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El artículo tiene como objetivo analizar la disponibilidad, acceso y asequibilidad de los medicamentos para niños con Enfermedad Renal Crónica (ERC) en tratamiento con hemodiálisis (HD) en un país de bajos a medianos ingresos. Se llevó a cabo un estudio transversal para determinar los medicamentos más utilizados en una unidad de hemodiálisis pediátrica, incluyendo el nombre del medicamento, dosis, frecuencia, forma farmacéutica y vía de administración. Dos farmacias dentro del perímetro del hospital, una pública y una privada, fueron consultadas para determinar el costoy disponibilidad de medicamentos genéricos y de marca. De un total de 30 pacientes de la unidad de hemodiálisis, 22 expedientes fueron revisados. En general 94% de marca se encontraban disponibles en las farmacias consultadas en comparación a un 52% de los medicamentos genéricos. En farmacias públicas, 41% de medicamentos de marca y 29% de medicamentos genéricos se encontraban disponibles. El costo promedio para un mes de tratamiento con medicamentos de marca adquiridos en una farmacia privada era de $495.00 vs $299.00 en una farmacia pública. Para medicamentos genéricos, el costo promedio correspondía a $414.00 y $239.00 en farmacias privadas y públicas respectivamente. En promedio, los medicamentos de marca adquiridos en una farmacia privada requieren 41 días de trabajo en un mes a comparación de 25 días si se adquieren en una farmacia pública. Los medicamentos genéricos adquiridos en farmacias privadas corresponden a 34 días de trabajo vs 20 días en farmacias públicas. En general existió un acceso limitado a medicamentos genéricos y los medicamentos poseen un costo general más elevado a comparación de otros países lo que implica un posible impacto en la adherencia terapéutica y los padecimientos secundarios de la ERC en los pacientes pediátricos en Guatemala. Esta realidad se puede aplicar a otros países de bajos a medianos ingresos.
This article aims to analyze the availability, access, and affordability of medications for children with advanced Chronic Kidney Disease (CKD) treated with hemodialysis (HD) in a low to middle income country (LMIC). A cross- sectional chart review was carried out to determine the most common medications used in an HD pediatric unit, including medication name, dose, frequency, dosage form, and route of administration. Two pharmacies within the hospital perimeter, one public and one private, were consulted to determine medication cost and availability for generic and brand-name equivalents. From 30 patients attending the HD unit, 22 records were reviewed. Overall, 94 % of brand name medications were available at pharmacies consulted, versus and 52% of generic medications. In public pharmacies, 41% of brand name, and 29% of generic medications were available. The average cost for a full month´s treatment for brand name drugs in the private pharmacy was 495.00 USD versus 299.00 USD in the public pharmacy. For generic drugs, the average cost was 414.00 USD, and 239.00 USD in private and public pharmacies respectively. On average, brand-name drugs in the private pharmacy cost 41 days' wages versus 25 in the public pharmacy. Generic drugs in the private pharmacy cost 34 days' wages versus 20 in the public pharmacy. Overall, there was limited access to generic medications, medications had an overall high cost compared to other countries both of which have the potential to impact treatment adherence and overall outcomes of CKD5 pediatric patients in Guatemala. This reality can be translated to other LMIC.
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Los pacientes con sepsis y shock séptico presentan una alta mortalidad, representan un importante problema de salud; en estos pacientes, la incidencia de depresión miocárdica es conocida y ha sido documentada. La presente investigación encuentra la mortalidad asociada a la disfunción ventricular derecha, diagnosticada por ecocardiografía transtorácica al pie de cama, que se presenta o desarrolla secundaria a sepsis y su relación con la mortalidad, destacando la utilidad diagnóstica de esta técnica de imagen a través de la evaluación de parámetros ecocardiográficos medíbles y de fácil para cuantificar al lado de la cama del paciente. Metodología: fue un estudio observacional prospectivo analítico de cohorte única, en el Hospital Militar Central, San Salvador, El Salvador, diseñado para incluir pacientes atendidos o ingresados al servicio de medicina interna y cuidados intensivos en un periodo de 4 meses durante el año 2022, que cumplan con los criterios de sepsis y shock séptico, según sepsis-3 utilizando el ecocardiograma transtorácico. Se realizo el ecocardiograma como técnica para evaluar la función del ventrículo derecho (VD) en dos momentos, en las primeras 48 horas y el día 4 desde el diagnóstico de sepsis o shock séptico, definimos disfunción ventricular derecha (DVD) según el tipo: disfunción sistólica VD: TAPSE <1,6 cm, disfunción diastólica VD: cociente E/A <0,8 >2, se cuantificó el balance hídrico acumulado hasta el día 4. Se dio seguimiento a cada paciente para registrar muerte o egreso hospitalario en condición de vivo, relacionando estos desenlaces con disfunción ventricular derecha y balance hídrico. Análisis y resultados: La mortalidad se expreso mediante la razón proporcional de mortalidad intrahospitalaria asociada a DVD, se registró en 46,15%, RR: 1,81, la mortalidad fue mayor en quienes presentaron disfunción diastólica del ventrículo derecho. Interpretación: La disfunción del ventrículo derecho de cualquier tipo o global y el balance de líquidos mayor a 1 litro, se asoció directamente con una mayor mortalidad intrahospitalaria.
Patients with sepsis and septic shock carry high mortality even today, which is why it represents an important health problem; In these patients, the incidence of myocardial depression is known and has been documented. Research: to know the associated mortality with right ventricular dysfunction, diagnosed by transthoracic echocardiography at the bedside, which presents or develops secondary to sepsis and its relationship with mortality, highlighting the diagnostic utility of this imaging technique through the evaluation of measurable echocardiographic parameters and easy to quantify at the patient's bedside. Study Design and Methods: The Study is Observational Prospective Analytical Single Cohort, Monocentric (Hospital Militar Central, San Salvador, El Salvador). designed to include a cohort treated or admitted to the internal medicine and intensive care service in a period of 4 months during the year 2022, who meet the criteria for sepsis and septic shock, according to sepsis-3, a transthoracic echocardiogram was used as a research technique for those who had their right ventricular(RV) function evaluated in two moments, the first 48 hours and on day 4 from the diagnosis of sepsis or septic shock, to To document the presence or absence of an exposure factor: Right Ventricular Dysfunction (DVD) of any type, we defined RV Systolic Dysfunction: TAPSE <1.6 cm, RV Diastolic Dysfunction: E/A ratio <0.8 >2, and the accumulated fluid balance was quantified up to the day 4. Consecutively, each patient was followed up to record in-hospital death or Discharge in Living Condition, relating these outcomes with Right ventricular dysfunction and fluid balance. Results: The proportional ratio of Intrahospital Mortality due to DVD was recorded at 46.15%, RR: 1.81, Mortality was higher in those who presented diastolic dysfunction of the Right Ventricle. Interpretation: Right ventricular dysfunction of any type or global together with fluid balance greater than 1 liter, was directly associated with increased in-hospital mortality.
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Ecocardiografia , Sepse , CoraçãoRESUMO
Carcinomas, such as colon cancer, initiate their invasion by rescuing the innate plasticity of both epithelial cells and stromal cells. Although Snail is a transcriptional factor involved in the Epithelial-Mesenchymal Transition, in recent years, many studies have also identified the major role of Snail in the activation of Cancer-Associated Fibroblast (CAF) cells and the remodeling of the extracellular matrix. In CAFs, Platelet-derived growth factor (PDGF) receptor signaling is a major functional determinant. High expression of both SNAI1 and PDGF receptors is associated with poor prognosis in cancer patients, but the mechanism(s) that underlie these connections are not understood. In this study, we demonstrate that PDGF-activated fibroblasts stimulate extracellular matrix (ECM) fiber remodeling and deposition. Furthermore, we describe how SNAI1, through the FAK pathway, is a necessary factor for ECM fiber organization. The parallel-oriented fibers are used by endothelial cells as "tracks", facilitating their activation and the creation of tubular structures mimicking in vivo capillary formation. Accordingly, Snail1 expression in fibroblasts was required for the co-adjuvant effect of these cells on matrix remodeling and neoangiogenesis when co-xenografted in nude mice. Finally, in tumor samples from colorectal cancer patients a direct association between stromal SNAI1 expression and the endothelial marker CD34 was observed. In summary, our results advance the understanding of PDGF/SNAI1-activated CAFs in matrix remodeling and angiogenesis stimulation.
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Exosome production from cancer-associated fibroblasts seems to be an important driver of tumor progression. We report the first in-depth biotype characterization of ncRNAs, analyzed by Next Generation Sequencing and Bioinformatics, expressed in established primary human normal and cancer-associated fibroblasts (CAFs) from cancer and normal mucosa tissues from 9 colorectal cancer patients, and/or packaged in their derived exosomes. Differential representation and enrichment analyses based on these ncRNAs revealed a significant number of differences between the ncRNA content of exosomes and the expression patterns of the normal and cancer-associated fibroblast cells. ncRNA regulatory elements are specifically packaged in CAF-derived exosomes, supporting a specific cross-talk between CAFs and colon cancer cells and/or other stromal cells, mediated by exosomes. These sncRNAs are potential biomarkers present in cancer-associated fibroblast-derived exosomes, which should thereby contribute to developing new non-invasive diagnostic, prognostic and predictive methods for clinical applications in management of cancer patients.
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Fibroblastos Associados a Câncer/citologia , Neoplasias Colorretais/genética , Exossomos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA não Traduzido/genética , Biomarcadores Tumorais/genética , Fibroblastos Associados a Câncer/química , Movimento Celular , Proliferação de Células , Células Cultivadas , Fibroblastos/química , Fibroblastos/citologia , Regulação Neoplásica da Expressão Gênica , Humanos , Prognóstico , Análise de Sequência de RNA , Microambiente TumoralRESUMO
Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC and 5-methylcytosine (5mC) in human primary samples from 12 non-tumoral brains and 53 gliomas. We found that the levels of 5hmC identified in non-tumoral samples were significantly reduced in gliomas. Strikingly, hypo-hydroxymethylation at 4627 (9.3%) CpG sites was associated with aberrant DNA hypermethylation and was strongly enriched in CpG island shores. The DNA regions containing these CpG sites were enriched in H3K4me2 and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. As this 5mC gain is inversely correlated with loss of 5hmC and has not been identified with classical sodium bisulfite-based technologies, we conclude that our data identifies a novel 5hmC-dependent type of aberrant DNA hypermethylation in glioma.
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5-Metilcitosina/análogos & derivados , Biomarcadores Tumorais/genética , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Glioma/patologia , 5-Metilcitosina/metabolismo , Estudos de Casos e Controles , Ilhas de CpG , Glioma/genética , Glioma/metabolismo , HumanosRESUMO
Snail1 transcriptional factor is essential for triggering epithelial-to-mesenchymal transition (EMT) and inducing tumor cell invasion. We report here an EMT-independent action of Snail1 on tumor invasion, as it is required for the activation of cancer-associated fibroblasts (CAF). Snail1 expression in fibroblasts requires signals derived from tumor cells, such as TGFß; reciprocally, in fibroblasts, Snail1 organizes a complex program that stimulates invasion of epithelial cells independent of the expression of Snail1 in these cells. Epithelial cell invasion is stimulated by the secretion by fibroblast of diffusible signaling molecules, such as prostaglandin E2 The capability of human or murine CAFs to promote tumor invasion is dependent on Snail1 expression. Inducible Snail1 depletion in mice decreases the invasion of breast tumors; moreover, epithelial tumor cells coxenografted with Snail1-depleted fibroblasts originated tumors with lower invasion than those transplanted with control fibroblasts. Therefore, these results demonstrate that the role of Snail1 in tumor invasion is not limited to EMT, but it is also dependent on its activity in stromal fibroblasts, where it orchestrates the cross-talk with epithelial tumor cells. Cancer Res; 76(21); 6205-17. ©2016 AACR.
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Fibroblastos Associados a Câncer/fisiologia , Neoplasias Epiteliais e Glandulares/patologia , Fatores de Transcrição da Família Snail/fisiologia , Animais , Linhagem Celular Tumoral , Dinoprostona/metabolismo , Transição Epitelial-Mesenquimal , Humanos , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Cancer cells efficiently transfer exosome contents (essentially mRNAs and microRNAs) to other cell types, modifying immune responses, cell growth, angiogenesis and metastasis. Here we analyzed the exosomes release by breast tumor cells with different capacities of stemness/metastasis based on CXCR4 expression, and evaluated their capacity to generate oncogenic features in recipient cells. Breast cancer cells overexpressing CXCR4 showed an increase in stemness-related markers, and in proliferation, migration and invasion capacities. Furthermore, recipient cells treated with exosomes from CXCR4-cells showed increased in the same abilities. Moreover, inoculation of CXCR4-cell-derived exosomes in immunocompromised mice stimulated primary tumor growth and metastatic potential. Comparison of nucleic acids contained into exosomes isolated from patients revealed a "stemness and metastatic" signature in exosomes of patients with worse prognosis. Finally, our data supported the view that cancer cells with stem-like properties show concomitant metastatic behavior, and their exosomes stimulate tumor progression and metastasis. Exosomes-derived nucleic acids from plasma of breast cancer patients are suitable markers in the prognosis of such patients.
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Neoplasias da Mama/patologia , Proliferação de Células , Transformação Celular Neoplásica/patologia , Exossomos/genética , Regulação Neoplásica da Expressão Gênica , Células-Tronco Neoplásicas/patologia , Receptores CXCR4/metabolismo , Animais , Apoptose , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Movimento Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Nus , Metástase Neoplásica , Células-Tronco Neoplásicas/metabolismo , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores CXCR4/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Cancer-associated fibroblasts (CAF) are major mediators in tumor microenvironment. We investigated the changes in protein expression in colon cancer-associated fibroblasts compared with normal fibroblasts (NF) in the context of searching for prognostic biomarkers, particularly for stage II patients. EXPERIMENTAL DESIGN: CAFs and NFs isolated from colon cancer patients were used to identify differentially expressed proteins using quantitative proteomics. Stromal expression of deregulated proteins was analyzed by IHC. Prognostic impact was studied using external gene-expression datasets for training, then quantitative PCR and IHC for validation in different cohorts of patients. Combined datasets were used for prediction of risk assessment at stages II and III. RESULTS: A desmoplastic signature composed of 32 proteins, highly specific for stromal components in colon cancer, was identified. These proteins were enriched for extracellular matrix organization components, TGFß signaling pathway, fibrosis, and wound-healing proteins. The expression in CAFs of 11 upregulated proteins and four downregulated proteins, selected for biomarker validation, was verified by orthogonal techniques. LOXL2 displayed a high prognostic impact by using external independent datasets and further validation in two different cohorts of patients. High expression of LOXL2 was associated with higher recurrence P = 0.001 HR, 5.38 [95% confidence interval (CI), 1.70-17.01] and overall survival P = 0.001 HR, 8.52 (95% CI, 1.90-38.29). IHC analysis revealed a prognostic value for LOXL2 in stage II patients. CONCLUSIONS: We identified LOXL2 to be associated with the outcome of colon cancer patients. Furthermore, it can be used to stratify patients at stages II and III for further therapeutic decisions.
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Aminoácido Oxirredutases/genética , Neoplasias do Colo/genética , Neoplasias do Colo/mortalidade , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Aminoácido Oxirredutases/metabolismo , Biomarcadores , Linhagem Celular Tumoral , Análise por Conglomerados , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Fibroblastos/patologia , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Estadiamento de Neoplasias , Prognóstico , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteômica/métodos , Reprodutibilidade dos Testes , Células Estromais/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral/genéticaRESUMO
BACKGROUND: The acquired resistance to chemotherapy represents the major limitation in the treatment of cancer. New strategies to solve this failure and improve patients' outcomes are necessary. The cancer preventive effect of ß-cryptoxanthin has been widely described in population studies. Few reports support its putative use as an antitumoral compound. Here we focus on the therapeutic potential of ß-cryptoxanthin individually or in combination with oxaliplatin in colon cancer and try to decipher the molecular basis underlying its effect. METHODS: Apoptosis, viability and proliferation assays, mouse models, and an intervention study in 20 healthy subjects were performed. A PCR array was carried out to unravel the molecular putative basis of the ß-cryptoxanthin effect, and further signaling experiments were conducted. Comet Assay was completed to evaluate the genotoxicity of the treatments. RESULTS: ß-Cryptoxanthin differentially regulates the expression of the P73 variants in vitro, in vivo, and in a human intervention study. This carotenoid decreases the proliferation of cancer cells and cooperates with oxaliplatin to induce apoptosis through the negative regulation of ΔNP73. The antitumoral concentrations of oxaliplatin decrease in the presence of ß-cryptoxanthin to achieve same percentage of growth inhibition. The genotoxicity in peripheral blood mononuclear cells of mice decreased in the combined treatment. CONCLUSIONS: We propose a putative novel therapeutic strategy for the treatment of colon cancer based on the combination of ß-cryptoxanthin and oxaliplatin. The combined regimen produced more benefit than either individual modality without increasing side effects. In addition, the concentration-limiting toxicity of oxaliplatin is reduced in the presence of the carotenoid.
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Antineoplásicos/farmacologia , Neoplasias do Colo/genética , Criptoxantinas/farmacologia , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Nucleares/genética , Compostos Organoplatínicos/farmacologia , Proteínas Supressoras de Tumor/genética , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Oxaliplatina , Isoformas de Proteínas , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Many studies have demonstrated that the endocannabinoid system (ECS) is altered in different tumor types, including colon cancer. However, little is known about the role of the ECS in tumor progression. Here we report the correlation between CB 2 expression and pathological data in a series of 175 colorectal cancer patients, as well as the response of the HT29 colon cancer-derived cell line upon CB 2 activation. CB 2 mRNA was detected in 28.6% of samples tested. It was more frequent in N+ patients and predicts disease free survival and overall survival in colon cancer. In positive samples, CB 2 was expressed with great intensity in tumor epithelial cells and correlated with tumor growth. Treatment of HT29 with CB 2 agonist revealed membrane loss of E-cadherin and SNAIL1 overexpression. A direct correlation between CB 2 and SNAIL1 expression was also found in human tumors. CB 2 receptor expression is a poor prognostic marker for colon cancer and the activation of this receptor, with non-apoptotic doses of agonists, could be collaborating with disease progression. These results raise the question whether the activation of CB 2 should be considered as anti-tumoral therapy.
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Crosstalk between tumor and stromal cells in the tumor microenvironment alter its properties in ways that facilitate the invasive behavior of tumor cells. Here, we demonstrate that cancer-associated fibroblasts (CAF) increase the stiffness of the extracellular matrix (ECM) and promote anisotropic fiber orientation, two mechanical signals generated through a Snail1/RhoA/αSMA-dependent mechanism that sustains oriented tumor cell migration and invasiveness. Snail1-depleted CAF failed to acquire myofibroblastic traits in response to TGFß, including RhoA activation, αSMA-positive stress fibers, increased fibronectin fibrillogenesis, and production of a stiff ECM with oriented fibers. Snail1 expression in human tumor-derived CAF was associated with an ability to organize the ECM. In coculture, a relatively smaller number of Snail1-expressing CAF were capable of imposing an anisotropic ECM architecture, compared with nonactivated fibroblasts. Pathologically, human breast cancers with Snail1(+) CAF tended to exhibit desmoplastic areas with anisotropic fibers, lymph node involvement, and poorer outcomes. Snail1 involvement in driving an ordered ECM was further confirmed in wound-healing experiments in mice, with Snail1 depletion preventing the anisotropic organization of granulation tissue and delaying wound healing. Overall, our results showed that inhibiting Snail1 function in CAF could prevent tumor-driven ECM reorganization and cancer invasion.
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Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fatores de Transcrição/biossíntese , Animais , Humanos , Camundongos , Camundongos Knockout , Metástase Neoplásica , Fatores de Transcrição da Família Snail , Microambiente Tumoral , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Adipogenesis requires a differentiation program driven by multiple transcription factors, where PPARγ and C/EBPα play a central role. Recent findings indicate that Snail inhibits adipocyte differentiation in 3T3-L1 and murine mesenchymal stem cells (mMSC). An in-depth quantitative SILAC analysis of the nuclear fraction of Snail-induced alterations of 3T3-L1 cells was carried out. In total, 2251 overlapping proteins were simultaneously quantified in forward and reverse experiments. We observed 574 proteins deregulated by Snail1 using a fold-change ≥1.5, with 111 up- and 463 down-regulated proteins, respectively. Among other proteins, multiple transcription factors such as Trip4, OsmR, Nr2f6, Cbx6, and Prrx1 were down-regulated. Results were validated in 3T3-L1 cells and mMSC cells by Western blot and quantitative PCR. Knock-down experiments in 3T3-L1 cells demonstrated that only Nr2f6 (and Trip4 at minor extent) was required for adipocyte differentiation. Ectopic expression of Nr2f6 reversed the effects of Snail1 and promoted adipogenesis. Because Nr2f6 inhibits the expression of IL-17, we tested the effect of Snail on IL-17 expression. IL-17 and TNFα were among the most up-regulated pro-inflammatory cytokines in Snail-transfected 3T3-L1 and mMSC cells. Furthermore, the blocking of IL-17 activity in Snail-transfected cells promoted adipocyte differentiation, reverting Snail inhibition. In summary, Snail inhibits adipogenesis through a down-regulation of Nr2f6, which in turn facilitates the expression of IL-17, an anti-adipogenic cytokine. These results would support a novel and important role for Snail and Nr2f6 in obesity control.
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Adipócitos/citologia , Adipócitos/metabolismo , Fatores de Transcrição COUP/metabolismo , Diferenciação Celular , Interleucina-17/metabolismo , Proteômica/métodos , Fatores de Transcrição/metabolismo , Células 3T3-L1 , Adipogenia , Animais , Extratos Celulares , Núcleo Celular/metabolismo , Regulação para Baixo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Modelos Biológicos , Proteínas Repressoras , Reprodutibilidade dos Testes , Transdução de Sinais , Fatores de Transcrição da Família Snail , TransfecçãoRESUMO
Snail1 transcriptional repressor is a major inducer of epithelial-to mesenchymal transition but is very limitedly expressed in adult animals. We have previously demonstrated that Snail1 is required for the maintenance of mesenchymal stem cells (MSCs), preventing their premature differentiation. Now, we show that Snail1 controls the tumorigenic properties of mesenchymal cells. Increased Snail1 expression provides tumorigenic capabilities to fibroblastic cells; on the contrary, Snail1 depletion decreases tumor growth. Genetic depletion of Snail1 in MSCs that are deficient in p53 tumor suppressor downregulates MSC markers and prevents the capability of these cells to originate sarcomas in immunodeficient SCID mice. Notably, an analysis of human sarcomas shows that, contrarily to epithelial tumors, these neoplasms display high Snail1 expression. This is particularly clear for undifferentiated tumors, which are associated with poor outcome. Together, our results indicate a role for Snail1 in the generation of sarcomas.
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Carcinogênese/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Sarcoma/metabolismo , Fatores de Transcrição/biossíntese , Animais , Western Blotting , Carcinogênese/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Estimativa de Kaplan-Meier , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos SCID , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma/genética , Sarcoma/mortalidade , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genéticaRESUMO
Tumor-derived exosomes mediate tumorigenesis by facilitating tumor growth, metastasis, development of drug resistance, and immunosuppression. However, little is known about the exosomes isolated from bronchoalveolar lavage (BAL) in patients with lung neoplasm. Exosomes isolated in plasma and BAL from 30 and 75 patients with tumor and nontumor pathology were quantified by acetylcholinesterase activity and characterized by Western Blot, Electron Microscopy, and Nanoparticle Tracking Analysis. Differences in exosome cargo were analyzed by miRNA quantitative PCR in pooled samples and validated in a second series of patients. More exosomes were detected in plasma than in BAL in both groups (P < 0.001). The most miRNAs evaluated by PCR array were detected in tumor plasma, tumor BAL, and nontumor BAL pools, but only 56% were detected in the nontumor plasma pool. Comparing the top miRNAs with the highest levels detected in each pool, we found close homology only between the BAL samples of the two pathologies. In tumor plasma, we found a higher percentage of miRNAs with increased levels than in tumor BAL or in nontumor plasma. The data reveal differences between BAL and plasma exosome amount and miRNA content.
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Adenocarcinoma/patologia , Sangue/metabolismo , Líquido da Lavagem Broncoalveolar/citologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/patologia , Exossomos/metabolismo , Neoplasias Pulmonares/patologia , Adenocarcinoma/sangue , Adenocarcinoma/química , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Líquido da Lavagem Broncoalveolar/química , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/química , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/química , Feminino , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/química , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Adulto JovemRESUMO
Tumor-derived exosomes are emerging as local and systemic cell-to-cell mediators of oncogenic information through the horizontal transfer of mRNAs, microRNAs and proteins during tumorigenesis. The exosomal content has been described as biologically active when taken up by the recipient cell. Identifying the specific molecular cargo of exosomes will help to determine their function in specific steps of the tumorigenic process. Here we evaluate whether ΔNp73 is selectively packaged in tumor-derived exosomes, its function in the acceptor cells in vitro and in vivo and its prognosis potential in cancer. ΔNp73 messenger is enriched in tumor-derived exosomes, suggesting its active sorting in these microvesicles. We observed the transmission of this exosome cargo to different cell types and how it confers proliferation potential and chemoresistance to the acceptor cells in vitro and in animal models. Finally, our data support the potential prognostic value of exosomal ΔNp73 in colon cancer patients.
Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Exossomos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Feminino , Células HCT116 , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Prognóstico , RNA Mensageiro/metabolismo , Taxa de Sobrevida , Proteína Tumoral p73RESUMO
Snail1 is a transcriptional factor that plays an important role in epithelial-mesenchymal transition and in the acquisition of invasive properties by epithelial cells. In colon tumors, Snail1 expression in the stroma correlates with lower specific survival of cancer patients. However, the role(s) of Snail1 expression in stroma and its association with patients' survival have not been determined. We used human primary carcinoma-associated fibroblasts (CAFs) or normal fibroblasts (NFs) and fibroblast cell lines to analyze the effects of Snail1 expression on the protumorigenic capabilities in colon cancer cells. Snail1 expression was higher in CAFs than in NFs and, as well as α-SMA, a classic marker of activated CAFs. Moreover, in tumor samples from 50 colon cancer patients, SNAI1 expression was associated with expression of other CAF markers, such as α-SMA and fibroblast activation protein. Interestingly, coculture of CAFs with colon cells induced a significant increase in epithelial cell migration and proliferation, which was associated with endogenous SNAI1 expression levels. Ectopic manipulation of Snail1 in fibroblasts demonstrated that Snail1 expression controlled migration as well as proliferation of cocultured colon cancer cells in a paracrine manner. Furthermore, expression of Snail1 in fibroblasts was required for the coadjuvant effect of these cells on colon cancer cell growth and invasion when coxenografted in nude mice. Finally, cytokine profile changes, particularly MCP-3 expression, in fibroblasts are put forward as mediators of Snail1-derived effects on colon tumor cell migration. In summary, these studies demonstrate that Snail1 is necessary for the protumorigenic effects of fibroblasts on colon cancer cells.
Assuntos
Carcinogênese , Neoplasias do Colo/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Ciclo Celular , Movimento Celular , Proliferação de Células , Técnicas de Cocultura , Neoplasias do Colo/genética , Citocinas/metabolismo , Endopeptidases , Feminino , Fibroblastos/patologia , Gelatinases/genética , Gelatinases/metabolismo , Expressão Gênica , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , RNA Mensageiro/biossíntese , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Fatores de Transcrição da Família Snail , Células Tumorais CultivadasRESUMO
PURPOSE: Cancer-associated fibroblasts (CAF) actively participate in reciprocal communication with tumor cells and with other cell types in the microenvironment, contributing to a tumor-permissive neighborhood and promoting tumor progression. The aim of this study is the characterization of how CAFs from primary human colon tumors promote migration of colon cancer cells. EXPERIMENTAL DESIGN: Primary CAF cultures from 15 primary human colon tumors were established. Their enrichment in CAFs was evaluated by the expression of various epithelial and myofibroblast specific markers. Coculture assays of primary CAFs with different colon tumor cells were performed to evaluate promigratory CAF-derived effects on cancer cells. Gene expression profiles were developed to further investigate CAF characteristics. RESULTS: Coculture assays showed significant differences in fibroblast-derived paracrine promigratory effects on cancer cells. Moreover, the association between CAFs' promigratory effects on cancer cells and classic fibroblast activation or stemness markers was observed. CAF gene expression profiles were analyzed by microarray to identify deregulated genes in different promigratory CAFs. The gene expression signature, derived from the most protumorogenic CAFs, was identified. Interestingly, this "CAF signature" showed a remarkable prognostic value for the clinical outcome of patients with colon cancer. Moreover, this prognostic value was validated in an independent series of 142 patients with colon cancer, by quantitative real-time PCR (qRT-PCR), with a set of four genes included in the "CAF signature." CONCLUSIONS: In summary, these studies show for the first time the heterogeneity of primary CAFs' effect on colon cancer cell migration. A CAF gene expression signature able to classify patients with colon cancer into high- and low-risk groups was identified.
Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica , Transcriptoma , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Análise por Conglomerados , Técnicas de Cocultura , Neoplasias do Colo/mortalidade , Neoplasias do Colo/patologia , Fibroblastos/patologia , Perfilação da Expressão Gênica , Complexo de Golgi/metabolismo , Humanos , Comunicação Parácrina , Fenótipo , PrognósticoRESUMO
PURPOSE: Cancer-associated fibroblasts (CAF) are essential components of the stroma that play a critical role in cancer progression. This study aimed to identify novel CAFs markers that might contribute to the invasion and the prognosis of colorectal cancer. EXPERIMENTAL DESIGN: The azoxymethane/dextran sodium sulfate mouse model of sporadic colon cancer represents an adequate source for the isolation of CAFs and normal fibroblasts. By using the explants technique, we purified CAFs and normal fibroblasts from colon tissues. Whole-cell extracts and supernatants were subjected to in-depth quantitative proteomic analysis by tandem mass spectrometry. Further validations of upregulated proteins in CAFs were carried out by chemokine microarray and immunohistochemical analyses of mouse and human tissues. RESULTS: Using a fold-change of 1.4 or more, we found 132 and 125 differentially expressed proteins in whole-cell extracts and supernatants, respectively. We found CAFs-associated proinflammatory and desmoplastic signatures. The proinflammatory signature was composed of several cytokines. Among them, CCL2 and CCL8 caused an increase in migration and invasion of colorectal cancer KM12 cells. The desmoplastic signature was composed of 30 secreted proteins. In mouse and human samples, expression of LTBP2, CDH11, OLFML3, and, particularly, FSTL1 was significantly increased in the tumoral stroma, without significant expression in the cancer epithelial cells. The combination of CALU and CDH11 stromal expression showed a significant association with disease-free survival and poor prognosis. CONCLUSION: We have identified LTBP2, CDH11, OLFML3, and FSTL1 as selective biomarkers of cancer stroma, and CALU and CDH11 as candidate stromal biomarkers of prognostic significance in colon cancer.
Assuntos
Neoplasias Colorretais/metabolismo , Fibroblastos/metabolismo , Mediadores da Inflamação/metabolismo , Proteoma , Animais , Biomarcadores , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Miofibroblastos/metabolismo , Prognóstico , Mapas de Interação de Proteínas , Reprodutibilidade dos Testes , Células Estromais/metabolismoRESUMO
TP73 is a member of the TP53 family, whose deregulated expression has been reported in a wide variety of cancers and linked to patients' outcome. The fact that TP73 encodes a complex number of isoforms (TAp73 and ΔTAp73) with opposing functions and the cross-talk with other members of the family (TP53 and TP63) make it difficult to determine its clinical relevance. Here, we review the molecular mechanisms driving TAp73 and ΔTAp73 expression and how these variants inhibit or promote carcinogenesis. We also highlight the intricate interplay between TP53 family members. In addition, we comment on current pharmacological approaches targeting the TP73 pathway and those affecting the TAp73/ΔTAp73 ratio. Finally, we discuss the current data available in the literature that provide evidence on the role of TP73 variants in predicting prognosis. To date, most of the studies that evaluate the status levels of TP73 isoforms have been based on limited-size series. Despite this limitation, these publications highlight the correlation between high levels of the oncogenic forms and failure to respond to chemotherapy and/or shorter survival. Finally, we emphasize the need for studies to evaluate the significance of combining the deregulation of various members of the TP53 family in order to define patient outcome or their responsiveness to specific therapies.
Assuntos
Proteínas de Ligação a DNA/genética , Redes Reguladoras de Genes , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Genes p53 , Humanos , Neoplasias/tratamento farmacológico , Oncogenes , Prognóstico , Isoformas de Proteínas/genética , Transdução de Sinais , Pesquisa Translacional Biomédica , Proteína Tumoral p73RESUMO
Fibroblast growth factor receptor 4 (FGFR4) is vital in early development and tissue repair. FGFR4 expression levels are very restricted in adult tissues, except in several solid tumors including colorectal cancer, which showed overexpression of FGFR4. Here, FGFR4 mutation analysis discarded the presence of activating mutations, other than Arg(388), in different colorectal cancer cell lines and tumoral samples. Stable shRNA FGFR4-silencing in SW480 and SW48 cell lines resulted in a significant decrease in cell proliferation, adhesion, cell migration and invasion. This decrease in the tumorigenic and invasive capabilities of colorectal cancer cells was accompanied by a decrease of Snail, Twist and TGFß gene expression levels and an increase of E-cadherin, causing a reversion to a more epithelial phenotype, in three different cell lines. In addition, FGFR4-signaling activated the oncogenic SRC, ERK1/2 and AKT pathways in colon cancer cells and promoted an increase in cell survival. The relevance of FGFR4 in tumor growth was supported by two different strategies. Kinase inhibitors abrogated FGFR4-related cell growth and signaling pathways at the same extent than FGFR4-silenced cells. Specific FGFR4-targeting using antibodies provoked a similar reduction in cell growth. Moreover, FGFR4 knock-down cells displayed a reduced capacity for in vivo tumor formation and angiogenesis in nude mice. Collectively, our data support a crucial role for FGFR4 in tumorigenesis, invasion and survival in colorectal cancer. In addition, FGFR4 targeting demonstrated its applicability for colorectal cancer therapy.