RESUMEN
Recruitment of fibroblasts to tumors and their activation into cancer-associated fibroblasts (CAFs) is a strategy used by tumor cells to direct extracellular matrix (ECM) remodeling, invasion, and metastasis, highlighting the need to investigate the molecular mechanisms driving CAF function. Endothelin-1 (ET-1) regulates the communication between cancer and stroma and facilitates the progression of serous ovarian cancer (SOC). By binding to Endothelin A (ETA) and B (ETB) receptors, ET-1 enables the recruitment of ß-arrestin1 (ß-arr1) and the formation of signaling complexes that coordinate tumor progression. However, how ET-1 receptors might "educate" human ovarian fibroblasts (HOFs) to produce altered ECM and promote metastasis remains to be elucidated. This study identifies ET-1 as a pivotal factor in the activation of CAFs capable of proteolytic ECM remodeling and the generation of heterotypic spheroids containing cancer cells with a propensity to metastasize. An autocrine/paracrine ET-1/ETA/BR/ß-arr1 loop enhances HOF proliferation, upregulates CAF marker expression, secretes pro-inflammatory cytokines, and increases collagen contractility, and cell motility. Furthermore, ET-1 facilitates ECM remodeling by promoting the lytic activity of invadosome and activation of integrin ß1. In addition, ET-1 signaling supports the formation of heterotypic HOF/SOC spheroids with enhanced ability to migrate through the mesothelial monolayer, and invade, representing metastatic units. The blockade of ETA/BR or ß-arr1 silencing prevents CAF activation, invadosome function, mesothelial clearance, and the invasive ability of heterotypic spheroids. In vivo, therapeutic inhibition of ETA/BR using bosentan (BOS) significantly reduces the metastatic potential of combined HOFs/SOC cells, associated with enhanced apoptotic effects on tumor cells and stromal components. These findings support a model in which ET-1/ß-arr1 reinforces tumor/stroma interaction through CAF activation and fosters the survival and metastatic properties of SOC cells, which could be counteracted by ETA/BR antagonists.
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Fibroblastos Asociados al Cáncer , Neoplasias Ováricas , Podosomas , beta-Arrestina 1 , Humanos , Femenino , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Ováricas/genética , beta-Arrestina 1/metabolismo , beta-Arrestina 1/genética , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/patología , Línea Celular Tumoral , Podosomas/metabolismo , Endotelina-1/metabolismo , Metástasis de la Neoplasia , Receptor de Endotelina A/metabolismo , Transducción de Señal , Matriz Extracelular/metabolismo , Movimiento Celular , Proliferación Celular , Animales , Fibroblastos/metabolismo , Invasividad NeoplásicaRESUMEN
Dissemination of high-grade serous ovarian cancer (HG-SOC) in the omentum and intercalation into a mesothelial cell (MC) monolayer depends on functional α5ß1 integrin (Intα5ß1) activity. Although the binding of Intα5ß1 to fibronectin drives these processes, other molecular mechanisms linked to integrin inside-out signaling might support metastatic dissemination. Here, we report a novel interactive signaling that contributes to Intα5ß1 activation and accelerates tumor cells toward invasive disease, involving the protein ß-arrestin1 (ß-arr1) and the activation of the endothelin A receptor (ETAR) by endothelin-1 (ET-1). As demonstrated in primary HG-SOC cells and SOC cell lines, ET-1 increased Intß1 and downstream FAK/paxillin activation. Mechanistically, ß-arr1 directly interacts with talin1 and Intß1, promoting talin1 phosphorylation and its recruitment to Intß1, thus fueling integrin inside-out activation. In 3D spheroids and organotypic models mimicking the omentum, ETAR/ß-arr1-driven Intα5ß1 signaling promotes the survival of cell clusters, with mesothelium-intercalation capacity and invasive behavior. The treatment with the antagonist of ETAR, Ambrisentan (AMB), and of Intα5ß1, ATN161, inhibits ET-1-driven Intα5ß1 activity in vitro, and tumor cell adhesion and spreading to intraperitoneal organs and Intß1 activity in vivo. As a prognostic factor, high EDNRA/ITGB1 expression correlates with poor HG-SOC clinical outcomes. These findings highlight a new role of ETAR/ß-arr1 operating an inside-out integrin activation to modulate the metastatic process and suggest that in the new integrin-targeting programs might be considered that ETAR/ß-arr1 regulates Intα5ß1 functional pathway.
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Integrina alfa5beta1 , Neoplasias Ováricas , Receptor de Endotelina A , Talina , beta-Arrestina 1 , Femenino , Humanos , beta-Arrestina 1/genética , beta-Arrestina 1/metabolismo , Carcinoma Epitelial de Ovario/genética , Línea Celular Tumoral , Endotelina-1/metabolismo , Neoplasias Ováricas/metabolismo , Receptor de Endotelina A/genética , Receptor de Endotelina A/metabolismo , Integrina alfa5beta1/metabolismo , Talina/genética , Talina/metabolismoRESUMEN
Conventional batch syntheses of polymer-based nanoparticles show considerable shortcomings in terms of scarce control over nanomaterials morphology and limited lot-to-lot reproducibility. Droplet-based microfluidics represents a valuable strategy to overcome these constraints, exploiting the formation of nanoparticles within discrete microdroplets. In this work, we synthesized nanogels (NGs) composed of hyaluronic acid and polyethyleneimine using a microfluidic flow-focusing device endowed with a pressure-driven micro-actuator. The actuator achieves real-time modulation of the junction orifice width, thereby regulating the microdroplet diameter and, as a result, the NG size. Acting on process parameters, NG hydrodynamic diameter could be tuned in the range 92-190 nm while preserving an extremely low polydispersity (0.015); those values are hardly achievable in batch syntheses and underline the strength of our toolbox for the continuous in-flow synthesis of nanocarriers. Furthermore, NGs were validated in vitro as a drug delivery system in a representative case study still lacking an effective therapeutic treatment: ovarian cancer. Using doxorubicin as a chemotherapeutic agent, we show that NG-mediated release of the drug results in an enhanced antiblastic effect vs. the non-encapsulated administration route even at sublethal dosages, highlighting the wide applicability of our microfluidics-enabled nanomaterials in healthcare scenarios.
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Nanopartículas , Nanoestructuras , Sistemas de Liberación de Medicamentos , Microfluídica/métodos , Nanogeles , Reproducibilidad de los ResultadosRESUMEN
Transcoelomic spread of serous ovarian cancer (SOC) results from the cooperative interactions between cancer and host components. Tumor-derived factors might allow the conversion of mesothelial cells (MCs) into tumor-associated MCs, providing a favorable environment for SOC cell dissemination. However, factors and molecular mechanisms involved in this process are largely unexplored. Here we investigated the tumor-related endothelin-1 (ET-1) as an inducer of changes in MCs supporting SOC progression. Here, we report a significant production of ET-1 from MCs associated with the expression of its cognate receptors, ETA and ETB, along with the protein ß-arrestin1. ET-1 triggers MC proliferation via ß-arrestin1-dependent MAPK and NF-kB pathways and increases the release of cancer-related factors. The ETA/ETB receptor activation supports the genetic reprogramming of mesothelial-to-mesenchymal transition (MMT), with upregulation of mesenchymal markers, as fibronectin, α-SMA, N-cadherin and vimentin, NF-kB-dependent Snail transcriptional activity and downregulation of E-cadherin and ZO-1, allowing to enhanced MC migration and invasion, and SOC transmesothelial migration. These effects are impaired by either blockade of ETAR and ETBR or by ß-arrestin1 silencing. Notably, in peritoneal metastases both ETAR and ETBR are co-expressed with MMT markers compared to normal control peritoneum. Collectively, our report shows that the ET-1 axis may contribute to the early stage of SOC progression by modulating MC pro-metastatic behaviour via MMT.
RESUMEN
BACKGROUND: Dietary factors have significant effects on the brain, modulating mood, anxiety, motivation and cognition. To date, no attention has been paid to the consequences that the combination of ethanol (EtOH) and a high-fat diet (HFD) have on learning and mood disorders during adolescence. The aim of the present work was to evaluate the biochemical and behavioral consequences of ethanol binge drinking and an HFD consumption in adolescent mice. METHODS: Animals received either a standard diet or an HFD (ad libitum vs. binge pattern) in combination with ethanol binge drinking and were evaluated in anxiety and memory. The metabolic profile and gene expression of leptin receptors and clock genes were also evaluated. RESULTS: Excessive white adipose tissue and an increase in plasma insulin and leptin levels were mainly observed in ad libitum HFD + EtOH mice. An upregulation of the Lepr gene expression in the prefrontal cortex and the hippocampus was also observed in ad libitum HFD groups. EtOH-induced impairment on spatial memory retrieval was absent in mice exposed to an HFD, although the aversive memory deficits persisted. Mice bingeing on an HFD only showed an anxiolytic profile, without other alterations. We also observed a mismatch between Clock and Bmal1 expression in ad libitum HFD animals, which were mostly independent of EtOH bingeing. CONCLUSIONS: Our results confirm the bidirectional influence that occurs between the composition and intake pattern of a HFD and ethanol consumption during adolescence, even when the metabolic, behavioral and chronobiological effects of this interaction are dissociated.
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Bulimia , Dieta Alta en Grasa , Etanol/toxicidad , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Adiposidad , Animales , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Hipocampo/metabolismo , Aprendizaje/fisiología , Leptina/sangre , Masculino , Ratones , Trastornos del Humor/etiología , Trastornos del Humor/metabolismo , Corteza Prefrontal/metabolismo , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Aumento de PesoRESUMEN
The options available for genetic modification of cells of the central nervous system (CNS) have greatly increased in the last decade. The current panoply of viral and nonviral vectors provides multifunctional platforms to deliver expression cassettes to many structures and nuclei. These cassettes can replace defective genes, modify a given pathway perturbed by diseases, or express proteins that can be selectively activated by drugs or light to extinguish or excite neurons. This review focuses on the use of canine adenovirus type 2 (CAV-2) vectors for gene transfer to neurons in the brain, spinal cord, and peripheral nervous system. We discuss (1) recent advances in vector production, (2) why CAV-2 vectors preferentially transduce neurons, (3) the mechanism underlying their widespread distribution via retrograde axonal transport, (4) how CAV-2 vectors have been used to address structure/function, and (5) their therapeutic applications.
RESUMEN
High-fat diets (HFD) impair hippocampal-dependent learning and memory and produce important changes in synaptic transmission by enhancing glutamate uptake, decreasing synaptic efficacy, and inhibiting plasticity mechanisms such as N-methyl-D-aspartate-mediated long-term depression (LTD) within the hippocampus. Adolescent animals seem to be particularly susceptible to the detrimental effect of HFD as dietary treatments carried out between weaning and early adulthood are much more efficient in terms of hippocampal damage that those carried out during the adult period. As palmitic acid is the most abundant saturated fatty acid in HFD, its effect on hippocampal function needs to be studied. However, glycogen synthase kinase-3 (GSK-3), a pleiotropic enzyme highly expressed in the central nervous system, modulates both hippocampal long-term potentiation (LTP) and LTD, and has been implicated in neurological disorders including Alzheimer's disease. In this study, we have characterized in mice hippocampus the effect of (i) a 48 h HFD intervention and (ii) in-vitro palmitic acid, as well as the possible involvement of GSK-3 in the above-mentioned plasticity mechanisms. Our results show that both 48 h HFD and palmitic acid inhibit LTP in hippocampal slices, whereas no effect on LTD was observed. Moreover, tideglusib, an ATP-noncompetitive inhibitor of GSK-3, induced hippocampal LTP and partially reversed the impairment of LTP induced by palmitic acid.
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Dieta Alta en Grasa/efectos adversos , Glucógeno Sintasa Quinasa 3/metabolismo , Hipocampo/fisiopatología , Potenciación a Largo Plazo/fisiología , Ácido Palmítico/metabolismo , Ácido Palmítico/toxicidad , Animales , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/efectos de los fármacos , Potenciación a Largo Plazo/efectos de los fármacos , Depresión Sináptica a Largo Plazo/efectos de los fármacos , Depresión Sináptica a Largo Plazo/fisiología , Masculino , Ratones Endogámicos C57BL , N-Metilaspartato/metabolismo , Fármacos Neuroprotectores/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Tiadiazoles/farmacología , Técnicas de Cultivo de TejidosRESUMEN
PURPOSE: Our aim was to characterize the effect of an unfamiliar high-fat diet (HFD) on circadian feeding behaviour, plasma parameters, body weight (BW), and gene expression in the prefrontal cortex (PFC) of adolescent male mice. To this end, mice were allowed to consume a HFD during 48 h, but one group was allowed a free choice of HFD or normal chow (FC-HFD), while the other was restricted to a non-optional unfamiliar HFD feeding (NOP-HFD). METHODS: Energy intake was monitored at 6-h intervals during 48 h. Mice cohorts were killed at 6-h intervals after 48-h dietary treatment, and PFC samples dissected for RT-PCR analysis. RESULTS: Mice on the FC-HFD protocol avoided eating the standard chow, showed lower energy intake and lower BW increase than NOP-HFD mice. All animals with access to HFD exhibited nocturnal overeating, but diurnal hyperphagia was more prominent in the FC-HFD cohort. A robust increase in tyrosine hydroxylase (Th) gene expression was detected specifically during the light period of the circadian cycle in FC-HFD mice. In contrast, both protocols similarly up-regulated the expression of cytosolic malic enzyme (Me1), which is very sensitive to HFD. CONCLUSION: Our data show that the PFC participates in driving motivational feeding during HFD-evoked hyperphagia and also suggest that sensory neural pathways might be relevant for the onset of eating disorders and overweight. Moreover, we have observed that animals that had the possibility of choosing between standard chow and HFD were more hyperphagic and specifically displayed an overexpression of the tyrosine hydroxylase gene.
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Conducta de Elección , Ritmo Circadiano , Dieta Alta en Grasa/efectos adversos , Ingestión de Energía , Corteza Prefrontal/fisiología , Animales , Glucemia/metabolismo , Peso Corporal , Carbohidratos de la Dieta/administración & dosificación , Grasas de la Dieta/administración & dosificación , Proteínas en la Dieta/administración & dosificación , Regulación de la Expresión Génica , Hiperfagia , Insulina/sangre , Leptina/sangre , Leptina/genética , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Sobrepeso/etiología , Sobrepeso/genética , Receptores de Leptina/sangre , Receptores de Leptina/genética , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo , Aumento de PesoRESUMEN
Recent studies point to dietary factors as important effectors in the brain and epidemiological studies suggest a direct relationship between mood and anxiety disorders, cognitive impairment and obesity. Nevertheless the link between the consumption of high-fat diets (HFD) and emotional disorders still remains unclear. This issue is of particular interest during adolescence, which is an important period for shaping learning and memory acquisition that can be particularly sensitive to the detrimental effects of HFD. Otherwise, major depressive disorder and anxiety crisis often emerge in adolescence. In the current study we have characterized in adolescent mice i) the onset of HFD-induced memory impairment using the novel location recognition (NLR) paradigm, and ii) the effect of HFD on depression- and anxiety-related behaviors by using the forced swimming and the elevated plus maze tests, respectively. Here we report that memory impairments induced by HFD were already perceptible after 4-weeks HFD whereas HFD induced already antidepressant-like effects after 48-h, that remained after long-term treatment (8 weeks). No effects in anxiety were found. These data indicate that the antidepressant-like effect of HFD is independent of memory deficits as it was already present after 48-h HFD, while no effects in memory were still observed at this time.
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Afecto , Dieta Alta en Grasa/efectos adversos , Aprendizaje , Afecto/efectos de los fármacos , Animales , Ansiolíticos/farmacología , Trastorno Depresivo/tratamiento farmacológico , Trastorno Depresivo/etiología , Diazepam/farmacología , Conducta Exploratoria/efectos de los fármacos , Aprendizaje/efectos de los fármacos , Masculino , Trastornos de la Memoria/tratamiento farmacológico , Trastornos de la Memoria/etiología , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Distribución Aleatoria , Reconocimiento en Psicología/efectos de los fármacos , Memoria Espacial/efectos de los fármacos , Factores de TiempoRESUMEN
We have investigated in adolescent mice the effect of subchronic leptin on (i) leptin receptor expression and functionality, and (ii) dopamine-related gene expression (tyrosine hydroxylase, Th; dopamine type-1 receptor, Drd1; dopamine type-2 receptor, Drd2) within the prefrontal cortex (PFC), which is involved in sensory perception of food and reward sensitivity, and the hippocampus, a brain area sensitive to food composition and pivotal in learning and memory processes related to feeding behaviour. Here, we show that leptin treatment triggered leptin resistance both in the hippocampus and in the PFC. In contrast, leptin induced the upregulation of dopamine-related genes in the PFC, whereas it failed to modify the expression of these genes in the hippocampus. The effect of leptin was similar irrespective of the time elapsed since the last leptin administration (either 2 or 14 h), indicating that the effect detected was not associated with leptin withdrawal. Our data show that leptin receptor desensitization is coincident with the upregulation of dopamine-related genes in the PFC of adolescent mice undergoing hyperleptinaemia triggered by exogenous leptin.
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Dopamina/genética , Leptina/administración & dosificación , Corteza Prefrontal/metabolismo , Receptores de Leptina/metabolismo , Tejido Adiposo/efectos de los fármacos , Animales , Ingestión de Alimentos/efectos de los fármacos , Expresión Génica , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Corteza Prefrontal/efectos de los fármacos , Receptores Dopaminérgicos/genética , Transducción de Señal/efectos de los fármacos , Tirosina 3-Monooxigenasa/genética , Regulación hacia ArribaRESUMEN
PURPOSE: Highly palatable foods behave as appetitive reinforcers and tend to be consumed compulsively. Nevertheless, the motivation for this kind of diets in experimental diet-induced obesity models has not been well established. Our hypothesis is that obesity caused by a regular consumption of high-fat diet (HFD) occurs concomitantly with the inhibition of food reward. The ultimate goal of our study was to further analyze the extent to which the perception of food as an appetitive reinforcer is a necessary condition for obesity. METHODS: We have evaluated the influence of HFD on operant food self-administration (FSA) during a whole light-dark (12-12-h) cycle in mice that consumed HFD either during 1, 4 or 8 weeks. The study has been complemented by a two-bottle free-choice assay between tap water and sweetened drinks. RESULTS: These data show that both 4- and 8-week HFD treatments induced a significant decrease in operant FSA rate. Moreover, HFD impaired the sweetened-conditioned flavor preference in the two-bottle choice assay. CONCLUSION: Our results, showing a reduction in how hard an animal is willing to work for food reinforcers, provide evidence that chronic consumption of HFD negatively contributes to the incentive motivation to acquire food/drink reinforcers. We demonstrate that energy homeostasis imbalance triggered by HFD is associated with the inhibition of hedonic feeding.
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Dieta Alta en Grasa , Grasas de la Dieta/administración & dosificación , Conducta Alimentaria , Recompensa , Animales , Conducta de Elección , Ansia/fisiología , Carbohidratos de la Dieta/administración & dosificación , Carbohidratos de la Dieta/análisis , Grasas de la Dieta/análisis , Fibras de la Dieta/administración & dosificación , Fibras de la Dieta/análisis , Proteínas en la Dieta/administración & dosificación , Proteínas en la Dieta/análisis , Ingestión de Energía , Preferencias Alimentarias , Homeostasis , Masculino , Ratones , Ratones Endogámicos C57BL , Edulcorantes Nutritivos/administración & dosificación , Edulcorantes Nutritivos/análisis , Obesidad/inducido químicamente , AutoadministraciónRESUMEN
Our hypothesis is that direct targeting of brain areas involved in the perception of food as a rewarding stimulus accounts for initial hyperphagia caused by high-fat food (HFD). Because adolescents are more sensitive than adults to HFD, studies were performed in five-week old male mice. We analyzed the effect of acute exposition to HFD on c-Fos immunolabeling and we observed that this diet selectively increased c-Fos immunolabeling in the dorsomedial prefrontal cortex (PFC). Furthermore HFD triggered strong and long-lasting conditioned place-preference (CPP) behavior. We also found that the strength of conditioning correlated with the up-regulation of the expression of genes involved in dopaminergic transmission together with a decreased expression of the Per2 gene in the CPF. Our data are coherent with the involvement of the dorsomedial PFC in the perception of HFD as a positive reinforcer and suggest that sensory stimuli activate this brain area after HFD intake.