RESUMEN
CD49d, the α4 chain of the VLA-4 integrin, is a negative prognosticator in chronic lymphocytic leukemia (CLL) with a key role in CLL cell-microenvironment interactions mainly occurring via its ligands VCAM-1 and fibronectin. In the present study, we focused on EMILIN-1 (Elastin-MIcrofibriL-INterfacer-1), an alternative VLA-4 ligand whose role has been so far reported only in non-hematological settings, by investigating: i) the distribution of EMILIN-1 in CLL-involved tissues; ii) the capability of EMILIN-1 to operate, via its globular C1q (gC1q) domain, as additional adhesion ligand in CLL; iii) the functional meaning of EMILIN-1 gC1q/VLA-4 interactions in CLL. EMILIN-1 is widely present in the CLL-involved areas of bone marrow biopsies (BMBs) without difference between CD49d negative and positive cases, displaying at least three different expression patterns: "fibrillar", "dot-like" and "mixed". The lack in CLL-BMB of neutrophil elastase, whose proteolytic activity degrades EMILIN-1 and impairs EMILIN-1 function, suggests full functional EMILIN-1 in CLL independently of its expression pattern. Functionally, EMILIN-1 gC1q domain promotes adhesion of CLL cells through specific interaction with VLA-4, and releases pro-survival signals for CLL cells, as demonstrated by enhanced ERK and AKT phosphorylation and impairment of in-vitro-induced apoptosis. EMILIN-1/VLA-4 interaction can efficiently contribute to the maintenance of the neoplastic clone in CLL.
Asunto(s)
Leucemia Linfocítica Crónica de Células B , Elastina , Humanos , Integrina alfa4beta1/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Ligandos , Glicoproteínas de Membrana , Microfibrillas/metabolismo , Microfibrillas/patología , Microambiente TumoralRESUMEN
A high platelet count is associated with a poor prognosis in ovarian cancer (OvCa). Despite good clinical responses with platinating agents in combination with taxanes, numerous OvCa patients relapse due to chemotherapy resistance. Here, we report that treatment of OvCa cells A2780, OVCAR5 and MDAH with releasate from activated platelets (PR) promoted multicellular tumor spheroid (MCTS) formation. These OvCa-MCTSs had increased percentages of CD133+ and aldehyde dehydrogenase (ALDH)+ cells, bona fide markers of OvCa cancer stem cells (CSCs). PR increased OVCAR5- and MDAH-MCTS viability and decreased the cytotoxic and pro-apoptotic effects of paclitaxel, cisplatin and carboplatin. PR increased the volume of spontaneously formed OVCAR8-MCTSs and counteracted their size reduction due to cisplatin, carboplatin and paclitaxel treatment. PR promoted the survival of ALDH+ and CD133+ OvCa cells during cisplatin, carboplatin and paclitaxel treatment. In conclusion, molecules and growth factors released by activated platelets (EGF, PDGF, TGF-ß, IGF and CCL5) may protect tumor cells from chemotherapy by promoting the expansion of ALDH+ and CD133+ OvCa-CSCs, favoring drug resistance and tumor relapse.
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Antígeno AC133/metabolismo , Aldehído Deshidrogenasa/metabolismo , Antineoplásicos/farmacología , Plaquetas/metabolismo , Citoprotección/efectos de los fármacos , Células Madre Neoplásicas/patología , Neoplasias Ováricas/patología , Esferoides Celulares/patología , Apoptosis/efectos de los fármacos , Plaquetas/efectos de los fármacos , Carboplatino/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Femenino , Humanos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Paclitaxel/farmacología , Esferoides Celulares/efectos de los fármacosRESUMEN
Elastin microfibril interface-located proteins (EMILINs) are extracellular matrix glycoproteins implicated in elastogenesis and cell proliferation. Recently, a missense mutation in the EMILIN1 gene has been associated with autosomal dominant connective tissue disorder and motor-sensory neuropathy in a single family. We identified by whole exome sequencing a novel heterozygous EMILIN1 mutation c.748C>T [p.R250C] located in the coiled coil forming region of the protein, in four affected members of an autosomal dominant family presenting a distal motor neuropathy phenotype. In affected patient a sensory nerve biopsy showed slight and unspecific changes in the number and morphology of myelinated fibers. Immunofluorescence study of a motor nerve within a muscle biopsy documented the presence of EMILIN-1 in nerve structures. Skin section and skin derived fibroblasts displayed a reduced extracellular deposition of EMILIN-1 protein with a disorganized network of poorly ramified fibers in comparison with controls. Downregulation of emilin1a in zebrafish displayed developmental delay, locomotion defects, and abnormal axonal arborization from spinal cord motor neurons. The phenotype was complemented by wild-type zebrafish emilin1a, and partially the human wild-type EMILIN1 cRNA, but not by the cRNA harboring the novel c.748C>T [p.R250C]. These data suggest a role of EMILIN-1 in the pathogenesis of diseases affecting the peripheral nervous system.
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Fibroblastos/patología , Glicoproteínas de Membrana/genética , Mutación/genética , Piel/patología , Adolescente , Animales , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Adulto Joven , Pez CebraRESUMEN
Classic Hodgkin lymphoma tumor cells express a functional CCR5 receptor, and tumor tissues express high CCL5 levels, suggesting that CCL5-CCR5 signaling is involved in tumor-microenvironment formation and tumor growth. Using the CCR5 antagonist, maraviroc, and a neutralizing anti-CCL5 antibody, we found that CCL5 secreted by classic Hodgkin lymphoma cells recruited mesenchymal stromal cells and monocytes. The "education" of mesenchymal stromal cells by tumor cell-conditioned medium enhanced mesenchymal stromal cells' proliferation and CCL5 secretion. In turn, educated mesenchymal stromal cell-conditioned medium increased the clonogenic growth of tumor cells and monocyte migration, but these effects were reduced by maraviroc. Monocyte education by tumor cell-conditioned medium induced their growth and reprogrammed them towards immunosuppressive tumor-associated macrophages that expressed IDO and PD-L1 and secreted IL-10, CCL17 and TGF-ß. Educated monocyte-conditioned medium slowed the growth of phytohemagglutinin-activated lymphocytes. Maraviroc decreased tumor cell growth and synergized with doxorubicin and brentuximab vedotin. A three-dimensional heterospheroid assay showed that maraviroc counteracted both the formation and viability of heterospheroids generated by co-cultivation of tumor cells with mesenchymal stromal cells and monocytes. In mice bearing tumor cell xenografts, maraviroc reduced tumor growth by more than 50% and inhibited monocyte accumulation, without weight loss. Finally, in classic Hodgkin lymphoma human tumor tissues, CCL5 and CD68 expression correlated positively, and patients with high CCL5 levels had poor prognosis. In conclusion, since the present challenges are to find molecules counteracting the formation of the immunosuppressive tumor microenvironment or new, less toxic drug combinations, the repurposed drug maraviroc may represent a new opportunity for classic Hodgkin lym phoma treatment.
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Antagonistas de los Receptores CCR5/farmacología , Enfermedad de Hodgkin/metabolismo , Enfermedad de Hodgkin/patología , Maraviroc/farmacología , Receptores CCR5/metabolismo , Microambiente Tumoral/efectos de los fármacos , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Reprogramación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Doxorrubicina/farmacología , Sinergismo Farmacológico , Enfermedad de Hodgkin/tratamiento farmacológico , Humanos , Ratones , Modelos Biológicos , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gastric cancer is a deadly tumor and a relatively common disease worldwide. Surgical resection and chemotherapy are the main clinical options to treat this type of disease, however the median overall survival rate is limited to one year. Thus, the development of new therapies is a highly necessary clinical need. Angiogenesis is a promising target for this tumor type, however clinical trials with the use of anti-angiogenic drugs have so far not met expectations. Therefore, it is important to better characterize the expression of molecules whose expression levels may impact on the efficacy of the treatments. In this study the characteristics of the gastric tumor associated blood vessels were first assessed by endomicroscopy. Next, we analyzed the expression of Multimerin-2, EMILIN-2 and EMILIN-1, three molecules of the EMI Domain ENdowed (EDEN) protein family. These molecules play important functions in the tumor microenvironment, affecting cancer progression both directly and indirectly impinging on angiogenesis and lymphangiogenesis. All the molecules were highly expressed in the normal mucosa whereas in a number of patients their expression was altered. We consider that better characterizing the gastric tumor microenvironment and the quality of the vasculature may achieve effective patient tailored therapies.
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Antígenos de Superficie/metabolismo , Glicoproteínas/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Glicoproteínas de Membrana/metabolismo , Neovascularización Patológica/metabolismo , Neoplasias Gástricas/metabolismo , Antígenos de Superficie/genética , Técnica del Anticuerpo Fluorescente , Glicoproteínas/genética , Humanos , Glicoproteínas de Membrana/genética , Neovascularización Patológica/genética , Neoplasias Gástricas/genéticaRESUMEN
Heritable connective tissue diseases are a highly heterogeneous family of over 200 disorders that affect the extracellular matrix. While the genetic basis of several disorders is established, the etiology has not been discovered for a large portion of patients, likely due to rare yet undiscovered disease genes. By performing trio-exome sequencing of a 55-year-old male proband presenting with multiple symptoms indicative of a connective disorder, we identified a heterozygous missense alteration in exon 1 of the Elastin Microfibril Interfacer 1 (EMILIN1) gene, c.64G>A (p.A22T). The proband presented with ascending and descending aortic aneurysms, bilateral lower leg and foot sensorimotor peripheral neuropathy, arthropathy, and increased skin elasticity. Sanger sequencing confirmed that the EMILIN1 alteration, which maps around the signal peptide cleavage site, segregated with disease in the affected proband, mother, and son. The impaired secretion of EMILIN-1 in cells transfected with the mutant p.A22T coincided with abnormal protein accumulation within the endoplasmic reticulum. In skin biopsy of the proband, we detected less EMILIN-1 with disorganized and abnormal coarse fibrils, aggregated deposits underneath the epidermis basal lamina, and dermal cells apoptosis. These findings collectively suggest that EMILIN1 may represent a new disease gene associated with an autosomal-dominant connective tissue disorder.
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Enfermedades del Tejido Conjuntivo/diagnóstico , Enfermedades del Tejido Conjuntivo/genética , Exoma , Genes Dominantes , Secuenciación de Nucleótidos de Alto Rendimiento , Glicoproteínas de Membrana/genética , Secuencia de Aminoácidos , Animales , Biopsia , Línea Celular , Análisis por Conglomerados , Biología Computacional/métodos , Análisis Mutacional de ADN , Femenino , Expresión Génica , Humanos , Imagen por Resonancia Magnética , Masculino , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Mutación , Linaje , Fenotipo , Alineación de Secuencia , Piel/patologíaRESUMEN
Lymphatic vasculature critically depends on the connections of lymphatic endothelial cells with the extracellular matrix (ECM), which are mediated by anchoring filaments (AFs). The ECM protein EMILIN1 is a component of AFs and is involved in the regulation of lymphatic vessel functions: accordingly, Emilin1(-/-) mice display lymphatic vascular morphological alterations, leading to functional defects such as mild lymphoedema, lymph leakage and compromised lymph drainage. In the present study, using a mouse post-surgical tail lymphoedema model, we show that the acute phase of acquired lymphoedema correlates with EMILIN1 degradation due to neutrophil elastase (NE) released by infiltrating neutrophils. As a consequence, the intercellular junctions of lymphatic endothelial cells are weakened and drainage to regional lymph nodes is severely affected. The local administration of sivelestat, a specific NE inhibitor, prevents EMILIN1 degradation and reduces lymphoedema, restoring a normal lymphatic functionality. The finding that, in human secondary lymphoedema samples, we also detected cleaved EMILIN1 with the typical bands of an NE-dependent pattern of fragmentation establishes a rationale for a powerful strategy that targets NE inhibition. In conclusion, the attempts to block EMILIN1 degradation locally represent the basis for a novel 'ECM' pharmacological approach to assessing new lymphoedema treatments.
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Vasos Linfáticos/fisiología , Linfedema/tratamiento farmacológico , Glicoproteínas de Membrana/fisiología , Proteínas Inhibidoras de Proteinasas Secretoras/farmacología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/fisiología , Femenino , Humanos , Vasos Linfáticos/efectos de los fármacos , Linfedema/metabolismo , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila , Proteínas Inhibidoras de Proteinasas Secretoras/uso terapéuticoRESUMEN
EMILIN2 is an extracellular matrix (ECM) protein that exerts contradictory effects within the tumour microenvironment: it induces apoptosis in a number of tumour cells, but it also enhances tumour neo-angiogenesis. In this study, we describe a new mechanism by which EMILIN2 attenuates tumour cell viability. Based on sequence homology with the cysteine-rich domain (CRD) of the Frizzled receptors, we hypothesized that EMILIN2 could affect Wnt signalling activation and demonstrate direct interaction with the Wnt1 ligand. This physical binding leads to decreased LRP6 phosphorylation and to the down-modulation of ß-catenin, TAZ and their target genes. As a consequence, EMILIN2 negatively affects the viability, migration and tumourigenic potential of MDA-MB-231 breast cancer cells in a number of two- and three-dimensional in vitro assays. EMILIN2 does not modulate Wnt signalling downstream of the Wnt-Frizzled interaction, since it does not affect the activation of the pathway following treatment with the GSK3 inhibitors LiCl and CHIR99021. The interaction with Wnt1 and the subsequent biological effects require the presence of the EMI domain, as there is no effect with a deletion mutant lacking this domain. Moreover, in vivo experiments show that the ectopic expression of EMILIN2, as well as treatment with the recombinant protein, significantly reduce tumour growth and dissemination of cancer cells in nude mice. Accordingly, the tumour samples are characterized by a significant down-regulation of the Wnt signalling pathway. Altogether, these findings provide further evidence of the complex regulations governed by EMILIN2 in the tumour microenvironment, and they identify a key extracellular regulator of the Wnt signalling pathway.
Asunto(s)
Neoplasias de la Mama/metabolismo , Movimiento Celular , Proliferación Celular , Glicoproteínas/metabolismo , Vía de Señalización Wnt , Proteína Wnt1/metabolismo , Aciltransferasas , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular , Regulación hacia Abajo , Femenino , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/genética , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Ratones Desnudos , Mutación , Invasividad Neoplásica , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Factores de Tiempo , Factores de Transcripción/metabolismo , Transfección , Carga Tumoral , Microambiente Tumoral , Proteína Wnt1/genética , beta Catenina/metabolismoRESUMEN
In this contribution, we investigated whether surface-enhanced Raman scattering (SERS) of serum can be a candidate method for detecting "luminal A" breast cancer (BC) at different stages. We selected three groups of participants aged over 50 years: 20 healthy women, 20 women with early localized small BC, and 20 women affected by BC with lymph node involvement. SERS revealed clear spectral differences between these three groups. A predictive model using principal component analysis (PCA) and linear discriminant analysis (LDA) was developed based on spectral data, and its performance was estimated with cross-validation. PCA-LDA of SERS spectra could distinguish healthy from BC subjects (sensitivity, 92 %; specificity, 85 %), as well as subjects with BC at different stages, with a promising diagnostic performance (sensitivity and specificity, ≥80 %; overall accuracy, 84 %). Our data suggest that SERS spectroscopy of serum, combined with multivariate data analysis, represents a minimally invasive, easy to use, and fast approach to discriminate healthy from BC subjects and even to distinguish BC at different clinical stages.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/sangre , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Humanos , Persona de Mediana EdadRESUMEN
Surface-enhanced Raman scattering (SERS) spectra were obtained from urine samples from subjects diagnosed with prostate cancer as well as from healthy controls, using Au nanoparticles as substrates. Principal component analysis (PCA) of the spectral data, followed by linear discriminant analysis (LDA), leads to a classification model with a sensitivity of 100 %, a specificity of 89 %, and an overall diagnostic accuracy of 95 %. Even considering the very limited number of samples involved in this report, preliminary results from this approach are extremely promising, encouraging further investigation.
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Neoplasias de la Próstata/orina , Espectrometría Raman/métodos , Anciano , Estudios de Casos y Controles , Análisis Discriminante , Oro/química , Humanos , Hipoxantina/orina , Masculino , Persona de Mediana Edad , Nanopartículas/química , Análisis de Componente Principal , Neoplasias de la Próstata/diagnóstico , Sensibilidad y EspecificidadRESUMEN
Surface-enhanced Raman spectroscopy (SERS) is a good candidate for the development of fast and easy-to-use diagnostic tools, possibly used on biofluids in point-of-care or screening tests. In particular, label-free SERS spectra of blood serum and plasma, two biofluids widely used in diagnostics, could be used as a metabolic fingerprinting approach for biomarker discovery. This study aims at a systematic evaluation of SERS spectra of blood serum and plasma, using various Ag and Au aqueous colloids, as SERS substrates, in combination with three excitation lasers of different wavelengths, ranging from the visible to the near-infrared. The analysis of the SERS spectra collected from 20 healthy subjects under a variety of experimental conditions revealed that intense and repeatable spectra are quickly obtained only if proteins are filtered out from samples, and an excitation in the near-infrared is used in combination with Ag colloids. Moreover, common plasma anticoagulants such as EDTA and citrate are found to interfere with SERS spectra; accordingly, filtered serum or heparin plasma are the samples of choice, having identical SERS spectra. Most bands observed in SERS spectra of these biofluids are assigned to uric acid, a metabolite whose blood concentration depends on factors such as sex, age, therapeutic treatments, and various pathological conditions, suggesting that, even when the right experimental conditions are chosen, great care must be taken in designing studies with the purpose of developing diagnostic tests.
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Análisis Químico de la Sangre , Plasma/química , Espectrometría Raman/métodos , Adulto , Factores de Edad , Femenino , Oro/química , Humanos , Masculino , Nanopartículas del Metal/química , Plata/química , Espectrometría Raman/instrumentación , Adulto JovenRESUMEN
Until recently, inflammatory chemokines were viewed mainly as indispensable "gate keepers" of immunity and inflammation. However, updated research indicates that cancer cells subvert the normal chemokine system and these molecules and their receptors become important constituents of the tumor microenvironment with very different ways to exert tumor-promoting roles. The CCR5 and the CCL5 ligand have been detected in some hematological malignancies, lymphomas, and a great number of solid tumors, but extensive studies on the role of the CCL5/CCR axis were performed only in a limited number of cancers. This review summarizes updated information on the role of CCL5 and its receptor CCR5 in cancer cell proliferation, metastasis, and the formation of an immunosuppressive microenvironment and highlights the development of newer therapeutic strategies aimed to inhibit the binding of CCL5 to CCR5, to inhibit CCL5 secretion, or to inhibit the interactions among tumor cells and the microenvironment leading to CCL5 secretion.
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Quimiocina CCL5/fisiología , Regulación Neoplásica de la Expresión Génica , Neoplasias/inmunología , Neoplasias/terapia , Receptores CCR5/fisiología , Animales , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , Femenino , Humanos , Inmunosupresores , Inflamación , Masculino , Metástasis de la Neoplasia , Neoplasias/metabolismo , Microambiente TumoralRESUMEN
Increasing evidence suggests that bone marrow derived mesenchymal stem cells (BM-MSCs) are recruited into the stroma of developing tumors where they contribute to progression by enhancing tumor growth and metastasis, or by inducing anticancer-drug resistance. Prostate cancer cells secrete ligands of epidermal growth factor receptor (EGFR) and EGFR signaling could play an important role in the cross-talk between mesenchymal stem cells and prostate cancer cells. In this study, we showed that treatment of human primary MSCs with conditioned medium (CM) derived from the bone metastatic PC3 carcinoma cells (PC3-CM) resulted in: a significant activation of EGFR; increased proliferation; increased osteoblastic but decreased adipocitic differentiation; inhibition of senescence induced by serum starvation; increased CCL5 secretion. These activities were significantly inhibited in the presence of the EGFR tyrosine kinase inhibitor gefitinib. PC3-CM directly inhibited osteoclastogenesis as well as the ability of osteoblasts to induce osteoclast differentiation. The increased MSCs migration by PC3-CM and PC3 cells was partially mediated by CCL5. MSC-CM increased the formation of colonies by PC3 cells and inhibited the anti-proliferative activity of Docetaxel. Activation of EGFR expressed on MSCs by PC3-CM enhanced their capability to increase PC3 cells proliferation and to inhibit Docetaxel activity. These findings, by showing that the tumor-promoting interactions between PC3 cells and MSCs are mediated, at least in part, by EGFR, suggest a novel application of the EGFR-tyrosine kinase inhibitors in the treatment of prostate cancer.
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Comunicación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/patología , Neoplasias de la Próstata/patología , Quinazolinas/farmacología , Taxoides/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Quimiocina CCL5/metabolismo , Quimiotaxis/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Docetaxel , Receptores ErbB/metabolismo , Gefitinib , Humanos , Masculino , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Osteoblastos/patología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoclastos/patología , Osteogénesis/efectos de los fármacos , Fosforilación/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
Sprouting of angiogenic perivascular cells is thought to be highly dependent upon autocrine and paracrine growth factor stimulation. Accordingly, we report that corneal angiogenesis induced by ectopic FGF implantation is strongly impaired in NG2/CSPG4 proteoglycan (PG) null mice known to harbour a putative deficit in pericyte proliferation/mobilization. Conversely, no significant differences were seen between wild type and knockout corneas when VEGF was used as an angiocrine factor. Perturbed responsiveness of NG2-deficient pericytes to paracrine and autocrine stimulation by several FGFs could be confirmed in cells isolated from NG2 null mice, while proliferation induced by other growth factors was equivalent in wild type and knockout cells. Identical results were obtained after siRNA-mediated knock-down of NG2 in human smooth muscle-like cell lines, as also demonstrated by the decreased levels of FGF receptor phosphorylation detected in these NG2 deprived cells. Binding assays with recombinant proteins and molecular interactions examined on live cells asserted that FGF-2 bound to NG2 in a glycosaminoglycan-independent, core protein-mediated manner and that the PG was alone capable of retaining FGF-2 on the cell membrane for subsequent receptor presentation. The use of dominant-negative mutant cells, engineered by combined transduction of NG2 deletion constructs and siRNA knock-down of the endogenous PG, allowed us to establish that the FGF co-receptor activity of NG2 is entirely mediated by its extracellular portion. In fact, forced overexpression of the NG2 ectodomain in human smooth muscle-like cells increased their FGF-2-induced mitosis and compensated for low levels of FGF receptor surface expression, in a manner equivalent to that produced by overexpression of the full-length NG2. Upon FGF binding, the cytoplasmic domain of NG2 is phosphorylated, but there is no evidence that this event elicits signal transductions that could bypass the FGFR-mediated ones. Pull-down experiments, protein-protein binding assays and flow cytometry FRET coherently revealed an elective ligand-independent association of NG2 with FGFR1 and FGFR3. The NG2 cooperation with these receptors was also corroborated functionally by the outcome of FGF-2 treatments of cells engineered to express diverse NG2/FGFR combinations. Comprehensively, the findings suggest that perivascular NG2 may serve as a dual modulator of the availability/accessibility of FGF at the cell membrane, as well as the resulting FGFR transducing activity.
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Antígenos/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Mitógenos/metabolismo , Pericitos/metabolismo , Proteoglicanos/metabolismo , Animales , Córnea/irrigación sanguínea , Transferencia Resonante de Energía de Fluorescencia , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de SeñalRESUMEN
OBJECTIVE: Cisplatin-based chemotherapy has been shown to improve survival in cervical cancer; however, treatment is associated with tumor resistance and significant toxicity. Lipoplatin is a new liposomal formulation of cisplatin, developed to reduce cisplatin toxicity, to improve drug accumulation at tumor sites and to overcome drug resistance. The aim of this study is to analyze the antitumoral activity of lipoplatin against cisplatin-resistant cervical cancer cells and to investigate its mechanism of action. METHODS: The activity and mechanism of action of lipoplatin were studied in the ME-180 cervical cancer cell line and its cisplatin-resistant clone R-ME-180 and HeLa cells using cell proliferation assays, flow cytometry, ELISA assay, cell migration, spheroids and tumor xenograft. RESULTS: We demonstrated that lipoplatin exhibited a potent antitumoral activity on HeLa, ME-180 cells and its cisplatin-resistant clone R-ME-180. Lipoplatin inhibited cell proliferation in a dose-dependent manner and was more active than the reference drug cisplatin in R-ME-180 cells and induced apoptosis, as evaluated by Annexin-V staining and DNA fragmentation, caspases 9 and 3 activation, Bcl-2, and Bcl-xL down-regulation, but Bax up-regulation inhibited thioredoxin reductase (TrxR) enzymatic activity and increased reactive oxygen species (ROS) accumulation; reduced EGFR expression and inhibited both migration and invasion. R-ME-180, but not ME-180 cells, generated three-dimensional (3D)-multicellular spheroids expressing the cancer stem cell marker ALDH. The ability of R-ME-180 cells to form spheroids in vitro and tumors in nude mice was also remarkably decreased by lipoplatin. CONCLUSIONS: Overall, our results suggest that lipoplatin has potential for the treatment of cisplatin-resistant cervical cancer.
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Cisplatino/administración & dosificación , Liposomas/administración & dosificación , Neoplasias del Cuello Uterino/tratamiento farmacológico , Aldehído Deshidrogenasa/metabolismo , Animales , Apoptosis/efectos de los fármacos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Cisplatino/farmacología , Resistencia a Antineoplásicos , Receptores ErbB/biosíntesis , Femenino , Células HeLa , Humanos , Ratones , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Especies Reactivas de Oxígeno/metabolismo , Esferoides Celulares , Reductasa de Tiorredoxina-Disulfuro/antagonistas & inhibidores , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: Emilin-1 is a protein of elastic extracellular matrix involved in blood pressure (BP) control by negatively affecting transforming growth factor (TGF)-ß processing. Emilin1 null mice are hypertensive. This study investigates how Emilin-1 deals with vascular mechanisms regulating BP. METHODS AND RESULTS: This study uses a phenotype rescue approach in which Emilin-1 is expressed in either endothelial cells or vascular smooth muscle cells of transgenic animals with the Emilin1(-/-) background. We found that normalization of BP required Emilin-1 expression in smooth muscle cells, whereas expression of the protein in endothelial cells did not modify the hypertensive phenotype of Emilin1(-/-) mice. We also explored the effect of treatment with anti-TGF-ß antibodies on the hypertensive phenotype of Emilin1(-/-) mice, finding that neutralization of TGF-ß in Emilin1 null mice normalized BP quite rapidly (2 weeks). Finally, we evaluated the vasoconstriction response of resistance arteries to perfusion pressure and neurohumoral agents in different transgenic mouse lines. Interestingly, we found that the hypertensive phenotype was coupled with an increased arteriolar myogenic response to perfusion pressure, while the vasoconstriction induced by neurohumoral agents remained unaffected. We further elucidate that, as for the hypertensive phenotype, the increased myogenic response was attributable to increased TGF-ß activity. CONCLUSIONS: Our findings clarify that Emilin-1 produced by vascular smooth muscle cells acts as a main regulator of resting BP levels by controlling the myogenic response in resistance arteries through TGF-ß.
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Presión Sanguínea , Hipertensión/metabolismo , Glicoproteínas de Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Vasoconstricción , Animales , Anticuerpos Neutralizantes/administración & dosificación , Arteriolas/metabolismo , Arteriolas/fisiopatología , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/genética , Monitoreo Ambulatorio de la Presión Arterial/métodos , Relación Dosis-Respuesta a Droga , Ecocardiografía Doppler , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Genotipo , Humanos , Hipertensión/genética , Hipertensión/fisiopatología , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiopatología , Óxido Nítrico Sintasa de Tipo III/genética , Óxido Nítrico Sintasa de Tipo III/metabolismo , Fenotipo , Telemetría , Factores de Tiempo , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Vasoconstricción/efectos de los fármacos , Vasoconstricción/genética , Vasoconstrictores/farmacologíaRESUMEN
Emerging evidences suggest that cyclin-dependent kinase inhibitors (CKIs) can regulate cellular functions other than cell cycle progression, such as differentiation and migration. Here, we report that cytoplasmic expression of p27(kip1) affects microtubule (MT) stability following cell adhesion on extracellular matrix (ECM) constituents. This p27(kip1) activity is due to its ability to bind and impair the function of the MT-destabilizing protein stathmin. Accordingly, upregulation of p27(kip1) or downregulation of stathmin expression results in the inhibition of mesenchymal cell motility. Moreover, high stathmin and low cytoplasmic p27(kip1) expression correlate with the metastatic phenotype of human sarcomas in vivo. This study provides a functional link between proliferation and invasion of tumor cells based on diverse activities of p27(kip1) in different subcellular compartments.
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Proteínas de Ciclo Celular/metabolismo , Movimiento Celular , Proteínas de Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Sarcoma/metabolismo , Sarcoma/patología , Proteínas Supresoras de Tumor/metabolismo , Animales , Adhesión Celular , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Inhibidores Enzimáticos/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/citología , Fibroblastos/fisiología , Fibronectinas/metabolismo , Genes Supresores de Tumor , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microtúbulos/genética , Microtúbulos/metabolismo , Metástasis de la Neoplasia , Fenotipo , Fosfoproteínas/genética , Estatmina , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Proteínas Supresoras de Tumor/genéticaAsunto(s)
Antineoplásicos/uso terapéutico , Auranofina/uso terapéutico , Enfermedad de Hodgkin/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Enfermedad de Hodgkin/metabolismo , Enfermedad de Hodgkin/patología , Humanos , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Colorectal cancer is one of the most frequent and deadly tumors. Among the key regulators of CRC growth and progression, the microenvironment has emerged as a crucial player and as a possible route for the development of new therapeutic opportunities. More specifically, the extracellular matrix acts directly on cancer cells and indirectly affecting the behavior of stromal and inflammatory cells, as well as the bioavailability of growth factors. Among the ECM molecules, EMILIN-2 is frequently down-regulated by methylation in CRC and the purpose of this study was to verify the impact of EMILIN-2 loss in CRC development and its possible value as a prognostic biomarker. METHODS: The AOM/DSS CRC protocol was applied to Emilin-2 null and wild type mice. Tumor development was monitored by endoscopy, the molecular analyses performed by IHC, IF and WB and the immune subpopulations characterized by flow cytometry. Ex vivo cultures of monocyte/macrophages from the murine models were used to verify the molecular pathways. Publicly available datasets were exploited to determine the CRC patients' expression profile; Spearman's correlation analyses and Cox regression were applied to evaluate the association with the inflammatory response; the clinical outcome was predicted by Kaplan-Meier survival curves. Pearson correlation analyses were also applied to a cohort of patients enrolled in our Institute. RESULTS: In preclinical settings, loss of EMILIN-2 associated with an increased number of tumor lesions upon AOM/DSS treatment. In addition, in the early stages of the disease, the Emilin-2 knockout mice displayed a myeloid-derived suppressor cells-rich infiltrate. Instead, in the late stages, lack of EMILIN-2 associated with a decreased number of M1 macrophages, resulting in a higher percentage of the tumor-promoting M2 macrophages. Mechanistically, EMILIN-2 triggered the activation of the Toll-like Receptor 4/MyD88/NF-κB pathway, instrumental for the polarization of macrophages towards the M1 phenotype. Accordingly, dataset and immunofluorescence analyses indicated that low EMILIN-2 expression levels correlated with an increased M2/M1 ratio and with poor CRC patients' prognosis. CONCLUSIONS: These novel results indicate that EMILIN-2 is a key regulator of the tumor-associated inflammatory environment and may represent a promising prognostic biomarker for CRC patients.
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Neoplasias Colorrectales/genética , Matriz Extracelular/metabolismo , Macrófagos/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Neoplasias Colorrectales/patología , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Microambiente TumoralRESUMEN
The control of bone resorption is crucial in osteolytic diseases. Once attached to bone, osteoclasts (OCs) initiate the resorption process through the activation of a complex cascade of morphological and biochemical changes. Hyaluronan (HA), an extracellular glycosaminoglycan long non-branching polysaccharide, is expressed in bone matrices. Here we demonstrate that HA counter-balances the erosion activity of human mature OCs by significantly reducing their degradative potential. HA treatment of fully differentiated OCs derived from human peripheral blood monocytes inhibited migration on collagen as well as bone resorption. HA-mediated effects were primarily due to TRAcP, MMP-9, and cathepsin K down-regulation and to the increased levels of TIMP-1, a natural MMP-9 inhibitor. Binding of HA to mature OCs was entirely mediated by CD44: function-blocking anti-CD44 antibodies fully abrogated HA effects, and the engagement of HA receptor caused a rapid de-phosphorylation of Ser325 in the CD44 cytoplasmic tail. The inhibitory action by HA was associated with a transient up-phosphorylation of Pyk2, a novel persistent phosphorylation of p38 and the down-regulation of NFATc1 transcription factor. Our results provide a direct evidence for the involvement of CD44 in the HA-dependent regulation of OC activity and suggest a signaling pathway that could be unique in OC function inhibition.