RESUMEN
Survival and expansion of malignant B cells in chronic lymphocytic leukemia (CLL) are highly dependent both on intrinsic defects in the apoptotic machinery and on the interactions with cells and soluble factors in the lymphoid microenvironment. The adaptor protein p66Shc is a negative regulator of antigen receptor signaling, chemotaxis and apoptosis whose loss in CLL B cells contributes to their extended survival and poor prognosis. Hence, the identification of compounds that restore p66Shc expression and function in malignant B cells may pave the way to a new therapeutic approach for CLL. Here we show that a novel oxazepine-based compound (OBC-1) restores p66Shc expression in primary human CLL cells by promoting JNK-dependent STAT4 activation without affecting normal B cells. Moreover, we demonstrate that the potent pro-apoptotic activity of OBC-1 in human leukemic cells directly correlates with p66Shc expression levels and is abrogated when p66Shc is genetically deleted. Preclinical testing of OBC-1 and the novel analogue OBC-2 in Eµ-TCL1 tumor-bearing mice resulted in a significantly longer overall survival and a reduction of the tumor burden in the spleen and peritoneum. Interestingly, OBCs promote leukemic cell mobilization from the spleen to the blood, which correlates with upregulation of sphingosine-1-phosphate receptor expression. In summary, our work identifies OBCs as a promising class of compounds that, by boosting p66Shc expression through the activation of the JNK/STAT4 pathway, display dual therapeutic effects for CLL intervention, namely the ability to mobilize cells from secondary lymphoid organs and a potent pro-apoptotic activity against circulating leukemic cells.
Asunto(s)
Antineoplásicos/uso terapéutico , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Oxazepinas/uso terapéutico , Animales , Antineoplásicos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Ratones Transgénicos , Oxazepinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Factor de Transcripción STAT4/genética , Factor de Transcripción STAT4/metabolismo , Receptores de Esfingosina-1-Fosfato/genética , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/genética , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/metabolismoRESUMEN
3D scaffolds used to repair damaged tissues should be able to mimic both composition and functions of natural extracellular matrix, which is mainly composed of polysaccharides and proteins. In our previous research new biomimetic sponges, based on blends of alginate with gelatin, were produced and characterized for myocardial tissue engineering applications. It was observed that these scaffolds can potentially function as a promising cardiac extracellular matrix substitute, but a reinforcement is required to improve their suturing properties. Aim of the present work was the development of a suturable biomimetic patch by the inclusion of a synthetic mesh within an alginate/gelatin scaffold. The mesh, produced by dry spinning, was made of eight superimposed layers of polycaprolactone microfibers, each one rotated of 45° with respect to the adjacent one. Reinforced scaffolds were obtained through the use of a mold, specially designed to place the fibrous mesh exactly in the center of the sponge. Both the reinforcement mesh and the reinforced scaffold were characterized. A perfect integration between the mesh and the sponge was observed. The fibrous mesh reduced the capacity of the sponge to absorb water, but the degree of hydrophilicity of the material was still comparable with that of natural cardiac tissue. The reinforced system showed a suitable stability in aqueous environment and it resulted much more resistant to suturing than not reinforced scaffold and even than human arteries. Polycaprolactone mesh was not cytotoxic and the reinforced scaffold was able to support cardiomyocytes adhesion and proliferation. Overall, the obtained results confirmed that the choice to modify the alginate/gelatin sponges through the insertion of an appropriate reinforcement system turned out to be correct in view of their potential use in myocardial tissue engineering.
Asunto(s)
Alginatos/química , Materiales Biomiméticos/química , Gelatina/química , Andamios del Tejido , Animales , Adhesión Celular , Línea Celular , Supervivencia Celular , Colorimetría , Humanos , Ratones , Ratas , Ingeniería de Tejidos/métodosRESUMEN
The Enzyme-Linked ImmunoSpot (ELISpot) assay detects cytokines secreted during T cell-specific immune responses against pathogens. As this assay has acquired importance in the clinical setting, standard bioanalytical evaluation of this method is required. Here, we describe a formal bioanalytical validation of a double-color ELISpot assay for the evaluation of IFN-γ and IL-4 released by T helper 1 and T helper 2 cells, respectively. As recommended by international guidelines, the parameters assessed were: range and detection limits (limit of detection, LOD; upper and lower limit of quantification, ULOQ and LLOQ), Linearity, Relative Accuracy, Repeatability, Intermediate Precision, Specificity and Robustness. The results obtained in this validation study demonstrate that this assay meets the established acceptability criteria. ELISpot is therefore a reliable technique for measuring T cell-specific immune responses against various antigens of interest.
Asunto(s)
Interleucina-4 , Leucocitos Mononucleares , Humanos , Interferón gamma , Ensayo de Immunospot Ligado a Enzimas/métodos , CitocinasRESUMEN
Autophagy is a lysosome dependent cell survival mechanism and is central to the maintenance of organismal homeostasis in both physiological and pathological situations. Targeting autophagy in cancer therapy attracted considerable attention in the past as stress-induced autophagy has been demonstrated to contribute to both drug resistance and malignant progression and recently interest in this area has re-emerged. Unlocking the therapeutic potential of autophagy modulation could be a valuable strategy for designing innovative tools for cancer treatment. Microtubule-targeting agents (MTAs) are some of the most successful anti-cancer drugs used in the clinic to date. Scaling up our efforts to develop new anti-cancer agents, we rationally designed multifunctional agents 5a-l with improved potency and safety that combine tubulin depolymerising efficacy with autophagic flux inhibitory activity. Through a combination of computational, biological, biochemical, pharmacokinetic-safety, metabolic studies and SAR analyses we identified the hits 5i,k. These MTAs were characterised as potent pro-apoptotic agents and also demonstrated autophagy inhibition efficacy. To measure their efficacy at inhibiting autophagy, we investigated their effects on basal and starvation-mediated autophagic flux by quantifying the expression of LC3II/LC3I and p62 proteins in oral squamous cell carcinoma and human leukaemia through western blotting and by immunofluorescence study of LC3 and LAMP1 in a cervical carcinoma cell line. Analogues 5i and 5k, endowed with pro-apoptotic activity on a range of hematological cancer cells (including ex-vivo chronic lymphocytic leukaemia (CLL) cells) and several solid tumor cell lines, also behaved as late-stage autophagy inhibitors by impairing autophagosome-lysosome fusion.
Asunto(s)
Antineoplásicos , Carcinoma de Células Escamosas , Neoplasias de la Boca , Antineoplásicos/metabolismo , Apoptosis , Autofagia , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Humanos , Microtúbulos , Neoplasias de la Boca/tratamiento farmacológicoRESUMEN
In this work we describe the synthesis of potent and selective quinolone-based histone deacetylase 6 (HDAC6) inhibitors. The quinolone moiety has been exploited as an innovative bioactive cap-group for HDAC6 inhibition; its synthesis was achieved by applying a multicomponent reaction. The optimization of potency and selectivity of these products was performed by employing computational studies which led to the discovery of the diethylaminomethyl derivatives 7g and 7k as the most promising hit molecules. These compounds were investigated in cellular studies to evaluate their anticancer effect against colon (HCT-116) and histiocytic lymphoma (U9347) cancer cells, showing good to excellent potency, leading to tumor cell death by apoptosis induction. The small molecules 7a, 7g and 7k were able to strongly inhibit the cytoplasmic and slightly the nuclear HDAC enzymes, increasing the acetylation of tubulin and of the lysine 9 and 14 of histone 3, respectively. Compound 7g was also able to increase Hsp90 acetylation levels in HCT-116 cells, thus further supporting its HDAC6 inhibitory profile. Cytotoxicity and mutagenicity assays of these molecules showed a safe profile; moreover, the HPLC analysis of compound 7k revealed good solubility and stability profile.
Asunto(s)
Histona Desacetilasa 6/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/farmacología , Quinolonas/farmacología , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Histona Desacetilasa 6/metabolismo , Inhibidores de Histona Desacetilasas/síntesis química , Inhibidores de Histona Desacetilasas/química , Humanos , Ratones , Modelos Moleculares , Estructura Molecular , Quinolonas/síntesis química , Quinolonas/química , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Background Recent data suggest that systemic inflammation can negatively affect atrioventricular conduction, regardless of acute cardiac injury. Indeed, gap-junctions containing connexin43 coupling cardiomyocytes and inflammation-related cells (macrophages) are increasingly recognized as important factors regulating the conduction in the atrioventricular node. The aim of this study was to evaluate the acute impact of systemic inflammatory activation on atrioventricular conduction, and elucidate underlying mechanisms. Methods and Results We analyzed: (1) the PR-interval in patients with inflammatory diseases of different origins during active phase and recovery, and its association with inflammatory markers; (2) the existing correlation between connexin43 expression in the cardiac tissue and peripheral blood mononuclear cells (PBMC), and the changes occurring in patients with inflammatory diseases over time; (3) the acute effects of interleukin(IL)-6 on atrioventricular conduction in an in vivo animal model, and on connexin43 expression in vitro. In patients with elevated C-reactive protein levels, atrioventricular conduction indices are increased, but promptly normalized in association with inflammatory markers reduction, particularly IL-6. In these subjects, connexin43 expression in PBMC, which is correlative of that measured in the cardiac tissue, inversely associated with IL-6 changes. Moreover, direct IL-6 administration increased atrioventricular conduction indices in vivo in a guinea pig model, and IL-6 incubation in both cardiomyocytes and macrophages in culture, significantly reduced connexin43 proteins expression. Conclusions The data evidence that systemic inflammation can acutely worsen atrioventricular conduction, and that IL-6-induced down-regulation of cardiac connexin43 is a mechanistic pathway putatively involved in the process. Though reversible, these alterations could significantly increase the risk of severe atrioventricular blocks during active inflammatory processes.
Asunto(s)
Bloqueo Atrioventricular , Conexina 43 , Animales , Nodo Atrioventricular , Citocinas , Cobayas , Humanos , Inflamación , Interleucina-6 , Leucocitos MononuclearesRESUMEN
Aim: Over the years, indole has proved to be a versatile scaffold for the design of molecules acting as anti-inflammatory agents. Materials & Methods: A small library of 3-amino-alkylated indoles has been obtained by an optimized Mannich green approach. The anti-inflammatory activity of the new 3-amino-alkylated indoles, GLYC 0-10, was evaluated in RAW 264.7 macrophages. Results: The anti-inflammatory activity of the new 3-amino-alkylated indoles, GLYC 0-10, was evaluatedn and, among them, GLYC 4, 5 and 9 displayed the greatest inhibitory effects on nitric oxide production, with IC50 values of 5.41, 4.22 and 6.3 µM, respectively. Conclusion: Our outcomes, overall, highlight the importance of the indole substitution in the anti-inflammatory activity of these compounds, exerted by acting on the interlinked NF-κB/ERK1/2 pathways.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Indoles/farmacología , Alquilación , Animales , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Relación Dosis-Respuesta a Droga , Indoles/síntesis química , Indoles/química , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Estructura Molecular , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Células RAW 264.7 , Relación Estructura-ActividadRESUMEN
BACKGROUND: During acute infections, the risk of malignant ventricular arrhythmias is increased, partly because of a higher propensity to develop QTc prolongation. Although it is generally believed that QTc changes almost exclusively result from concomitant treatment with QT-prolonging antimicrobials, direct effects of inflammatory cytokines on ventricular repolarization are increasingly recognized. We hypothesized that systemic inflammation per se can significantly prolong QTc during acute infections, via cytokine-mediated changes in K+ channel expression. METHODS: We evaluated (1) the frequency of QTc prolongation and its association with inflammatory markers, in patients with different types of acute infections, during active disease and remission; (2) the prevalence of acute infections in a cohort of consecutive patients with Torsades de Pointes; (3) the relationship between K+ channel mRNA levels in ventricles and peripheral blood mononuclear cells and their changes in patients with acute infection over time. RESULTS: In patients with acute infections, regardless of concomitant QT-prolonging antimicrobial treatments, QTc was significantly prolonged but rapidly normalized in parallel to CRP (C-reactive protein) and cytokine level reduction. Consistently in the Torsades de Pointes cohort, concomitant acute infections were highly prevalent (30%), despite only a minority (25%) of these cases were treated with QT-prolonging antimicrobials. KCNJ2 K+ channel expression in peripheral blood mononuclear cell, which strongly correlated to that in ventricles, inversely associated to CRP and IL (interleukin)-1 changes in acute infection patients. CONCLUSIONS: During acute infections, systemic inflammation rapidly induces cytokine-mediated ventricular electrical remodeling and significant QTc prolongation, regardless concomitant antimicrobial therapy. Although transient, these changes may significantly increase the risk of life-threatening ventricular arrhythmia in these patients. It is timely and warranted to transpose these findings to the current coronavirus disease 2019 (COVID-19) pandemic, in which both increased amounts of circulating cytokines and cardiac arrhythmias are demonstrated along with a frequent concomitant treatment with several QT-prolonging drugs. Graphic Abstract: A graphic abstract is available for this article.
Asunto(s)
Enfermedades Transmisibles/metabolismo , Citocinas/metabolismo , Paro Cardíaco/metabolismo , Frecuencia Cardíaca , Ventrículos Cardíacos/metabolismo , Inflamación/metabolismo , Leucocitos Mononucleares/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Torsades de Pointes/metabolismo , Potenciales de Acción , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Antiinfecciosos/efectos adversos , Enfermedades Transmisibles/tratamiento farmacológico , Enfermedades Transmisibles/epidemiología , Enfermedades Transmisibles/fisiopatología , Femenino , Paro Cardíaco/epidemiología , Paro Cardíaco/fisiopatología , Frecuencia Cardíaca/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/fisiopatología , Humanos , Inflamación/epidemiología , Inflamación/fisiopatología , Leucocitos Mononucleares/efectos de los fármacos , Masculino , Persona de Mediana Edad , Canales de Potasio de Rectificación Interna/genética , Prevalencia , Factores de Riesgo , Transducción de Señal , Factores de Tiempo , Torsades de Pointes/epidemiología , Torsades de Pointes/fisiopatología , Adulto JovenRESUMEN
Histone deacetylase inhibitors (HDACi) have emerged as promising therapeutics for the treatment of neurodegeneration, cancer, and rare disorders. Herein, we report the development of a series of spiroindoline-based HDAC6 isoform-selective inhibitors based on the X-ray crystal studies of the hit 6a. We identified compound 6j as the most potent and selective hHDAC6 inhibitor of the series. Biological investigation of compounds 6b, 6h, and 6j demonstrated their antiproliferative activity against several cancer cell lines. Western blotting studies indicated that they were able to increase tubulin acetylation, without significant variation in histone acetylation state, and induced PARP cleavage indicating their apoptotic potential at the molecular level. 6j induced HDAC6-dependent pSTAT3 inhibition.
RESUMEN
Background Systemic inflammation is a strong predictor of atrial fibrillation. A key role for electrical remodeling is increasingly recognized, and experimental data suggest that inflammatory cytokines can directly affect connexins resulting in gap-junction dysfunction. We hypothesized that systemic inflammation, regardless of its origin, promotes atrial electric remodeling in vivo, as a result of cytokine-mediated changes in connexin expression. Methods and Results Fifty-four patients with different inflammatory diseases and elevated C-reactive protein were prospectively enrolled, and electrocardiographic P-wave dispersion indices, cytokine levels (interleukin-6, tumor necrosis factor-α, interleukin-1, interleukin-10), and connexin expression (connexin 40, connexin 43) were measured during active disease and after reducing C-reactive protein by >75%. Moreover, peripheral blood mononuclear cells and atrial tissue specimens from an additional sample of 12 patients undergoing cardiac surgery were evaluated for atrial and circulating mRNA levels of connexins. Finally, in vitro effects of interleukin-6 on connexin expression were studied in HL-1 mouse atrial myocytes. In patients with active inflammatory diseases, P-wave dispersion indices were increased but rapidly decreased within days when C-reactive protein normalizes and interleukin-6 levels decline. In inflammatory disease patients, both P-wave dispersion indices and interleukin-6 changes were inversely associated with circulating connexin levels, and a positive correlation between connexin expression in peripheral blood mononuclear cells and atrial tissue was demonstrated. Moreover, interleukin-6 significantly reduced connexin expression in HL-1 cells. Conclusions Our data suggest that regardless of specific etiology and organ localization, systemic inflammation, via interleukin-6 elevation, rapidly induces atrial electrical remodeling by down-regulating cardiac connexins. Although transient, these changes may significantly increase the risk for atrial fibrillation and related complications during active inflammatory processes.
Asunto(s)
Remodelación Atrial/inmunología , Conexinas/genética , Inflamación/inmunología , Interleucina-6/inmunología , Miocitos Cardíacos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antibacterianos/uso terapéutico , Antiinflamatorios/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/inmunología , Remodelación Atrial/genética , Proteína C-Reactiva/inmunología , Procedimientos Quirúrgicos Cardíacos , Conexina 43/efectos de los fármacos , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/efectos de los fármacos , Conexinas/metabolismo , Electrocardiografía , Femenino , Regulación de la Expresión Génica , Atrios Cardíacos/citología , Humanos , Infecciones/tratamiento farmacológico , Infecciones/inmunología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/fisiopatología , Interleucina-1/inmunología , Interleucina-10/inmunología , Interleucina-6/farmacología , Masculino , Ratones , Persona de Mediana Edad , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Adulto Joven , Proteína alfa-5 de Unión ComunicanteRESUMEN
Background: Torsades de pointes (TdP) is a life-threatening ventricular tachycardia occurring in long QT-syndrome patients. It usually develops when multiple QT-prolonging factors are concomitantly present, more frequently drugs and electrolyte imbalances. Since proton-pump inhibitors (PPIs)-associated hypomagnesemia is an increasingly recognized adverse event, PPIs were recently included in the list of drugs with conditional risk of TdP, despite only few cases of TdP in PPI users have been reported so far. Objectives: Aim of the present study is to evaluate whether PPI-induced hypomagnesemia actually has a significant clinical impact on the risk of TdP in the general population. Methods: Forty-eight unselected patients who experienced TdP were consecutively enrolled (2008-2017). Shortly after the first TdP episode, in those patients who did not receive magnesium sulfate and/or potassium or calcium replacement therapy, serum electrolytes were measured and their relationship with PPI usage analyzed. Results: Many patients (28/48, 58%) were under current PPI treatment when TdP occurred. Among TdP patients in whom serum electrolyte determinations were obtained before replacement therapy (27/48), those taking PPIs had significantly lower serum magnesium levels than those who did not. Hypomagnesemia occurred in ~40% of patients receiving PPIs (6/14), in all cases after an extended treatment (>2 weeks). In patients taking PPIs the mean QT-prolonging risk factor number was significantly higher than in those who did not, a difference which was mainly driven by lower magnesium levels. Conclusions: In unselected TdP patients, PPI-induced hypomagnesemia was common and significantly contributed to their cumulative arrhythmic risk. By providing clinical support to current recommendations, our data confirm that more awareness is needed when a PPI is prescribed, specifically as regards the risk of life-threatening arrhythmias.
RESUMEN
OBJECTIVE: Increasing evidence indicates systemic inflammation as a new potential cause of acquired long QT syndrome (LQTS), via cytokine-mediated changes in cardiomyocyte ion channels. Torsade de pointes (TdP) is a life-threatening polymorphic ventricular tachycardia occurring in patients with LQTS, usually when multiple QT-prolonging factors are simultaneously present. Since classical risk factors cannot fully explain TdP events in a number of patients, we hypothesised that systemic inflammation may represent a currently overlooked risk factor contributing to TdP development in the general population. METHODS: Forty consecutive patients who experienced TdP (TdP cohort) were consecutively enrolled and circulating levels of C-reactive protein (CRP) and proinflammatory cytokines (interleukin-6 (IL-6), tumour necrosis factor alpha (TNFα), interleukin-1 (IL-1)) were compared with patients with active rheumatoid arthritis (RA), comorbidity or healthy controls. An additional 46 patients with different inflammatory conditions (acute infections, n=31; immune-mediated diseases, n=12; others, n=3) and elevated CRP (inflammatory cohort) were prospectively enrolled, and corrected QT (QTc) and cytokine levels were measured during active disease and after a CRP decrease of >75% subsequent to therapy. RESULTS: In the TdP cohort, 80% of patients showed elevated CRP levels (median: ~3 mg/dL), with a definite inflammatory disease identifiable in 18/40 cases (acute infections, n=12; immune-mediated diseases, n=5; others, n=1). In these subjects, IL-6, but not TNFα and IL-1, was ~15-20 times higher than in controls, and comparable to RA patients. In the inflammatory cohort, where QTc prolongation was common (mean values: 456.6±30.9 ms), CRP reduction was associated with IL-6 level decrease and significant QTc shortening (-22.3 ms). CONCLUSION: The data are first to show that systemic inflammation via elevated IL-6 levels may represent a novel QT-prolonging risk factor contributing to TdP occurrence in the presence of other classical risk factors. If confirmed, this could open new avenues in antiarrhythmic therapy.
Asunto(s)
Mediadores de Inflamación/sangre , Inflamación/complicaciones , Interleucina-6/sangre , Torsades de Pointes/etiología , Anciano , Anciano de 80 o más Años , Biomarcadores/sangre , Proteína C-Reactiva/análisis , Estudios de Casos y Controles , Electrocardiografía , Femenino , Humanos , Inflamación/sangre , Inflamación/diagnóstico , Interleucina-1/sangre , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Prospectivos , Factores de Riesgo , Torsades de Pointes/sangre , Torsades de Pointes/diagnóstico , Torsades de Pointes/fisiopatología , Factor de Necrosis Tumoral alfa/sangre , Regulación hacia ArribaRESUMEN
Objectives: Systemic sclerosis (SSc) is a connective tissue disorder presenting fibrosis of the skin and internal organs, for which no effective treatments are currently available. Increasing evidence indicates that the P2X7 receptor (P2X7R), a nucleotide-gated ionotropic channel primarily involved in the inflammatory response, may also have a key role in the development of tissue fibrosis in different body districts. This study was aimed at investigating P2X7R expression and function in promoting a fibrogenic phenotype in dermal fibroblasts from SSc patients, also analyzing putative underlying mechanistic pathways. Methods: Fibroblasts were isolated by skin biopsy from 9 SSc patients and 8 healthy controls. P2X7R expression, and function (cytosolic free Ca2+ fluxes, α-smooth muscle actin [α-SMA] expression, cell migration, and collagen release) were studied. Moreover, the role of cytokine (interleukin-1ß, interleukin-6) and connective tissue growth factor (CTGF) production, and extracellular signal-regulated kinases (ERK) activation in mediating P2X7R-dependent pro-fibrotic effects in SSc fibroblasts was evaluated. Results: P2X7R expression and Ca2+ permeability induced by the selective P2X7R agonist 2'-3'-O-(4-benzoylbenzoyl)ATP (BzATP) were markedly higher in SSc than control fibroblasts. Moreover, increased αSMA expression, cell migration, CTGF, and collagen release were observed in lipopolysaccharides-primed SSc fibroblasts after BzATP stimulation. While P2X7-induced cytokine changes did not affect collagen production, it was completely abrogated by inhibition of the ERK pathway. Conclusion: In SSc fibroblasts, P2X7R is overexpressed and its stimulation induces Ca2+-signaling activation and a fibrogenic phenotype characterized by increased migration and collagen production. These data point to the P2X7R as a potential, novel therapeutic target for controlling exaggerated collagen deposition and tissue fibrosis in patients with SSc.
RESUMEN
BACKGROUND: Results from the BEVA2007 trial, suggest that the metronomic chemotherapy regimen with dose-fractioned cisplatin and oral etoposide (mPE) +/- bevacizumab, a monoclonal antibody to the vascular endothelial growth factor (VEGF), shows anti-angiogenic and immunological effects and is a safe and active treatment for metastatic non-small cell lung cancer (mNSCLC) patients. We carried out a retrospective analysis aimed to evaluate the antitumor effects of this treatment in a subset of patients with squamous histology. METHODS: Retrospective analysis was carried out in a subset of 31 patients with squamous histology enrolled in the study between September 2007 and September 2015. All of the patients received chemotherapy with cisplatin (30 mg/sqm, days 1-3q21) and oral etoposide (50 mg, days 1-15q21) (mPE) and 14 of them also received bevacizumab 5 mg/kg on the day 3q21 (mPEBev regimen). RESULTS: This treatment showed a disease control rate of 71% with a mean progression free survival (PFS) and overall survival (OS) of 13.6 and 17 months respectively. After 4 treatment courses, 6 patients showing a remarkable tumor shrinkage, underwent to radical surgery, attaining a significant advantage in term of survival (P=0.048). Kaplan-Meier and log-rank test identified the longest survival in patients presenting low baseline levels in neutrophil-to-lymphocyte ratio (NLR) (P=0.05), interleukin (IL) 17A (P=0.036), regulatory-T-cells (Tregs) (P=0.020), and activated CD83+ dendritic cells (DCs) (P=0.03). CONCLUSIONS: These results suggest that the mPE +/- bevacizumab regimen is feasible and should be tested in comparative trials in advanced squamous-NSCLC (sqNSCLC). Moreover, its immune-biological effects strongly suggest the investigation in sequential combinations with immune check-point inhibitors.