RESUMO
Cancers are heterogeneous at the cell level, and the mechanisms leading to cancer heterogeneity could be clonal evolution or cancer stem cells. Cancer stem cells are resistant to most anti-cancer treatments and could be preferential targets to reverse this resistance, either targeting stemness pathways or cancer stem cell surface markers. Gold nanoparticles have emerged as innovative tools, particularly for photo-thermal therapy since they can be excited by laser to induce hyperthermia. Gold nanoparticles can be functionalized with antibodies to specifically target cancer stem cells. Preclinical studies using photo-thermal therapy have demonstrated the feasibility of targeting chemo-resistant cancer cells to reverse clinical chemoresistance. Here, we review the data linking cancer stem cells and chemoresistance and discuss the way to target them to reverse resistance. We particularly focus on the use of functionalized gold nanoparticles in the treatment of chemo-resistant metastatic cancers.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ouro/uso terapêutico , Neoplasias/terapia , Células-Tronco Neoplásicas/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Sinergismo Farmacológico , Feminino , Ouro/farmacologia , Humanos , Hipertermia Induzida , Masculino , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Células-Tronco Neoplásicas/patologia , Resultado do TratamentoRESUMO
Cancer is now the most severe complication in the long term in transplant recipients. As most solid-organ or hematopoietic stem-cell transplantations are allogeneic, chimerism studies can be performed on cancers occurring in recipients. We summarize here the different methods used to study chimerism in cancers developing in allogeneic-transplant recipients, analyze their respective advantages and report the main results obtained from these studies. Chimerism analyses of cancers in transplant recipients require methods suited to tissue samples. In the case of gender-mismatched transplantation, the XY chromosomes can be explored using fluorescent in situ hybridization on whole-tissue sections or Y-sequence-specific PCR after the laser microdissection of tumor cells. For cancers occurring after gender-matched transplantation, laser microdissection of tumor cells enables studies of microsatellite markers and high-resolution melting analysis of mitochondrial DNA on genes with marked polymorphism, provided these are different in the donor and the recipient. The results of different studies address the cancers that develop in both recipients and in transplants. The presence of chimeric cells in these two types of cancer implies an exchange of progenitor/stem-cells between transplant and recipient, and the plasticity of these progenitor/stem-cells contributes to epithelial cancers. The presence of chimeric cells in concomitant cancers and preneoplastic lesions implies that the oncogenesis of these cancers progresses through a multistep process.
Assuntos
Quimerismo , Neoplasias Epiteliais e Glandulares/genética , Transplantados , DNA Mitocondrial/genética , Feminino , Humanos , Masculino , Repetições de Microssatélites/genética , Transplante HomólogoRESUMO
BACKGROUND: Increased permeability, predominantly controlled by endothelial junction stability, is an early event in the deterioration of vascular integrity in ischemic disorders. Hemorrhage, edema, and inflammation are the main features of reperfusion injuries, as observed in acute myocardial infarction (AMI). Thus, preservation of vascular integrity is fundamental in ischemic heart disease. Angiopoietins are pivotal modulators of cell-cell junctions and vascular integrity. We hypothesized that hypoxic induction of angiopoietin-like protein 4 (ANGPTL4) might modulate vascular damage, infarct size, and no-reflow during AMI. METHODS AND RESULTS: We showed that vascular permeability, hemorrhage, edema, inflammation, and infarct severity were increased in angptl4-deficient mice. We determined that decrease in vascular endothelial growth factor receptor 2 (VEGFR2) and VE-cadherin expression and increase in Src kinase phosphorylation downstream of VEGFR2 were accentuated after ischemia-reperfusion in the coronary microcirculation of angptl4-deficient mice. Both events led to altered VEGFR2/VE-cadherin complexes and to disrupted adherens junctions in the endothelial cells of angptl4-deficient mice that correlated with increased no-reflow. In vivo injection of recombinant human ANGPTL4 protected VEGF-driven dissociation of the VEGFR2/VE-cadherin complex, reduced myocardial infarct size, and the extent of no-reflow in mice and rabbits. CONCLUSIONS: These data showed that ANGPTL4 might constitute a relevant target for therapeutic vasculoprotection aimed at counteracting the effects of VEGF, thus being crucial for preventing no-reflow and conferring secondary cardioprotection during AMI.
Assuntos
Angiopoietinas/uso terapêutico , Endotélio Vascular/metabolismo , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/prevenção & controle , Fenômeno de não Refluxo/metabolismo , Fenômeno de não Refluxo/prevenção & controle , Proteína 4 Semelhante a Angiopoietina , Angiopoietinas/deficiência , Animais , Cardiotônicos/metabolismo , Cardiotônicos/uso terapêutico , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Infarto do Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Coelhos , Distribuição AleatóriaRESUMO
Although rodent graft-versus-host disease (GVHD) models have suggested that indoleamine 2,3-dioxygenase (IDO) is a critical regulator of gastrointestinal GVHD, parallel human studies on IDO expression have not been reported. IDO expression was assessed in 20 patients who underwent duodenal biopsy. IDO was upregulated in epithelial cells. In situ analyses reveal that macrophages and dendritic cells stain positive for IDO, but that most of the IDO(+) cells were a novel population of CD3(+)CD4(+)IDO(+) cells. The proportion of CD4(+)IDO(+) T cells was significantly higher in patients with moderate GVHD. In situ regulatory T cell and Th17 numbers correlated with overall severity. Although needing confirmatory results from larger sample sets, these data are consistent with the hypothesis that IDO is involved in regulating gastrointestinal GVHD.
Assuntos
Duodeno/enzimologia , Doença Enxerto-Hospedeiro/enzimologia , Indolamina-Pirrol 2,3,-Dioxigenase/biossíntese , Adulto , Duodeno/imunologia , Duodeno/patologia , Células Epiteliais/enzimologia , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Masculino , Pessoa de Meia-Idade , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Células Th17/enzimologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
The angiogenic microenvironment has been known to be a component of angioimmunoblastic T-cell lymphoma since its initial characterization. We have shown that angioimmunoblastic T-cell lymphoma endothelial cells produce vascular endothelial growth factor-A (VEGFA), and participate in lymphoma progression. In squamous cell carcinoma, endothelial BCL2 expression induces a crosstalk with tumor cells through VEGFA, a major mediator of tumoral angiogenesis. In the present study, we analyzed BCL2 and VEGFA in 30 angioimmunoblastic T-cell lymphomas, using triple immunofluorescence to identify protein coexpression in well-characterized lymphoma cells and microenvironment neoangiogenic endothelial cells. Using quantitative real-time PCR, we assessed mRNA expression levels in laser-microdissected endothelial and lymphoma cells. In lymphoma cells, as in endothelial cells, BCL2 and VEGFA proteins were coexpressed. BCL2 was expressed only in neoangiogenic CD34(+)CD105(+) endothelial cells. In laser-microdissected cells, mRNA studies showed a significant relationship between BCL2 and VEGFA levels in CD34(+) endothelial cells, but not in CD3(+)CD10(+)lymphoma cells, or in CD34(+) endothelial cells from lymph node hyperplasia. Further study showed that, in AITL, BCL2 mRNA levels in CD34(+)CD105(+) neoangiogenic endothelial cells also correlated with microvessel density, International Prognostic Index, Ann Arbor stage, bone marrow involvement and elevated LDH. BCL2 expression by CD105(+) neoangiogenic endothelial cells is related to tumor progression in angioimmunoblastic T-cell lymphoma.
Assuntos
Antígenos CD/análise , Biomarcadores Tumorais/análise , Células Endoteliais/química , Linfadenopatia Imunoblástica/metabolismo , Linfonodos/química , Linfoma de Células T/química , Microvasos/química , Proteínas Proto-Oncogênicas c-bcl-2/análise , Receptores de Superfície Celular/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD34/análise , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Progressão da Doença , Intervalo Livre de Doença , Endoglina , Células Endoteliais/imunologia , Células Endoteliais/patologia , Feminino , Imunofluorescência , Humanos , Linfadenopatia Imunoblástica/genética , Linfadenopatia Imunoblástica/imunologia , Linfadenopatia Imunoblástica/mortalidade , Linfadenopatia Imunoblástica/patologia , Linfadenopatia Imunoblástica/terapia , Estimativa de Kaplan-Meier , Microdissecção e Captura a Laser , Linfonodos/irrigação sanguínea , Linfonodos/imunologia , Linfonodos/patologia , Linfoma de Células T/genética , Linfoma de Células T/imunologia , Linfoma de Células T/mortalidade , Linfoma de Células T/patologia , Linfoma de Células T/terapia , Masculino , Microvasos/imunologia , Microvasos/patologia , Pessoa de Meia-Idade , Análise Multivariada , Neovascularização Patológica , Paris , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase em Tempo Real , Medição de Risco , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/análise , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Th17 cells have never been explored in human graft-versus-host disease (GVHD). We studied the correlation between the presence of Th17 cells with histologic and clinical parameters. We first analyzed a cohort of 40 patients with GVHD of the gastrointestinal tract. Tumor necrosis factor (TNF), TNF receptors, and Fas expression, and apoptotic cells, CD4(+)IL-17(+) cells (Th17), and CD4(+)Foxp3(+) cells (Treg) were quantified. A Th17/Treg ratio less than 1 correlated both with the clinical diagnosis (P < .001) and more than 2 pathologic grades (P < .001). A Th17/Treg ratio less than 1 also correlated with the intensity of apoptosis of epithelial cells (P = .03), Fas expression in the cellular infiltrate (P = .003), TNF, and TNF receptor expression (P < .001). We then assessed Th17/Treg ratio in 2 other independent cohorts; a second cohort of 30 patients and confirmed that Th17/Treg ratio less than 1 correlated with the pathologic grade of GI GVHD. Finally, 15 patients with skin GVHD and 11 patients with skin rash but without pathologic GVHD were studied. Results in this third cohort of patients with skin disease confirmed those found in patients with GI GVHD. These analyses in 96 patients suggest that Th17/Treg ratio could be a sensitive and specific pathologic in situ biomarker of GVHD.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Neoplasias Hematológicas/imunologia , Interleucina-17/imunologia , Dermatopatias/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Estudos de Coortes , Feminino , Doença Enxerto-Hospedeiro/metabolismo , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/terapia , Humanos , Interleucina-17/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Dermatopatias/metabolismo , Dermatopatias/terapia , Linfócitos T Reguladores/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In animal models, tissue stem cells were proposed to exhibit an unexpected level of plasticity, although issues on cell fusions have lead to some controversies. Only transplantation experiments using genetically distinct recipients and donors can unequivocally show these changes in cell fate. We have analyzed oral squamous cell carcinomas arising in 8 long-term survivors of allogeneic bone marrow transplantation, in whom chronic graft-versus-host disease greatly favors development of squamous cell carcinomas, possibly as a consequence of lichenoid mucosal inflammation. With the use of 2 independent methods, (1) combined immunostaining and fluorescent in situ hybridization (FISH) analysis for X and Y chromosomes sequences in sex-mismatched grafts and (2) comparison of microsatellite typing of laser-microdissected tumor, donor, and recipient cells, in all tumors, we showed that 4 of these 8 epithelial tumors actually arose from the engrafted allogeneic bone marrow. Thus, donor-derived bone marrow cells, whether hematopoietic or mesenchymal, recruited to sites of chronic mucosal inflammation yielded epithelial tumors. Our observations therefore show that marrow cells in humans have a major role in epithelial cancer formation after allogeneic transplantation.
Assuntos
Transplante de Medula Óssea/efeitos adversos , Carcinoma de Células Escamosas/etiologia , Doença Enxerto-Hospedeiro/complicações , Neoplasias Bucais/etiologia , Quimeras de Transplante , Adulto , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Pré-Escolar , Feminino , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Humanos , Hibridização in Situ Fluorescente , Masculino , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Fatores Sexuais , Doadores de Tecidos , Transplante Homólogo , Adulto JovemRESUMO
Abdominal aortic aneurysms (AAA) are defined by an increased aortic diameter and characterized by impairment of the extracellular matrix, macrophages infiltration and decreased density of smooth muscle cells. Our aim is to identify the key molecules involved in the pathogenesis of AAAs. This study investigated transcriptomic and proteomic profiles of macrophages from AAA patients (>50 mm aortic diameter) (n = 24) and peripheral arterial occlusion (PAO) patients without AAA detected (n = 18), who both needed a surgery. An antibody protein microarray, generated by printing antibodies onto membranes against proteins selected from the transcriptomic and proteomic analysis, was performed to validate the proteins differentially expressed specifically in macrophages and plasma from the same patients. We found a restricted number of proteins differentially expressed between AAA and PAO patients: TIMP-3, ADAMTS5, and ADAMTS8 that differ significantly in plasma of AAA patients compared to PAO patients, as found in the macrophages. In contrast to plasma MMP-9, soluble glycoprotein V (sGPV) and plasmin-antiplasmin complex levels, plasma TIMP-3 levels were not correlated to AAA size but interestingly correlated to sGPV, a platelet activation marker. Combining transcriptomic and proteomic is a valid approach to identify diseases causing proteins and potential biomarkers.
Assuntos
Aneurisma da Aorta Abdominal/metabolismo , Perfilação da Expressão Gênica/métodos , Macrófagos/metabolismo , Análise Serial de Proteínas/métodos , Proteoma/análise , Anticorpos/metabolismo , Biomarcadores , Proteínas Sanguíneas/análise , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Eletroforese em Gel Bidimensional , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Doenças Vasculares Periféricas , Proteínas/análise , Proteínas/genética , Proteínas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Reprodutibilidade dos Testes , Estatísticas não ParamétricasRESUMO
The major long-term prognostic factor for breast cancer patients treated by first-line chemotherapy is response to treatment. We have previously shown that complete responses to high doses epirubicin-cyclophosphamide were observed only in human tumors bearing a TP53 mutation. Three xenografted human breast tumors, 2 of them with a TP53 mutation and one of them without, were studied for their immediate response to this drug association. Cell cycle, cellular senescence and cell death were characterized and quantified on tissue section before and after treatment. The TP53 wild-type tumor showed a strong early induction of senescence-like phenotype with overexpression of SA-beta-gal and p21(CIP1). In contrast both TP53 mutated tumors showed no sign of cell cycle arrest or senescence. Conversely, abnormal mitoses strongly increased, only in TP53 mutated tumors. Thus, in these in vivo models, epirubicin-cyclophosphamide treatment induces senescence-like features in TP53 wild-type tumor, likely accounting for cell cycle arrest and subsequent resistance to treatment. Conversely in TP53 mutated tumors, chemotherapy induces mitotic catastrophe and tumor death, accounting for complete response to this association exclusively in patients with TP53 mutated tumors.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Genes p53 , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Mutação , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ciclo Celular , Senescência Celular , Ciclofosfamida/administração & dosagem , Análise Mutacional de DNA , DNA de Neoplasias , Epirubicina/administração & dosagem , Humanos , Camundongos , Transplante de Neoplasias , Proteína Supressora de Tumor p53/genéticaRESUMO
Endotheliitis is a major sign of graft rejection. Recipient-derived endothelial cells found in two series of liver and kidney transplants were related to graft rejection. Here, we assessed the presence and the number of chimeric endothelial cells in lung transplants, and their relation with graft rejection. In six males grafted with female lungs out of 193 lung transplantations, endothelial chimerism was studied by combined XY-fluorescent in situ hybridization with CD31 and CD45 immunostainings and blood group antigens. On samples graded according to the revised working formulation for lung allograft rejection, we found chimeric macrophages (73.1 to 87.2%) in all cases and chimeric endothelial cells (1.3 to 2.1%) in four patients. Another method using ABO blood group also showed endothelial cells positive for recipient-type blood group antigens in three patients. By both methods, presence of chimeric endothelial cells was related to pathological signs of acute rejection (P<0.05).
Assuntos
Quimerismo , Células Endoteliais/patologia , Rejeição de Enxerto/patologia , Transplante de Pulmão/patologia , Sistema ABO de Grupos Sanguíneos/metabolismo , Idoso , Biópsia , Método Duplo-Cego , Células Endoteliais/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Antígenos Comuns de Leucócito/metabolismo , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Tolerância ao TransplanteRESUMO
In this study, we investigated the in vivo effects of red wine polyphenol compounds (RWPC) in rats that were submitted to middle cerebral occlusion as an experimental model of stroke. Male Wistar rats were given RWPC [30 mg/(kg x d) dissolved in drinking water] or water for 1 wk before being subjected to transient middle cerebral artery occlusion followed by reperfusion. Sham-operated rats were subjected to transient occlusion in which the filament was not completely introduced. The release of amino acids and energy metabolites were monitored by intracerebral microdialysis. The volume of the ischemic lesion was assessed 24 h after reperfusion. Proteomic analysis of brain tissue was performed to study the effects of ischemia and RWPC on specific protein expression. Treatment with RWPC completely prevented the burst of excitatory amino acids that occurred in response to ischemia in untreated rats and significantly reduced brain infarct volumes. Rats chronically treated with RWPC, however, had lower basal concentrations of energy metabolites, including glucose and lactate in the brain parenchyma, compared with untreated rats. Chronic RWPC treatment significantly enhanced the residual cerebral blood flow during occlusion and reperfusion in rats subjected to transient occlusion compared with untreated rats. This effect resulted from arterial vasodilatation, as the internal diameters of several arteries were significantly enlarged after RWPC treatment. Proteomic studies revealed the modulation by RWPC of the expression of proteins involved in the maintenance of neuronal caliber and axon formation, in the protection against oxidative stress, and in energy metabolism. These findings provide an experimental basis for the beneficial effects of RWPC on the neurovascular unit during stroke.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Flavonoides/química , Flavonoides/farmacologia , Fenóis/química , Fenóis/farmacologia , Acidente Vascular Cerebral/tratamento farmacológico , Vinho/análise , Aminoácidos/metabolismo , Animais , Velocidade do Fluxo Sanguíneo , Infarto Encefálico/patologia , Córtex Cerebral/irrigação sanguínea , Córtex Cerebral/metabolismo , Metabolismo Energético/efeitos dos fármacos , Sequestradores de Radicais Livres/metabolismo , Masculino , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Polifenóis , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Digital microscopy enables several observers to look at any field of one microscopical section, at any magnification, through an Internet connexion. An overview of the systems used to digitize microscopy slides and to put them on line is presented. This technique is already used in many fields of pathology, for teaching, research and, to a lesser extent, diagnostic purposes. Several examples are given in this review, some of them with a true evaluation process, and strong points and weaker points are addressed. While conventional microscopy remains the keystone method in 2007 and for the coming years, it is also obvious that digital microscopy will be playing an increasing role. It is our task to make it evolve according to our needs.
Assuntos
Microscopia/métodos , Microscopia/normas , Tecnologia/tendências , Computadores , Humanos , Internet , Patologia/métodos , Patologia/tendências , Telepatologia/métodos , Telepatologia/tendências , Interface Usuário-ComputadorRESUMO
Allogeneic hematopoietic stem cell transplantation is a curative treatment for numerous hematological malignancies. However, acute graft-versus-host disease (aGvHD) is a major complication affecting 40-70% of all transplant patients, whereby the earliest and most frequent presentation is in the skin. MicroRNAs play a role in varied biological process and have been reported as potential biomarkers for aGvHD. More recently, microRNAs have received added attention as circulatory biomarkers that can be detected in biofluids. In this study, we performed global microRNA expression profiling using a discovery cohort of diagnostic cutaneous aGvHD biopsies (n = 5, stages 1-3) and healthy volunteers (n = 4), in order to identify a signature list of microRNAs that could be used as diagnostic biomarkers for cutaneous aGvHD. Candidate microRNAs (n = 8) were then further investigated in a validation cohort of post-HSCT skin biopsies (n = 17), pre-HSCT skin biopsies (n = 6) and normal controls (n = 6) for their association with aGvHD. Expression of let-7c (p = 0.014), miR-503-5p (p = 0.003), miR-365a-3p (p = 0.02), miR-34a-5p (p < 0.001) and miR-34a-3p (p = 0.006) were significantly differentially expressed between groups and significantly associated with survival outcome in post-HSCT patients (miR-503-5p ROC AUC = 0.83 p = 0.021, Log Rank p = 0.003; miR-34a-3p ROC AUC = 0.93, p = 0.003, Log Rank p = 0.004). There was no association with relapse. A statistical interaction between miR-34a-3p and miR-503-5p (p = 0.016) was diagnostic for aGvHD. Expression levels of the miR-34a-5p protein target p53 were assessed in the epidermis of the skin, and an inverse correlation was identified (r2 = 0.44, p = 0.039). Expression of the validated candidate microRNAs was also assessed at day 28 post-HSCT in the sera of transplant recipients, in order to investigate their potential as circulatory microRNA biomarkers. Expression of miR-503-5p (p = 0.001), miR-34a-5p (p = 0.005), and miR-34a-3p (p = 0.004) was significantly elevated in the sera of patients who developed aGvHD versus no-aGvHD (n = 30) and miR-503-5p was associated with overall survival (OS) (ROC AUC = 0.80, p = 0.04, Log Rank p = 0.041). In conclusion, this investigation reports that microRNA expression levels in clinical skin biopsies, obtained at the time of cutaneous aGvHD onset, show potential as diagnostic biomarkers for aGvHD and as predictive biomarkers for OS. In addition, the same microRNAs can be detected in the circulation and show predictive association with post-HSCT outcomes.
RESUMO
BACKGROUND: Primary mediastinal large B-cell lymphoma (PMBL) shares pathological features with diffuse large B-cell lymphoma (DLBCL), and molecular features with classical Hodgkin lymphoma (cHL). The miR-17~92 oncogenic cluster, located at chromosome 13q31, is a region that is amplified in DLBCL. METHODS: Here we compared the expression of each member of the miR-17~92 oncogenic cluster in samples from 40 PMBL patients versus 20 DLBCL and 20 cHL patients, and studied the target genes linked to deregulated miRNA in PMBL. RESULTS: We found a higher level of miR-92a in PMBL than in DLBCL, but not in cHL. A combination of in silico prediction and transcriptomic analyses enabled us to identify FOXP1 as a main miR-92a target gene in PMBL, a result so far not established. This was confirmed by 3'UTR, and RNA and protein expressions in transduced cell lines. In vivo studies using the transduced cell lines in mice enabled us to demonstrate a tumor suppressor effect of miR-92a and an oncogenic effect of FOXP1.A higher expression of miR-92a and the down-regulation of FOXP1 mRNA and protein expression were also found in human samples of PMBL, while miR-92a expression was low and FOXP1 was high in DLBCL. CONCLUSIONS: We concluded to a post-transcriptional regulation by miR-92a through FOXP1 targeting in PMBL, with a clinico-pathological relevance for better characterisation of PMBL.
Assuntos
Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Linfoma Difuso de Grandes Células B/genética , Neoplasias do Mediastino/genética , MicroRNAs/genética , Proteínas Repressoras/genética , Regiões 3' não Traduzidas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Masculino , Neoplasias do Mediastino/metabolismo , Neoplasias do Mediastino/patologia , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Adulto JovemRESUMO
There is growing evidence for the role of cancer stem-cells in drug resistance, but with few in situ studies on human tumor samples to decipher the mechanisms by which they resist anticancer agents.Triple negative breast cancer (TNBC) is the most severe sub-type of breast cancer, occurring in younger women and associated with poor prognosis even when treated at a localized stage.We investigated here the relationship between complete pathological response after chemotherapy and breast cancer stem-cell characteristics in pre-treatment biopsies of 78 women with triple negative breast carcinoma (TNBC).We found that chemoresistance was associated with large numbers of breast cancer stem-cells, and that these cancer stem-cells were neither proliferative nor apoptotic, but in an autophagic state related to hypoxia. Using relevant pharmacological models of patient-derived TNBC xenografts, we further investigated the role of autophagy in chemoresistance of breast cancer stem-cells. We demonstrated that hypoxia increased drug resistance of autophagic TNBC stem-cells, and showed that molecular or chemical inhibition of autophagic pathway was able to reverse chemoresistance.Our results support breast cancer stem-cell evaluation in pre-treatment biopsies of TNBC patients, and the need for further research on autophagy inhibition to reverse resistance to chemotherapy.
Assuntos
Antineoplásicos/administração & dosagem , Autofagia/efeitos dos fármacos , Cloroquina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cloroquina/farmacologia , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Gradação de Tumores , Estadiamento de Neoplasias , Células-Tronco Neoplásicas/efeitos dos fármacos , Estudos Prospectivos , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/cirurgia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: An emerging concept is that a neuronal isoform of nitric oxide synthase (NOS1) may regulate myocardial contractility. However, a role for NOS1-derived nitric oxide (NO) in heart failure (HF) has not been defined. METHODS AND RESULTS: Using a model of myocardial infarction-induced HF, we demonstrated that cardiac NOS1 expression and activity increased in HF rats (P<0.05 and P<0.001 versus shams, respectively). This was associated with translocation of NOS1 from the ryanodine receptor to the sarcolemma through interactions with caveolin-3 in HF hearts. With ex vivo and in vivo pressure-volume analysis, cardiac NOS1-derived NO was found to be negatively inotropic in shams but not HF hearts. Ventricular elastance (E(es)) was significantly reduced in HF rats (P<0.05), and tau, the time constant of left ventricular relaxation, was prolonged (both P<0.05). Acute NOS1 inhibition significantly increased E(es) by 33+/-3% and tau by 17+/-2% (P<0.05) in shams, although these effects were significantly attenuated in HF hearts. beta-Adrenergic stimulation induced a marked increase in systolic performance in sham hearts, with the responses being significantly blunted in HF hearts. E(es) increased by 163+/-42% (P<0.01) in sham hearts and 56+/-9% in HF hearts, and LV +dP/dt increased by 97+/-9% (P<0.01) in shams and 37+/-7% (P<0.05) in the HF group. Interestingly, preferential NOS1 inhibition enhanced the blunted responses of LV +dP/dt and E(es) to beta-adrenergic stimulation in HF rats but had no effect in shams. CONCLUSIONS: These results provide the first evidence that increased NOS1-derived NO production may play a role in the autocrine regulation of myocardial contractility in HF.
Assuntos
Insuficiência Cardíaca/enzimologia , Contração Miocárdica/fisiologia , Infarto do Miocárdio/complicações , Miocárdio/enzimologia , Proteínas do Tecido Nervoso/fisiologia , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico/fisiologia , Receptores Adrenérgicos beta/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Comunicação Autócrina , Caveolina 3 , Caveolinas/metabolismo , Dobutamina/farmacologia , Indução Enzimática , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/etiologia , Isoproterenol/farmacologia , Masculino , Contração Miocárdica/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I , Ratos , Ratos Wistar , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sarcolema/metabolismo , Disfunção Ventricular Esquerda/enzimologia , Disfunção Ventricular Esquerda/etiologiaRESUMO
Experimental data suggest that nitric oxide (NO) generated from neuronal NO synthase (nNOS) modulates the myocardial inotropic state. To assess the contribution of NO, derived from endothelial and neuronal isoforms, to the pathophysiology of congestive heart failure in human beings, we compared expression, localisation, and specific activity of NOS isoforms in myocardium from patients with dilated cardiomyopathy with those in controls who had died from head trauma or intracranial bleeds. Diseased hearts had a significant increase in nNOS mRNA and protein expression, and activity associated with the translocation of nNOS to the sarcolemma through interactions with caveolin 3. Enhanced nNOS activity counteracted a decrease in eNOS expression and activity. Our results provide evidence of increased nNOS-derived NO in the failing human heart. Such altered regulation may be important in the pathophysiology of cardiac dysfunction in human congestive heart failure.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Miocárdio/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/metabolismo , Western Blotting , Cálcio/metabolismo , Caveolina 3 , Caveolinas/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismoRESUMO
OBJECTIVES: This study was designed to investigate whether nicotinamide adenine dinucleotide 3-phosphate (reduced form) (NADPH) oxidase is expressed in the human heart and whether it contributes to reactive oxygen species (ROS) production in heart failure. BACKGROUND: A phagocyte-type NADPH oxidase complex is a major source of ROS in the vasculature and is implicated in the pathophysiology of hypertension and atherosclerosis. An increase in myocardial oxidative stress due to excessive production of ROS may be involved in the pathophysiology of congestive heart failure. Recent studies have suggested an important role for myocardial NADPH oxidase in experimental models of cardiac disease. However, it is unknown whether NADPH oxidase is expressed in the human myocardium or if it has any role in human heart failure. METHODS: Myocardium of explanted nonfailing (n = 9) and end-stage failing (n = 13) hearts was studied for the expression of NADPH oxidase subunits and oxidase activity. RESULTS: The NADPH oxidase subunits p22(phox), gp91(phox), p67(phox), and p47(phox) were all expressed at messenger ribonucleic acid and protein level in cardiomyocytes of both nonfailing and failing hearts. NADPH oxidase activity was significantly increased in end-stage failing versus nonfailing myocardium (5.86 +/- 0.41 vs. 3.72 +/- 0.39 arbitrary units; p < 0.01). The overall level of oxidase subunit expression was unaltered in failing compared with nonfailing hearts. However, there was increased translocation of the regulatory subunit, p47(phox), to myocyte membranes in failing myocardium. CONCLUSIONS: This is the first report of the presence of NADPH oxidase in human myocardium. The increase in NADPH oxidase activity in the failing heart may be important in the pathophysiology of cardiac dysfunction by contributing to increased oxidative stress.
Assuntos
Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/genética , Miocárdio/enzimologia , Miocárdio/patologia , NADP/análise , NADP/genética , Adulto , Expressão Gênica/genética , Expressão Gênica/fisiologia , Insuficiência Cardíaca/patologia , Humanos , Técnicas In Vitro , Medições Luminescentes , Pessoa de Meia-Idade , NADP/fisiologia , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Nitric oxide (NO) has been implicated in the development of heart failure, although the source, significance, and functional role of the different NO synthase (NOS) isoforms in this pathology are controversial. The presence of a neuronal-type NOS isoform (NOS1) in the cardiac sarcoplasmic reticulum has been recently discovered, leading to the hypothesis that NOS1-derived NO may notably alter myocardial inotropy. However, the regulation and role(s) of NOS1 in cardiac diseases remain to be determined. Using an experimental model of myocardial infarction (MI) in senescent rats, we demonstrated a significant increase in cardiac NOS1 expression and activity in MI, coupled with the translocation of this enzyme to the sarcolemma through interactions with caveolin-3. The enhanced NOS1 activity counteracts the decrease in cardiac NOS3 expression and activity observed in heart failure. We demonstrated an increased interaction between NOS1 and its regulatory protein HSP90 in post-MI hearts, a potential mechanism for the higher NOS1 activity in this setting. Finally, preferential in vivo inhibition of NOS1 activity enhanced basal post-MI left ventricular dysfunction in senescent rats. These results provide the first evidence that increased NOS1-derived NO production may play a significant role in the autocrine regulation of myocardial contractility after MI in aging rats.
Assuntos
Infarto do Miocárdio/enzimologia , Miocárdio/enzimologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/biossíntese , Envelhecimento , Animais , Proteínas de Choque Térmico HSP90/fisiologia , Modelos Cardiovasculares , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Óxido Nítrico Sintase Tipo I , Transporte Proteico , Ratos , Sarcolema/enzimologia , Regulação para Cima , Disfunção Ventricular Esquerda/etiologiaRESUMO
Caveolins modulate signaling pathways involved in cardiac development. Caveolin-1 exists in two isoforms: the beta-isoform derivates from an alternative translational start site that creates a protein truncated by 31 amino acids, mainly expressed in endothelial cells, whereas caveolin-3 is present in muscle cells. Our aim was to define caveolin distribution and expression during cardiac postnatal development using immunofluorescence and Western blotting. Caveolin-3 sarcolemmal labeling appeared as dotted lines from days 1 to 5 and as continuous lines after 14 days of age. Caveolin-3 expression, low at birth, increased (4-fold) to reach a maximum (P < 0.05) by day 5 and then decreased to stabilize in adults. Total caveolin-1 and its alpha-isoform were codistributed at birth in endothelial and smooth muscle cells; afterward, only the caveolin-1alpha labeling became limited to endothelium. Quantitative analysis indicated a similar temporal pattern of both total caveolin-1 and caveolin-1alpha expression, suggesting that caveolin-1alpha and -1beta are coregulated; the caveolin-1alpha levels increased fourfold by day 5 to reach a maximum by day 14 (P < 0.05). Tyrosine-14-caveolin-1 phosphorylation, low at birth, increased suddenly around day 14 (8-fold vs. day 1) and returning afterward to basal level. Because the T3/T4 level is maximal by day 14, caveolin-1 expression/phosphorylation profiles were analyzed in hypothyroid heart. The levels of caveolin-1alpha and consequently tyrosine-14-caveolin-1 phosphorylation, but not that of caveolin-3, decreased (50%) in hypothyroid 14-day-old rats. Our data demonstrate that, during postnatal cardiac growth, 1) caveolins are distinctly regulated, and 2) thyroid hormones are involved in caveolin-1alpha expression.