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
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
Ether lipids are composed of alkyl lipids with an ether bond at the sn-1 position of a glycerol backbone and alkenyl lipids, which possess a vinyl ether bond at the sn-1 position of the glycerol. These ether glycerolipids are present either as polar glycerophospholipids or neutral glycerolipids. Before studying the biological role of molecular species of ether glycerolipids, there is a need to separate and quantify total alkyl and alkenyl glycerolipids from biological samples in order to determine any variation depending on tissue or physiopathological conditions. Here, we detail the development of the first high-performance thin-layer chromatography method for the quantification of total alkyl and alkenyl glycerolipids thanks to the separation of their corresponding alkyl and alkenyl glycerols. This method starts with a reduction of all lipids after extraction, resulting in the reduction of neutral and polar ether glycerolipids into alkyl and alkenyl glycerols, followed by an appropriate purification and, finally, the linearly ascending development of alkyl and alkenyl glycerols on high-performance thin-layer chromatography plates, staining, carbonization and densitometric analysis. Calibration curves were obtained with commercial alkyl and alkenyl glycerol standards, enabling the quantification of alkyl and alkenyl glycerols in samples and thus directly obtaining the quantity of alkyl and alkenyl lipids present in the samples. Interestingly, we found a differential quantity of these lipids in shark liver oil compared to chimera. We quantified alkyl and alkenyl glycerolipids in periprostatic adipose tissues from human prostate cancer and showed the feasibility of this method in other biological matrices (muscle, tumor).
Asunto(s)
Aceites de Pescado , Lípidos , Tiburones , Animales , Cromatografía en Capa Delgada , Éter , Éteres , Glicerol , Aceites de Plantas , Lípidos/análisisRESUMEN
The modulation of SK3 ion channels can be efficiently and selectively achieved by using the amphiphilic compound Ohmline (a glyco-glycero-ether-lipid). We report herein a series of Ohmline analogues featuring the replacement of one ether function by a thioether function located at the same position or shifted close to its initial position. The variation of the lipid chain length and the preparation of two analogues featuring either one sulfoxide or one sulfone moiety complete this series. Patch clamp measurements indicate that the presence of the thioether function (compounds 7 and 17a) produces strong activators of SK3 channels, whereas the introduction of a sulfoxide or a sulfone function at the same place produces amphiphiles devoid of an effect on SK3 channels. Compounds 7 and 17a are the first amphiphilic compounds featuring strong activation of SK3 channels (close to 200% activation). The cytosolic calcium concentration determined from fluorescence at 3 different times for compound 7b (13 min, 1 h, 24 h) revealed that the effect is different suggesting that the compound could be metabolized over time. This compound could be used as a strong SK3 activator for a short time. The capacity of 7b to activate SK3 was then used to induce vasorelaxation via an endothelium-derived hyperpolarization (EDH) pathway. For the first time, we report that an amphiphilic compound can affect the endothelium dependent vasorelaxation.
Asunto(s)
Éteres/farmacología , Glucolípidos/farmacología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Compuestos de Sulfhidrilo/farmacología , Tensoactivos/farmacología , Animales , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Éteres/química , Glucolípidos/química , Humanos , Masculino , Ratas , Ratas Wistar , Compuestos de Sulfhidrilo/química , Tensoactivos/síntesis química , Tensoactivos/química , Vasodilatación/efectos de los fármacosRESUMEN
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
Natural O-alkyl-glycerolipids, also known as alkyl-ether-lipids (AEL), feature a long fatty alkyl chain linked to the glycerol unit by an ether bond. AEL are ubiquitously found in different tissues but, are abundant in shark liver oil, breast milk, red blood cells, blood plasma, and bone marrow. Only a few AEL are commercially available, while many others with saturated or mono-unsaturated alkyl chains of variable length are not available. These compounds are, however, necessary as standards for analytical methods. Here, we investigated different reported procedures and we adapted some of them to prepare a series of 1-O-alkyl-glycerols featuring mainly saturated alkyl chains of various lengths (14:0, 16:0, 17:0, 19:0, 20:0, 22:0) and two monounsaturated chains (16:1, 18:1). All of these standards were fully characterized by NMR and GC-MS. Finally, we used these standards to identify the AEL subtypes in shark and chimera liver oils. The distribution of the identified AEL were: 14:0 (20-24%), 16:0 (42-54%) and 18:1 (6-16%) and, to a lesser extent, (0.2-2%) for each of the following: 16:1, 17:0, 18:0, and 20:0. These standards open the possibilities to identify AEL subtypes in tumours and compare their composition to those of non-tumour tissues.
Asunto(s)
Cromatografía de Gases/normas , Aceites de Pescado/química , Glicéridos/síntesis química , Hígado/química , Tiburones , AnimalesRESUMEN
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
Oct-1 (POU2f1) and Oct-2 (POU2f2) are members of the POU family of transcription factors. They recognize the same DNA sequence but fulfil distinct functions: Oct-1 is ubiquitous and regulates a variety of genes while Oct-2 is restricted to B-cells and neurones. Here we examine the interplay and regulatory mechanisms of these factors to control the inducible nitric oxide synthase (iNOS, NOS2). Using two breast cancer cell lines as a comparative model, we found that MCF-7 express iNOS upon cytokine stimulation while MDA-MB-231 do not. Oct-1 is present in both cell lines but MDA-MB-231 also express high levels of Oct-2. Manipulation of Oct-2 expression in these cell lines demonstrates that it is directly responsible for the repression of iNOS in MDA-MB-231. In MCF-7 cells Oct-1 binds the iNOS promoter, recruits RNA PolII and triggers initiation of transcription. In MDA-MB-231 cells, both Oct-1 and Oct-2 bind the iNOS promoter, forming a higher-order complex which fails to recruit RNA PolII, and as a consequence iNOS transcription does not proceed. Unravelling the mechanisms of transcription factor activity is paramount to the understanding of gene expression patterns that determine cell behaviour.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Óxido Nítrico Sintasa de Tipo II/genética , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Factor 2 de Transcripción de Unión a Octámeros/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa III/metabolismo , Proteínas Represoras/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Células MCF-7 , Transcripción GenéticaRESUMEN
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
Despite the recent advances in the treatment of tumors with intrinsic chemotherapy resistance, such as melanoma and renal cancers, their prognosis remains poor and new chemical agents with promising activity against these cancers are urgently needed. Sphaeropsidin A, a fungal metabolite whose anticancer potential had previously received little attention, was isolated from Diplodia cupressi and found to display specific anticancer activity in vitro against melanoma and kidney cancer subpanels in the National Cancer Institute (NCI) 60-cell line screen. The NCI data revealed a mean LC50 of ca. 10 µM and a cellular sensitivity profile that did not match that of any other agent in the 765,000 compound database. Subsequent mechanistic studies in melanoma and other multidrug-resistant in vitro cancer models showed that sphaeropsidin A can overcome apoptosis as well as multidrug resistance by inducing a marked and rapid cellular shrinkage related to the loss of intracellular Cl(-) and the decreased HCO3 (-) concentration in the culture supernatant. These changes in ion homeostasis and the absence of effects on the plasma membrane potential were attributed to the sphaeropsidin A-induced impairment of regulatory volume increase (RVI). Preliminary results also indicate that depending on the type of cancer, the sphaeropsidin A effects on RVI could be related to Na-K-2Cl electroneutral cotransporter or Cl(-)/HCO3 (-) anion exchanger(s) targeting. This study underscores the modulation of ion-transporter activity as a promising therapeutic strategy to combat drug-resistant cancers and identifies the fungal metabolite, sphaeropsidin A, as a lead to develop anticancer agents targeting RVI in cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Diterpenos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Animales , Western Blotting , Línea Celular Tumoral , Diterpenos/química , Células HEK293 , Humanos , Ratones , Microscopía por Video , Estructura Molecular , Propidio , Azul de TripanoRESUMEN
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 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
Expression of the human inducible nitric oxide synthase (hiNOS) is generally undetectable in resting cells, but stimulation by a variety of signals including cytokines induces transcription in most cell types. The tight transcriptional regulation of the enzyme is a complex mechanism many aspects of which remain unknown. Here, we describe an octamer (Oct) element in hiNOS proximal promoter, located close to the TATA box. This site constitutively binds Oct-1 and its deletion abrogates cytokine-induced transcription, showing that it is indispensable though not sufficient for transcription. Increasing the distance between Oct and the TATA box by inserting inert DNA sequence inhibits transcription, and footprinting of this region shows no other protein binding in resting cells, suggesting an interaction between the two complexes. Chromatin immunoprecipitation assays detect the presence of Oct-1, RNA polymerase II and trimethyl K4 histone H3 on the proximal promoter in resting cells, confirming that the gene is primed for transcription before stimulation. RT-PCR of various fragments along the hiNOS gene shows that transcription is initiated in resting cells and this is inhibited by interference with Oct-1 binding to the proximal site of the promoter. We propose that, through interaction with the initiation complex, Oct-1 regulates hiNOS transcription by priming the gene for the rapid response required in an immune response.
Asunto(s)
Óxido Nítrico Sintasa de Tipo II/genética , Factor 1 de Transcripción de Unión a Octámeros/metabolismo , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , Células HCT116 , Humanos , Modelos Biológicos , Mutagénesis Sitio-Dirigida , Factor 1 de Transcripción de Unión a Octámeros/genética , Regiones Promotoras Genéticas , TATA Box , Transcripción GenéticaRESUMEN
Ether-lipids (EL) are specific lipids bearing a characteristic sn-1 ether bond. Depending on the ether or vinyl-ether nature of this bond, they are present as alkyl- or alkenyl-EL, respectively. Among EL, alkenyl-EL, also referred as plasmalogens in the literature, attract most of the scientific interest as they are the predominant EL species in eukaryotic cells, thus less is known about alkyl-EL. EL have been implicated in various signaling pathways and alterations in their quantity are frequently observed in pathologies such as neurodegenerative and cardiovascular diseases or cancer. However, it remains unknown whether both alkyl- and alkenyl-EL play the same roles in these processes. This review summarizes the roles and mechanisms of action of EL in cellular signaling and tries to discriminate between alkyl- and alkenyl-EL. We also focus on the involvement of EL-mediated alterations of cellular signaling in diseases and discuss the potential interest for EL in therapy.
Asunto(s)
Éter , Éteres , Éteres/química , Plasmalógenos/metabolismoRESUMEN
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
The emerging concept of small conductance Ca2+-activated potassium channels (SKCa) as pharmacological target for cancer treatment has significantly increased in recent years. In this study, we isolated the P01 toxin from Androctonus australis (Aa) scorpion venom and investigated its effect on biological properties of glioblastoma U87, breast MDA-MB231 and colon adenocarcinoma LS174 cancer cell lines. Our results showed that P01 was active only on U87 glioblastoma cells. It inhibited their proliferation, adhesion and migration with IC50 values in the micromolar range. We have also shown that P01 reduced the amplitude of the currents recorded in HEK293 cells expressing SK2 channels with an IC50 value of 3 pM, while it had no effect on those expressing SK3 channels. The investigation of the SKCa channels expression pattern showed that SK2 transcripts were expressed differently in the three cancer cell lines. Particularly, we highlighted the presence of SK2 isoforms in U87 cells, which could explain and rely on the specific activity of P01 on this cell line. These experimental data highlighted the usefulness of scorpion peptides to decipher the role of SKCa channels in the tumorigenesis process, and develop potential therapeutic molecules targeting glioblastoma with high selectivity.
RESUMEN
Therapeutic strategies for metastatic castration-resistant prostate cancer aim to target androgen receptor signaling. Despite initial survival benefits, treatment resistance invariably occurs, leading to lethal disease. Therapies targeting the androgen receptor can induce the emergence of a neuroendocrine phenotype and reactivate embryonic programs associated with epithelial to mesenchymal transition. We recently reported that dysregulation of the calcium signal can induce the transcription factor Zeb1, a key determinant of cell plasticity during tumor progression. The aim of this study was to determine whether the androgen receptor-targeted treatment Enzalutamide could induce dysregulation of the calcium signal involved in the progression toward epithelial to mesenchymal transition and neuroendocrine differentiation, contributing to therapeutic escape. Our results show that Zeb1 and the SK3 potassium channel are overexpressed in vivo in neuroendocrine castration-resistant prostate cancer and in vitro in LNCaP cells neurodifferentiated after Enzalutamide treatment. Moreover, the neuroendocrine phenotype is associated with a deregulation of the expression of Orai calcium channels. We showed that Zeb1 and SK3 are critical drivers of neuroendocrine differentiation. Interestingly, Ohmline, an SK3 inhibitor, can prevent the expression of Zeb1 and neuroendocrine markers induced by Enzalutamide. This study offers new perspectives to increase hormone therapy efficacy and improve clinical outcomes.
RESUMEN
We report the synthesis of three bioactive pyrene-based fluorescent analogues of Ohmline which is the most efficient and selective inhibitor of SK3 ion channel. The interaction of these Ohmline-pyrene (OP1-3) with liposomes of different composition reveals that only OP2 and OP3 are readily integrated into liposomes. Fluorescence measurements indicate that, depending on their concentration, OP2 and OP3 exist either as monomer or as a mixture of monomer and excimers within the liposome bilayer. Among the three Ohmline Pyrene compounds (OP1-3) only OP2 is able to reduce SK3 currents and is the first efficient fluorescent modulator of SK3 channel as revealed by patch clamp measurements (- 71.3 ± 13.3% at 10 µM) and by its inhibition of SK3-dependent cancer cell migration at (-32.5% ± 4.8% at 1 µM). We also report the first fluorescence study on living breast cancer cells (MDA-MB-231) showing that OP2 is rapidly integrated in bio-membranes followed by cell internalization.