RESUMO
Objectives: Gemcitabine, a first-line chemotherapeutic nucleoside analog drug (NAD) for pancreatic cancer, faces limitations due to drug resistance. Characterizing pancreatic cancer cells' transport characteristics may help identify the mechanisms behind drug resistance, and develop more effective therapeutic strategies. Therefore, in this study, we aimed to determine the nucleoside transport properties of Panc-1 cells, one of the commonly used pancreatic adenocarcinoma cell lines. Materials and Methods: To assess the presence of equilibrative nucleoside transporter-1 (ENT-1) in Panc-1 cells, we performed immunofluorescence staining, western blot analysis, and S-(4-nitrobenzyl)-6-thioinosine (NBTI) binding assays. We also conducted standard uptake assays to measure the sodium-independent uptake of [3H]-labeled chloroadenosine, hypoxanthine, and uridine. In addition, we determined the half-maximal inhibitory concentration (IC50) of gemcitabine. Statistical analyses were performed using GraphPad Prism version 8.0 for Windows. Results: The sodium-independent uptake of [3H]-labeled chloroadenosine, hypoxanthine, and uridine was measured using standard uptake assays, and the transport rates were determined as 111.1 ± 3.4 pmol/mg protein/10 s, 62.5 ± 4.8 pmol/mg protein/10 s, and 101.3 ± 2.5 pmol/mg protein/10 s, respectively. Furthermore, the presence of ENT-1 protein was confirmed using NBTI binding assays (Bmax 1.52 ± 0.1 pmol/mg protein; equilibrium dissociation constant 0.42 ± 0.1 nM). Immunofluorescence assays and western blot analysis also revealed ENT-1 in Panc-1 cells. The determined IC50 of gemcitabine in Panc-1 cells was 2 µM, indicating moderate sensitivity. Conclusion: These results suggest that Panc-1 is a suitable preclinical cellular model for studying NAD transport properties and potential therapies in pancreatic cancer and pharmaceutical research.
RESUMO
Human equilibrative nucleoside transporter 1 (hENT1) is a major route of entry of nucleosides and nucleoside analog drugs. The regulation of hENT1 is poorly understood in spite of its clinical importance as a drug transporter. Immunofluorescence microscopy and fluorescence-activated cell sorting suggested that cytidine pre-treatment (40 µM, 6 h) promotes hENT1 internalization in a way that does not affect either hENT1-mediated nucleoside uptake or gemcitabine-induced cytotoxicity. The Scatchard plot analyses of our NBTI binding data support previous speculations that hENT1 proteins exist as two sub-populations, and suggest that cytidine pre-treatment leads to the internalization of one population.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/fisiologia , Antimetabólitos Antineoplásicos/farmacologia , Sítios de Ligação , Citidina/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Células HEK293 , Humanos , Transporte Proteico , Tioinosina/análogos & derivados , Tioinosina/metabolismo , GencitabinaRESUMO
Equilibrative nucleoside transporters (ENTs) facilitate the flux of nucleosides, such as adenosine, and nucleoside analog (NA) drugs across cell membranes. A correlation between adenosine flux and calcium-dependent signaling has been previously reported; however, the mechanistic basis of these observations is not known. Here we report the identification of the calcium signaling transducer calmodulin (CaM) as an ENT1-interacting protein, via a conserved classic 1-5-10 motif in ENT1. Calcium-dependent human ENT1-CaM protein interactions were confirmed in human cell lines (HEK293, RT4, U-87 MG) using biochemical assays (HEK293) and the functional assays (HEK293, RT4), which confirmed modified nucleoside uptake that occurred in the presence of pharmacological manipulations of calcium levels and CaM function. Nucleoside and NA drug uptake was significantly decreased (â¼12% and â¼39%, respectively) by chelating calcium (EGTA, 50 µM; BAPTA-AM, 25 µM), whereas increasing intracellular calcium (thapsigargin, 1.5 µM) led to increased nucleoside uptake (â¼26%). Activation of N-methyl-d-aspartate (NMDA) receptors (in U-87 MG) by glutamate (1 mM) and glycine (100 µM) significantly increased nucleoside uptake (â¼38%) except in the presence of the NMDA receptor antagonist, MK-801 (50 µM), or CaM antagonist, W7 (50 µM). These data support the existence of a previously unidentified novel receptor-dependent regulatory mechanism, whereby intracellular calcium modulates nucleoside and NA drug uptake via CaM-dependent interaction of ENT1. These findings suggest that ENT1 is regulated via receptor-dependent calcium-linked pathways resulting in an alteration of purine flux, which may modulate purinergic signaling and influence NA drug efficacy.
Assuntos
Cálcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/química , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sítios de Ligação , Cálcio/química , Células HEK293 , Humanos , Ligação Proteica , Receptores de N-Metil-D-Aspartato/químicaRESUMO
The adenosine transporter 1 (ENT1) transports nucleosides, such as adenosine, and cytotoxic nucleoside analog drugs. ENT1 is well established to play a role in adenosinergic signaling in the cardiovascular system by modulating adenosine levels. Moderate ethanol consumption is cardioprotective and underlying mechanisms of action are not clear although adenosinergic signaling has been implicated. Here, we show that ethanol (5-200 mM) significantly reduces ENT1-dependent [(3)H] 2-chloroadenosine uptake (by up to 27 %) in the cardiomyocyte cell line, HL-1. Inhibition or absence of ENT1 is known to be cardioprotective, suggesting that the interaction of ethanol with ENT1 may promote adenosinergic cardioprotective pathways in the cardiovasculature.Ethanol sensitivity of adenosine uptake is altered by pharmacological activation of PKA and PKC. Primary cardiomyocytes from PKCε-null mice have significantly greater sensitivity to inhibition (by approximately 37 %) of adenosine uptake by ethanol than controls. These data suggest that the presence of ethanol may compromise ENT1-dependent nucleoside analog drug cytotoxicity, and indeed, ethanol (5 mM) reduces the cytotoxic effects of gemcitabine (2 nM), an anti-cancer drug, in the human cancer cell line, HTB2. Thus, the pharmacological inhibition of ENT1 by ethanol may contribute to ethanol-dependent cardioprotection but compromise gemcitabine cytotoxicity.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Etanol/farmacologia , Miócitos Cardíacos/metabolismo , Proteína Quinase C-épsilon/metabolismo , Adenosina/metabolismo , Animais , Antimetabólitos Antineoplásicos/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/toxicidade , Humanos , Camundongos , Camundongos Knockout , Miócitos Cardíacos/efeitos dos fármacos , GencitabinaRESUMO
AIMS: Equilibrative nucleoside transporters (ENT) modulate the flux of adenosine. The ENT1-null (KO) mouse heart is endogenously cardioprotected but the cellular basis of this phenotype is unknown. Therefore, we investigated the cellular mechanisms underlying ENT1-mediated cardioprotection. MAIN METHODS: Circulating adenosine levels were measured in WT and KO mice. Cellular levels of nucleosides and nucleotides were investigated in isolated adult cardiomyocytes from WT and KO mice using HPLC following hypoxic challenge (30 min, 2% O(2)). Changes in hypoxic gene expression were analyzed by PCR arrays and cAMP levels were measured to investigate contributions from adenosine receptors. KEY FINDINGS: Circulating adenosine levels were significantly higher in KO (416±42nmol/l, n=12) compared to WT animals (208±21, n=13, p<0.001). Absence of ENT1 led to an elevated expression of genes involved in cardioprotective pathways compared to WT cardiomyocytes. Following hypoxic challenge, extracellular adenosine levels were significantly elevated in KO (4360±1840 pmol/mg protein) versus WT cardiomyocytes (3035±730 pmol/mg protein, n≥12, p<0.05). This effect was enhanced in the presence of dipyridamole (30 µM), which inhibits ENT1 and ENT2. Enhanced extracellular adenosine levels in ENT1-null cardiomyocytes appeared to come from a pool of extracellular nucleotides including IMP, AMP and ADP. Adenosine receptor (AR) activation mimicked increases in cAMP levels due to hypoxic challenge suggesting that ENT1 modulates AR-dependent signaling. SIGNIFICANCE: ENT1 contributes to modulation of extracellular adenosine levels and subsequent purinergic signaling via ARs. ENT1-null mice possess elevated circulating adenosine levels and reduced cellular uptake resulting in a perpetually cardioprotected phenotype.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/genética , Miócitos Cardíacos/metabolismo , Nucleosídeos/metabolismo , Adenosina/metabolismo , Animais , Hipóxia Celular , Células Cultivadas , AMP Cíclico/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Feminino , Deleção de Genes , Regulação da Expressão Gênica , Inosina/metabolismo , Camundongos , Camundongos Knockout , Miócitos Cardíacos/citologia , Proteínas Quinases/metabolismo , Receptores Purinérgicos/metabolismoRESUMO
Equilibrative nucleoside transporters (ENTs) are integral membrane proteins that facilitate the movement of nucleosides and hydrophilic nucleoside analog (NA) drugs across cell membranes. ENTs are also targets for cardioprotectant drugs, which block re-uptake of the purine nucleoside adenosine, thereby enhancing purinergic receptor signaling pathways. ENTs are therefore important contributors to drug bioavailability and efficacy. Despite this important clinical role, very little is known about the structure and regulation of ENTs. Biochemical and structural studies on ENT proteins have been limited by their low endogenous expression levels, hydrophobicity and labile nature. To address these issues, we developed an approach whereby tagged mammalian ENT1 protein was over-expressed in mammalian cell lines, confirmed to be functional and isolated by affinity purification to sufficient levels to be analyzed using MALDI-TOF and tandem MS mass spectrometry. This proteomic approach will allow for a more detailed analysis of the structure, function and regulation of ENTs in the future.
Assuntos
Proteínas de Transporte de Nucleosídeo Equilibrativas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Células COS , Chlorocebus aethiops , Proteínas de Transporte de Nucleosídeo Equilibrativas/genética , Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Camundongos , Oligopeptídeos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tioinosina/análogos & derivados , Tioinosina/metabolismo , TripsinaRESUMO
To better understand the role of equilibrative nucleoside transporters (ENT) in purine nucleoside-dependent physiology of the cardiovascular system, we investigated whether the ENT1-null mouse heart was cardioprotected in response to ischemia (coronary occlusion for 30 min followed by reperfusion for 2 h). We observed that ENT1-null mouse hearts showed significantly less myocardial infarction compared with wild-type littermates. We confirmed that isolated wild-type adult mouse cardiomyocytes express predominantly ENT1, which is primarily responsible for purine nucleoside uptake in these cells. However, ENT1-null cardiomyocytes exhibit severely impaired nucleoside transport and lack ENT1 transcript and protein expression. Adenosine receptor expression profiles and expression levels of ENT2, ENT3, and ENT4 were similar in cardiomyocytes isolated from ENT1-null adult mice compared with cardiomyocytes isolated from wild-type littermates. Moreover, small interfering RNA knockdown of ENT1 in the cardiomyocyte cell line, HL-1, mimics findings in ENT1-null cardiomyocytes. Taken together, our data demonstrate that ENT1 plays an essential role in cardioprotection, most likely due to its effects in modulating purine nucleoside-dependent signaling and that the ENT1-null mouse is a powerful model system for the study of the role of ENTs in the physiology of the cardiomyocyte.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/fisiologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Transportador Equilibrativo 1 de Nucleosídeo/genética , Feminino , Camundongos , Camundongos Transgênicos , Modelos Animais , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Nucleosídeos de Purina/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores Purinérgicos P1/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Human Equilibrative Nucleoside Transporter 1 (hENT1) is an integral membrane protein that transports nucleosides and analog drugs across cellular membranes. Very little is known about intracellular processing and localization of hENT1. Here we show that disruption of a highly conserved triplet (PWN) near the N-terminus, or the last eight C-terminal residues (two hydrophobic triplets separated by a positive arginine) result in loss of plasma membrane localization and/or transport function. To understand the role of specific residues within these regions, we studied the localization patterns of N- or C-terminal deletion and/or substitution mutants of GFP-hENT1 using confocal microscopy. Quantification of GFP-hENT1 (mutant and wildtype) protein at the plasma membrane was conducted using nitrobenzylthioinosine (NBTI) binding. Functionality of the GFP-hENT1 mutants was determined by heterologous expression in Xenopus laevis oocytes followed by measurement of uridine uptake. Mutation of the proline within the PWN motif disrupts plasma membrane localization. C-terminal mutations (primarily within the hydrophobic triplets) lead to hENT1 retention within the cell (e.g. in the ER). Some mutants still localize to the plasma membrane but show reduced transport activity. These data suggest that these two regions contribute to the structural integrity and thus correct processing and function of hENT1.
Assuntos
Membrana Celular/metabolismo , Transportador Equilibrativo 1 de Nucleosídeo/química , Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Transporte Biológico , Neoplasias da Mama/metabolismo , Células COS , Células Cultivadas , Chlorocebus aethiops , Primers do DNA/química , Transportador Equilibrativo 1 de Nucleosídeo/genética , Feminino , Humanos , Dados de Sequência Molecular , Mutação , Oócitos/citologia , Oócitos/metabolismo , Homologia de Sequência de Aminoácidos , Uridina/metabolismo , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismoRESUMO
The purine nucleoside adenosine is a physiologically important molecule in the heart. Brief exposure of cardiomyocytes to hypoxic challenge results in the production of extracellular adenosine, which then interacts with adenosine receptors to activate compensatory signaling pathways that lead to cellular resistance to subsequence hypoxic challenge. This phenomenon is known as preconditioning (PC), and, while adenosine is clearly involved, other components of the response are less well understood. Flux of nucleosides, such as adenosine and inosine, across cardiomyocyte membranes is dependent on equilibrative nucleoside transporters 1 and 2 (ENT1 and ENT2). We have previously shown in the murine cardiomyocyte HL-1 cell line that hypoxic challenge leads to an increase in intracellular adenosine, which exits the cell via ENT1 and preconditions via A1 and A3 adenosine receptor-dependent mechanisms. However, the role and contribution of inosine and ENT2 are unclear. In this study, we confirmed that ENT1 and ENT2 are both capable of transporting inosine. Moreover, we found that hypoxic challenge leads to a significant increase in levels of intracellular inosine, which exits the cell via both ENT1 and ENT2. Exogenously added inosine (5 microM) preconditions cardiomyocytes in an A1 adenosine receptor-dependent manner since preconditioning can be blocked by the A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 microM) but not the A3 adenosine receptor antagonist MRS-1220 (200 nM). These data suggest that cardiomyocyte responses to hypoxic PC are more complex than previously thought, involving both adenosine and inosine and differing, but overlapping, contributions of the two ENT isoforms.
Assuntos
Transportador Equilibrativo 2 de Nucleosídeo/metabolismo , Inosina/metabolismo , Miócitos Cardíacos/metabolismo , Adenosina/metabolismo , Antagonistas do Receptor A1 de Adenosina , Antagonistas do Receptor A3 de Adenosina , Animais , Hipóxia Celular , Linhagem Celular , Sobrevivência Celular , Transportador Equilibrativo 1 de Nucleosídeo , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Proteínas de Transporte de Nucleosídeos/antagonistas & inibidores , Proteínas de Transporte de Nucleosídeos/metabolismo , Quinazolinas/farmacologia , Receptor A1 de Adenosina/metabolismo , Receptor A3 de Adenosina/metabolismo , Tioinosina/análogos & derivados , Tioinosina/farmacologia , Triazóis/farmacologia , Regulação para Cima , Xantinas/farmacologiaRESUMO
Brief exposure of the heart to hypoxia results in less cellular damage after subsequent hypoxia, an effect known as preconditioning (PC). PC has been widely studied but is still not fully understood. Adenosine (Ado), adenosine receptors, and protein kinase C (PKC) have been implicated as integral components of PC. Adenosine (nucleoside) transporters (NTs) facilitate flux of Ado across cell membranes, but their role in PC is unknown. Therefore, we used the murine cardiomyocyte cell line, HL-1, and asked if there was feedback regulation of NTs by Ado, Ado receptors, and PKC following either hypoxic or pharmacological PC. Activation (by specific agonists) of A1 or A3 Ado receptors or PKC resulted in PC in HL-1. The A1 (but not A3) receptor is coupled to PKCepsilon, and activation of PKCepsilon (by specific peptide agonist) resulted in PC. Moreover, PKCepsilon stimulates Ado uptake via the predominant NT in HL-1, mouse equilibrative nucleoside transporter 1 (mENT1). Studies in primary neonatal mouse cardiomyocytes confirmed our observations in HL-1 cells. Hypoxic challenge led to a rapid increase in, and efflux of, intracellular Ado from cells, which was blocked by NT inhibitors (dipyridamole/nitrobenzylthioinosine). Moreover, NT inhibition during hypoxia or PC was highly protective, suggesting that Ado loss contributes to decreased cell viability. Our data suggest that hypoxic challenge causes an efflux of Ado via ENTs, activation of A1 and/or A3 receptors, signaling through PKCepsilon, and activation of ENT1. Since Ado is required for ATP synthesis on reperfusion, this feedback regulation of mENT1 would promote reuptake of Ado.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Miócitos Cardíacos/metabolismo , Proteína Quinase C/metabolismo , Receptores Purinérgicos P1/fisiologia , Transdução de Sinais/fisiologia , Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Hipóxia Celular , Linhagem Celular , Células Cultivadas , Transportador Equilibrativo 1 de Nucleosídeo , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Proteínas de Transporte de Nucleosídeos , Proteína Quinase C-deltaRESUMO
OBJECTIVE: Adenosine is an important paracrine hormone in the cardiovascular system. Adenosine flux across cardiomyocyte membranes occurs mainly via equilibrative nucleoside transporters (ENTs). The role of the ENTs in adenosine physiology is poorly understood, particularly in response to metabolic stress such as hypoxia. Therefore, we investigated the effects of chronic hypoxia on ENT1, the predominant ENT isoform in cardiomyocytes. METHODS: HL-1 cells (immortalized murine cardiomyocytes) were exposed to hypoxia (2% O2) for 0-20 h. Cell viability, lactate dehydrogenase (LDH) release, glucose uptake, GLUT1 and GLUT4 protein, adenosine uptake, PKC activity, translocation profiles of PKCdelta and, nitrobenzylthioinosine (NBTI) binding and mENT1 mRNA levels were measured. The role of PKC in regulating mENT1 was further investigated using phorbol ester (100 nM, 18 h) and a dominant negative PKC construct, pSVK3PKC1-401. RESULTS: HL-1 cells have typical cardiomyocyte responses to hypoxia based on cell viability, LDH release, glucose uptake and GLUT protein levels. Hypoxia (8-20 h) down-regulates mENT1-dependent adenosine uptake, NBTI-binding and PKC but not PKCdelta in HL-1 cells. Abrogation of PKC activity using chronic phorbol ester or a dominant negative PKC mimicked the effect of hypoxia on adenosine uptake suggesting that PKC is involved in regulation of mENT1. Hypoxia (4 h) decreases mENT1 mRNA, which returns to basal levels by 20 h. CONCLUSIONS: Chronic hypoxia down-regulates mENT1 activity possibly via PKC. Hypoxia and PKC also regulate mENT1 RNA levels. Cardiomyocytes may regulate mENT1 (via PKC) to modulate release and/or uptake of adenosine. However, the relationship between mENT1 mRNA levels, protein levels and functional transport is complex.
Assuntos
Transportador Equilibrativo 1 de Nucleosídeo/metabolismo , Hipóxia/metabolismo , Miócitos Cardíacos/metabolismo , Angiotensina I/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Transportador Equilibrativo 1 de Nucleosídeo/análise , Camundongos , Camundongos Transgênicos , Proteína Quinase C/metabolismoRESUMO
Regulation of nucleoside transporters is poorly understood. We show that acute stimulation of protein kinase C (PKC) causes a rapid increase in S-(4-nitrobenzyl)-6-thioinosine-sensitive (human equilibrative nucleoside transporter 1, hENT1) nucleoside uptake, in human cultured cells, which is not due to increased metabolism and which can be blocked by PKC inhibitors. Use of isoform-specific inhibitors indicates that PKC delta and/or epsilon (but not alpha, beta or gamma) are responsible for the acute effects. Down-regulation of PKC decreases hENT1-dependent uridine uptake. These are the first data to show rapid PKC delta/epsilon-dependent stimulation of hENT1 transport by a mechanism that may involve activation of transporters at the membrane possibly by post-translational modification of the protein.