteicos Ricos em Prolina/genética , Inibidores da Tripsina/metabolismoRESUMO
BACKGROUND/AIMS: The therapeutic potential of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) in kidney injury has been largely reported. However, new approaches are necessary to optimize the efficacy in the treatment of renal diseases. MSCs physiologically are under a low O2 partial pressure (pO2), and culturing adipose-derived MSCs (ADMSCs) in hypoxia alters their secretory paracrine properties. The aim of this study was to evaluate whether hypoxia preconditioning of ADMSCs alters the properties of secreted EVs to improve renal recovery after ischemia-reperfusion injury (IRI). METHODS: The supernatants of ADMSCs cultivated under 21% pO2 (control) or 1% pO2 (hypoxia) were ultracentrifuged for EVs isolation that were posteriorly characterized by flow cytometry and electron microscopy. The uptake and effects of these EVs were analyzed by using in vitro and in vivo models. HK-2 renal tubule cell line was submitted do ATP depletion injury model. Proteomic analyses of these cells treated with EVs after injury were performed by nano-UPLC tandem nano-ESI-HDMSE method. For in vivo analyses, male Wistar rats were submitted to 45 min bilateral ischemia, followed by renal intracapsular administration of ADMSC-EVs within a 72 h reperfusion period. Histological, immunohistochemical and qRT-PCR analysis of these kidneys were performed to evaluate cell death, inflammation and oxidative stress. Kidney function was evaluated by measuring the blood levels of creatinine and urea. RESULTS: The results demonstrate that hypoxia increases the ADMSCs capacity to secrete EVs that trigger different energy supply, antiapoptotic, immunomodulatory, angiogenic and anti-oxidative stress responses in renal tissue compared with EVs secreted in normoxia. Proteomic analyses of renal tubule cells treated with EVs from ADMSCs in normoxia and hypoxia give a specific signature of modulated proteins for each type of EVs, indicating regulation of distinct biological processes. CONCLUSION: In summary, hypoxia potentially offers an interesting strategy to enhance the properties of EVs in the treatment of acute kidney disease.
Assuntos
Injúria Renal Aguda/terapia , Vesículas Extracelulares/transplante , Células-Tronco Mesenquimais/metabolismo , Traumatismo por Reperfusão/terapia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Tecido Adiposo/citologia , Animais , Hipóxia Celular , Linhagem Celular , Células Cultivadas , Vesículas Extracelulares/metabolismo , Humanos , Rim/metabolismo , Rim/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Masculino , Células-Tronco Mesenquimais/citologia , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologiaRESUMO
Mesenchymal stromal cells (hMSCs) are key components of the bone marrow microenvironment (BMM). A molecular signature in hMSCs from Acute myeloid leukemia patients (hMSC-AML) has been proposed where BMP4 is decreased and could be regulated by WNT signaling pathway. Therefore, the aim of this work was to verify whether the WNT signaling pathway can regulate the BMP4 gene in hMSCs. The results showed differentially expressed genes in the WNT canonical pathway between hMSC-AML and hMSCs from healthy donors and a real-time quantitative assay corroborated with these findings. Moreover, the main WNT canonical pathway regulators were decreased in hMSC-AML, such as LEF-1, ß-catenin and the ß-catenin/TCF-LEF regulatory complex in the nucleus. This result, together with functional assays, suggests that the induction of BMP4 expression by the WNT signaling pathway is decreased in hMSC-AML. Overall, the WNT canonical pathway is able to regulate the BMP4 gene in hMSC-AML and its reduced activation could also lead to the lower expression of BMP4 in hMSC-AML. Due to the important role of the BMM, changes in BMP4 expression through the WNT canonical pathway may be a potential mechanism of leukemogenesis.
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Classical Hodgkin lymphoma (cHL) cells overexpress heat-shock protein 90 (HSP90), an important intracellular signaling hub regulating cell survival, which is emerging as a promising therapeutic target. Here, we report the antitumor effect of celastrol, an anti-inflammatory compound and a recognized HSP90 inhibitor, in Hodgkin and Reed-Sternberg cell lines. Two disparate responses were recorded. In KM-H2 cells, celastrol inhibited cell proliferation, induced G0/G1 arrest, and triggered apoptosis through the activation of caspase-3/7. Conversely, L428 cells exhibited resistance to the compound. A proteomic screening identified a total of 262 differentially expressed proteins in sensitive KM-H2 cells and revealed that celastrol's toxicity involved the suppression of the MAPK/ERK (extracellular signal regulated kinase/mitogen activated protein kinase) pathway. The apoptotic effects were preceded by a decrease in RAS (proto-oncogene protein Ras), p-ERK1/2 (phospho-extracellular signal-regulated Kinase-1/2), and c-Fos (proto-oncogene protein c-Fos) protein levels, as validated by immunoblot analysis. The L428 resistant cells exhibited a marked induction of HSP27 mRNA and protein after celastrol treatment. Our results provide the first evidence that celastrol has antitumor effects in cHL cells through the suppression of the MAPK/ERK pathway. Resistance to celastrol has rarely been described, and our results suggest that in cHL it may be mediated by the upregulation of HSP27. The antitumor properties of celastrol against cHL and whether the disparate responses observed in vitro have clinical correlates deserve further research.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Doença de Hodgkin/metabolismo , Células de Reed-Sternberg/metabolismo , Triterpenos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Triterpenos Pentacíclicos , Proteoma , Proto-Oncogene Mas , Células de Reed-Sternberg/efeitos dos fármacos , Proteínas ras/metabolismoRESUMO
Cryptococcosis is a fungal disease of global distribution with a higher incidence in developing countries, where it represents one of the main causes of meningitis, associated with high morbidity and mortality. It mainly occurs in patients with immunosuppression (due to HIV infection, glucocorticoid treatment, transplants, cancer, etc.). However, in recent years there has been an increase in cases in immunocompetent, which is as serious or more severe than in immunocompromised. We report two cases of cryptococcal meningitis. A 48-year-old male with no pathological history, in whom HIV infection or another cause of immunosuppression was ruled out, and a 67-year-old woman with a previous diagnosis of liver cirrhosis. The above mentioned highlights the importance of always considering Cryptococcus spp. as possible causative agent of meningitis, in both immunocompromised and immunocompetent patients.
Assuntos
Cryptococcus neoformans/genética , Meningite Criptocócica/diagnóstico , Idoso , Feminino , Humanos , Hospedeiro Imunocomprometido , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Tomografia Computadorizada por Raios XRESUMO
A wide variety of cellular processes and signaling events are regulated by the proteolytic enzyme γ-secretase. Notch-1 is one of the substrates of γ-secretase and its role in the regulation of muscle differentiation has been well described. Importantly, besides Notch-1, a number of proteins have been identified to undergo proteolysis by γ-secretase. To date, the specific role of γ-secretase during embryonic skeletal muscle differentiation has not been studied. Therefore, we address this question through the analysis of in vitro grown chick myogenic cells during the formation of multinucleated myotubes. The γ-secretase inhibitor DAPT (N-N[-(3,5-Difluorophenacetyl-l-alanyl)]-S-328 phenylglycine-t-butyl-ester) induces muscle hypertrophy. Knockdown of Notch-1 using siRNA specific to chick shows no significant effect in myotube size, suggesting that γ-secretase-dependent effects on muscle hypertrophy in chick myogenic cells are Notch-1-independent. We also investigate the effects of γ-secretase inhibition in the whole proteomic profile of chick myogenic cells. We identified 276 differentially expressed proteins from Label-free proteomic approach. Data overview of interaction network obtained from STRING show that after γ-secretase inhibition cells exhibited imbalance in protein metabolism, cytoskeleton/adhesion, and Sonic Hedgehog signaling. The collection of these results provides new insights into the role of γ-secretase in skeletal muscle hypertrophy.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Proteínas Aviárias/metabolismo , Diaminas/toxicidade , Hipertrofia/veterinária , Proteínas Musculares/metabolismo , Doenças Musculares/veterinária , Receptores Notch/metabolismo , Tiazóis/toxicidade , Animais , Células Cultivadas , Embrião de Galinha , Hipertrofia/induzido quimicamente , Hipertrofia/fisiopatologia , Doenças Musculares/induzido quimicamente , Doenças Musculares/fisiopatologia , Mapas de Interação de Proteínas , Proteômica , Transdução de SinaisRESUMO
Resumen La criptococosis es una enfermedad fúngica de distribución global con una mayor incidencia en países en vía de desarrollo, donde constituye una de las principales causas de meningitis, asociado a una alta morbi-mortalidad. Afecta principalmente a pacientes con inmunosupresión (por infección por VIH, tratamiento con glucocorticoides, sometidos a trasplantes, cáncer, etc). Sin embargo, en los últimos años se ha presentado un incremento de casos en inmunocompetentes, siendo tanto o más grave que en inmunocomprometidos. Presentamos dos casos de meningitis criptocóccica. Un varón de 48 años, en quien se descartó una infección por VIH u otra causa de inmunosupresión y una mujer de 67 años con antecedentes de cirrosis hepática. Debe considerarse Cryptococcus spp. como posible agente causal de meningitis, tanto en pacientes inmunocomprometidos como inmunocompetentes.
Cryptococcosis is a fungal disease of global distribution with a higher incidence in developing countries, where it represents one of the main causes of meningitis, associated with high morbidity and mortality. It mainly occurs in patients with immunosuppression (due to HIV infection, glucocorticoid treatment, transplants, cancer, etc.). However, in recent years there has been an increase in cases in immunocompetent, which is as serious or more severe than in immunocompromised. We report two cases of cryptococcal meningitis. A 48-year-old male with no pathological history, in whom HIV infection or another cause of immunosuppression was ruled out, and a 67-year-old woman with a previous diagnosis of liver cirrhosis. The above mentioned highlights the importance of always considering Cryptococcus spp. as possible causative agent of meningitis, in both immunocompromised and immunocompetent patients.
Assuntos
Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Meningite Criptocócica/diagnóstico , Cryptococcus neoformans/genética , Tomografia Computadorizada por Raios X , Reação em Cadeia da Polimerase , Hospedeiro ImunocomprometidoRESUMO
BACKGROUND: Among the many challenges in cancer diagnosis is the early distinction between metastatic cancer and a secondary tumor. This difficulty stems from the lack of markers that offer high sensitivity and specificity and can be easily applied in routine laboratory work. An example of this challenge is distinguishing gastric metastases originating from breast cancer from a gastric primary tumor. Hepatocyte nuclear factor 4 alpha (HNF4A) has been suggested as a potential marker in these cases. The aim of this study was to analyze the expression of HNF4A, estrogen receptor (ER), progesterone receptor (PR) and gross cystic disease fluid protein 15 (GCDFP-15) in a Brazilian cohort. METHODS: We performed immunohistochemistry analysis of HNF4A, ER, PR and GCDFP-15 in 126 patients divided into three cohorts: primary breast cancer, primary gastric cancer and both types of tumors. RESULTS: Our data confirmed the sensitivity and specificity of the HNF4A marker compared to other currently used clinical markers. CONCLUSION: HNF4A alone could be a gold standard marker for distinguishing primary gastric cancer from breast metastasis, thus validating its potential clinical use, especially in populations with high genetic diversity.
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Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Fator 4 Nuclear de Hepatócito/metabolismo , Neoplasias Gástricas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/secundário , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/patologiaRESUMO
The metastatic process in breast cancer is related to the expression of the epithelial-to-mesenchymal transition transcription factors (EMT-TFs) SNAIL, SLUG, SIP1 and TWIST1. EMT-TFs and nuclear factor-κB (NF-κB) activation have been associated with aggressiveness and metastatic potential in carcinomas. Here, we sought to examine the role of NF-κB in the aggressive properties and regulation of EMT-TFs in human breast cancer cells. Blocking NF-κB/p65 activity by reducing its transcript and protein levels (through siRNA-strategy and dehydroxymethylepoxyquinomicin [DHMEQ] treatment) in the aggressive MDA-MB-231 and HCC-1954 cell lines resulted in decreased invasiveness and migration, a downregulation of SLUG, SIP1, TWIST1, MMP11 and N-cadherin transcripts and an upregulation of E-cadherin transcripts. No significant changes were observed in the less aggressive cell line MCF-7. Bioinformatics tools identified several NF-κB binding sites along the promoters of SNAIL, SLUG, SIP1 and TWIST1 genes. Through chromatin immunoprecipitation and luciferase reporter assays, the NF-κB/p65 binding on TWIST1, SLUG and SIP1 promoter regions was confirmed. Thus, we suggest that NF-κB directly regulates the transcription of EMT-TF genes in breast cancer. Our findings may contribute to a greater understanding of the metastatic process of this neoplasia and highlight NF-κB as a potential target for breast cancer treatment.
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Neoplasias da Mama/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , NF-kappa B/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) is an important transcriptional factor frequently associated with the proliferation and survival of a large number of distinct cancer types. However, the signaling pathways and mechanisms that regulate STAT3 activation remain to be elucidated. METHODS: In this study we took advantage of existing cellular models for chronic myeloid leukemia resistance, western blot, in vitro signaling, real time PCR, flow cytometry approaches for cell cycle and apoptosis evaluation and siRNA assay in order to investigate the possible relationship between STATIP1, STAT3 and CML resistance. RESULTS: Here, we report the characterization of STAT3 protein regulation by STAT3-interacting protein (STATIP1) in the leukemia cell line K562, which demonstrates constitutive BCR-ABL TK activity. K562 cells exhibit high levels of phosphorylated STAT3 accumulated in the nucleus and enhanced BCR-ABL-dependent STAT3 transcriptional activity. Moreover, we demonstrate that STATIP1 is not involved in either BCR-ABL or STAT3 signaling but that STATIP1 is involved in the down-regulation of STAT3 transcription levels; STATIP1-depleted K562 cells display increased proliferation and increased levels of the anti-apoptosis STAT3 target genes CCND1 and BCL-XL, respectively. Furthermore, we demonstrated that Lucena, an Imatinib (IM)-resistant cell line, exhibits lower STATIP1 mRNA levels and undergoes apoptosis/cell cycle arrest in response to STAT3 inhibition together with IM treatment. We provide evidence that STATIP1 siRNA could confer therapy resistance in the K562 cells. Moreover, analysis of CML patients showed an inverse expression of STAIP1 and STAT3 mRNA levels, ratifying that IM-resistant patients present low STATIP1/high STAT3 mRNA levels. CONCLUSIONS: Our data suggest that STATIP1 may be a negative regulator of STAT3 and demonstrate its involvement in IM therapy resistance in CML.
Assuntos
Benzamidas/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Fator de Transcrição STAT3/metabolismo , Adulto , Idoso , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Masculino , Pessoa de Meia-Idade , Fosforilação , Fator de Transcrição STAT3/genética , Regulação para Cima , Adulto JovemRESUMO
To better characterize the cellular pathways involved in breast cancer molecular subtypes, we performed a proteomic study using a label-free LC-MS strategy for determining the proteomic profile of Luminal A, Luminal-HER2, HER2-positive, and triple-negative (TN) breast tumors compared with healthy mammary tissue. This comparison aimed to identify the aberrant processes specific for each subtype and might help to refine our understanding regarding breast cancer biology. Our results address important molecular features (both specific and commonly shared) that explain the biological behavior of each subtype. Changes in proteins related to cytoskeletal organization were found in all tumor subtypes, indicating that breast tumors are under constant structural modifications to invade and metastasize. We also found changes in cell-adhesion processes in all molecular subtypes, corroborating that invasiveness is a common property of breast cancer cells. Luminal-HER2 and HER2 tumors also presented altered cell cycle regulation, as shown by the several DNA repair-related proteins. An altered immune response was also found as a common process in the Luminal A, Luminal-HER2, and TN subtypes, and complement was the most important pathway. Analysis of the TN subtype revealed blood coagulation as the most relevant biological process.
Assuntos
Neoplasias da Mama/classificação , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Proteoma/metabolismo , Western Blotting , Adesão Celular/genética , Adesão Celular/fisiologia , Cromatografia Líquida , Proteínas do Citoesqueleto/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imuno-Histoquímica , Hibridização In Situ , Espectrometria de Massas , Invasividade Neoplásica/genética , Invasividade Neoplásica/fisiopatologia , Proteoma/genética , Proteômica/métodosRESUMO
One of the potential mechanisms of imatinib mesylate (IM) resistance in chronic myeloid leukemia (CML) is increased level of P-glycoprotein (Pgp). Pgp is an efflux pump capable of activating the multidrug resistance (MDR) phenotype. The gene encoding Pgp (ABCB1) has several binding sites in its promoter region, along with CpG islands and GC boxes, involved in its epigenetic control. In previous work, we performed a proteomic study to identify proteins involved in IM cross-resistance in acute leukemia. Among these proteins, we identified LRPPRC as a potential regulator of ABCB1 transcription via an invMED1 binding site in ABCB1. Interestingly, this invMED1 binding site overlaps with the GC -100 box. In this work, we investigated the potential role of LRPPRC in the regulation of ABCB1 transcriptional activity in CML resistance. In addition, we evaluated the potential connection between this regulation and the methylation status of the ABCB1 promoter in its GC -100 box. Our results show that LRPPRC binds prominently to the ABCB1 promoter in Lucena cells, an IM-resistant cell line. Luciferase assays showed that ABCB1 transcription is positively regulated by LRPPRC upon its knockdown. Pyrosequencing analysis showed that the ABCB1 promoter is differentially methylated at its GC -100 box in K562 cells compared with Lucena cells, and in CML patients with different response to IM. Chromatin immunoprecipitation and Pgp expression after DNA demethylation treatment showed that LRPPRC binding is affected by the methylation status of ABCB1 GC -100 box. Taken together, our findings indicate that LRPPRC is a transcription factor related to ABCB1 expression and highlight the importance of epigenetic regulation in CML resistance.