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Glioblastoma (GBM) is still a deadly tumor due to its highly infiltrative growth behavior and its resistance to therapy. Evidence is accumulating that sphingosine-1-phosphate (S1P) acts as an important tumor-promoting molecule that is involved in the activation of the S1P receptor subtype 1 (S1PR1). Therefore, we investigated the effect of ACT-209905 (a putative S1PR1 modulator) on the growth of human (primary cells, LN-18) and murine (GL261) GBM cells. The viability and migration of GBM cells were both reduced by ACT-209905. Furthermore, co-culture with monocytic THP-1 cells or conditioned medium enhanced the viability and migration of GBM cells, suggesting that THP-1 cells secrete factors which stimulate GBM cell growth. ACT-209905 inhibited the THP-1-induced enhancement of GBM cell growth and migration. Immunoblot analyses showed that ACT-209905 reduced the activation of growth-promoting kinases (p38, AKT1 and ERK1/2), whereas THP-1 cells and conditioned medium caused an activation of these kinases. In addition, ACT-209905 diminished the surface expression of pro-migratory molecules and reduced CD62P-positive GBM cells. In contrast, THP-1 cells increased the ICAM-1 and P-Selectin content of GBM cells which was reversed by ACT-209905. In conclusion, our study suggests the role of S1PR1 signaling in the growth of GBM cells and gives a partial explanation for the pro-tumorigenic effects that macrophages might have on GBM cells.
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Coproporphyrin I (CPI) and III (CPIII) are discussed as biomarkers for organic anion transporting polypeptides (OATPs). We report on CPI and CPIII levels in wildtype, rSlco2b1-knockout, and SLCO2B1-humanized rats at baseline and after administration of atorvastatin, an inhibitor of the CPIII-specific rOATP2B1/hOATP2B1 and the CPI/CPIII-transporting rOATP1B2. OATP-inhibition by atorvastatin leads to significantly increased CPI and CPIII serum levels. However, basal CP serum levels in rSlco2b1-knockout animals were significantly lower (CPI), or unaffected (CPIII). In the presence of atorvastatin, this genotype effect was abolished. In conclusion, our results indicate an unexpected impact of OATP2B1 on CP serum levels in rats.
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In recent years, the identification of endogenous substrates as biomarkers became an uprising topic. Particularly coproporphyrins (CPs), byproducts of heme biosynthesis, are intensely investigated as biomarkers for predicting interactions with the organic anion transporting polypeptide (OATP) 1B transporters. In the context of drug-drug interactions, several preclinical and clinical studies assessed the effect of the OATP1B-index inhibitor rifampin on CPI levels. However, rifampin is not only a "perpetrator" drug of transporters but is also known for its interaction with the nuclear receptor pregnane X receptor (PXR) leading to the efficient induction of PXR-target genes. These include hemoproteins like cytochrome P450 enzymes but also the δ-aminolevulinate synthase 1, which is the rate-limiting enzyme in heme biosynthesis. In this study, we showed that quantification of CPs in clinical serum samples was possible after long-term storage at -20°C. We quantified CPI, CPIII, and heme levels in clinical serum samples (at selected timepoints) that originated from a trial investigating the interaction potential of repeated rifampin administration in 12 healthy participants. In samples collected at the assumed time to maximum concentration of rifampin, higher CP levels were observed compared to baseline. Increased levels persisted even 14 h after discontinuation of rifampin. No impact on heme serum levels was observed. We found a correlation between CP isomers at baseline and at 14 h after rifampin intake. In summary, we show that multiple doses of rifampin affect CP levels. However, besides inhibition of hepatic OATP function there is evidence for an interaction with CP levels beyond the transporter level.
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Transportadores de Anión Orgánico , Rifampin , Humanos , Rifampin/farmacología , Coproporfirinas , Proteínas de Transporte de Membrana , Interacciones Farmacológicas , Biomarcadores , HemoRESUMEN
The human drug transporter Organic Anion Transporting Polypeptide (hOATP)2B1 facilitates cellular uptake of its substrates. Various studies suggest that hOATP2B1 is involved in intestinal absorption, but preclinical evaluations performed in rodents do not support this. Thus, our study aimed to compare the expression and function of hOATP2B1 with its orthologue in rats (rOatp2b1). Even if the general expression pattern was comparable, the transporters exhibited substantial differences on functional level. While bromosulfophthalein and atorvastatin were substrates of both transporters, the steroid sulfate conjugates estrone 3-sulfate (E1S), progesterone sulfate and dehydroepiandrosterone sulfate were only transported by hOATP2B1. To further elucidate these functional differences, experiments searching for the E1S substrate recognition site were conducted generating human-rat chimera as well as partly humanized variants of rOatp2b1. The rOatp2b1-329-hOATP2B1 chimera led to a significant increase in E1S uptake suggesting the C-terminal part of the human transporter is involved. However, humanization of various regions within this part, namely of the transmembrane domain (TMD)-9, TMD-10 or the extracellular loop-5 did not significantly change E1S transport function. Replacement of the intracellular loop-3, slightly enhanced cellular accumulation of sulfated steroids. Taken together, we report that OATP2B1 exhibited differences in recognition of steroid sulfate conjugates comparing the rat and human orthologues.
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Transportadores de Anión Orgánico , Animales , Atorvastatina , Transporte Biológico , Estrona , Humanos , Absorción Intestinal , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , RatasRESUMEN
Despite comprehensive therapy and extensive research, glioblastoma (GBM) still represents the most aggressive brain tumor in adults. Glioma stem cells (GSCs) are thought to play a major role in tumor progression and resistance of GBM cells to radiochemotherapy. The PIM1 kinase has become a focus in cancer research. We have previously demonstrated that PIM1 is involved in survival of GBM cells and in GBM growth in a mouse model. However, little is known about the importance of PIM1 in cancer stem cells. Here, we report on the role of PIM1 in GBM stem cell behavior and killing. PIM1 inhibition negatively regulates the protein expression of the stem cell markers CD133 and Nestin in GBM cells (LN-18, U-87 MG). In contrast, CD44 and the astrocytic differentiation marker GFAP were up-regulated. Furthermore, PIM1 expression was increased in neurospheres as a model of GBM stem-like cells. Treatment of neurospheres with PIM1 inhibitors (TCS PIM1-1, Quercetagetin, and LY294002) diminished the cell viability associated with reduced DNA synthesis rate, increased caspase 3 activity, decreased PCNA protein expression, and reduced neurosphere formation. Our results indicate that PIM1 affects the glioblastoma stem cell behavior, and its inhibition kills glioblastoma stem-like cells, pointing to PIM1 targeting as a potential anti-glioblastoma therapy.
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Antineoplásicos/farmacología , Neoplasias Encefálicas/patología , Glioblastoma/patología , Células Madre Neoplásicas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Cromonas/farmacología , Cromonas/uso terapéutico , Ensayos de Selección de Medicamentos Antitumorales , Flavonas/farmacología , Flavonas/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Morfolinas/farmacología , Morfolinas/uso terapéutico , Células Madre Neoplásicas/patología , Proteínas Proto-Oncogénicas c-pim-1/genética , Células Tumorales CultivadasRESUMEN
The central nervous system (CNS) is an important pharmacological target, but it is very effectively protected by the blood-brain barrier (BBB), thereby impairing the efficacy of many potential active compounds as they are unable to cross this barrier. Among others, membranous efflux transporters like P-Glycoprotein are involved in the integrity of this barrier. In addition to these, however, uptake transporters have also been found to selectively uptake certain compounds into the CNS. These transporters are localized in the BBB as well as in neurons or in the choroid plexus. Among them, from a pharmacological point of view, representatives of the organic anion transporting polypeptides (OATPs) are of particular interest, as they mediate the cellular entry of a variety of different pharmaceutical compounds. Thus, OATPs in the BBB potentially offer the possibility of CNS targeting approaches. For these purposes, a profound understanding of the expression and localization of these transporters is crucial. This review therefore summarizes the current state of knowledge of the expression and localization of OATPs in the CNS, gives an overview of their possible physiological role, and outlines their possible pharmacological relevance using selected examples.
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The online version of the original article can be found at https://doi.org/10.1007/s00784-020-03594-w.
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BACKGROUND: The anesthetic ketamine after intravenous dosing is nearly completely metabolized to R- and S-stereoisomers of the active norketamine (analgesic, psychoactive) and 2,6-hydroxynorketamine (potential analgesic, antidepressant) as well as the inactive dehydronorketamine. Oral administration favors the formation of 2,6-hydroxynorketamines via extensive presystemic metabolism. The authors hypothesized that plasma exposure to 2,6-hydroxynorketamines relative to the psychoactive ketamine is greater after prolonged-release ketamine tablets than it is after intravenous ketamine. METHODS: Pharmacokinetics of ketamine after intravenous infusion (5.0 mg) and single-dose administrations of 10, 20, 40, and 80 mg prolonged-released tablets were evaluated in 15 healthy white human subjects by means of a controlled, ascending-dose study. The stereoisomers of ketamine and metabolites were quantified in serum and urine by validated tandem mass-spectrometric assays and evaluated by noncompartmental pharmacokinetic analysis. RESULTS: After 40 mg prolonged-release tablets, the mean ± SD area under the concentrations-time curve ratios for 2,6-hydroxynorketamine/ketamine were 18 ± 11 (S-stereoisomers) and 30 ± 16 (R-stereoisomers) compared to 1.7 ± 0.8 and 3.1 ± 1.4 and after intravenous infusion (both P < 0.001). After 10 and 20 mg tablets, the R-ratios were even greater. The distribution volumes at steady state of S- and R-ketamine were 6.6 ± 2.2 and 5.6 ± 2.1 l/kg, terminal half-lives 5.2 ± 3.4 and 6.1 ± 3.1 h, and metabolic clearances 1,620 ± 380 and 1,530 ± 380 ml/min, respectively. Bioavailability of the 40 mg tablets was 15 ± 8 (S-isomer) and 19 ± 10% (R-isomer) and terminal half-life 11 ± 4 and 10 ± 4 h. About 7% of the dose was renally excreted as S-stereoisomers and 17% as R-stereoisomers. CONCLUSIONS: Prolonged-release ketamine tablets generate a high systemic exposure to 2,6-hydroxynorketamines and might therefore be an efficient and safer pharmaceutical dosage form for treatment of patients with chronic neuropathic pain compared to intravenous infusion.
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Analgésicos/metabolismo , Analgésicos/farmacocinética , Ketamina/metabolismo , Ketamina/farmacocinética , Administración Oral , Adulto , Analgésicos/administración & dosificación , Preparaciones de Acción Retardada , Femenino , Voluntarios Sanos , Humanos , Ketamina/administración & dosificación , Masculino , Valores de Referencia , Adulto JovenRESUMEN
The organic anion transporting polypeptide 2B1 (OATP2B1) was one of the first cloned members of the SLCO family. However, its physiological and pharmacological role is still poorly understood, and object of a current debate on the transporter's relevance. Within this commentary, we summarize the data currently available on the transporter's expression and its substrates and highlight the strength and difficulties of the methods that have been applied to gather these data. The conclusion drawn from these findings was that OATP2B1 due to its intestinal expression is most likely involved in oral drug absorption of its substrate and therefore prone for interactions. This has been tested in in vivo drug interaction and/or pharmacogenetic studies. While some of these support the notion of OATP2B1 being of relevance in drug absorption, the pharmacogenetic findings are rather inconclusive. We will explain our thoughts why OATP2B1 may not influence the general systemic pharmacokinetic of certain substrates, but possibly local distribution processes, like the transfer across the blood-brain-barrier. Besides the pharmacokinetic aspects, there are data on endogenous molecules like coproporphyrins and sulfated steroids. Therefore, we will also highlight possible physiological roles of OATP2B1, which are driven by its expression pattern in the tubular cells of the kidney as well as its expression in the blood brain barrier. Finally we also deal with the advantages and disadvantages in the use of animal models to decipher the role of OATP2B1 in pharmacokinetics of its substrates and beyond.
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Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Transportadores de Anión Orgánico/metabolismo , Preparaciones Farmacéuticas/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Interacciones Farmacológicas/fisiología , Humanos , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/fisiología , Preparaciones Farmacéuticas/administración & dosificación , Distribución Tisular/efectos de los fármacos , Distribución Tisular/fisiologíaRESUMEN
OBJECTIVE: SLC22A4/5 single nucleotide polymorphisms (SNPs) have been reported to affect inflammatory diseases. We report the relationship of these polymorphisms with adiposity and tooth loss as elucidated in a 10-year follow-up study. METHODS: Participants of the Study of Health in Pomerania (SHIP, N = 4105) were genotyped for the polymorphisms c.1507C > T in SLC22A4 (rs1050152) and -207C > G in SLC22A5 (rs2631367) using allele-specific real-time PCR assays. A total of 1817 subjects, 934 female and 883 male aged 30-80 years, underwent follow-up 10 years later (SHIP-2) and were assessed for adiposity and tooth loss. RESULTS: The frequencies of the rarer SLC22A4 TT and SLC22A5 CC alleles were 16.7% and 20.3%, respectively. In women, tooth loss was associated with genotype TT vs. CC with incidence rate ratio IRR = 0.74 (95%C.I. 0.60-0.92) and CC vs. GG IRR = 0.79 (0.65-0.96) for SLC22A4 and SLC22A5 SNPs, respectively. In men, no such associations were observed. In the follow-up examination, the relationship between tooth loss and these SNPs was in parallel with measures of body shape such as BMI, body weight, waist circumference, or body fat accumulation. The association between muscle strength and body fat mass was modified by the genotypes studied. CONCLUSIONS: SLC22A4 c.150C > T and SLC22A5 -207C > G polymorphisms are associated with tooth loss and markers of body shape in women but not in men. CLINICAL RELEVANCE: Tooth loss may be related to obesity beyond inflammatory mechanisms, conceivably with a genetic background.
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Carnitina , Pérdida de Diente , Adiposidad/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Estudios de Seguimiento , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Obesidad/genética , Proteínas de Transporte de Catión Orgánico/genética , Polimorfismo de Nucleótido Simple , Miembro 5 de la Familia 22 de Transportadores de Solutos , Simportadores/genética , Pérdida de Diente/genéticaRESUMEN
The family of Organic Anion Transporting Polypeptides are known to facilitate the transmembrane transport. OATP1B3-1B7 is a novel member of the OATP1B-subfamily, and is encoded by SLCO1B3-SLCO1B7 readthrough deriving from the genes SLCO1B3 and SLCO1B7 on chromosome 12. The resulting protein is expressed in the smooth endoplasmatic reticulum of hepatocytes, is functional, and transports dehydroepiandrosterone-sulfate (DHEAS). In the gene area encoding for the 1B7-part of the protein, there are coding polymorphisms. It was the aim of this study to test the frequency and the impact of these genetic variants on transport activity. The minor allele frequency (MAF) of the coding polymorphisms was determined in a cohort of 192 individuals. DHEAS transport function was determined by applying the vTF-7 based heterologous expression system using plasmids encoding for OATP1B3-1B7 or the respective variants. The genetic variants 641 T (MAF 0.021), 1073 G (MAF 0.169) and 1775 A (MAF 0.013) significantly reduced DHEAS accumulation in cells transfected with OATP1B3-1B7, albeit without significantly influencing expression of the transporter as determined by Western blot analysis and immunofluorescence after heterologous expression. Genotyping revealed complete linkage of the variants 884A, 1073 G and 1501C. Presence of the haplotype abolished the DHEAS-transport function of OATP1B3-1B7. Naturally and frequently occurring genetic variants located within the gene region of SLCO1B7 encoding for the 1B7-part of OATP1B3-1B7 influence the in vitro function of this member of the OATP1B-family. With their functional characterisation, we provide the basis for pharmacogenetic studies, which may help to understand the in vivo relevance of this transporter.
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Sulfato de Deshidroepiandrosterona/metabolismo , Transportadores de Anión Orgánico/genética , Variantes Farmacogenómicas , Polimorfismo de Nucleótido Simple , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos/genética , Proteínas Transportadoras de Solutos/genética , Transporte Biológico , Bases de Datos Genéticas , Frecuencia de los Genes , Haplotipos , Células HeLa , Humanos , Cinética , Transportadores de Anión Orgánico/metabolismo , Fenotipo , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos/metabolismo , Proteínas Transportadoras de Solutos/metabolismoRESUMEN
Interaction with the dopaminergic system in the central nervous system is either therapeutically intended or it is a side effect. In both cases, dopamine-receptor agonists (DRA) like the ergoline derivative bromocriptine and dopamine-receptor antagonists (DRAn) like metoclopramide have to cross the blood-brain barrier (BBB). The organic anion transporting polypeptides (OATP) 1A2 and 2B1 are cellular uptake carriers for a variety of endogenous and xenobiotic compounds. As both transporters are expressed in endothelial cells of the BBB, the aim of the present study was to determine whether the DRA bromocriptine, cabergoline, and pergolide and the DRAn metoclopramide and domperidone are interacting with OATP1A2 and 2B1 and could therefore be candidate genes modifying wanted and unwanted effects of these drugs. Localization of both transporters in the brain was confirmed using LC-MS/MS and immunofluorescence stainings. For the functional studies, MDCKII cells stably expressing OATP1A2 or 2B1 were used. Initial interaction studies with the well-characterized transporter substrate estrone 3-sulfate revealed that all tested compounds except pergolide inhibit the transport function of both proteins with the most potent effect for bromocriptine (IC50 = 2.2 µM (OATP1A2) and IC50 = 2.5 µM (OATP2B1)). Further studies using the indirect competitive counterflow method identified bromocriptine, cabergoline, and domperidone as substrates of both transporters, whereas metoclopramide was only transported by OATP1A2. These findings were verified for domperidone by direct measurements using its tritium-labeled form as a tracer. Moreover, the transporter-mediated uptake of this compound was sensitive to the OATP1A2 and OATP2B1 inhibitor naringin. In conclusion, this study suggests that OATP1A2 and 2B1 may play a role in the uptake of DR agonists and antagonists into the brain.
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Agonistas de Dopamina/metabolismo , Antagonistas de Dopamina/metabolismo , Transportadores de Anión Orgánico/metabolismo , Animales , Encéfalo/metabolismo , Bromocriptina/metabolismo , Línea Celular , Perros , Domperidona/metabolismo , Dopamina , Humanos , Adenohipófisis/metabolismo , Espectrometría de Masas en TándemRESUMEN
PURPOSE: Apoptotic dysregulation, redox adaptive mechanisms, and resilience to hypoxia are major causes of glioblastoma (GBM) resistance to therapy. Commonly known as crucial factors in energy metabolism, OCTN2 (SLC22A5) and its substrate L-carnitine (LC) are increasingly recognized as actors in cytoprotection. This study provides a comprehensive expression and survival analysis of the OCTN2/LC system in GBM and clarifies the system's impact on GBM progression. EXPERIMENTAL DESIGN: OCTN2 expression and LC content were measured in 121 resected human GBM specimens and 10 healthy brain samples and analyzed for prognostic significance. Depending on LC administration, the effects of hypoxic, metabolic, and cytotoxic stress on survival and migration of LN18 GBM cells were further studied in vitro. Finally, an orthotopic mouse model was employed to investigate inhibition of the OCTN2/LC system on in vivo GBM growth. RESULTS: Compared with healthy brain, OCTN2 expression was increased in primary and even more so in recurrent GBM on mRNA and protein level. High OCTN2 expression was associated with a poor overall patient survival; the unadjusted HR for death was 2.7 (95% CI, 1.47-4.91; P < 0.001). LC administration to GBM cells increased their tolerance toward cytotoxicity, whereas siRNA-mediated OCTN2 silencing led to a loss of tumor cell viability. In line herewith, OCTN2/LC inhibition by meldonium resulted in reduced tumor growth in an orthotopic GBM mouse model. CONCLUSIONS: Our data indicate a potential role of the OCTN2/LC system in GBM progression and resistance to therapy, and suggests OCTN2 as a prognostic marker in patients with primary GBM.
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Biomarcadores de Tumor/metabolismo , Carnitina/metabolismo , Proliferación Celular , Citoprotección , Glioblastoma/mortalidad , Recurrencia Local de Neoplasia/mortalidad , Miembro 5 de la Familia 22 de Transportadores de Solutos/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Movimiento Celular , Niño , Preescolar , Femenino , Estudios de Seguimiento , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Glioblastoma/patología , Glioblastoma/cirugía , Humanos , Lactante , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/cirugía , Pronóstico , Miembro 5 de la Familia 22 de Transportadores de Solutos/genética , Tasa de Supervivencia , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto JovenRESUMEN
The protein family of Organic Anion Transporting Polypeptides (OATPs) summarizes various transporters known to facilitate cellular uptake of xenobiotics. One member of this family is OATP2B1. This transporter is ubiquitously expressed and possesses a PDZ-binding motif at the C-terminus. PDZK1 (PDZ domain-containing 1) is a scaffold protein that influences function of different membrane proteins by sorting/stabilization of their membrane localization. It was aim of the herein reported study to investigate whether there is an interaction between OATP2B1 and PDZK1, and to further characterize its impact on transport function. At first expression of both OATP2B1 and PDZK1 was evaluated in liver, kidney and intestine. Based on the existence of a C-terminal PDZ-class I binding motif in OATP2B1 and the co-expression in all tested tissues an interaction was likely. Testing the influence of PDZK1 on OATP2B1 transport function revealed enhanced transport capacity for estrone 3-sulfate, thereby suggesting a change in OATP2B1 amount in the membrane. This assumption was validated by Western blot analysis. Finally, deletion of the C-terminal PDZ-binding motif in OATP2B1 lowered the impact of PDZK1 on transport function. Taken together, we report an interaction of PDZK1 with OATP2B1, which influences localization and function of the transporter. Changes in PDZK1 expression may therefore be one factor contributing to interindividual differences in OATP2B1 mediated pharmacokinetic processes.
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Proteínas Portadoras/metabolismo , Transportadores de Anión Orgánico/metabolismo , Animales , Sitios de Unión , Proteínas Portadoras/genética , Perros , Estrona/análogos & derivados , Estrona/metabolismo , Humanos , Intestino Delgado/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Células de Riñón Canino Madin Darby , Proteínas de la Membrana , Transportadores de Anión Orgánico/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de ProteínasRESUMEN
Neurosteroids, comprising pregnane, androstane, and sulfated steroids can alter neuronal excitability through interaction with ligand-gated ion channels and other receptors and have therefore a therapeutic potential in several brain disorders. They can be formed in brain cells or are synthesized by an endocrine gland and reach the brain by penetrating the blood-brain barrier (BBB). Especially sulfated steroids such as pregnenolone sulfate (PregS) and dehydroepiandrosterone sulfate (DHEAS) depend on transporter proteins to cross membranes. In this review, we discuss the involvement of ATP-binding cassette (ABC)- and solute carrier (SLC)-type membrane proteins in the transport of these compounds at the BBB and in the choroid plexus (CP), but also in the secretion from neurons and glial cells. Among the ABC transporters, especially BCRP (ABCG2) and several MRP/ABCC subfamily members (MRP1, MRP4, MRP8) are expressed in the brain and known to efflux conjugated steroids. Furthermore, several SLC transporters have been shown to mediate cellular uptake of steroid sulfates. These include members of the OATP/SLCO subfamily, namely OATP1A2 and OATP2B1, as well as OAT3 (SLC22A3), which have been reported to be expressed at the BBB, in the CP and in part in neurons. Furthermore, a role of the organic solute transporter OSTα-OSTß (SLC51A/B) in brain DHEAS/PregS homeostasis has been proposed. This transporter was reported to be localized especially in steroidogenic cells of the cerebellum and hippocampus. To date, the impact of transporters on neurosteroid homeostasis is still poorly understood. Further insights are desirable also with regard to the therapeutic potential of these compounds.
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The anthracycline-mediated cardiotoxicity is still not completely understood. To examine the impact of cholesterol metabolism and transport in this context, cholesterol and oxysterol levels as well as the expression of the cholesterol transporters ABCA1 and ABCG1 were analyzed in doxorubicin-treated HL-1 murine cardiomyocytes as well as in mouse model for acute doxorubicin-induced cardiotoxicity. Doxorubicin-treated HL-1 cells exhibited enhanced cholesterol (153±20% of control), oxysterol (24S-hydroxycholesterol: 206±29% of control) and cholesterol precursor levels (lathosterol: 122±12% of control; desmosterol: 188±10% of control) indicating enhanced cholesterol synthesis. Moreover, abca1 and abcg1 were upregulated on mRNA, protein and functional level caused by a doxorubicin-mediated activation of the nuclear receptor LXR. In addition, the oxysterols not only induced the abca1 and abcg1 in HL-1 cells but also enhanced the expression of endothelin-1 and transforming growth factor-ß, which have already been identified as important factors in doxorubicin-induced cardiotoxicity. These in vitro findings were verified in a murine model for acute doxorubicin-induced cardiotoxicity, demonstrating elevated cardiac (2.1±0.2vs. 3.6±1.0ng/mg) and systemic cholesterol levels (105.0±8.4vs. 130.0±4.3mg/dl), respectively, as well as enhanced oxysterol levels such as cardiac 24S-hydroxycholesterol (2.1±0.2vs. 3.6±1.0ng/mg). In line with these findings cardiac mRNA expression of abca1 (303% of control) and abcg1 (161% of control) was induced. Taken together, our data demonstrate enhanced cholesterol and oxysterol levels by doxorubicin, resulting in a LXR-dependent upregulation of abca1 and abcg1. In this context, the cytotoxic effects of oxysterols and their impact on cardiac gene expression should be considered as an important factor in doxorubicin-induced cardiotoxicity.
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Transportador 1 de Casete de Unión a ATP/biosíntesis , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/biosíntesis , Doxorrubicina/farmacología , Receptores X del Hígado/fisiología , Miocitos Cardíacos/metabolismo , Oxiesteroles/metabolismo , Animales , Células Cultivadas , Colesterol/metabolismo , Relación Dosis-Respuesta a Droga , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
The organic anion-transporting polypeptide (OATP) 2B1 which is ubiquitously expressed in the human body is assumed to play an important role in the cellular uptake of many drugs. Although the expression and function of this solute carrier transporter is well characterized in the human liver and other tissues, little is known about its localization and functional relevance in the intestine. Thus, it was the aim of this study to investigate its localization and function in the human jejunum and in the frequently used intestinal Caco-2 cell line. The basolateral membrane of jejunal tissue from 6 individuals showed a significant enrichment of OATP2B1 (17-fold) and the known basolateral proteins ABCC3 and Na/K-ATPase compared to the apical membrane as derived from targeted proteomics analysis. On the contrary, apical localization could be confirmed for ABCB1, ABCC2, and PEPT1. Basolateral localization of OATP2B1 could also be verified in Caco-2 cells. Bidirectional transport studies with established OATP2B1 substrates (sulfasalazine and pravastatin) across freshly exercised human jejunum and Caco-2 cell monolayers demonstrated a markedly higher transport from the basal to the apical compartment than in the opposite direction. Our data provide evidence for a basolateral localization of OATP2B1 which may improve our understanding of intestinal drug absorption.
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Membrana Celular/metabolismo , Yeyuno/metabolismo , Transportadores de Anión Orgánico/metabolismo , Péptidos/metabolismo , Adenosina Trifosfatasas/metabolismo , Anciano , Anciano de 80 o más Años , Transporte Biológico , Células CACO-2 , Calibración , Femenino , Humanos , Absorción Intestinal/fisiología , Masculino , Persona de Mediana Edad , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Péptidos/química , Proteómica/métodosRESUMEN
BACKGROUND: Immunization against beta-amyloid (Aß) reduces cerebral Aß deposits and improves cognitive capacities in transgenic mouse models, and thus has been considered a promising disease- modifying therapeutic approach for Alzheimer's disease (AD). Although clinical trials in AD patients have yielded evidence for clearance of parenchymal Aß plaques, Aß increases in blood vessels of treated patients. We hypothesize that an age-related decline in the mechanisms that clear Aß from the brain might be at least in part responsible for the failure to purge and re-distribute Aß. The expulsion of Aß via the blood-brain barrier is mediated by specialized transport proteins such as P-glycoprotein (P-gp, ABCB1/MDR1). OBJECTIVE: The objective of this study is to investigate the influence of the absence of P-gp at the bloodbrain barrier on the effectiveness of Aß peptide immunization in APP/PS1+/- P-gp ko mice. METHODS: Male APP/PS1+/- P-gp wt (n = 8) and APP/PS1+/- P-gp ko (n = 8) mice were actively immunized with human Aß42. After behavioral testing animals were sacrificed at the age of 395 days (+/- 5 days) and antibody titres against Aß were measured. Brains were dissected and soluble/insoluble cerebral Aß was quantified, additionally the number of amyloid plaques and severity of amyloid angiopathy were evaluated. RESULTS: In immunized mice with intact P-gp, our results showed a significant reduction of soluble and insoluble Aß40 and Aß42. Furthermore, immunization significantly reduced Aß plaque burden. In contrast, immunized APP/PS1+/- P-gp ko mice lacking functional P-gp did not show a reduction of Aß40 or Aß42 accumulation in the brain except for the soluble form of Aß42. Furthermore, after active immunization these mice displayed a stronger intracerebral amyloid angiopathy. CONCLUSION: The results show that the absence of P-gp results in a significant disturbance of Aß removal from the brain and increased intraparenchymal cerebral amyloid angiopathy after immunization against Aß. Our data indicate that the selective up-regulation of P-gp could enhance the efficacy of Aß immunization in the treatment or prevention of AD.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/deficiencia , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/metabolismo , Angiopatía Amiloide Cerebral/etiología , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Enfermedad de Alzheimer/inmunología , Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/toxicidad , Precursor de Proteína beta-Amiloide/genética , Animales , Anticuerpos/sangre , Modelos Animales de Enfermedad , Conducta Exploratoria/fisiología , Adyuvante de Freund/toxicidad , Hemorragia/etiología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/toxicidad , Presenilina-1/genéticaRESUMEN
The multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit ß3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knock-down of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leukaemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.