RESUMO
Infections remain a major cause of morbidity and mortality among hematopoietic stem cell transplant (HSCT) recipients. Unlike Epstein-Barr Virus (EBV) and Human Cytomegalovirus (HCMV), Human Herpesvirus (HHV) 6, HHV7 and HHV8 are not routinely monitored in many centers, especially in the pediatric population of low-medium income countries. We screened EBV, HCMV, HHV6, HHV7 and HHV8 in 412 leukocytes-plasma paired samples from 40 pediatric patients assisted in a tertiary hospital in Mexico. Thirty-two underwent allo-HSCT, whereas eight received auto-HSCT. Overall viral detection frequencies in allo- and auto-HSCT were: EBV = 43.7% and 30.0%, HCMV = 5.0% and 6.7%, HHV6 = 7.9% and 20.0% and HHV7 = 9.7% and 23.3%. HHV8 was not detected in any sample. Interestingly, HHV6 and HHV7 were more frequent in auto-HSCT, and HHV6 was observed in all episodes of multiple detection in auto-HSCT patients. We found EBV DNA in plasma samples, whereas HCMV, HHV6 and HHV7 DNA were predominantly observed in leukocytes, indicative of their expansion in cellular compartments. We also found that IL-1ß, IL-2, IL-6 and IL-8 were significantly increased in episodes in which multiple viruses were simultaneously detected, and samples positive for EBV DNA and graft-versus-host disease had a further increase of IL-1ß and IL-8. In conclusion, the EBV, HCMV, HHV6 and HHV7 burdens were frequently detected in allo- and auto-HSCT, and their presence associated with systemic inflammation.
RESUMO
Yin-Yang transcription factor 1 (YY1) is involved in tumor progression, metastasis and has been shown to be elevated in different cancers, including leukemia. The regulatory mechanism underlying YY1 expression in leukemia is still not understood. Bioinformatics analysis reveal three Hypoxia-inducible factor 1-alpha (HIF-1α) putative binding sites in the YY1 promoter region. The regulation of YY1 by HIF-1α in leukemia was analyzed. Mutation of the putative YY1 binding sites in a reporter system containing the HIF-1α promoter region and CHIP analysis confirmed that these sites are important for YY1 regulation. Leukemia cell lines showed that both proteins HIF-1α and YY1 are co-expressed under hypoxia. In addition, the expression of mRNA of YY1 was increased after 3 h of hypoxia conditions and affect several target genes expression. In contrast, chemical inhibition of HIF-1α induces downregulation of YY1 and sensitizes cells to chemotherapeutic drugs. The clinical implications of HIF-1α in the regulation of YY1 were investigated by evaluation of expression of HIF-1α and YY1 in 108 peripheral blood samples and by RT-PCR in 46 bone marrow samples of patients with pediatric acute lymphoblastic leukemia (ALL). We found that the expression of HIF-1α positively correlates with YY1 expression in those patients. This is consistent with bioinformatic analyses of several databases. Our findings demonstrate for the first time that YY1 can be transcriptionally regulated by HIF-1α, and a correlation between HIF-1α expression and YY1 was found in ALL clinical samples. Hence, HIF-1α and YY1 may be possible therapeutic target and/or biomarkers of ALL.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Fator de Transcrição YY1/metabolismo , Adolescente , Linhagem Celular Tumoral , Criança , Pré-Escolar , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Recém-NascidoRESUMO
Accumulating evidence strongly indicates that the presence of cancer stem cells (CSCs) leads to the emergence of worse clinical scenarios, such as chemo- and radiotherapy resistance, metastasis, and cancer recurrence. CSCs are a highly tumorigenic population characterized by self-renewal capacity and differentiation potential. Thus, CSCs establish a hierarchical intratumor organization that enables tumor adaptation to evade the immune response and resist anticancer therapy. YY1 functions as a transcription factor, RNA-binding protein, and 3D chromatin regulator. Thus, YY1 has multiple effects and regulates several molecular processes. Emerging evidence indicates that the development of lethal YY1-mediated cancer phenotypes is associated with the presence of or enrichment in cancer stem-like cells. Therefore, it is necessary to investigate whether and to what extent YY1 regulates the CSC phenotype. Since CSCs mirror the phenotypic behavior of stem cells, we initially describe the roles played by YY1 in embryonic and adult stem cells. Next, we scrutinize evidence supporting the contributions of YY1 in CSCs from a number of various cancer types. Finally, we identify new areas for further investigation into the YY1-CSCs axis, including the participation of YY1 in the CSC niche.
Assuntos
Células-Tronco Neoplásicas , Carcinogênese/patologia , Humanos , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismoRESUMO
BACKGROUND: Some reports have demonstrated the role of the G Protein-coupled Estrogen Receptor (GPER) in growth and proliferation of breast cancer cells. OBJECTIVE: In an effort to develop new therapeutic strategies against breast cancer, we employed an in silico study to explore the binding modes of tetrahydroquinoline 2 and 4 to be compared with the reported ligands G1 and G1PABA. METHODS: This study aimed to design and filter ligands by in silico studies determining their Lipinski's rule, toxicity and binding properties with GPER to achieve experimental assays as anti-proliferative compounds of breast cancer cell lines. RESULTS: In silico studies suggest as promissory two tetrahydroquinoline 2 and 4 which contain a carboxyl group instead of the acetyl group (as is needed for G1 synthesis), which add low (2) and high hindrance (4) chemical moieties to explore the polar, hydrophobic and hindrance effects. Docking and molecular dynamics simulations of the target compounds were performed with GPER to explore their binding mode and free energy values. In addition, the target small molecules were synthesized and assayed in vitro using breast cancer cells (MCF-7 and MDA-MB-231). Experimental assays showed that compound 2 decreased cell proliferation, showing IC50 values of 50µM and 25µM after 72h of treatment of MCF-7 and MDA-MB-231 cell lines, respectively. Importantly, compound 2 showed a similar inhibitory effect on proliferation as G1 compound in MDA-MB-231 cells, suggesting that both ligands reach the GPER-binding site in a similar way, as was demonstrated through in silico studies. CONCLUSION: A concentration-dependent inhibition of cell proliferation occurred with compound 2 in the two cell lines regardless of GPER.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Quinolinas/farmacologia , Receptores de Estrogênio/antagonistas & inibidores , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Modelos Moleculares , Estrutura Molecular , Quinolinas/síntese química , Quinolinas/química , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Relação Estrutura-Atividade , Termodinâmica , Células Tumorais CultivadasRESUMO
Resistance to chemotherapy hinders the successful treatment of acute lymphoblastic leukemia (ALL). The multi-drug resistance-1 (MDR1/ABCB1) gene encodes P-glycoprotein (P-gp), which plays an important role in chemoresistance; however, its transcriptional regulation remains unclear. We investigated the role of YY1 in the regulation of MDR1 and its relation to ALL outcomes. Analysis of the MDR1 promoter revealed four putative YY1-binding sites, which we analyzed using a reporter system and ChIP analysis. YY1 silencing resulted in the inhibition of MDR1 expression and function. The clinical roles of YY1 and MDR1 expression were evaluated in children with ALL. Expression of both proteins was increased in ALL patients compared to controls. We identified a positive correlation between YY1 and MDR1 expression. High levels of YY1 were associated with decreased overall survival. Our results demonstrated that YY1 regulates the transcription of MDR1. Therefore, YY1 may serve as a useful prognostic and/or therapeutic target.
Assuntos
Biomarcadores Tumorais/análise , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Fator de Transcrição YY1/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Adolescente , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Proliferação de Células , Criança , Pré-Escolar , Estudos de Coortes , Etoposídeo/farmacologia , Feminino , Seguimentos , Humanos , Lactente , Recém-Nascido , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , Regiões Promotoras Genéticas , Taxa de Sobrevida , Células Tumorais Cultivadas , Fator de Transcrição YY1/genéticaRESUMO
Tumor-initiating cells possess the capacity for self-renewal and to create heterogeneous cell lineages within a tumor. Therefore, the identification and isolation of cancer stem cells is an essential step in the analysis of their biology. The aim of the present study was to determine whether the cell surface protein neuropilin 1 (NRP1) can be used as a biomarker of stem-like cells in lung cancer tumors. For this purpose, NRP1-negative (NRP1-) and NRP1-positive (NRP1+) cell subpopulations from two lung cancer cell lines were sorted by flow cytometry. The NRP1+ cell subpopulation showed an increased expression of pluripotency markers OCT-4, Bmi-1 and NANOG, as well as higher cell migration, clonogenic and self-renewal capacities. NRP1 gene knockdown resulted not only in a decreased expression of stemness markers but also in a decrease in the clonogenic, cell migration and self-renewal potential. In addition, the NRP1+ cell subpopulation exhibited dysregulated expression of epithelial-to-mesenchymal transition-associated genes, including the ΔNp63 isoform protein, a previously reported characteristic of cancer stem cells. Notably, a genome-wide expression analysis of NRP1-knockdown cells revealed a potential new NRP1 pathway involving OLFML3 and genes associated with mitochondrial function. In conclusion, we demonstrated that NRP1+ lung cancer cells have tumor-initiating properties. NRP1 could be a useful biomarker for tumor-initiating cells in lung cancer tumors.
Assuntos
Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/citologia , Neuropilina-1/genética , Neuropilina-1/metabolismo , Células A549 , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Células Tumorais CultivadasRESUMO
Abstract: In mammals, apoptosis is the main mechanism to eliminate unwanted cells, securing tissue homeostasis and consequently maintaining the health in the organism. Classically, apoptosis culminates with the activation of caspases, which are enzymes that display cysteine protease activity to degrade specific substrates implied in essential cellular processes. This process is highly regulated. A key regulation mechanism is mediated by the Inhibitor of Apoptosis Proteins (IAPs) family members, which inhibit the activated forms of caspases through physical interaction with them. Smac/DIABLO, a mitochondrial protein that is translocated to the cytoplasm in apoptotic conditions, derepresses the IAP-mediated caspase inhibition through physical interaction with IAPs. The first four amino acids (AVPI) of Smac/DIABLO mediate the interaction with IAPs and subsequent apoptosis induction. This interaction has lead to the creation of small molecules mimicking the AVPI segment for potential anticancer therapy. Nevertheless, several studies have pointed out the existence of AVPI-independent functions of Smac/DIABLO. The aim of this review was to provide a landscape of these underestimated AVPI-independent biological functions that have been observed using different approaches, such as the study of endogenous splice variant isoforms and truncated and mutated artificial proteins.
Resumen: La apoptosis es uno de los principales mecanismos en los mamíferos para eliminar células no deseadas, asegurando la homeostasis de los tejidos y, consecuentemente, la salud de los mismos. De forma clásica, la apoptosis finaliza con la activación de las caspasas, enzimas que despliegan actividad de proteasas de cisteína, involucradas en la degradación de sustratos específicos implicados en procesos celulares esenciales. El proceso apoptótico se encuentra altamente regulado. Un mecanismo de regulación es el mediado por los miembros de la familia de las Proteínas Inhibidoras de la Apoptosis (PIA), las cuales inhiben a las formas activas de las caspasas a través de la interacción física con estas. Smac/DIABLO, proteína mitocondrial que es translocada al citoplasma en condiciones apoptóticas, antagoniza la inhibición de las caspasas mediante su interacción física con las PIA. Los cuatro primeros aminoácidos (AVPI) de Smac/DIABLO intervienen en su asociación con las PIA y la subsecuente inducción apoptótica. Esto ha guiado a la generación de pequeñas moléculas miméticas del segmento AVPI para el uso potencial como una terapia anti-cancerígena. Sin embargo, varios estudios han indicado la presencia de funciones en Smac/DIABLO independientes del AVPI. El objetivo de esta revisión fue proporcionar un panorama de estas funciones biológicas desestimadas —independientes al AVPI— las cuales se han observado utilizando diferentes aproximaciones, como el estudio de las isoformas generadas por el procesamiento alternativo del gen y la síntesis de proteínas artificialmente mutadas.
RESUMO
In mammals, apoptosis is the main mechanism to eliminate unwanted cells, securing tissue homeostasis and consequently maintaining the health in the organism. Classically, apoptosis culminates with the activation of caspases, which are enzymes that display cysteine protease activity to degrade specific substrates implied in essential cellular processes. This process is highly regulated. A key regulation mechanism is mediated by the Inhibitor of Apoptosis Proteins (IAPs) family members, which inhibit the activated forms of caspases through physical interaction with them. Smac/DIABLO, a mitochondrial protein that is translocated to the cytoplasm in apoptotic conditions, derepresses the IAP-mediated caspase inhibition through physical interaction with IAPs. The first four amino acids (AVPI) of Smac/DIABLO mediate the interaction with IAPs and subsequent apoptosis induction. This interaction has lead to the creation of small molecules mimicking the AVPI segment for potential anticancer therapy. Nevertheless, several studies have pointed out the existence of AVPI-independent functions of Smac/DIABLO. The aim of this review was to provide a landscape of these underestimated AVPI-independent biological functions that have been observed using different approaches, such as the study of endogenous splice variant isoforms and truncated and mutated artificial proteins.
RESUMO
XAF1 is a tumour suppressor gene that compromises cell viability by modulating different cellular events such as mitosis, cell cycle progression and apoptosis. In cancer, the XAF1 gene is commonly silenced by CpG-dinucleotide hypermethylation of its promoter. DNA demethylating agents induce transcriptional reactivation of XAF1, sensitizing cancer cells to therapy. The molecular mechanisms that mediate promoter CpG methylation have not been previously studied. Here, we demonstrate that CTCF interacts with the XAF1 promoter in vivo in a methylation-sensitive manner. By transgene assays, we demonstrate that CTCF mediates the open-chromatin configuration of the XAF1 promoter, inhibiting both CpG-dinucleotide methylation and repressive histone posttranslational modifications. In addition, the absence of CTCF in the XAF1 promoter inhibits transcriptional activation induced by well-known apoptosis activators. We report for the first time that epigenetic silencing of the XAF1 gene is a consequence of the loss of CTCF binding.
Assuntos
Metilação de DNA , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/genética , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Western Blotting , Fator de Ligação a CCCTC , Sobrevivência Celular , Imunoprecipitação da Cromatina , Humanos , Imunoprecipitação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Smac-α is a mitochondrial protein that, during apoptosis, is translocated to the cytoplasm, where it negatively regulates members of the inhibitor of apoptosis (IAP) family via the IAP-binding motif (IBM) contained within its amino-terminus. Here, we describe a new alternative splice variant from Smac gene, which we have named Smac-ε. Smac-ε lacks both an IBM and a mitochondrial-targeting signal (MTS) element. Smac-ε mRNA exhibits a tissue-specific expression pattern in healthy human tissues as well as in several cancer cell lines. The steady-state levels of endogenous Smac-ε protein is regulated by the proteasomal pathway. When ectopically expressed, this isoform presents a cytosolic localization and is unable to associate with or to regulate the expression of X-linked Inhibitor of apoptosis protein, the best-studied member of IAP family. Nevertheless, over-expression of Smac-ε increases mammosphere formation. Whole genome expression analyses from these mammospheres show activation of several pro-survival and growth pathways, including Estrogen-Receptor signaling. In conclusion, our results support the functionality of this new Smac isoform.
Assuntos
Neoplasias da Mama/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Reguladoras de Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Citosol/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células MCF-7 , Proteínas Mitocondriais/genética , Análise de Sequência com Séries de Oligonucleotídeos , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas , Proteólise , Esferoides Celulares , Transcrição Gênica , Transfecção , Regulação para CimaRESUMO
B cell leukemia-3 (Bcl-3) has been defined as an anti-apoptotic gene; however, the exact mechanisms through which Bcl-3 influences apoptosis have been elusive. To determine the specific role of Bcl-3 in apoptosis, we evaluated the effect of its silencing on the expression of proteins involved in either the extrinsic or intrinsic apoptotic pathways induced by ultraviolet light B-mediated DNA damage. We found that, in Bcl-3-silenced cells, caspase-3, caspase-8 and caspase-9 activation is accelerated and tBid mitochondrial content is increased. It is important to note that, although mitochondrial Smac levels were reduced after UV exposure, the rate of reduction was slightly higher in Bcl-3 silenced cells than in control cells. Additionally, p53 levels diminished in Bcl-3 silenced cells compared to control cells, as did those of DNA-PK, a DNA repair protein. Altogether, our data indicate that Bcl-3 protects cells from apoptosis by regulating both apoptotic pathways.