RESUMEN
Ion channels are key regulators of cancer cell pathophysiology. They contribute to a variety of processes such as maintenance of cellular osmolarity and membrane potential, motility (via interactions with the cytoskeleton), invasion, signal transduction, transcriptional activity and cell cycle progression, leading to tumour progression and metastasis. Ion channels thus represent promising targets for cancer therapy. Ion channels are attractive targets because many of them are expressed at the plasma membrane and a broad range of existing inhibitors are already in clinical use for other indications. However, many of the ion channels identified in cancer cells are also active in healthy normal cells, so there is a risk that certain blockers may have off-target effects on normal physiological function. This review describes recent research advances into ion channel inhibitors as anticancer therapeutics. A growing body of evidence suggests that a range of existing and novel Na+, K+, Ca2+ and Cl- channel inhibitors may be effective for suppressing cancer cell proliferation, migration and invasion, as well as enhancing apoptosis, leading to suppression of tumour growth and metastasis, either alone or in combination with standard-of-care therapies. The majority of evidence to date is based on preclinical in vitro and in vivo studies, although there are several examples of ion channel-targeting strategies now reaching early phase clinical trials. Given the strong links between ion channel function and regulation of tumour growth, metastasis and chemotherapy resistance, it is likely that further work in this area will facilitate the development of new therapeutic approaches which will reach the clinic in the future.
Asunto(s)
Neoplasias , Membrana Celular/metabolismo , Membrana Celular/patología , Proliferación Celular , Humanos , Canales Iónicos , Neoplasias/patología , Transducción de SeñalRESUMEN
Cancers of epithelial origin such as breast, prostate, cervical, gastric, colon and lung cancer account for a large proportion of deaths worldwide. Better treatment of metastasis, the main cause of cancer deaths, is therefore urgently required. Several of these tumours have been shown to have an abnormally high concentration of Na+ ([Na+ ]) and emerging evidence points to this accumulation being due to elevated intracellular [Na+ ]. This poses intriguing questions about the cellular mechanisms underlying Na+ dysregulation in cancer, and its pathophysiological significance. Elevated intracellular [Na+ ] may be due to alterations in activity of the Na+ /K+ -ATPase, and/or increased influx via Na+ channels and Na+ -linked transporters. Maintenance of the electrochemical Na+ gradient across the plasma membrane is vital to power many cellular processes that are highly active in cancer cells, including glucose and glutamine import. Na+ channels are also upregulated in cancer cells, which in turn promotes tumour growth and metastasis. For example, ENaC and ASICs are overexpressed in cancers, increasing invasion and proliferation. In addition, voltage-gated Na+ channels are also upregulated in a range of tumour types, where they promote metastatic cell behaviours via various mechanisms, including membrane potential depolarisation and altered pH regulation. Together, recent findings relating to elevated Na+ in the tumour microenvironment and how this may be regulated by several classes of Na+ channels provide a link between altered Na+ handling and poor clinical outcome. There are new opportunities to leverage this altered Na+ microenvironment for therapeutic benefit, as exemplified by several ongoing clinical trials.
Asunto(s)
Neoplasias de la Mama , Canales de Sodio , Humanos , Femenino , Canales de Sodio/metabolismo , Membrana Celular/metabolismo , Neoplasias de la Mama/metabolismo , Microambiente TumoralRESUMEN
Despite extensive basic and clinical research on immune checkpoint regulatory pathways, little is known about the effects of the ionic tumor microenvironment on immune checkpoint expression and function. Here we describe a mechanistic link between Na+/K+-ATPase (NKA) inhibition and activity of the immune checkpoint protein indoleamine-pyrrole 2',3'-dioxygenase 1 (IDO1). We found that IDO1 was necessary and sufficient for production of kynurenine, a downstream tryptophan metabolite, in cancer cells. We developed a spectrophotometric assay to screen a library of 31 model ion transport-targeting compounds for potential effects on IDO1 function in A549 lung and MDA-MB-231 breast cancer cells. This revealed that the cardiac glycosides ouabain and digoxin inhibited kynurenine production at concentrations that did not affect cell survival. NKA inhibition by ouabain and digoxin resulted in increased intracellular Na+ levels and downregulation of IDO1 mRNA and protein levels, which was consistent with the reduction in kynurenine levels. Knockdown of ATP1A1, the É1 subunit of the NKA and target of cardiac glycosides, increased Na+ levels to a lesser extent than cardiac glycoside treatment and did not affect IDO1 expression. However, ATP1A1 knockdown significantly enhanced the effect of cardiac glycosides on IDO1 expression and kynurenine production. Mechanistically, we show that cardiac glycoside treatment resulted in curtailing the length of phosphorylation-mediated stabilization of STAT1, a transcriptional regulator of IDO1 expression, an effect enhanced by ATP1A1 knockdown. Our findings reveal cross talk between ionic modulation via cardiac glycosides and immune checkpoint protein expression in cancer cells with broad mechanistic and clinical implications.
Asunto(s)
Glicósidos Cardíacos , Indolamina-Pirrol 2,3,-Dioxigenasa , Neoplasias , Factor de Transcripción STAT1 , ATPasa Intercambiadora de Sodio-Potasio , Células A549 , Glicósidos Cardíacos/farmacología , Línea Celular Tumoral , Digoxina/farmacología , Humanos , Proteínas de Punto de Control Inmunitario , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Indolamina-Pirrol 2,3,-Dioxigenasa/biosíntesis , Quinurenina/metabolismo , Neoplasias/patología , Ouabaína/metabolismo , Ouabaína/farmacología , Factor de Transcripción STAT1/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
The voltage-gated Na+ channel ß1 subunit, encoded by SCN1B, regulates cell surface expression and gating of α subunits and participates in cell adhesion. ß1 is cleaved by α/ß and γ-secretases, releasing an extracellular domain and intracellular domain (ICD), respectively. Abnormal SCN1B expression/function is linked to pathologies including epilepsy, cardiac arrhythmia, and cancer. In this study, we sought to determine the effect of secretase cleavage on ß1 function in breast cancer cells. Using a series of GFP-tagged ß1 constructs, we show that ß1-GFP is mainly retained intracellularly, particularly in the endoplasmic reticulum and endolysosomal pathway, and accumulates in the nucleus. Reduction in endosomal ß1-GFP levels occurred following γ-secretase inhibition, implicating endosomes and/or the preceding plasma membrane as important sites for secretase processing. Using live-cell imaging, we also report ß1ICD-GFP accumulation in the nucleus. Furthermore, ß1-GFP and ß1ICD-GFP both increased Na+ current, whereas ß1STOP-GFP, which lacks the ICD, did not, thus highlighting that the ß1-ICD is necessary and sufficient to increase Na+ current measured at the plasma membrane. Importantly, although the endogenous Na+ current expressed in MDA-MB-231 cells is tetrodotoxin (TTX)-resistant (carried by Nav1.5), the Na+ current increased by ß1-GFP or ß1ICD-GFP was TTX-sensitive. Finally, we found ß1-GFP increased mRNA levels of the TTX-sensitive α subunits SCN1A/Nav1.1 and SCN9A/Nav1.7. Taken together, this work suggests that the ß1-ICD is a critical regulator of α subunit function in cancer cells. Our data further highlight that γ-secretase may play a key role in regulating ß1 function in breast cancer.
Asunto(s)
Neoplasias de la Mama , Canales de Sodio , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Femenino , Humanos , Canal de Sodio Activado por Voltaje NAV1.7 , Sodio/metabolismo , Canales de Sodio/metabolismo , Tetrodotoxina/farmacología , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/genéticaRESUMEN
The development of therapies that can suppress invasion and prevent metastasis, 'anti-metastatic drugs', is an important area of unmet therapeutic need. The new results of a recent open-label, multicentre randomised trial published in J Clin Oncol showed a significant disease-free survival (DFS) benefit for breast cancer patients receiving presurgical, peritumoral injection of lidocaine, an amide local anaesthetic, which blocks voltage-gated sodium channels (VGSCs). VGSCs are expressed on electrically excitable cells, including neurons and cardiomyocytes, where they sustain rapid membrane depolarisation during action potential firing. As a result of this key biophysical function, VGSCs are important drug targets for excitability-related disorders, including epilepsy, neuropathic pain, affective disorders and cardiac arrhythmia. A growing body of preclinical evidence highlights VGSCs as key protagonists in regulating altered sodium influx in breast cancer cells, thus driving invasion and metastasis. Furthermore, prescription of certain VGSC-inhibiting medications has been associated with reduced cancer incidence and improved survival in several observational studies. Thus, VGSC-inhibiting drugs already in clinical use may be ideal candidates for repurposing as possible anti-metastatic therapies. While these results are promising, further work is required to establish whether other VGSC inhibitors may afford superior metastasis suppression. Finally, increasing preclinical evidence suggests that several other ion channels are also key drivers of cancer hallmarks; thus, there are undoubtedly further opportunities to harness ion transport inhibition that should also be explored.
Asunto(s)
Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/patología , Transporte Iónico , Supervivencia sin Enfermedad , Sodio/uso terapéutico , Ensayos Clínicos Controlados Aleatorios como Asunto , Estudios Multicéntricos como AsuntoRESUMEN
Sodium (Na+) concentration in solid tumours of different origin is highly dysregulated, and this corresponds to the aberrant expression of Na+ transporters. In particular, the α subunits of voltage gated Na+ channels (VGSCs) raise intracellular Na+ concentration ([Na+]i) in malignant cells, which influences the progression of solid tumours, predominantly driving cancer cells towards a more aggressive and metastatic phenotype. Conversely, re-expression of VGSC ß subunits in cancer cells can either enhance tumour progression or promote anti-tumourigenic properties. Metastasis is the leading cause of cancer-related mortality, highlighting an important area of research which urgently requires improved therapeutic interventions. Here, we review the extent to which VGSC subunits are dysregulated in solid tumours, and consider the implications of such dysregulation on solid tumour progression. We discuss current understanding of VGSC-dependent mechanisms underlying increased invasive and metastatic potential of solid tumours, and how the complex relationship between the tumour microenvironment (TME) and VGSC expression may further drive tumour progression, in part due to the interplay of infiltrating immune cells, cancer-associated fibroblasts (CAFs) and insufficient supply of oxygen (hypoxia). Finally, we explore past and present clinical trials that investigate utilising existing VGSC modulators as potential pharmacological options to support adjuvant chemotherapies to prevent cancer recurrence. Such research demonstrates an exciting opportunity to repurpose therapeutics in order to improve the disease-free survival of patients with aggressive solid tumours.
Asunto(s)
Neoplasias , Canales de Sodio Activados por Voltaje , Humanos , Canales de Sodio Activados por Voltaje/metabolismo , Neoplasias/metabolismo , Fenotipo , Sodio/metabolismo , Microambiente TumoralRESUMEN
BACKGROUND: Breast cancer remains a leading cause of death in women and novel imaging biomarkers are urgently required. Here, we demonstrate the diagnostic and treatment-monitoring potential of non-invasive sodium (23Na) MRI in preclinical models of breast cancer. METHODS: Female Rag2-/- Il2rg-/- and Balb/c mice bearing orthotopic breast tumours (MDA-MB-231, EMT6 and 4T1) underwent MRI as part of a randomised, controlled, interventional study. Tumour biology was probed using ex vivo fluorescence microscopy and electrophysiology. RESULTS: 23Na MRI revealed elevated sodium concentration ([Na+]) in tumours vs non-tumour regions. Complementary proton-based diffusion-weighted imaging (DWI) linked elevated tumour [Na+] to increased cellularity. Combining 23Na MRI and DWI measurements enabled superior classification accuracy of tumour vs non-tumour regions compared with either parameter alone. Ex vivo assessment of isolated tumour slices confirmed elevated intracellular [Na+] ([Na+]i); extracellular [Na+] ([Na+]e) remained unchanged. Treatment with specific inward Na+ conductance inhibitors (cariporide, eslicarbazepine acetate) did not affect tumour [Na+]. Nonetheless, effective treatment with docetaxel reduced tumour [Na+], whereas DWI measures were unchanged. CONCLUSIONS: Orthotopic breast cancer models exhibit elevated tumour [Na+] that is driven by aberrantly elevated [Na+]i. Moreover, 23Na MRI enhances the diagnostic capability of DWI and represents a novel, non-invasive biomarker of treatment response with superior sensitivity compared to DWI alone.
Asunto(s)
Neoplasias de la Mama , Sodio , Animales , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/tratamiento farmacológico , Medios de Contraste , Imagen de Difusión por Resonancia Magnética/métodos , Femenino , Humanos , Imagen por Resonancia Magnética/métodos , RatonesRESUMEN
Ion channels can regulate the plasma membrane potential (Vm ) and cell migration as a result of altered ion flux. However, the mechanism by which Vm regulates motility remains unclear. Here, we show that the Nav 1.5 sodium channel carries persistent inward Na+ current which depolarizes the resting Vm at the timescale of minutes. This Nav 1.5-dependent Vm depolarization increases Rac1 colocalization with phosphatidylserine, to which it is anchored at the leading edge of migrating cells, promoting Rac1 activation. A genetically encoded FRET biosensor of Rac1 activation shows that depolarization-induced Rac1 activation results in acquisition of a motile phenotype. By identifying Nav 1.5-mediated Vm depolarization as a regulator of Rac1 activation, we link ionic and electrical signaling at the plasma membrane to small GTPase-dependent cytoskeletal reorganization and cellular migration. We uncover a novel and unexpected mechanism for Rac1 activation, which fine tunes cell migration in response to ionic and/or electric field changes in the local microenvironment.
Asunto(s)
Neoplasias de la Mama/genética , Microambiente Celular/genética , Canal de Sodio Activado por Voltaje NAV1.5/genética , Proteína de Unión al GTP rac1/genética , Técnicas Biosensibles , Neoplasias de la Mama/patología , Línea Celular Tumoral , Membrana Celular/genética , Membrana Celular/metabolismo , Movimiento Celular/genética , Citoesqueleto/química , Citoesqueleto/genética , Femenino , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Humanos , Canales Iónicos/genética , Potenciales de la Membrana/genética , Canal de Sodio Activado por Voltaje NAV1.5/química , Transducción de Señal/genética , Proteína de Unión al GTP rac1/químicaRESUMEN
BACKGROUND: An emerging problem in the treatment of breast cancer is the increasing incidence of metastases to the brain. Metastatic brain tumours are incurable and can cause epileptic seizures and cognitive impairment, so better understanding of this niche, and the cellular mechanisms, is urgently required. Microglia are the resident brain macrophage population, becoming "activated" by neuronal injury, eliciting an inflammatory response. Microglia promote proliferation, angiogenesis and invasion in brain tumours and metastases. However, the mechanisms underlying microglial involvement appear complex and better models are required to improve understanding of function. METHODS: Here, we sought to address this need by developing a model to study metastatic breast cancer cell-microglial interactions using intravital imaging combined with ex vivo electrophysiology. We implanted an optical window on the parietal bone to facilitate observation of cellular behaviour in situ in the outer cortex of heterozygous Cx3cr1GFP/+ mice. RESULTS: We detected GFP-expressing microglia in Cx3cr1GFP/+ mice up to 350 µm below the window without significant loss of resolution. When DsRed-expressing metastatic MDA-MB-231 breast cancer cells were implanted in Matrigel under the optical window, significant accumulation of activated microglia around invading tumour cells could be observed. This inflammatory response resulted in significant cortical disorganisation and aberrant spontaneously-occurring local field potential spike events around the metastatic site. CONCLUSIONS: These data suggest that peritumoral microglial activation and accumulation may play a critical role in local tissue changes underpinning aberrant cortical activity, which offers a possible mechanism for the disrupted cognitive performance and seizures seen in patients with metastatic breast cancer.
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Neoplasias Encefálicas/secundario , Neoplasias de la Mama/patología , Modelos Animales de Enfermedad , Microscopía Intravital/métodos , Microglía , Animales , Línea Celular Tumoral , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microambiente Tumoral/fisiologíaRESUMEN
Currently, cell separation occurs almost exclusively by density gradient methods and by fluorescence- and magnetic-activated cell sorting (FACS/MACS). These variously suffer from lack of specificity, high cell loss, use of labels, and high capital/operating cost. We present a dielectrophoresis (DEP)-based cell-separation method, using 3D electrodes on a low-cost disposable chip; one cell type is allowed to pass through the chip whereas the other is retained and subsequently recovered. The method advances usability and throughput of DEP separation by orders of magnitude in throughput, efficiency, purity, recovery (cells arriving in the correct output fraction), cell losses (those which are unaccounted for at the end of the separation), and cost. The system was evaluated using three example separations: live and dead yeast; human cancer cells/red blood cells; and rodent fibroblasts/red blood cells. A single-pass protocol can enrich cells with cell recovery of up to 91.3% at over 300,000 cells per second with >3% cell loss. A two-pass protocol can process 300,000,000 cells in under 30 min, with cell recovery of up to 96.4% and cell losses below 5%, an effective processing rate >160,000 cells per second. A three-step protocol is shown to be effective for removal of 99.1% of RBCs spiked with 1% cancer cells while maintaining a processing rate of â¼170,000 cells per second. Furthermore, the self-contained and low-cost nature of the separator device means that it has potential application in low-contamination applications such as cell therapies, where good manufacturing practice compatibility is of paramount importance.
Asunto(s)
Separación Celular/métodos , Electroforesis/métodos , Fenómenos Electrofisiológicos , Eritrocitos/citología , Neoplasias/patología , Saccharomyces cerevisiae/citología , Línea Celular Tumoral , Separación Celular/economía , Electroforesis/economía , HumanosRESUMEN
Voltage-gated Na(+) channel (VGSC) ß1 subunits, encoded by SCN1B, are multifunctional channel modulators and cell adhesion molecules (CAMs). Mutations in SCN1B are associated with the genetic epilepsy with febrile seizures plus (GEFS+) spectrum disorders in humans, and Scn1b-null mice display severe spontaneous seizures and ataxia from postnatal day (P)10. The goal of this study was to determine changes in neuronal pathfinding during early postnatal brain development of Scn1b-null mice to test the hypothesis that these CAM-mediated roles of Scn1b may contribute to the development of hyperexcitability. c-Fos, a protein induced in response to seizure activity, was up-regulated in the Scn1b-null brain at P16 but not at P5. Consistent with this, epileptiform activity was observed in hippocampal and cortical slices prepared from the P16 but not from the P5-P7 Scn1b-null brain. On the basis of these results, we investigated neuronal pathfinding at P5. We observed disrupted fasciculation of parallel fibers in the P5 null cerebellum. Further, P5 null mice showed reduced neuron density in the dentate gyrus granule cell layer, increased proliferation of granule cell precursors in the hilus, and defective axonal extension and misorientation of somata and processes of inhibitory neurons in the dentate gyrus and CA1. Thus, Scn1b is critical for neuronal proliferation, migration, and pathfinding during the critical postnatal period of brain development. We propose that defective neuronal proliferation, migration, and pathfinding in response to Scn1b deletion may contribute to the development of hyperexcitability.
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Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/deficiencia , Factores de Edad , Animales , Ataxia/etiología , Ataxia/metabolismo , Ataxia/patología , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Encéfalo/citología , Movimiento Celular , Proliferación Celular , Fenómenos Electrofisiológicos , Hipocampo/crecimiento & desarrollo , Hipocampo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/citología , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Convulsiones/etiología , Convulsiones/metabolismo , Convulsiones/patología , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/genética , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/metabolismoRESUMEN
BACKGROUND: Voltage-gated Na(+) channels (VGSCs) are heteromeric protein complexes containing pore-forming α subunits and smaller, non-pore-forming ß subunits. VGSCs are classically expressed in electrically excitable cells, e.g. neurons. VGSCs are also expressed in tumour cells, including breast cancer (BCa) cells, where they enhance cellular migration and invasion. However, despite extensive work defining in detail the molecular mechanisms underlying the expression of VGSCs and their pro-invasive role in cancer cells, there has been a notable lack of clinically relevant in vivo data exploring their value as potential therapeutic targets. FINDINGS: We have previously reported that the VGSC-blocking antiepileptic drug phenytoin inhibits the migration and invasion of metastatic MDA-MB-231 cells in vitro. The purpose of the present study was to establish whether VGSCs might be viable therapeutic targets by testing the effect of phenytoin on tumour growth and metastasis in vivo. We found that expression of Nav1.5, previously detected in MDA-MB-231 cells in vitro, was retained on cells in orthotopic xenografts. Treatment with phenytoin, at a dose equivalent to that used to treat epilepsy (60 mg/kg; daily), significantly reduced tumour growth, without affecting animal weight. Phenytoin also reduced cancer cell proliferation in vivo and invasion into surrounding mammary tissue. Finally, phenytoin significantly reduced metastasis to the liver, lungs and spleen. CONCLUSIONS: This is the first study showing that phenytoin reduces breast tumour growth and metastasis in vivo. We propose that pharmacologically targeting VGSCs, by repurposing antiepileptic or antiarrhythmic drugs, should be further studied as a potentially novel anti-cancer therapy.
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Anticonvulsivantes/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Fenitoína/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/metabolismo , Animales , Anticonvulsivantes/administración & dosificación , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Metástasis de la Neoplasia , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Fenitoína/administración & dosificación , Bloqueadores de los Canales de Sodio/administración & dosificación , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Voltage-gated Na(+) channels (VGSCs) are heteromeric proteins composed of pore-forming α subunits and smaller ß subunits. The ß subunits are multifunctional channel modulators and are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs). ß1, encoded by SCN1B, is best characterized in the central nervous system (CNS), where it plays a critical role in regulating electrical excitability, neurite outgrowth and migration during development. ß1 is also expressed in breast cancer (BCa) cell lines, where it regulates adhesion and migration in vitro. In the present study, we found that SCN1B mRNA/ß1 protein were up-regulated in BCa specimens, compared with normal breast tissue. ß1 upregulation substantially increased tumour growth and metastasis in a xenograft model of BCa. ß1 over-expression also increased vascularization and reduced apoptosis in the primary tumours, and ß1 over-expressing tumour cells had an elongate morphology. In vitro, ß1 potentiated outgrowth of processes from BCa cells co-cultured with fibroblasts, via trans-homophilic adhesion. ß1-mediated process outgrowth in BCa cells required the presence and activity of fyn kinase, and Na(+) current, thus replicating the mechanism by which ß1 regulates neurite outgrowth in CNS neurons. We conclude that when present in breast tumours, ß1 enhances pathological growth and cellular dissemination. This study is the first demonstration of a functional role for ß1 in tumour growth and metastasis in vivo. We propose that ß1 warrants further study as a potential biomarker and targeting ß1-mediated adhesion interactions may have value as a novel anti-cancer therapy.
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Neoplasias de la Mama/patología , Neoplasias Hepáticas/secundario , Neoplasias Pulmonares/secundario , Neuritas/patología , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Western Blotting , Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proliferación Celular , Femenino , Humanos , Técnicas para Inmunoenzimas , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Ratones Desnudos , Persona de Mediana Edad , Neuritas/metabolismo , Neuronas/metabolismo , Neuronas/patología , Proteínas Proto-Oncogénicas c-fyn/genética , Proteínas Proto-Oncogénicas c-fyn/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Subunidad beta-1 de Canal de Sodio Activado por Voltaje/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: To study the reproducibility of 23Na magnetic resonance imaging (MRI) measurements from breast tissue in healthy volunteers. METHODS: Using a dual-tuned bilateral 23Na/1H breast coil at 3-T MRI, high-resolution 23Na MRI three-dimensional cones sequences were used to quantify total sodium concentration (TSC) and fluid-attenuated sodium concentration (FASC). B1-corrected TSC and FASC maps were created. Two readers manually measured mean, minimum and maximum TSC and mean FASC values using two sampling methods: large regions of interest (LROIs) and small regions of interest (SROIs) encompassing fibroglandular tissue (FGT) and the highest signal area at the level of the nipple, respectively. The reproducibility of the measurements and correlations between density, age and FGT apparent diffusion coefficient (ADC) values were evaluatedss. RESULTS: Nine healthy volunteers were included. The inter-reader reproducibility of TSC and FASC using SROIs and LROIs was excellent (intraclass coefficient range 0.945-0.979, p < 0.001), except for the minimum TSC LROI measurements (p = 0.369). The mean/minimum LROI TSC and mean LROI FASC values were lower than the respective SROI values (p < 0.001); the maximum LROI TSC values were higher than the SROI TSC values (p = 0.009). TSC correlated inversely with age but not with FGT ADCs. The mean and maximum FGT TSC and FASC values were higher in dense breasts in comparison to non-dense breasts (p < 0.020). CONCLUSIONS: The chosen sampling method and the selected descriptive value affect the measured TSC and FASC values, although the inter-reader reproducibility of the measurements is in general excellent. RELEVANCE STATEMENT: 23Na MRI at 3 T allows the quantification of TSC and FASC sodium concentrations. The sodium measurements should be obtained consistently in a uniform manner. KEY POINTS: ⢠23Na MRI allows the quantification of total and fluid-attenuated sodium concentrations (TSC/FASC). ⢠Sampling method (large/small region of interest) affects the TSC and FASC values. ⢠Dense breasts have higher TSC and FASC values than non-dense breasts. ⢠The inter-reader reproducibility of TSC and FASC measurements was, in general, excellent. ⢠The results suggest the importance of stratifying the sodium measurements protocol.
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Mama , Imagen por Resonancia Magnética , Sodio , Humanos , Femenino , Reproducibilidad de los Resultados , Adulto , Imagen por Resonancia Magnética/métodos , Mama/diagnóstico por imagen , Persona de Mediana Edad , Isótopos de Sodio , Voluntarios Sanos , Variaciones Dependientes del Observador , Adulto JovenRESUMEN
Solid tumours have abnormally high intracellular [Na+]. The activity of various Na+ channels may underlie this Na+ accumulation. Voltage-gated Na+ channels (VGSCs) have been shown to be functionally active in cancer cell lines, where they promote invasion. However, the mechanisms involved, and clinical relevance, are incompletely understood. Here, we show that protein expression of the Nav1.5 VGSC subtype strongly correlates with increased metastasis and shortened cancer-specific survival in breast cancer patients. In addition, VGSCs are functionally active in patient-derived breast tumour cells, cell lines, and cancer-associated fibroblasts. Knockdown of Nav1.5 in a mouse model of breast cancer suppresses expression of invasion-regulating genes. Nav1.5 activity increases ATP demand and glycolysis in breast cancer cells, likely by upregulating activity of the Na+/K+ ATPase, thus promoting H+ production and extracellular acidification. The pH of murine xenograft tumours is lower at the periphery than in the core, in regions of higher proliferation and lower apoptosis. In turn, acidic extracellular pH elevates persistent Na+ influx through Nav1.5 into breast cancer cells. Together, these findings show positive feedback between extracellular acidification and the movement of Na+ into cancer cells which can facilitate invasion. These results highlight the clinical significance of Nav1.5 activity as a potentiator of breast cancer metastasis and provide further evidence supporting the use of VGSC inhibitors in cancer treatment.
RESUMEN
Voltage-gated Na(+) channel (VGSC) beta1 subunits regulate cell-cell adhesion and channel activity in vitro. We previously showed that beta1 promotes neurite outgrowth in cerebellar granule neurons (CGNs) via homophilic cell adhesion, fyn kinase, and contactin. Here we demonstrate that beta1-mediated neurite outgrowth requires Na(+) current (I(Na)) mediated by Na(v)1.6. In addition, beta1 is required for high-frequency action potential firing. Transient I(Na) is unchanged in Scn1b (beta1) null CGNs; however, the resurgent I(Na), thought to underlie high-frequency firing in Na(v)1.6-expressing cerebellar neurons, is reduced. The proportion of axon initial segments (AIS) expressing Na(v)1.6 is reduced in Scn1b null cerebellar neurons. In place of Na(v)1.6 at the AIS, we observed an increase in Na(v)1.1, whereas Na(v)1.2 was unchanged. This indicates that beta1 is required for normal localization of Na(v)1.6 at the AIS during the postnatal developmental switch to Na(v)1.6-mediated high-frequency firing. In agreement with this, beta1 is normally expressed with alpha subunits at the AIS of P14 CGNs. We propose reciprocity of function between beta1 and Na(v)1.6 such that beta1-mediated neurite outgrowth requires Na(v)1.6-mediated I(Na), and Na(v)1.6 localization and consequent high-frequency firing require beta1. We conclude that VGSC subunits function in macromolecular signaling complexes regulating both neuronal excitability and migration during cerebellar development.
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Cerebelo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Canales de Sodio/metabolismo , Potenciales de Acción/fisiología , Animales , Axones/metabolismo , Cerebelo/citología , Cerebelo/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Neurológicos , Complejos Multiproteicos , Canal de Sodio Activado por Voltaje NAV1.6 , Proteínas del Tejido Nervioso/química , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Transducción de Señal , Canales de Sodio/química , Canales de Sodio/deficiencia , Canales de Sodio/genética , Tetrodotoxina/toxicidad , Subunidad beta-1 de Canal de Sodio Activado por VoltajeRESUMEN
Hypoxia in disease describes persistent low oxygen conditions, observed in a range of pathologies, including cancer. In the discovery of biomarkers in biological models, pathophysiological traits present a source of translatable metabolic products for the diagnosis of disease in humans. Part of the metabolome is represented by its volatile, gaseous fraction; the volatilome. Human volatile profiles, such as those found in breath, are able to diagnose disease, however accurate volatile biomarker discovery is required to target reliable biomarkers to develop new diagnostic tools. Using custom chambers to control oxygen levels and facilitate headspace sampling, the MDA-MB-231 breast cancer cell line was exposed to hypoxia (1% oxygen) for 24 h. The maintenance of hypoxic conditions in the system was successfully validated over this time period. Targeted and untargeted gas chromatography mass spectrometry approaches revealed four significantly altered volatile organic compounds when compared to control cells. Three compounds were actively consumed by cells: methyl chloride, acetone and n-Hexane. Cells under hypoxia also produced significant amounts of styrene. This work presents a novel methodology for identification of volatile metabolisms under controlled gas conditions with novel observations of volatile metabolisms by breast cancer cells.
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With phenotypic heterogeneity in whole cell populations widely recognised, the demand for quantitative and temporal analysis approaches to characterise single cell morphology and dynamics has increased. We present CellPhe, a pattern recognition toolkit for the unbiased characterisation of cellular phenotypes within time-lapse videos. CellPhe imports tracking information from multiple segmentation and tracking algorithms to provide automated cell phenotyping from different imaging modalities, including fluorescence. To maximise data quality for downstream analysis, our toolkit includes automated recognition and removal of erroneous cell boundaries induced by inaccurate tracking and segmentation. We provide an extensive list of features extracted from individual cell time series, with custom feature selection to identify variables that provide greatest discrimination for the analysis in question. Using ensemble classification for accurate prediction of cellular phenotype and clustering algorithms for the characterisation of heterogeneous subsets, we validate and prove adaptability using different cell types and experimental conditions.
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Algoritmos , Rastreo Celular , Imagen de Lapso de Tiempo , Rastreo Celular/métodosRESUMEN
Intracellular Ca2+ signaling and Na+ homeostasis are inextricably linked via ion channels and co-transporters, with alterations in the concentration of one ion having profound effects on the other. Evidence indicates that intracellular Na+ concentration ([Na+ ]i ) is elevated in breast tumors, and that aberrant Ca2+ signaling regulates numerous key cancer hallmark processes. The present study therefore aimed to determine the effects of Na+ depletion on intracellular Ca2+ handling in metastatic breast cancer cell lines. The relationship between Na+ and Ca2+ was probed using fura-2 and SBFI fluorescence imaging and replacement of extracellular Na+ with equimolar N-methyl-D-glucamine (0Na+ /NMDG) or choline chloride (0Na+ /ChoCl). In triple-negative MDA-MB-231 and MDA-MB-468 cells and Her2+ SKBR3 cells, but not ER+ MCF-7 cells, 0Na+ /NMDG and 0Na+ /ChoCl resulted in a slow, sustained depletion in [Na+ ]i that was accompanied by a rapid and sustained increase in intracellular Ca2+ concentration ([Ca2+ ]i ). Application of La3+ in nominal Ca2+ -free conditions had no effect on this response, ruling out reverse-mode NCX activity and Ca2+ entry channels. Moreover, the Na+ -linked [Ca2+ ]i increase was independent of membrane potential hyperpolarization (NS-1619), but was inhibited by pharmacological blockade of IP3 receptors (2-APB), phospholipase C (PLC, U73122) or following depletion of endoplasmic reticulum Ca2+ stores (cyclopiazonic acid). Thus, Na+ is linked to PLC/IP3 -mediated activation of endoplasmic reticulum Ca2+ release in metastatic breast cancer cells and this may have an important role in breast tumors where [Na+ ]i is perturbed.
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Neoplasias de la Mama , Señalización del Calcio , Humanos , Femenino , Señalización del Calcio/fisiología , Sodio/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Canales Iónicos/metabolismo , Calcio/metabolismoRESUMEN
Scn1b-null mice have a severe neurological and cardiac phenotype. Human mutations in SCN1B result in epilepsy and cardiac arrhythmia. SCN1B is expressed as two developmentally regulated splice variants, ß1 and ß1B, that are each expressed in brain and heart in rodents and humans. Here, we studied the structure and function of ß1B and investigated a novel human SCN1B epilepsy-related mutation (p.G257R) unique to ß1B. We show that wild-type ß1B is not a transmembrane protein, but a soluble protein expressed predominantly during embryonic development that promotes neurite outgrowth. Association of ß1B with voltage-gated Na+ channels Na(v)1.1 or Na(v)1.3 is not detectable by immunoprecipitation and ß1B does not affect Na(v)1.3 cell surface expression as measured by [(3)H]saxitoxin binding. However, ß1B coexpression results in subtle alteration of Na(v)1.3 currents in transfected cells, suggesting that ß1B may modulate Na+ current in brain. Similar to the previously characterized p.R125C mutation, p.G257R results in intracellular retention of ß1B, generating a functional null allele. In contrast, two other SCN1B mutations associated with epilepsy, p.C121W and p.R85H, are expressed at the cell surface. We propose that ß1B p.G257R may contribute to epilepsy through a mechanism that includes intracellular retention resulting in aberrant neuronal pathfinding.