RESUMO
Pancreatic stellate cells (PSCs) that can co-metastasize with cancer cells shape the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) by producing an excessive amount of extracellular matrix. This leads to a TME characterized by increased tissue pressure, hypoxia, and acidity. Moreover, cells within the tumor secrete growth factors. The stimuli of the TME trigger Ca2+ signaling and cellular Na+ loading. The Na+/Ca2+ exchanger (NCX) connects the cellular Ca2+ and Na+ homeostasis. The NCX is an electrogenic transporter, which shuffles 1 Ca2+ against 3 Na+ ions over the plasma membrane in a forward or reverse mode. Here, we studied how the impact of NCX activity on PSC migration is modulated by cues from the TME. NCX expression was revealed with qPCR and Western blot. [Ca2+]i, [Na+]i, and the cell membrane potential were determined with the fluorescent indicators Fura-2, Asante NaTRIUM Green-2, and DiBAC4(3), respectively. PSC migration was quantified with live-cell imaging. To mimic the TME, PSCs were exposed to hypoxia, pressure, acidic pH (pH 6.6), and PDGF. NCX-dependent signaling was determined with Western blot analyses. PSCs express NCX1.3 and NCX1.9. [Ca2+]i, [Na+]i, and the cell membrane potential are 94.4 nmol/l, 7.4 mmol/l, and - 39.8 mV, respectively. Thus, NCX1 usually operates in the forward (Ca2+ export) mode. NCX1 plays a differential role in translating cues from the TME into an altered migratory behavior. When NCX1 is operating in the forward mode, its inhibition accelerates PSC migration. Thus, NCX1-mediated extrusion of Ca2+ contributes to a slow mode of migration of PSCs.
Assuntos
Células Estreladas do Pâncreas , Trocador de Sódio e Cálcio , Humanos , Trocador de Sódio e Cálcio/metabolismo , Células Estreladas do Pâncreas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transdução de Sinais , Hipóxia , Cálcio/metabolismoRESUMO
Loss of apical-basal polarity and downregulation of cell-cell contacts is a critical step during the pathogenesis of cancer. Both processes are regulated by the scaffolding protein Pals1, however, it is unclear whether the expression of Pals1 is affected in cancer cells and whether Pals1 is implicated in the pathogenesis of the disease.Using mRNA expression data and immunostainings of cancer specimen, we show that Pals1 is frequently downregulated in colorectal cancer, correlating with poorer survival of patients. We further found that Pals1 prevents cancer cell metastasis by controlling Rac1-dependent cell migration through inhibition of Arf6, which is independent of the canonical binding partners of Pals1. Loss of Pals1 in colorectal cancer cells results in increased Arf6 and Rac1 activity, enhanced cell migration and invasion in vitro and increased metastasis of transplanted tumor cells in mice. Thus, our data reveal a new function of Pals1 as a key inhibitor of cell migration and metastasis of colorectal cancer cells. Notably, this new function is independent of the known role of Pals1 in tight junction formation and apical-basal polarity.
Assuntos
Fator 6 de Ribosilação do ADP/metabolismo , Neoplasias Colorretais/patologia , Proteínas de Membrana/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Movimento Celular/fisiologia , Células HCT116 , Xenoenxertos , Humanos , Camundongos , Invasividade Neoplásica/patologiaRESUMO
Small-molecule probes for the inâ vitro imaging of KCa 3.1 channel-expressing cells were developed. Senicapoc, showing high affinity and selectivity for the KCa 3.1 channels, was chosen as the targeting component. BODIPY dyes 15-20 were synthesized and connected by a CuI -catalyzed azide-alkyne [3+2]cycloaddition with propargyl ether senicapoc derivative 8, yielding fluorescently labeled ligands 21-26. The dimethylpyrrole-based imaging probes 25 and 26 allow staining of KCa 3.1 channels in NSCLC cells. The specificity was shown by removing the punctate staining pattern by pre-incubation with senicapoc. The density of KCa 3.1 channels detected with 25 and by immunostaining was identical. The punctate structure of the labeled channels could also be observed in living cells. Molecular modeling showed binding of the senicapoc-targeting component towards the binding site within the ion channel and orientation of the linker with the dye along the inner surface of the ion channel.
Assuntos
Corantes Fluorescentes/química , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Células A549 , Sítios de Ligação , Compostos de Boro/química , Reação de Cicloadição , Corantes Fluorescentes/síntese química , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/química , Ligantes , Microscopia de Fluorescência , Simulação de Dinâmica MolecularRESUMO
The article describes the development of Buchwald-Hartwig amination of different bromoflavones with amino acid and peptide derivatives as nitrogen source giving unique structures. The previously observed racemization, which occurred during the synthesis of flavone-amino acid hybrids, was successfully prevented in most cases. The biological assays of these novel structures showed cytotoxic effects on different cancer cell lines.
Assuntos
Aminoácidos/química , Dipeptídeos/química , Flavonas/química , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Cinética , Testes de Sensibilidade Microbiana , Análise Espectral/métodosRESUMO
In vitro manipulation of membrane sterol level affects the regulation of ion channels and consequently certain cellular functions; however, a comprehensive study that confirms the pathophysiological significance of these results is missing. The malfunction of 7-dehydrocholesterol (7DHC) reductase in Smith-Lemli-Opitz syndrome (SLOS) leads to the elevation of the 7-dehydrocholesterol level in the plasma membrane. T lymphocytes were isolated from SLOS patients to assess the effect of the in vivo altered membrane sterol composition on the operation of the voltage-gated Kv1.3 channel and the ion channel-dependent mitogenic responses. We found that the kinetic and equilibrium parameters of Kv1.3 activation changed in SLOS cells. Identical changes in Kv1.3 operation were observed when control/healthy T cells were loaded with 7DHC. Removal of the putative sterol binding sites on Kv1.3 resulted in a phenotype that was not influenced by the elevation in membrane sterol level. Functional assays exhibited impaired activation and proliferation rate of T cells probably partially due to the modified Kv1.3 operation. We concluded that the altered membrane sterol composition hindered the operation of Kv1.3 as well as the ion channel-controlled T cell functions.
Assuntos
Canal de Potássio Kv1.3/metabolismo , Síndrome de Smith-Lemli-Opitz/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Estudos de Casos e Controles , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Criança , Desidrocolesteróis/metabolismo , Humanos , FenótipoRESUMO
Voltage-clamp fluorometry (VCF) enables the study of voltage-sensitive proteins through fluorescent labeling accompanied by ionic current measurements for voltage-gated ion channels. The heterogeneity of the fluorescent signal represents a significant challenge in VCF. The VCF signal depends on where the cysteine mutation is incorporated, making it difficult to compare data among different mutations and different studies and standardize their interpretation. We have recently shown that the VCF signal originates from quenching amino acids in the vicinity of the attached fluorophores, together with the effect of the lipid microenvironment. Based on these, we performed experiments to test the hypothesis that the VCF signal could be altered by amphiphilic quenching molecules in the cell membrane. Here we show that a phenylalanine-conjugated flavonoid (4-oxo-2-phenyl-4H-chromene-7-yl)-phenylalanine, (later Oxophench) has potent effects on the VCF signals of the Ciona intestinalis HV1 (CiHv1) proton channel. Using spectrofluorimetry, we showed that Oxophench quenches TAMRA (5(6)-carboxytetramethylrhodamine-(methane thiosulfonate)) fluorescence. Moreover, Oxophench reduces the baseline fluorescence in oocytes and incorporates into the cell membrane while reducing the membrane fluidity of HEK293 cells. Our model calculations confirmed that Oxophench, a potent membrane-bound quencher, modifies the VCF signal during conformational changes. These results support our previously published model of VCF signal generation and point out that a change in the VCF signal may not necessarily indicate an altered conformational transition of the investigated protein.
Assuntos
Membrana Celular , Ciona intestinalis , Fluorometria , Técnicas de Patch-Clamp , Fenilalanina , Animais , Membrana Celular/metabolismo , Membrana Celular/química , Fluorometria/métodos , Ciona intestinalis/metabolismo , Ciona intestinalis/química , Ciona intestinalis/genética , Fenilalanina/química , Fenilalanina/análogos & derivados , Oócitos/metabolismo , Flavonoides/química , Flavonoides/farmacologia , Xenopus laevis , Canais Iônicos/metabolismo , Canais Iônicos/química , Corantes Fluorescentes/química , HumanosRESUMO
Pancreatic stellate cells (PSCs) are primarily responsible for producing the stiff tumor tissue in pancreatic ductal adenocarcinoma (PDAC). Thereby, PSCs generate a stiffness gradient between the healthy pancreas and the tumor. This gradient induces durotaxis, a form of directional cell migration driven by differential stiffness. The molecular sensors behind durotaxis are still unclear. To investigate the role of mechanosensitive ion channels in PSC durotaxis, we established a two-dimensional stiffness gradient mimicking PDAC. Using pharmacological and genetic methods, we investigated the role of the ion channels Piezo1, TRPC1, and TRPV4 in PSC durotaxis. We found that PSC migration towards a stiffer substrate is diminished by altering Piezo1 activity. Moreover, disrupting TRPC1 along with TRPV4 abolishes PSC durotaxis even when Piezo1 is functional. Hence, PSC durotaxis is optimal with an intermediary level of mechanosensitive channel activity, which we simulated using a numerically discretized mathematical model. Our findings suggest that mechanosensitive ion channels, particularly Piezo1, detect the mechanical microenvironment to guide PSC migration.
RESUMO
Atherosclerosis involves immune mechanisms: T lymphocytes are found in atherosclerotic plaques, suggesting their activation during atherogenesis. The predominant voltage-gated potassium channel of T cells, Kv1.3 is a key regulator of the Ca(2+)-dependent activation pathway. In the present experiments we studied the proliferation capacity and functional changes of Kv1.3 channels in T cells from healthy and hypercholestaeremic patients. By means of CFSE-assay (carboxyfluorescein succinimidyl ester) we showed that spontaneous activation rate of lymphocytes in hypercholesterolemia was elevated and the antiCD3/antiCD28 co-stimulation was less effective as compared to the healthy group. Using whole-cell patch-clamping we obtained that the activation and deactivation kinetics of Kv1.3 channels were faster in hypercholesterolemic state but no change in other parameters of Kv1.3 were found (inactivation kinetics, steady-state activation, expression level). We suppose that incorporation of oxLDL species via its raft-rupturing effect can modify proliferative rate of T cells as well as the gating of Kv1.3 channels.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Hipercolesterolemia/metabolismo , Canal de Potássio Kv1.3/metabolismo , Potássio/metabolismo , Adulto , Antígenos CD28/imunologia , Complexo CD3/imunologia , Cálcio/metabolismo , Proliferação de Células , Feminino , Fluoresceínas , Humanos , Hipercolesterolemia/imunologia , Ativação do Canal Iônico/fisiologia , Canal de Potássio Kv1.3/biossíntese , Ativação Linfocitária , Masculino , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Técnicas de Patch-Clamp , SuccinimidasRESUMO
Pancreatic ductal adenocarcinoma (PDAC) stands as a highly aggressive and lethal cancer, characterized by a grim prognosis and scarce treatment alternatives. Within this context, TRPV6, a calcium-permeable channel, emerges as a noteworthy candidate due to its overexpression in various cancers, capable of influencing the cell behavior in different cancer entities. Nonetheless, the exact expression pattern and functional significance of TRPV6 in the context of PDAC remains enigmatic. This study scrutinizes the expression of TRPV6 in tissue specimens obtained from 46 PDAC patients across distinct stages and grades. We manipulated TRPV6 expression (knockdown, overexpression) in the human PDAC cell lines Panc-1 and Capan-1. Subsequently, we analyzed its impact on multiple facets, encompassing Ca2+ influx, proliferation, apoptosis, migration, chemoresistance, and tumor growth, both in vitro and in vivo. Notably, the data indicate a direct correlation between TRPV6 expression levels, tumor stage, and grade, establishing a link between TRPV6 and PDAC proliferation in tissue samples. Decreasing TRPV6 expression via knockdown hampered Ca2+ influx, resulting in diminished proliferation and viability in both cell lines, and cell cycle progression in Panc-1. The knockdown simultaneously led to an increase in apoptotic rates and increased the susceptibility of cells to 5-FU and gemcitabine treatments. Moreover, it accelerated migration and promoted collective movement among Panc-1 cells. Conversely, TRPV6 overexpression yielded opposing outcomes in terms of proliferation in Panc-1 and Capan-1, and the migration of Panc-1 cells. Intriguingly, both TRPV6 knockdown and overexpression diminished the process of tumor formation in vivo. This intricate interplay suggests that PDAC aggressiveness relies on a fine-tuned TRPV6 expression, raising its profile as a putative therapeutic target.
RESUMO
Pancreatic ductal adenocarcinoma (PDAC) progresses in an organ with a unique pH landscape, where the stroma acidifies after each meal. We hypothesized that disrupting this pH landscape during PDAC progression triggers pancreatic stellate cells (PSCs) and cancer-associated fibroblasts (CAFs) to induce PDAC fibrosis. We revealed that alkaline environmental pH was sufficient to induce PSC differentiation to a myofibroblastic phenotype. We then mechanistically dissected this finding, focusing on the involvement of the Na+/H+ exchanger NHE1. Perturbing cellular pH homeostasis by inhibiting NHE1 with cariporide partially altered the myofibroblastic PSC phenotype. To show the relevance of this finding in vivo, we targeted NHE1 in murine PDAC (KPfC). Indeed, tumor fibrosis decreased when mice received the NHE1-inhibitor cariporide in addition to gemcitabine treatment. Moreover, the tumor immune infiltrate shifted from granulocyte rich to more lymphocytic. Taken together, our study provides mechanistic evidence on how the pancreatic pH landscape shapes pancreatic cancer through tuning PSC differentiation.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Camundongos , Animais , Células Estreladas do Pâncreas/patologia , Linhagem Celular Tumoral , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Fenótipo , Homeostase , Fibrose , Neoplasias PancreáticasRESUMO
Pancreatic stellate cell (PSC) activation is a major event occurring during pancreatic ductal adenocarcinoma (PDAC) development. Up to now mechanisms underlying their activation by mechanical cues such as the elevated tissue pressure in PDAC remain poorly understood. Here we investigate the role of one potential mechano-transducer, TRPC1 ion channel, in PSC activation. Using pre-activated human siTRPC1 and murine TRPC1-KO PSCs, we show that TRPC1 promotes αSMA (α-smooth muscle actin) expression, the main activation marker, in cooperation with the phosphorylated SMAD2, under normal and elevated pressure. Functional studies following TRPC1 silencing demonstrate the dual role of TRPC1 in the modulation of PSC proliferation and IL-6 secretion through the activation of ERK1/2 and SMAD2 pathways. Moreover, pressurization changes the mechanical behavior of PSCs by increasing their cellular stiffness and emitted traction forces in a TRPC1-dependent manner. In summary, these results point to a role of TRPC1 channels in sensing and transducing the characteristic mechanical properties of the PDAC microenvironment in PSCs.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , MAP Quinases Reguladas por Sinal Extracelular , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/metabolismo , Proteína Smad2/metabolismo , Canais de Cátion TRPC , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
Glioblastoma (GBM) is the most aggressive glial tumor, where ion channels, including KCa1.1, are candidates for new therapeutic options. Since the auxiliary subunits linked to KCa1.1 in GBM are largely unknown we used electrophysiology combined with pharmacology and gene silencing to address the functional expression of KCa1.1/ß subunits complexes in both primary tumor cells and in the glioblastoma cell line U-87 MG. The pattern of the sensitivity (activation/inhibition) of the whole-cell currents to paxilline, lithocholic acid, arachidonic acid, and iberiotoxin; the presence of inactivation of the whole-cell current along with the loss of the outward rectification upon exposure to the reducing agent DTT collectively argue that KCa1.1/ß3 complex is expressed in U-87 MG. Similar results were found using human primary glioblastoma cells isolated from patient samples. Silencing the ß3 subunit expression inhibited carbachol-induced Ca2+ transients in U-87 MG thereby indicating the role of the KCa1.1/ß3 in the Ca2+ signaling of glioblastoma cells. Functional expression of the KCa1.1/ß3 complex, on the other hand, lacks cell cycle dependence. We suggest that the KCa1.1/ß3 complex may have diagnostic and therapeutic potential in glioblastoma in the future.
Assuntos
Glioblastoma , Humanos , Glioblastoma/genética , Transdução de Sinais/fisiologia , CarbacolRESUMO
Voltage-clamp fluorometry (VCF) supplies information about the conformational changes of voltage-gated proteins. Changes in the fluorescence intensity of the dye attached to a part of the protein that undergoes a conformational rearrangement upon the alteration of the membrane potential by electrodes constitute the signal. The VCF signal is generated by quenching and dequenching of the fluorescence as the dye traverses various local environments. Here we studied the VCF signal generation, using the Hv1 voltage-gated proton channel as a tool, which shares a similar voltage-sensor structure with voltage-gated ion channels but lacks an ion-conducting pore. Using mutagenesis and lipids added to the extracellular solution we found that the signal is generated by the combined effects of lipids during movement of the dye relative to the plane of the membrane and by quenching amino acids. Our 3-state model recapitulates the VCF signals of the various mutants and is compatible with the accepted model of two major voltage-sensor movements.
Assuntos
Ativação do Canal Iônico , Prótons , Canais Iônicos/metabolismo , Fluorometria , Aminoácidos , LipídeosRESUMO
Ewing sarcoma (EwS) is a rare and highly malignant bone tumor occurring mainly in childhood and adolescence. Physiologically, the bone is a central hub for Ca2+ homeostasis, which is severely disturbed by osteolytic processes in EwS. Therefore, we aimed to investigate how ion transport proteins involved in Ca2+ homeostasis affect EwS pathophysiology. We characterized the expression of 22 candidate genes of Ca2+-permeable or Ca2+-regulated ion channels in three EwS cell lines and found the Ca2+-activated K+ channel KCa2.1 (KCNN1) to be exceptionally highly expressed. We revealed that KCNN1 expression is directly regulated by the disease-driving oncoprotein EWSR1-FL1. Due to its consistent overexpression in EwS, KCNN1 mRNA could be a prognostic marker in EwS. In a large cohort of EwS patients, however, KCNN1 mRNA quantity does not correlate with clinical parameters. Several functional studies including patch clamp electrophysiology revealed no evidence for KCa2.1 function in EwS cells. Thus, elevated KCNN1 expression is not translated to KCa2.1 channel activity in EwS cells. However, we found that the low K+ conductance of EwS cells renders them susceptible to hypoosmotic solutions. The absence of a relevant K+ conductance in EwS thereby provides an opportunity for hypoosmotic therapy that can be exploited during tumor surgery.
RESUMO
Interaction of the long control region (LCR) and the E2 protein of HPV11s was studied by in silico modelling and in vitro functional analysis. Genomes of HPV11s from fifteen (six known and nine novel) patients (two solitary papillomas, eleven respiratory papillomatoses of different severity, one condyloma acuminatum and one cervical atypia) were sequenced; E2 polymorphisms were analysed in silico by protein modelling. E2 and LCR variants were cloned into pcDNA3.1+ expression vector and into pALuc reporter vector, respectively, transfected to HEp2 cells alone or in different combinations and the luciferase activity was measured. In the E2, the ubiquitous polymorphism K308R caused stronger binding between the dimers but did not alter DNA binding; E2s with this polymorphism were significantly less efficient than the reference in promoting LCR activity. The unique polymorphism Q86K changed the negative surface charge of E2 (Q86) to positive (K86). The unique polymorphisms S245F and N247T in the hinge region disrupt a probable phosphorylation site in a RXXS motif targeted by protein kinase A and B, but do not affect directly the amino acids critical to nuclear transport. Both unique patterns partly restored the LCR activating potential disrupted by K308R. A unique E2/E4 ORF with a 58-bp deletion leading to a frameshift and an early stop codon resulted in a practically nonfunctional E2, and was associated with a papillomatosis with dysplasia. When testing existing LCR-E2 combinations, LCR with intrinsically lower enhancer capacity was only marginally activated by its E2 (R308 and the deletion mutant), and did not significantly exceed the activity of the reference LCR without E2. Combined with more potent LCRs associated with more severe disease, the activity was significantly higher, but still significantly lower than LCRs with reference E2. In summary, LCR-E2 interaction determined by their polymorphisms may explain, at least partly, differences in disease severity.
Assuntos
Papillomavirus Humano 11/genética , Papiloma/virologia , Infecções por Papillomavirus/virologia , Polimorfismo Genético , Proteínas Virais/genética , Condiloma Acuminado/virologia , Feminino , Humanos , Masculino , Infecções Respiratórias/virologia , Índice de Gravidade de DoençaRESUMO
Orai3 calcium (Ca2+) channels are implicated in multiple breast cancer processes, such as proliferation and survival as well as resistance to chemotherapy. However, their involvement in the breast cancer cell migration processes remains vague. In the present study, we exploited MDA-MB-231 and MDA-MB-231 BrM2 basal-like estrogen receptor-negative (ER-) cell lines to assess the direct role of Orai3 in cell migration. We showed that Orai3 regulates MDA-MB-231 and MDA-MB-231 BrM2 cell migration in two distinct ways. First, we showed that Orai3 remodels cell adhesive capacities by modulating the intracellular Ca2+ concentration. Orai3 silencing (siOrai3) decreased calpain activity, cell adhesion and migration in a Ca2+-dependent manner. In addition, Orai3 interacts with focal adhesion kinase (FAK) and regulates the actin cytoskeleton, in a Ca2+-independent way. Thus, siOrai3 modulates cell morphology by altering F-actin polymerization via a loss of interaction between Orai3 and FAK. To summarize, we demonstrated that Orai3 regulates cell migration through a Ca2+-dependent modulation of calpain activity and, in a Ca2+-independent manner, the actin cytoskeleton architecture via FAK.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Movimento Celular , Calpaína/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Forma Celular , Feminino , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Ligação ProteicaRESUMO
Nuclear pore complexes (NPCs) selectively mediate all nucleocytoplasmic transport and engage in fundamental cell-physiological processes. It is hypothesized that NPCs are critical for malignant transformation and survival of lung cancer cells, and test the hypothesis in lowly and highly metastatic non-small human lung cancer cells (NSCLCs). It is shown that malignant transformation is paralleled by an increased NPCs density, and a balanced pathological weakening of the physiological stringency of the NPC barrier. Pharmacological interference using barrier-breaking compounds collapses the stringency. Concomitantly, it induces drastic overall structural changes of NSCLCs, terminating their migration. Moreover, the degree of malignancy is found to be paralleled by substantially decreased lamin A/C levels. The latter provides crucial structural and mechanical stability to the nucleus, and interacts with NPCs, cytoskeleton, and nucleoskeleton for cell maintenance, survival, and motility. The recent study reveals the physiological importance of the NPC barrier stringency for mechanical and structural resilience of normal cell nuclei. Hence, reduced lamin A/C levels in conjunction with controlled pathological weakening of the NPC barrier stringency may facilitate deformability of NSCLCs during the metastasis steps. Modulation of the NPC barrier presents a potential strategy for suppressing the malignant phenotype or enhancing the effectiveness of currently existing chemotherapeutics.
Assuntos
Neoplasias Pulmonares/metabolismo , Membrana Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Sobrevivência Celular , Modelos Animais de Doenças , Humanos , Camundongos , Poro Nuclear/metabolismoRESUMO
The Ca2+ activated potassium channel 3.1 (KCa 3.1) is involved in critical steps of the metastatic cascade, such as proliferation, migration, invasion and extravasation. Therefore, a fast and efficient protocol for imaging of KCa 3.1 channels was envisaged. The novel fluorescently labeled small molecule imaging probes 1 and 2 were synthesized by connecting a dimethylpyrrole-based BODIPY dye with a derivative of the KCa 3.1 channel inhibitor senicapoc via linkers of different length. Patch-clamp experiments revealed the inhibition of KCa 3.1 channels by the probes confirming interaction with the channel. Both probes 1 and 2 were able to stain KCa 3.1 channels in non-small-cell lung cancer (NSCLC) cells following a simple, fast and efficient protocol. Pre-incubation with unlabeled senicapoc removed the punctate staining pattern showing the specificity of the new probes 1 and 2. Staining of the channel with the fluorescently labeled senicapoc derivatives 1 or 2 or with antibody-based indirect immunofluorescence yielded identical or very similar densities of stained KCa 3.1 channels. However, co-staining using both methods did not lead to the expected overlapping punctate staining pattern. This observation was explained by docking studies showing that the antibody used for indirect immunofluorescence and the probes 1 and 2 label different channel populations. Whereas the antibody binds at the closed channel conformation, the probes 1 and 2 bind within the open channel.
Assuntos
Acetamidas/farmacologia , Compostos de Boro/farmacologia , Corantes Fluorescentes/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Compostos de Tritil/farmacologia , Células A549 , Acetamidas/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Compostos de Boro/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Corantes Fluorescentes/metabolismo , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/imunologia , Camundongos , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Ligação Proteica , Coloração e Rotulagem , Compostos de Tritil/metabolismoRESUMO
The importance of the intracellular Ca2+ concentration ([Ca2+]i) in neutrophil function has been intensely studied. However, the role of the intracellular Na+ concentration ([Na+]i) which is closely linked to the intracellular Ca2+ regulation has been largely overlooked. The [Na+]i is regulated by Na+ transport proteins such as the Na+/Ca2+-exchanger (NCX1), Na+/K+-ATPase, and Na+-permeable, transient receptor potential melastatin 2 (TRPM2) channel. Stimulating with either N-formylmethionine-leucyl-phenylalanine (fMLF) or complement protein C5a causes distinct changes of the [Na+]i. fMLF induces a sustained increase of [Na+]i, surprisingly, reaching higher values in TRPM2-/- neutrophils. This outcome is unexpected and remains unexplained. In both genotypes, C5a elicits only a transient rise of the [Na+]i. The difference in [Na+]i measured at t = 10 min after stimulation is inversely related to neutrophil chemotaxis. Neutrophil chemotaxis is more efficient in C5a than in an fMLF gradient. Moreover, lowering the extracellular Na+ concentration from 140 to 72 mM improves chemotaxis of WT but not of TRPM2-/- neutrophils. Increasing the [Na+]i by inhibiting the Na+/K+-ATPase results in disrupted chemotaxis. This is most likely due to the impact of the altered Na+ homeostasis and presumably NCX1 function whose expression was shown by means of qPCR and which critically relies on proper extra- to intracellular Na+ concentration gradients. Increasing the [Na+]i by a few mmol/l may suffice to switch its transport mode from forward (Ca2+-efflux) to reverse (Ca2+-influx) mode. The role of NCX1 in neutrophil chemotaxis is corroborated by its blocker, which also causes a complete inhibition of chemotaxis.