RESUMEN
SK3 channels are potassium channels found to promote tumor aggressiveness. We have previously demonstrated that SK3 is regulated by synthetic ether lipids, but the role of endogenous ether lipids is unknown. Here, we have studied the role of endogenous alkyl- and alkenyl-ether lipids on SK3 channels and on the biology of cancer cells. Experiments revealed that the suppression of alkylglycerone phosphate synthase or plasmanylethanolamine desaturase 1, which are key enzymes for alkyl- and alkenyl-ether-lipid synthesis, respectively, decreased SK3 expression by increasing micro RNA (miR)-499 and miR-208 expression, leading to a decrease in SK3-dependent calcium entry, cell migration, and matrix metalloproteinase 9-dependent cell adhesion and invasion. We identified several ether lipids that promoted SK3 expression and found a differential role of alkyl- and alkenyl-ether lipids on SK3 activity. The expressions of alkylglycerone phosphate synthase, SK3, and miR were associated in clinical samples emphasizing the clinical consistency of our observations. To our knowledge, this is the first report showing that ether lipids differentially control tumor aggressiveness by regulating an ion channel. This insight provides new possibilities for therapeutic interventions, offering clinicians an opportunity to manipulate ion channel dysfunction by adjusting the composition of ether lipids.
Asunto(s)
Canales de Potasio de Pequeña Conductancia Activados por el Calcio , Humanos , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Movimiento Celular , MicroARNs/metabolismo , MicroARNs/genética , Lípidos/química , Línea Celular Tumoral , Invasividad Neoplásica , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias/genéticaRESUMEN
T-cell receptor stimulation triggers cytosolic Ca2+ signaling by inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum (ER) and Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels gated by ER-located stromal-interacting molecules (STIM1/2). Physiologically, cytosolic Ca2+ signaling manifests as regenerative Ca2+ oscillations, which are critical for nuclear factor of activated T-cells-mediated transcription. In most cells, Ca2+ oscillations are thought to originate from IP3 receptor-mediated Ca2+ release, with CRAC channels indirectly sustaining them through ER refilling. Here, experimental and computational evidence support a multiple-oscillator mechanism in Jurkat T-cells whereby both IP3 receptor and CRAC channel activities oscillate and directly fuel antigen-evoked Ca2+ oscillations, with the CRAC channel being the major contributor. KO of either STIM1 or STIM2 significantly reduces CRAC channel activity. As such, STIM1 and STIM2 synergize for optimal Ca2+ oscillations and activation of nuclear factor of activated T-cells 1 and are essential for ER refilling. The loss of both STIM proteins abrogates CRAC channel activity, drastically reduces ER Ca2+ content, severely hampers cell proliferation and enhances cell death. These results clarify the mechanism and the contribution of STIM proteins to Ca2+ oscillations in T-cells.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio , Señalización del Calcio , Humanos , Calcio/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Señalización del Calcio/genética , Células Jurkat , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/genética , Molécula de Interacción Estromal 2/metabolismo , Técnicas de Inactivación de Genes , Modelos Biológicos , Isoformas de Proteínas , Transporte de Proteínas/genética , Proliferación Celular/genética , Supervivencia Celular/genéticaRESUMEN
The intracellular Ca2+ concentration is mainly controlled by Ca2+ channels. These channels form complexes with K+ channels, which function to amplify Ca2+ flux. In cancer cells, voltage-gated/voltage-dependent Ca2+ channels and non-voltage-gated/voltage-independent Ca2+ channels have been reported to interact with K+ channels such as Ca2+-activated K+ channels and voltage-gated K+ channels. These channels are activated by an increase in cytosolic Ca2+ concentration or by membrane depolarization, which induces membrane hyperpolarization, increasing the driving force for Ca2+ flux. These complexes, composed of K+ and Ca2+ channels, are regulated by several molecules including lipids (ether lipids and cholesterol), proteins (e.g. STIM), receptors (e.g. S1R/SIGMAR1), and peptides (e.g. LL-37) and can be targeted by monoclonal antibodies, making them novel targets for cancer research.
Asunto(s)
Neoplasias , Canales de Potasio con Entrada de Voltaje , Calcio/metabolismo , Canales de Calcio/metabolismo , Humanos , Lípidos , Neoplasias/tratamiento farmacológico , Potasio/metabolismo , Canales de Potasio/metabolismoRESUMEN
Glioblastoma is the most aggressive and invasive form of central nervous system tumors due to the complexity of the intracellular mechanisms and molecular alterations involved in its progression. Unfortunately, current therapies are unable to stop its neoplastic development. In this context, we previously identified and characterized AaTs-1, a tetrapeptide (IWKS) from Androctonus autralis scorpion venom, which displayed an anti-proliferative effect against U87 cells with an IC50 value of 0.57 mM. This peptide affects the MAPK pathway, enhancing the expression of p53 and altering the cytosolic calcium concentration balance, likely via FPRL-1 receptor modulation. In this work, we designed and synthesized new dendrimers multi-branched molecules based on the sequence of AaTs-1 and showed that the di-branched (AaTs-1-2B), tetra-branched (AaTs-1-4B) and octo-branched (AaTs-1-8B) dendrimers displayed 10- to 25-fold higher effects on the proliferation of U87 cells than AaTs-1. We also found that the effects of the newly designed molecules are mediated by the enhancement of the ERK1/2 and AKT phosphorylated forms and by the increase in p53 expression. Unlike AaTs-1, AaTs-1-8B and especially AaTs-1-4B affected the migration of the U87 cells. Thus, the multi-branched peptide synthesis strategy allowed us to make molecules more active than the linear peptide against the proliferation of U87 glioblastoma cells.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Oligopéptidos/farmacología , Venenos de Escorpión/farmacología , Animales , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dendrímeros/química , Dendrímeros/farmacología , Humanos , Oligopéptidos/química , Venenos de Escorpión/química , EscorpionesRESUMEN
Glioblastoma is an aggressive cancer, against which medical professionals are still quite helpless, due to its resistance to current treatments. Scorpion toxins have been proposed as a promising alternative for the development of effective targeted glioblastoma therapy and diagnostic. However, the exploitation of the long peptides could present disadvantages. In this work, we identified and synthetized AaTs-1, the first tetrapeptide from Androctonus australis scorpion venom (Aa), which exhibited an antiproliferative effect specifically against human glioblastoma cells. Both the native and synthetic AaTs-1 were endowed with the same inhibiting effect on the proliferation of U87 cells with an IC50 of 0.56 mM. Interestingly, AaTs-1 was about two times more active than the anti-glioblastoma conventional chemotherapeutic drug, temozolomide (TMZ), and enhanced its efficacy on U87 cells. AaTs-1 showed a significant similarity with the synthetic peptide WKYMVm, an agonist of a G-coupled formyl-peptide receptor, FPRL-1, known to be involved in the proliferation of glioma cells. Interestingly, the tetrapeptide triggered the dephosphorylation of ERK, p38, and JNK kinases. It also enhanced the expression of p53 and FPRL-1, likely leading to the inhibition of the store operated calcium entry. Overall, our work uncovered AaTs-1 as a first natural potential FPRL-1 antagonist, which could be proposed as a promising target to develop new generation of innovative molecules used alone or in combination with TMZ to improve glioblastoma treatment response. Its chemical synthesis in non-limiting quantity represents a valuable advantage to design and develop low-cost active analogues to treat glioblastoma cancer.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma , Oligopéptidos/farmacología , Receptores de Formil Péptido/biosíntesis , Receptores de Lipoxina/biosíntesis , Venenos de Escorpión/química , Proteína p53 Supresora de Tumor/biosíntesis , Regulación hacia Arriba/efectos de los fármacos , Animales , Antineoplásicos/química , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Humanos , Oligopéptidos/química , EscorpionesRESUMEN
Ether lipids (ELs) are lipids characterized by the presence of either an ether linkage (alkyl lipids) or a vinyl ether linkage [i.e., plasmalogens (Pls)] at the sn1 position of the glycerol backbone, and they are enriched in PUFAs at the sn2 position. In this review, we highlight that ELs have various biological functions, act as a reservoir for second messengers (such as PUFAs) and have roles in many diseases. Some of the biological effects of ELs may be associated with their ability to regulate ion channels that control excitation-contraction/secretion/mobility coupling and therefore cell physiology. These channels are embedded in lipid membranes, and lipids can regulate their activities directly or indirectly as second messengers or by incorporating into membranes. Interestingly, ELs and EL-derived PUFAs have been reported to play a key role in several pathologies, including neurological disorders, cardiovascular diseases, and cancers. Investigations leading to a better understanding of their mechanisms of action in pathologies have opened a new field in cancer research. In summary, newly identified lipid regulators of ion channels, such as ELs and PUFAs, may represent valuable targets to improve disease diagnosis and advance the development of new therapeutic strategies for managing a range of diseases and conditions.
Asunto(s)
Éter/química , Ácidos Grasos Insaturados/metabolismo , Canales Iónicos/metabolismo , Animales , HumanosRESUMEN
In prostate cancer research, there is a lack of valuable preclinical models. Tumor cell heterogeneity and sensitivity to microenvironment signals, such as hypoxia or extracellular calcium concentration, are difficult to reproduce. Here, we developed and characterized an ex vivo tissue culture model preserving these properties. Prostate tissue slices from 26 patients were maintained ex vivo under optimized culture conditions. The expression of markers associated with proliferation, androgen-receptor signaling, and hypoxia was assessed by immunostaining. A macroscope was used to achieve real-time calcium fluorescence optical imaging. Tissue morphology was maintained successfully without necrosis for 5 days. Compared with native tumors and tissue cultured with androgens, androgen deprivation in the medium led to decreased expression of both androgen receptor and its target gene products, prostate specific antigen (PSA) and ETS-related gene (ERG). Ex vivo cultured slices also were sensitive to hypoxia because carbonic anhydrase IX and zinc finger E-box binding homeobox 1 (Zeb1) protein levels increased in 1% oxygen. Exposure of slices to supraphysiological extracellular Ca2+ concentration induced a robust and rapid Ca2+ entry, with a greater response in tumor compared with nontumor tissue. This ex vivo model reproduces the morphologic and functional characteristics of human prostate cancer, including sensitivity to androgen deprivation and induced response to hypoxia and extracellular Ca2+. It therefore could become an attractive tool for drug response prediction studies.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Modelos Biológicos , Neoplasias de la Próstata/metabolismo , Microambiente Tumoral , Anciano , Antígenos de Neoplasias/metabolismo , Anhidrasa Carbónica IX/metabolismo , Hipoxia de la Célula , Humanos , Calicreínas/metabolismo , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/terapia , Técnicas de Cultivo de Tejidos , Regulador Transcripcional ERG/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismoRESUMEN
Hypoxia is a well-established feature of prostate cancer (PCa) and is associated with disease aggressiveness. The hypoxic microenvironment initiates multiple adaptive responses including epithelial-to-mesenchymal transition (EMT) and a remodeling of calcium homeostasis involved in cancer progression. In the present study, we identified a new hypoxia signaling pathway with a positive feedback loop between the EMT transcription factor Zeb1 and SK3, a Ca2+-activated K+ channel, which leads to amplifying store-operated Ca2+ entry. Zeb1 and SK3 channel were strongly upregulated by hypoxia both in vitro and ex vivo in organotypic cultures of human PCa. Taking into account the sensitivity of the SK3 channel to the membrane lipid composition, we identified lipids such as Ohmline (an alkyl ether lipid and SK3 inhibitor), linoleic acid (LA) and eicosapentaenoic acid (EPA) (fatty acids associated with indolent PCa), which were able to completely abrogate the hypoxia-induced changes in Zeb1 expression. Ultimately, better understanding of this new hypoxia-induced EMT pathway may allow to develop adjuvant therapeutic strategies, in order to control PCa aggressiveness and improve treatment outcomes.
Asunto(s)
Transición Epitelial-Mesenquimal , Hipoxia/fisiopatología , Neoplasias de la Próstata/patología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Microambiente Tumoral , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Línea Celular Tumoral , Movimiento Celular , Ácido Eicosapentaenoico/farmacología , Glucolípidos/farmacología , Humanos , Ácido Linoleico/farmacología , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/antagonistas & inhibidoresRESUMEN
Tight control of basal cytosolic Ca2+ concentration is essential for cell survival and to fine-tune Ca2+-dependent cell functions. A way to control this basal cytosolic Ca2+ concentration is to regulate membrane Ca2+ channels including store-operated Ca2+ channels and secondary messenger-operated channels linked to G-protein-coupled or tyrosine kinase receptor activation. Orai, with or without its reticular STIM partner and Transient Receptor Potential (TRP) proteins, were considered to be the main Ca2+ channels involved. It is well accepted that, in response to cell stimulation, opening of these Ca2+ channels contributes to Ca2+ entry and the transient increase in cytosolic Ca2+ concentration involved in intracellular signaling. However, in various experimental conditions, Ca2+ entry and/or Ca2+ currents can be recorded at rest, without application of any experimental stimulation. This led to the proposition that some plasma membrane Ca2+ channels are already open/activated in basal condition, contributing therefore to constitutive Ca2+ entry. This article focuses on direct and indirect observations supporting constitutive activity of channels belonging to the Orai and TRP families and on the mechanisms underlying their basal/constitutive activities.
Asunto(s)
Calcio/metabolismo , Neoplasias/metabolismo , Animales , Señalización del Calcio , Humanos , Neoplasias/patologíaRESUMEN
Membrane lipid rafts are distinct plasma membrane nanodomains that are enriched with cholesterol, sphingolipids and gangliosides, with occasional presence of saturated fatty acids and phospholipids containing saturated acyl chains. It is well known that they organize receptors (such as Epithelial Growth Factor Receptor), ion channels and their downstream acting molecules to regulate intracellular signaling pathways. Among them are Ca2+ signaling pathways, which are modified in tumor cells and inhibited upon membrane raft disruption. In addition to protein components, lipids from rafts also contribute to the organization and function of Ca2+ signaling microdomains. This article aims to focus on the lipid raft KCa/ClCa/Ca2+ channel complexes that regulate Ca2+ and EGFR signaling in cancer cells, and discusses the potential modification of these complexes by lipids as a novel therapeutic approach in tumor development. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.
Asunto(s)
Antineoplásicos/uso terapéutico , Calcio/metabolismo , Regulación Neoplásica de la Expresión Génica , Lípidos de la Membrana/antagonistas & inhibidores , Microdominios de Membrana/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Canales de Calcio/genética , Canales de Calcio/metabolismo , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Ácidos Grasos Omega-3/uso terapéutico , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ácidos Linoleicos Conjugados/uso terapéutico , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/metabolismo , Microdominios de Membrana/ultraestructura , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Canales de Potasio/genética , Canales de Potasio/metabolismo , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Cystic fibrosis (CF) is an inherited disease associated with chronic severe lung inflammation, leading to premature death. To develop innovative anti-inflammatory treatments, we need to characterize new cellular and molecular components contributing to the mechanisms of lung inflammation. Here, we focused on the potential role of "transient receptor potential vanilloid-4" (TRPV4), a nonselective calcium channel. We used both in vitro and in vivo approaches to demonstrate that TRPV4 expressed in airway epithelial cells triggers the secretion of major proinflammatory mediators such as chemokines and biologically active lipids, as well as a neutrophil recruitment in lung tissues. We characterized the contribution of cytosolic phospholipase A2, MAPKs, and NF-κB in TRPV4-dependent signaling. We also showed that 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids, i.e., four natural lipid-based TRPV4 agonists, are present in expectorations of CF patients. Also, TRPV4-induced calcium mobilization and inflammatory responses were enhanced in cystic fibrosis transmembrane conductance regulator-deficient cellular and animal models, suggesting that TRPV4 is a promising target for the development of new anti-inflammatory treatments for diseases such as CF.
Asunto(s)
Células Epiteliales Alveolares/metabolismo , Fibrosis Quística/metabolismo , Canales Catiónicos TRPV/fisiología , Células A549 , Animales , Señalización del Calcio , Fibrosis Quística/inmunología , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Femenino , Humanos , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas Sprague-DawleyRESUMEN
Carney complex (CNC) is a hereditary disease associating cardiac myxoma, spotty skin pigmentation and endocrine overactivity. CNC is caused by inactivating mutations in the PRKAR1A gene encoding PKA type I alpha regulatory subunit (RIα). Although PKA activity is enhanced in CNC, the mechanisms linking PKA dysregulation to endocrine tumorigenesis are poorly understood. In this study, we used Förster resonance energy transfer (FRET)-based sensors for cAMP and PKA activity to define the role of RIα in the spatiotemporal organization of the cAMP/PKA pathway. RIα knockdown in HEK293 cells increased basal as well as forskolin or prostaglandin E1 (PGE1)-stimulated total cellular PKA activity as reported by western blots of endogenous PKA targets and the FRET-based global PKA activity reporter, AKAR3. Using variants of AKAR3 targeted to subcellular compartments, we identified similar increases in the response to PGE1 in the cytoplasm and at the outer mitochondrial membrane. In contrast, at the plasma membrane, the response to PGE1 was decreased along with an increase in basal FRET ratio. These results were confirmed by western blot analysis of basal and PGE1-induced phosphorylation of membrane-associated vasodilator-stimulated phosphoprotein. Similar differences were observed between the cytoplasm and the plasma membrane in human adrenal cells carrying a RIα inactivating mutation. RIα inactivation also increased cAMP in the cytoplasm, at the outer mitochondrial membrane and at the plasma membrane, as reported by targeted versions of the cAMP indicator Epac1-camps. These results show that RIα inactivation leads to multiple, compartment-specific alterations of the cAMP/PKA pathway revealing new aspects of signaling dysregulation in tumorigenesis.
Asunto(s)
Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Alprostadil/farmacología , Complejo de Carney/genética , Complejo de Carney/metabolismo , Membrana Celular/metabolismo , Colforsina/farmacología , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Silenciador del Gen , Células HEK293 , Humanos , Espacio Intracelular/metabolismo , Transporte de Proteínas , Interferencia de ARN , Transducción de SeñalRESUMEN
Potassium channels belong to the largest and the most diverse super-families of ion channels. Among them, Ca(2+)-activated K(+) channels (KCa) comprise many members. Based on their single channel conductance they are divided into three subfamilies: big conductance (BKCa), intermediate conductance (IKCa) and small conductance (SKCa; SK1, SK2 and SK3). Ca(2+) channels are divided into two main families, voltage gated/voltage dependent Ca(2+) channels and non-voltage gated/voltage independent Ca(2+) channels. Based on their electrophysiological and pharmacological properties and on the tissue where there are expressed, voltage gated Ca(2+) channels (Cav) are divided into 5 families: T-type, L-type, N-type, P/Q-type and R-type Ca(2+). Non-voltage gated Ca(2+) channels comprise the TRP (TRPC, TRPV, TRPM, TRPA, TRPP, TRPML and TRPN) and Orai (Orai1 to Orai3) families and their partners STIM (STIM1 to STIM2). A depolarization is needed to activate voltage-gated Ca(2+) channels while non-voltage gated Ca(2+) channels are activated by Ca(2+) depletion of the endoplasmic reticulum stores (SOCs) or by receptors (ROCs). These two Ca(2+) channel families also control constitutive Ca(2+) entries. For reducing the energy consumption and for the fine regulation of Ca(2+), KCa and Ca(2+) channels appear associated as complexes in excitable and non-excitable cells. Interestingly, there is now evidence that KCa-Ca(2+) channel complexes are also found in cancer cells and contribute to cancer-associated functions such as cell proliferation, cell migration and the capacity to develop metastases. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Células Eucariotas/metabolismo , Canales de Potasio Calcio-Activados/metabolismo , Subunidades de Proteína/metabolismo , Animales , Canales de Calcio/clasificación , Canales de Calcio/genética , Señalización del Calcio , Movimiento Celular , Proliferación Celular , Retículo Endoplásmico/metabolismo , Células Eucariotas/citología , Regulación de la Expresión Génica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Especificidad de Órganos , Canales de Potasio Calcio-Activados/clasificación , Canales de Potasio Calcio-Activados/genética , Subunidades de Proteína/clasificación , Subunidades de Proteína/genéticaRESUMEN
SK3 channel mediates the migration of various cancer cells. When expressed in breast cancer cells, SK3 channel forms a complex with Orai1, a voltage-independent Ca(2+) channel. This SK3-Orai1 complex associates within lipid rafts where it controls a constitutive Ca(2+) entry leading to cancer cell migration and bone metastases development. Since cAMP was found to modulate breast cancer cell migration, we hypothesized that this could be explained by a modulation of SK3 channel activity. Herein, we study the regulation of SK3 channel by the cAMP-PKA pathway and the consequences for SK3-dependent Ca(2+) entry and cancer cell migration. We established that the beta-adrenergic receptor agonist, isoprenaline, or the direct adenylyl cyclase activator forskolin alone or in combination with the PDE4 inhibitor, CI-1044, decreased SK3 channel activity without modifying the expression of SK3 protein at the plasma membrane. Forskolin and CI-1044 reduced the SK3-dependent constitutive Ca(2+) entry and the SK3-dependent migration of MDA-MB-435s cells. PKA inhibition with KT 5720 reduced: (1) the effect of forskolin and CI-1044 by 50 % on Ca(2+) entry and (2) SK3 activity by inhibiting the serine phosphorylation of SK3. These cAMP-elevating agents displaced Orai1 protein outside lipid rafts in contrast to SK3, which remained in the lipid rafts fractions. All together, these results show that activation of the cAMP-PKA pathway decreases SK3 channel and SK3-Orai1 complex activities, leading to a decrease in both Ca(2+) entry and cancer cell migration. This work supports the potential use of cAMP-elevating agents to reduce cancer cell migration and may provide novel opportunities to address/prevent bone metastasis.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Movimiento Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Agonistas Adrenérgicos beta/farmacología , Azepinas/farmacología , Carbazoles/farmacología , Línea Celular Tumoral , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Células HEK293 , Humanos , Isoproterenol/farmacología , Microdominios de Membrana/metabolismo , Niacinamida/análogos & derivados , Niacinamida/farmacología , Proteína ORAI1 , Inhibidores de Fosfodiesterasa 4/farmacología , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Pirroles/farmacologíaRESUMEN
No data are currently available on the functional role of small conductance Ca2+-activated K+ channels (SKCa) in ovarian cancer. Here, we characterized the role of SK2 (KCa2.2) in ovarian cancer cell migration and chemosensitivity. Using the selective non-cell-permeant SK2 inhibitor Lei-Dab7, we identified functional SK2 channels at the plasma membrane, regulating store-operated Ca2+ entry (SOCE) in both cell lines tested (COV504 and OVCAR3). Silencing KCNN2 with short interfering RNA (siRNA), or blocking SK2 activity with Lei-Dab7, decreased cell migration. The more robust effect of KCNN2 knockdown compared to Lei-Dab7 treatment suggested the involvement of functional intracellular SK2 channels in both cell lines. In cells treated with lysophosphatidic acid (LPA), an ovarian cancer biomarker of progression, SK2 channels are a key player of LPA pro-migratory activity but their role in SOCE is abolished. Concerning chemotherapy, SK2 inhibition increased chemoresistance to Taxol® and low KCNN2 mRNA expression was associated with the worst prognosis for progression-free survival in patients with serous ovarian cancer. The dual roles of SK2 mean that SK2 activators could be used as an adjuvant chemotherapy to potentiate treatment efficacy and SK2 inhibitors could be administrated as monotherapy to limit cancer cell dissemination.
Asunto(s)
Membrana Celular , Movimiento Celular , Neoplasias Ováricas , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , Humanos , Femenino , Neoplasias Ováricas/patología , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/tratamiento farmacológico , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/genética , Línea Celular Tumoral , Membrana Celular/metabolismo , Resistencia a Antineoplásicos/genética , Lisofosfolípidos/metabolismo , Calcio/metabolismoRESUMEN
The blood-brain barrier (BBB) maintains brain homeostasis, regulates influx and efflux transport, and provides protection to the brain tissue. Ultrasound (US) and microbubble (MB)-mediated blood-brain barrier opening is an effective and safe technique for drug delivery in-vitro and in-vivo. However, the exact mechanism underlying this technique is still not fully elucidated. The aim of the study is to explore the contribution of transcytosis in the BBB transient opening using an in-vitro model of BBB. Utilizing a diverse set of techniques, including Ca2+ imaging, electron microscopy, and electrophysiological recordings, our results showed that the combined use of US and MBs triggers membrane deformation within the endothelial cell membrane, a phenomenon primarily observed in the US + MBs group. This deformation facilitates the vesicles transportation of 500 kDa fluorescent Dextran via dynamin-/caveolae-/clathrin- mediated transcytosis pathway. Simultaneously, we observed increase of cytosolic Ca2+ concentration, which is related with increased permeability of the 500 kDa fluorescent Dextran in-vitro. This was found to be associated with the Ca2+-protein kinase C (PKC) signaling pathway. The insights provided by the acoustically-mediated interaction between the microbubbles and the cells delineate potential mechanisms for macromolecular substance permeability.
Asunto(s)
Barrera Hematoencefálica , Dextranos , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Acústica , Microburbujas , Colorantes , Sistemas de Liberación de Medicamentos/métodos , Comunicación CelularRESUMEN
The recent discoveries of the involvement of SK3 channel in some cell motility mechanisms occurring in cancer disease have opened up the way to the synthesis of inhibitors that could reduce metastasis formation. On the basis of our recent previous works showing that both lactose-glycero-ether lipid (Ohmline) and some phosphate analogues (GPGEL) were efficient compounds to modulate SK3 channel activity, the present study, which found its inspiration in the structure of the natural glycolipid DiGalactosylDiacylGlycerol (DGDG), reports the incorporation of a digalactosyl moiety (α-galactopyranosyl-(1â6)-ß-galactopyranosyl-) as the polar head of a glycero ether lipid. For the construction of the digalactosyl fragment, two synthetic approaches were compared. The standard strategy which is based on the use of the benzyl protecting group to produce 1â6 disaccharide unit, was compared with a second method that made use of the trimethylsilyl moiety as a protecting group. This second strategy, which is applied for the first time to the synthesis of (1â6)-disaccharide unit, presents a net advantage in terms of efficacy (better global yield) and cost. Finally, compound 16, which is characterized by a (1â6) DiGalactosyl unit (DG) as the polar head of the amphiphilic structure, was tested as a modulator of the SK3 channel activity. Patch-clamp experiments have shown that compound 16 reduced SK3 currents (-28.2 ± 2.0% at 5 µM) and cell migration assays performed at 300 nM have shown a reduction of cell migration (SK3 + HEK293T) by 19.6 ± 2.7%.
Asunto(s)
Glucolípidos/química , Glucolípidos/farmacología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/antagonistas & inhibidores , Movimiento Celular/efectos de los fármacos , Células HEK293 , Humanos , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismoRESUMEN
CD20 monoclonal antibodies (mAbs) eliminate B cells in several clinical contexts. At least two of these Abs, obinutuzumab (OBI) and rituximab (RTX), induce quick elimination of targets and put cancer patients at risk of tumor lysis syndrome (TLS) within 12-24 h of the first dose. The mechanisms of killing can require the recruiting of effector mechanisms from the patient's immune system, but they can induce direct killing as well. This can be more rapid than recruiting cellular effectors and/or complement. We showed here that OBI and RTX induce quick (<1 h) and high (up to 60% for OBI) killing of two different B cell lines. This was unveiled by using two different techniques that circumvent cell centrifugation steps: a Muse® Cell Analyzer-based approach and a direct examination of the cells' physical properties by using forward scatter (FS) area and side scatter (SS) area by flow cytometry. These results excluded the presence of aggregates and were also confirmed by developing a normalized survival ratio based on the co-incubation of RTX- and OBI-sensitive cells with MOLM-13, an insensitive cell line. Finally, this normalized survival ratio protocol confirmed the RTX- and OBI-direct killing on primary tumor B cells from B cell chronic lymphocytic leukemia (B-CLL) and Non-Hodgkin's lymphoma (NHL) patients. Moreover, we unveiled that direct killing is higher than previously expected and absent in patients' samples at relapse. We also observed that these mAbs, prior to increasing intracellular calcium levels, decrease calcium entry, although manipulating calcium levels did not affect their cytotoxicity. Altogether, our results show that direct killing is a major mechanism to induce cell death by RTX and OBI mAbs.
RESUMEN
Ion channels are transmembrane structures that allow the passage of ions across cell membranes such as the plasma membrane or the membranes of various organelles like the nucleus, endoplasmic reticulum, Golgi apparatus or mitochondria. Aberrant expression of various ion channels has been demonstrated in several tumor cells, leading to the promotion of key functions in tumor development, such as cell proliferation, resistance to apoptosis, angiogenesis, invasion and metastasis. The link between ion channels and these key biological functions that promote tumor development has led to the classification of cancers as oncochannelopathies. Among all ion channels, the most varied and numerous, forming the largest family, are the potassium channels, with over 70 genes encoding them in humans. In this context, this review will provide a non-exhaustive overview of the role of plasma membrane potassium channels in cancer, describing 1) the nomenclature and structure of potassium channels, 2) the role of these channels in the control of biological functions that promotes tumor development such as proliferation, migration and cell death, and 3) the role of two particular classes of potassium channels, the SKCa- and Kv1- type potassium channels in cancer progression.
Asunto(s)
Neoplasias , Canales de Potasio de la Superfamilia Shaker , Humanos , Neoplasias/patología , Apoptosis , Canales Iónicos , Canales de PotasioRESUMEN
Prostate cancer (PCa) represents one of the most frequent diagnosed cancer in males worldwide. Due to routine screening tests and the efficiency of available treatments, PCa-related deaths have significantly decreased over the past decades. However, PCa remains a critical threat if detected at a late stage in which, cancer cells would have already detached from the primary tumor to spread and invade other parts of the body. Calcium (Ca2+) channels and their protein regulators are now considered as hallmarks of cancer and some of them have been well examined in PCa. Among these Ca2+ channels, isoform 3 of the ORAI channel family has been shown to regulate the proliferation of PCa cells via the Arachidonic Acid-mediated Ca2+ entry, requiring the involvement of STIM1 (Stromal Interaction Molecule 1). Still, no study has yet demonstrated a role of the "neglected" STIM2 isoform in PCa or if it may interact with ORAI3 to promote an oncogenic behavior. In this study, we demonstrate that ORAI3 and STIM2 are upregulated in human PCa tissues. In old KIMAP (Knock-In Mouse Prostate Adenocarcinoma) mice, ORAI3 and STIM2 mRNA levels were significantly higher than ORAI1 and STIM1. In vitro, we show that ORAI3-STIM2 interact under basal conditions in PC-3 cells. ORAI3 silencing increased Store Operated Ca2+ Entry (SOCE) and induced a significant increase of the cell population in G2/M phase of the cell cycle, consistent with the role of ORAI3 as a negative regulator of SOCE. Higher expression levels of CDK1-Y15/Cyclin B1 were detected and mitotic arrest-related death occurred after ORAI3 silencing, which resulted in activating Bax/Bcl-2-mediated apoptotic pathway and caspase-8 activation and cleavage. STIM2 and ORAI3 expression increased in M phase while STIM1 expression and SOCE amplitude significantly decreased. Taken together, ORAI3 -STIM2 complex allows a successful progression through mitosis of PCa cells by evading mitotic catastrophe.