RESUMO
Intestinal drug transporters are crucial determinants for absorption and oral bioavailability of drugs. In healthy tissue donors, a recent study revealed profound discrepancies between mRNA expression and protein abundance as well as differences in the protein content between small and large intestine for clinically relevant multidrug transporters as the ATP binding cassette transporter subfamily B member 1 (ABCB1) and subfamily C member 3 (ABCC3) and the solute carrier family 15 member 1 (SLC15A1, PEPT1). As the mechanisms underlying these observations remained unclear, the aim of the present study was to elucidate the intestinal regiospecific microRNA profile under physiological conditions and identify specific microRNAs contributing to the post-transcriptional regulation of major drug transporters. For this purpose, tissue samples were collected from six intestinal sites obtained from six healthy tissue donors. The expression of 754 microRNAs was determined using qRT-PCR based low density arrays, and microRNA expression levels were correlated with transporter protein abundance quantified by targeted proteomics. A total of 241 microRNA-transporter pairs were identified, showing significant negative correlations to protein abundance (p < 0.05). Out of these, for nine pairs, the binding of the microRNA to the respective transporter 3'-UTR was predicted in silico. Besides the already known interactions of miR-27a-3p-ABCB1 and miR-193a-3p-PEPT1, reporter gene assays confirmed binding of miR-192-5p to the ABCC3 3'-UTR (reduction of reporter gene activity by 31%; p = 0.0012), miR-409-3p to the ABCB1 3'-UTR (reduction by 38%; p = 0.0006), and miR-193b-3p as well as miR-27a-3p to PEPT1 3'-UTR (reduction by 49% (p = 0.0012) and 20% (p = 0.0043), respectively). These results suggest that mucosal microRNA expression contributes to the explanation of discrepancies between mRNA expression and protein abundance as well as site-dependent differences in protein content along the human intestine under physiological conditions, as exemplified for ABCB1, ABCC3, and PEPT1.
Assuntos
Mucosa Intestinal/metabolismo , MicroRNAs/metabolismo , Adulto , Humanos , MicroRNAs/genética , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutagênese Sítio-Dirigida , Transportador 1 de Peptídeos/genética , Transportador 1 de Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Adulto JovemRESUMO
MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally control the expression of their target genes via RNA interference. There is increasing evidence that expression of miRNAs is dysregulated in neuronal disorders, including epilepsy, a chronic neurological disorder characterized by spontaneous recurrent seizures. Mesial temporal lobe epilepsy (MTLE) is a common type of focal epilepsy in which disease-induced abnormalities of hippocampal neurogenesis in the subgranular zone as well as gliosis and neuronal cell loss in the cornu ammonis area are reported. We hypothesized that in MTLE altered miRNA-mediated regulation of target genes could be involved in hippocampal cell remodeling. A miRNA screen was performed in hippocampal focal and non-focal brain tissue samples obtained from the temporal neocortex (both n=8) of MTLE patients. Out of 215 detected miRNAs, two were differentially expressed (hsa-miR-34c-5p: mean increase of 5.7 fold (p=0.014), hsa-miR-212-3p: mean decrease of 76.9% (p=0.0014)). After in-silico target gene analysis and filtering, reporter gene assays confirmed RNA interference for hsa-miR-34c-5p with 3'-UTR sequences of GABRA3, GRM7 and GABBR2 and for hsa-miR-212-3p with 3'-UTR sequences of SOX11, MECP2, ADCY1 and ABCG2. Reporter gene assays with mutated 3'-UTR sequences of the transcription factor SOX11 identified two different binding sites for hsa-miR-212-3p and its primary transcript partner hsa-miR-132-3p. Additionally, there was an inverse time-dependent expression of Sox11 and miR-212-3p as well as miR-132-3p in rat neonatal cortical neurons. Transfection of neurons with anti-miRs for miR-212-3p and miR-132-3p suggest that both miRNAs work synergistically to control Sox11 expression. Taken together, these results suggest that differential miRNA expression in neurons could contribute to an altered function of the transcription factor SOX11 and other genes in the setting of epilepsy, resulting not only in impaired neural differentiation, but also in imbalanced neuronal excitability and accelerated drug export.
Assuntos
Encéfalo/metabolismo , Epilepsia Resistente a Medicamentos/genética , Epilepsia Resistente a Medicamentos/patologia , MicroRNAs/metabolismo , Fatores de Transcrição SOXC/genética , Fatores de Transcrição SOXC/metabolismo , Adulto , Animais , Linhagem Celular Tumoral , Células Cultivadas , Córtex Cerebral/citologia , Simulação por Computador , Feminino , Perfilação da Expressão Gênica , Hepatoblastoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Componente Principal , Ratos , Fatores de Tempo , Adulto JovemRESUMO
BACKGROUND: The functional influence of single-nucleotide polymorphisms (SNPs) of the ATP-binding cassette (ABC) transporter ABCC2 (MRP2) has been characterized in numerous studies. The aim of this study was to address the question of whether distinct ABCC2 haplotypes, which differ in their mRNA secondary structures, show an influence on the degree of mRNA and protein downregulation through miRNA interaction. METHODS: A model using human peripheral blood monocytic cells (PBMCs) isolated from healthy Caucasian volunteers, with three defined ABCC2 haplotypes comprising the 5'-UTR SNP -24C>T, the 1249G>A SNP (V417I), and the silent 3972C>T SNP, was outlined. Cells were transiently transfected with miRNA-379, already known to target ABCC2 in HepG2 cells. RESULTS: ABCC2 was downregulated through miR-379 in a haplotype-dependent manner: the wild-type CGC/CGC was modestly affected (mRNA: -12.7±4.2%, protein: -9.9±0.1%), whereas variant haplotypes were more strongly suppressed: CGT/CGT (mRNA: -36.7±2.4%, protein: -21.6±0.4%) and TGT/TGT (mRNA: -55.7±1.2%, protein: -46.3±4.0%). In addition, glutathione-methylfluorescein efflux was significantly reduced in miR-379-transfected peripheral blood monocytic cells corresponding to ABCC2 protein expression. CONCLUSION: This observation may suggest a differential suppression of ABCC2 by miR-379 caused by haplotype-dependent differences in mRNA secondary structures, resulting in changes in mRNA target accessibility or mRNA stability.
Assuntos
MicroRNAs/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , RNA Mensageiro/genética , Adulto , Feminino , Regulação da Expressão Gênica , Haplótipos , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Polimorfismo de Nucleotídeo Único , Estabilidade de RNA/genéticaRESUMO
MicroRNAs (miRNAs) are small noncoding RNAs, which regulate the expression of their target genes post-transcriptionally by RNA interference. They are involved in almost all cellular processes, including proliferation, differentiation, apoptosis, cell survival and the maintenance of tissue specificity. Recent findings also suggest that efflux pumps of the ABC (ATP-binding cassette) transporter family are subject to miRNA-mediated gene regulation. Moreover, it seems that ABC transporters are embedded in a concerted and miRNA-guided network of concurrently regulated proteins that mediate altered drug transport and cell survival in changing environmental conditions. In this review, we summarize recent findings of miRNAs interacting with ABC transporters, which have been connected with drug distribution as well as with drug resistance. Additionally, we specify findings of complex miRNA-protein pathways conferring increased drug export and cell survival.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , MicroRNAs/fisiologia , Farmacogenética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Sobrevivência Celular/genética , Resistência a Medicamentos/genética , Regulação da Expressão Gênica/genética , HumanosRESUMO
BACKGROUND: Despite the enormous success of imatinib in chronic myeloid leukemia (CML), therapy resistance has emerged in a significant proportion of patients, partly because of the overexpression of ABC efflux transporters. METHODS: Using an array comprising 667 miRNAs, we investigated whether the expression of microRNAs (miRNAs) is altered in CML K-562 cells becoming resistant to increasing concentrations of imatinib. ABCB1 and ABCG2 mRNA (quantitative real-time PCR) and protein expression (western blot) were quantified under short-term and 4 months' imatinib treatment. Interaction of miR-212 and miR-328 with ABCG2 was investigated by transfection experiments and reporter gene assays using respective miRNA precursors or miRNA inhibitors. RESULTS: Although ABCB1 protein was not expressed, ABCG2 protein was 7.2-fold elevated after long-term treatment with 0.3 µmol/l imatinib and decreased gradually at higher concentrations. miRNAs miR-212 and miR-328 were identified to correlate inversely with ABCG2 expression under these conditions. Short-term treatment also induced ABCG2 protein concentration dependently and caused a downregulation of miR-212, but not of miR-328 at all tested concentrations (P=0.050). Reporter gene assays confirmed miR-212 to target the 3'-UTR region of ABCG2. In contrast, transfection of anti-miR-212 revealed an upregulation of ABCG2 protein expression, whereas the effect of anti-miR-328 was weak. CONCLUSION: Our study suggests an association of imatinib treatment, miRNA downregulation and ABCG2 overexpression, possibly contributing to the mechanisms involved in imatinib distribution and response in CML therapy.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , MicroRNAs/metabolismo , Proteínas de Neoplasias/metabolismo , Piperazinas/administração & dosagem , Pirimidinas/administração & dosagem , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Benzamidas , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Proteínas de Neoplasias/genéticaRESUMO
PURPOSE: The cytotoxic drug cyclophosphamide (CP) is bioactivated into 4-hydroxy-cyclophosphamide (4-OH-CP) through cytochrome P450 enzymes and cleared through aldehyde dehydrogenase and glutathione S-transferase. This prospective study analyzes the influence of drug metabolizing enzyme genotype on (1) plasma 4-OH-CP:CP ratio and (2) myelotoxicity in breast cancer patients on 500 mg/m(2) cyclophosphamide. METHODS: Sixty-eight female breast cancer patients on FAC (fluorouracil, adriamycin, cyclophosphamide) were included. Genotyping of cytochrome P450 enzymes CYP2B6, CYP2C9, CYP2C19, CYP3A5, aldehyde dehydrogenase (ALDH3A1), and glutathione S-transferase (GSTA1) was done either through RFLP or pyrosequencing. Plasma CP and 4-OH-CP were measured immediately and 1 and 2 h after the end of infusion through LC-MS. The leukocyte count was determined on day 10 and 20 after chemotherapy. RESULTS: At CP dose of 500 mg/m(2), the 4-OH-CP:CP ratio was negatively affected by CYP2C19*2 genotype (p = 0.039) showing a gene-dose effect. Moreover ALDH3A1*2 genotype increased 4-OH-CP:CP ratio (p = 0.037). These effects did not remain significant in a univariate analysis of variance including all genotypes. GSTA1*B carriers were at increased risk of severe leucopenia (OR 6.94; 95% CI 1.75-27.6, p = 0.006). CONCLUSION: The myelotoxicity in patients receiving FAC is related to the activity of the phase-II enzyme GSTA1 but is independent of the formation of 4-OH-CP.
Assuntos
Aldeído Desidrogenase/genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Mama/genética , Sistema Enzimático do Citocromo P-450/genética , Glutationa Transferase/genética , Adulto , Idoso , Antineoplásicos Alquilantes/administração & dosagem , Antineoplásicos Alquilantes/sangue , Antineoplásicos Alquilantes/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Ciclofosfamida/administração & dosagem , Ciclofosfamida/análogos & derivados , Ciclofosfamida/sangue , Ciclofosfamida/farmacocinética , Ciclofosfamida/uso terapêutico , Doxorrubicina/farmacocinética , Doxorrubicina/uso terapêutico , Feminino , Fluoruracila/farmacocinética , Fluoruracila/uso terapêutico , Genótipo , Humanos , Contagem de Leucócitos , Pessoa de Meia-IdadeRESUMO
microRNAs (miRNAs), which contribute to the post-transcriptional processing through 3'-untranslated region-interference, have been shown to be involved in the regulation of ATP-binding cassette (ABC) membrane transporters. The aim of this study was to investigate whether ABCC2, an important efflux transporter for various endogenous and exogenous compounds at several compartment barriers, is subject to miRNA-mediated post-transcriptional gene regulation. We screened the expression of 377 human miRNAs in HepG2 cells after 48 h of treatment with 5 µM rifampicin [a pregnane X receptor (PXR) ligand] or vehicle using reverse transcription-polymerase chain reaction-based low-density arrays. Specific miRNA, ABCC2 mRNA, and protein expression were monitored in HepG2 cells undergoing rifampicin treatment for 72 h. Loss- and gain-of-function experiments and reporter gene assays were performed for further confirmation. Highly deregulated miRNAs compared with in silico data revealed miRNA (miR) 379 as candidate miRNA targeting ABCC2 mRNA. Under rifampicin treatment, ABCC2 mRNA increased significantly, with a maximal fold change of 1.56 ± 0.43 after 24 h. In addition, miR-379 increased (maximally 4.10 ± 1.33-fold after 48 h), whereas ABCC2 protein decreased with a maximal fold change of 0.47 ± 0.08 after 72 h. In contrast, transfection of miR-379 inhibitor led to an elevation of ABCC2 protein expression after rifampicin incubation for 48 h. We identify a miRNA negatively regulating ABCC2 on the post-transcriptional level and provide evidence that this miRNA impedes overexpression of ABCC2 protein after a PXR-mediated external transcriptional stimulus in HepG2 cells.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , MicroRNAs/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Rifampina/farmacologia , Sítios de Ligação/genética , Células Hep G2 , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Interferência de RNA/fisiologiaRESUMO
BACKGROUND: Antiepileptic treatment response has been suggested to be modulated by genetic polymorphisms of drug efflux transporters, in particular ABCB1. Recently, we found a significant association of ABCC2 -24C>T with nonresponse, primarily in the context of generalized epilepsy. Moreover, ABCC2 1249G>A was reported to alter transmembranal carbamazepine transport. Therefore, we aimed to confirm the association of ABCC2 variants with pharmacotherapy-resistance in Caucasians mainly affected by partial epilepsy. PATIENTS AND METHODS: A total of 208 patients (114 male; age: 11.3±5.9 years) were genotyped for three putatively functionally relevant polymorphisms of ABCC2 (-24C>T, 1249G>A, 3972C>T). Genotype and haplotype frequencies were compared between responders and nonresponders to first-line antiepileptic treatment. RESULTS: Carriers of the ABCC2 1249G>A variant (417V>I) were more frequent among responders [odds ratio (OR)=2.68 (1.25-5.78); P=0.010]. This association remained significant after adjusting for age, sex and seizure type, [OR=2.88 (1.23-6.73); P=0.015]. The impact of 1249G>A was more pronounced among 64 patients receiving carbamazepine or oxcarbazepine (P=0.005), but nonsignificant in patients receiving other anticonvulsants. ABCC2 -24C>T and 3972C>T showed lack of association to therapy response. Haplotype analyses revealed that haplotype H2 containing solely the 1249A variant allele was more frequent in the responder group [OR=2.98 (1.38-6.44); P=0.004]. DISCUSSION: These data argue for a greater probability of antiepileptic drug response among carriers of the ABCC2 1249A variant that is associated with reduced carbamazepine transport. Although we could not confirm an impact of ABCC2 -24C>T, these results suggest that ABCC2 genotype may also modulate the response to anticonvulsants besides the extensively studied ABCB1 (P-glycoprotein).
Assuntos
Anticonvulsivantes/uso terapêutico , Carbamazepina/análogos & derivados , Carbamazepina/uso terapêutico , Resistência a Medicamentos/genética , Epilepsias Parciais/tratamento farmacológico , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Adolescente , Alelos , Biomarcadores Farmacológicos , Criança , Feminino , Estudos de Associação Genética , Genótipo , Haplótipos , Humanos , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Oxcarbazepina , Polimorfismo de Nucleotídeo Único , Ácido Valproico/uso terapêutico , População Branca/genéticaRESUMO
BACKGROUND: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by constitutive activity of the tyrosine kinase BCR-ABL1. Although the introduction of tyrosine kinase inhibitors (TKIs) has substantially improved patients' prognosis, drug resistance remains one of the major challenges in CML therapy. MicroRNAs (miRNAs), a class of short non-coding RNAs acting as post-transcriptional regulators, are implicated in CML progression and drug resistance. The aim of the present study was to analyze the miRNA expression profiles of 45 treatment-naïve CML patients in chronic phase (28 peripheral blood and 17 bone marrow samples) with respect to future response to imatinib therapy. METHODS: TaqMan low density arrays were used to analyze the miRNA expression pattern of the patient samples. For selected microRNAs, reporter gene assays were performed to study their ability to regulate CML associated target genes. RESULTS: Significant lower expression levels of miR-142-5p were identified in both, peripheral blood and bone marrow samples of future non-responders suggesting a potential tumor suppressor role of this miRNA. This was supported by reporter gene assays that identified the survival, proliferation and invasion promoting CML related genes ABL2, cKIT, MCL1 and SRI as targets of miR-142-5p and miR-365a-3p, the latter identified as potential biomarker in peripheral blood samples. CONCLUSION: MiR-142-5p and to a certain extend also miR-365a-3p were able to discriminate treatment-naïve CML patients not responding to imatinib in the course of their treatment from patients, who responded to therapy. However, further large-scale studies should clarify if the identified miRNAs have the potential as predictive biomarkers for TKI resistance.
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Pain sensitivity is characterized by interindividual variability, determined by factors including genetic variation of nociceptive receptors and pathways. The sigma-1 receptor (SIGMAR1) is involved in pain modulation especially under pre-sensitized conditions. However, the contribution of SIGMAR1 genetic variants to pain generation and sensitivity is unknown yet. This study aimed to identify effects of 5 SIGMAR1 variants on the somatosensory phenotype of neuropathic pain patients (nâ¯=â¯228) characterized by standardized quantitative sensory testing. Principal component analysis revealed that the SIGMAR1 variants -297G>T (rs10814130) and 5A>C (rs1800866) significantly lowered thermal detection and heat/pressure nociception in particular in neuropathic pain patients with mainly preserved somatosensory function. Compared to wild-type, the variant allele -297T was associated with loss of warm detection (Pâ¯=â¯.049), lower heat-pain sensitivity (Pâ¯=â¯.027) and wind-up ratio (Pâ¯=â¯.023) as well as increased paradoxical heat sensation (Pâ¯=â¯.020). Likewise for 5A>C the strongest genotype-associated differences observed were reduced peripheral (less heat hyperalgesia; Pâ¯=â¯.026) and central sensitization (lower mechanical pain sensitivity; Pâ¯=â¯.026) in variant compared to wild-type carriers. This study indicates lack of association of SIGMAR1 -297G>T and 5A>C genetic variants to susceptibility to develop chronic pain, but significant modulation of somatosensory function in neuropathic pain patients. PERSPECTIVE: This article presents the first study indicating a modulation of somatosensory function in neuropathic pain patients by selected genetic variants in SIGMAR1. As our findings could contribute to the explanation of interindividual differences in drug response they might help to improve the treatment of neuropathic pain.
Assuntos
Sensibilização do Sistema Nervoso Central/fisiologia , Hiperalgesia/genética , Hiperalgesia/fisiopatologia , Neuralgia/genética , Neuralgia/fisiopatologia , Nociceptividade/fisiologia , Receptores sigma/genética , Percepção do Tato/fisiologia , Adulto , Idoso , Sensibilização do Sistema Nervoso Central/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Percepção do Tato/genética , Receptor Sigma-1RESUMO
Intestinal transporters and metabolizing enzymes are the important factors of the intestinal absorption barrier. Because there is evidence that their expression and function may be affected during inflammatory conditions, we investigated gene expression, protein abundance, and regulation of relevant intestinal transporters and metabolizing enzymes in the intestinal mucosa of patients with ulcerative colitis (UC). Specimens from inflamed and noninflamed tissues of 10 patients with UC as well as colonic control tissues of 10 patients without inflammation were subjected to gene (9 enzymes, 15 transporters, 9 cytokines) and microRNA (N = 54) expression analysis. Protein abundance was quantified by liquid chromatography-tandem mass spectrometry-based targeted proteomics. Gene expression of several metabolizing enzymes (e.g., CYP2C9, UGT1A1) and transporters such as ABCB1 (ABCB1), ABCG2 (ABCG2), and monocarboxylate transporter 1 (MCT1, SLC16A1) were significantly decreased during inflammation and negatively correlated to microRNAs. On contrary, multidrug resistance-protein 4 (MRP4, ABCC4), organic anion-transporting polypeptide 2B1 (OATP2B1, SLCO2B1), and organic cation transporter-like 2 (ORCTL2, SLC22A18) were significantly elevated in inflamed tissue. However, at protein level, these findings could only be confirmed for MCT1. UC is associated with complex changes in the intestinal expression of enzymes, transporters, cytokines, and microRNAs, which may affect efficacy of anti-inflammatory drug therapy or the disease state itself.
Assuntos
Colite Ulcerativa/genética , Sistema Enzimático do Citocromo P-450/genética , Regulação da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Preparações Farmacêuticas/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adulto , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Sistema Enzimático do Citocromo P-450/metabolismo , Feminino , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Intestinos/patologia , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Pessoa de Meia-Idade , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Adulto JovemRESUMO
BCR-ABL-independent resistance against tyrosine kinase inhibitor is an emerging problem in therapy of chronic myeloid leukemia. Such drug resistance can be linked to dysregulation of ATP-binding cassette (ABC)-transporters leading to increased tyrosine kinase inhibitor efflux, potentially caused by changes in microRNA expression or DNA-methylation. In an in vitro-imatinib-resistance model using K-562 cells, microRNA-212 was found to be dysregulated and inversely correlated to ABC-transporter ABCG2 expression, targeting its 3'-UTR. However, the functional impact on drug sensitivity remained unknown. Therefore, we performed transfection experiments using microRNA-mimics and -inhibitors and investigated their effect on imatinib-susceptibility in sensitive and resistant leukemic cell lines. Under imatinib-treatment, miR-212 inhibition led to enhanced cell viability (p = 0.01), reduced apoptosis (p = 0.01) and cytotoxicity (p = 0.03). These effects were limited to treatment-naïve cells and were not observed in cells, which were resistant to various imatinib-concentrations (0.1 µM to 2 µM). Further analysis in treatment-naïve cells revealed that miR-212 inhibition resulted in ABCG2 upregulation and increased ABCG2-dependent efflux. Furthermore, we observed miR-212 promoter hypermethylation in 0.5 and 2 µM IM-resistant sublines, whereas ABCG2 methylation status was not altered. Taken together, the miR-212/ABCG2-axis influences imatinib-susceptibility contributing to development of imatinib-resistance. Our data reveal new insights into mechanisms initiating imatinib-resistance in leukemic cells.
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BACKGROUND AND AIMS: Ezetimibe is an inhibitor of the cholesterol uptake transporter Niemann-Pick C1-like protein (NPC1L1). Target concentrations can be influenced by intestinal uridine diphosphate-glucuronosyltransferases (UGTs) and the efflux transporters P-glycoprotein (P-gp) (ABCB1) and multidrug resistance associated protein 2 (MRP2) (ABCC2). This study evaluates the contribution of these factors to the disposition and cholesterol-lowering effect of ezetimibe before and after induction of UGT1A1, P-gp, and MRP2 with rifampin (INN, rifampicin). METHODS: Serum concentrations of ezetimibe, as well as its glucuronide, and the plant sterols campesterol and sitosterol (surrogate for cholesterol absorption) were studied in 12 healthy subjects before and after rifampin comedication. In parallel, duodenal expression of UGT1A1, P-gp, MRP2, and NPC1L1 was quantified by use of real-time reverse transcriptase-polymerase chain reaction and quantitative immunohistochemical evaluation. The affinity of ezetimibe and its glucuronide to P-gp and MRP2 was assessed in P-gp- overexpressing Madin-Darby canine kidney II cells and P-gp-containing or MRP2-containing inside-out vesicles. RESULTS: Up-regulation of intestinal P-gp, MRP2, and UGT1A1 (but not of NPC1L1) by rifampin was associated with markedly decreased areas under the curve of ezetimibe and its glucuronide (116 +/- 78.1 ng.h/mL versus 49.9 +/- 31.0 ng.h/mL and 635 +/- 302 ng.h/mL versus 225 +/- 86.4 ng.h/mL, respectively; both P = .002) and increased intestinal clearances (2400 +/- 1560 mL/min versus 5500 +/- 4610 mL/min [P = .003] and 76.6 +/- 113 mL/min versus 316 +/- 457 mL/min [P = .010], respectively) and nearly abolished sterol-lowering effects. Intestinal expression of UGT1A1, ABCB1, and ABCC2 was inversely correlated with the effects of ezetimibe on plant sterol serum concentrations. Parallel in vitro studies confirmed that ezetimibe glucuronide is a high-affinity substrate of MRP2 and has a low affinity to P-gp whereas ezetimibe interacts with P-gp and MRP2. CONCLUSIONS: The disposition and sterol-lowering effects of ezetimibe are modified by metabolic degradation of the drug via intestinal UGT1A1 and either intestinal or hepatic secretion (or both) via P-gp and MRP2.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Anticolesterolemiantes/farmacocinética , Azetidinas/farmacocinética , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/genética , Absorção Intestinal/genética , Mucosa Intestinal/metabolismo , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adulto , Antibióticos Antituberculose/farmacologia , Área Sob a Curva , Interações Medicamentosas , Ezetimiba , Feminino , Humanos , Intestinos/enzimologia , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Rifampina/farmacologia , Esteróis/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
AIM: To identify the exact length and possible length variations of the ABCB1 3'-UTR as important regulatory site for miRNA interaction of this drug transporter and its possible contribution to drug resistance. MATERIALS & METHODS: 3'-RACE and various standard PCR experiments were performed using cDNA of different human cell lines and liver tissue. The abundance of 3'-UTR fragments was analyzed using quantitative RT-PCR. RESULTS: Five different ABCB1 3'-UTR length variants were identified. miRNA binding sites were located only on the three longer fragments. Imatinib-resistant leukemia cells expressed predominantly shorter 3'-UTRs, where miRNA binding sites are absent. CONCLUSION: Shortening of the ABCB1 3'-UTR causes loss of miRNA-dependent translational control leading to elevated ABCB1 protein levels.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Mesilato de Imatinib/farmacologia , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Sítios de Ligação , Linhagem Celular Tumoral , Simulação por Computador , Variação Genética , Humanos , MicroRNAs/genética , Reação em Cadeia da PolimeraseRESUMO
Elafin is a potent reversible inhibitor of the pro-inflammatory proteases leukocyte elastase and protease 3. It is currently in clinical development for the use in postoperative inflammatory diseases. We investigated the pharmacokinetics of (99m)Tc-labeled elafin ((99m)Tc-Elafin) in blood and individual organs in rat after bolus intravenous injection using the single photon emission tomography (SPECT). (99m)Tc-Elafin predominantly accumulated in the kidney reaching a maximum of 8.5% ± 0.1% of the injected dose per gram (ID/g) at 5 min post injection (p.i) and decreased only slowly during 24 h. In contrast, the initially high radio activity recorded in the other organs rapidly decreased parallel to the radioactivity detected in blood. The blood kinetics fits to a two compartment kinetics model. The radio activity in the dissected kidney was 4.98 ± 1.24%ID/g 24 h p.i, while in other organs, including the brain, no accumulation of (99m)Tc-Elafin was detected. At this time point 30% of the detected radioactivity in the kidney was identified to be not metabolized (99m)Tc-Elafin. In conclusion, the blood and organ-specific kinetic data provide a basis for planning of adequate dosing regimens and the high accumulation of intact elafin in the kidney favors clinical developments targeting inflammatory kidney diseases, such as chronic allograft nephropathy after kidney transplantation.
Assuntos
Elafina/farmacocinética , Inibidores Enzimáticos/farmacocinética , Tecnécio/química , Animais , Elafina/química , Elafina/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Elastase Pancreática/antagonistas & inibidores , Elastase Pancreática/metabolismo , Ratos , Distribuição TecidualRESUMO
AIM: Correlation of outcomes of cyclophosphamide (CP) therapy in antineutrophil cytoplasmic antibody-associated vasculitis with genotype polymorphisms in prodrug activating cytochrome P450 enzyme genes CYP2C9 and CYP2C19. PATIENTS & METHODS: One hundred and ninety six patients with antineutrophil cytoplasmic antibody-associated vasculitis treated with CP, either as intravenous pulse or as daily oral medication, were included. Genotypes of CYP2C9 and CYP2C19 were correlated with clinical outcomes (leukopenia, infection, urotoxicity and treatment response). RESULTS: Sixty five (33.2%) patients had variant CYP2C9 and 55 (28.1%) had variant CYP2C19 genotype. In patients bearing variant CYP2C9, leukopenia was documented significantly more frequent than in carriers of wild-type CYP2C9 (55.4 vs 37.4%; odds ratio: 2.08; 95% CI: 1.14-3.80; p = 0.017). The impact of the CYP2C9 genotype was stronger in patients treated with oral CP (69.6 vs 45.6%; odds ratio: 2.73; 95% CI: 1.27-5.89; p = 0.009), but was not present in patients treated with intravenous pulsed CP. We observed less refractory disease courses in patients with variant CYP2C9, not reaching statistical significance. CONCLUSION: Patients with variant CYP2C9 are at increased risk for cyclophosphamide-induced leukopenia but may have a better chance to respond to treatment.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/tratamento farmacológico , Ciclofosfamida/efeitos adversos , Citocromo P-450 CYP2C9/genética , Imunossupressores/efeitos adversos , Leucopenia/induzido quimicamente , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Resultado do Tratamento , Adulto JovemRESUMO
Regulatory RNAs play a key role in the regulation of protein expression patterns in neurological diseases. Here we studied the regulation of miRNAs in a chronic rat model of temporal lobe epilepsy. The analysis was focused on a putative link with pharmacoresponsiveness as well as the functional implications of the regulation of a selected miRNA. The findings did not reveal a difference in hippocampal miRNA expression between phenobarbital responders and nonresponders. However, when comparing rats following status epilepticus with control rats we identified 13 differentially expressed miRNAs with miRNA-187-3p being most strongly regulated. mRNAs encoding KCNK10/TREK-2 as well as DYRK2 were confirmed as targets of miRNA-187-3p. Expression of the potassium channel protein KCNK10/TREK-2 negatively correlated with hippocampal miRNA-187-3p expression and proved to be upregulated in the chronic phase of the epilepsy model. In conclusion, our data do not suggest a relevant impact of miRNA expression patterns on pharmacoresponsiveness. However, we confirmed regulation of miRNA-187-3p and demonstrated that it impacts the expression of the two-pore domain potassium channel protein KCNK10/TREK-2. Considering evidence from brain ischemia models, KCNK10/TREK-2 upregulation might serve a protective function with a beneficial impact on astrocytic potassium and glutamate homeostasis.
Assuntos
Epilepsia do Lobo Temporal/metabolismo , Hipocampo/metabolismo , MicroRNAs/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Anticonvulsivantes/farmacologia , Modelos Animais de Doenças , Epilepsia Resistente a Medicamentos/tratamento farmacológico , Epilepsia Resistente a Medicamentos/metabolismo , Estimulação Elétrica , Epilepsia do Lobo Temporal/tratamento farmacológico , Feminino , Expressão Gênica , Células Hep G2 , Hipocampo/efeitos dos fármacos , Humanos , Neuroestimuladores Implantáveis , MicroRNAs/genética , Mutação , Fenobarbital/farmacologia , Canais de Potássio de Domínios Poros em Tandem/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Ratos Sprague-Dawley , Estado Epiléptico/tratamento farmacológico , Estado Epiléptico/metabolismo , Quinases DyrkRESUMO
AIM: A number of studies have demonstrated that ABCB1 and BCRP (ABCG2) actively transport Aß. We aimed to investigate the association of genetic variants of selected multidrug transporters with Alzheimer's disease (AD) in histopathologically confirmed AD cases and controls. MATERIALS & METHODS: DNA from brain tissue of 71 AD cases with Consortium to Establish a Registry for Alzheimer's Disease (CERAD) neuropathological stages B/C and 81 controls was genotyped for selected variants in ABCA1, ABCA7, ABCB1, ABCC2 and ABCG2. In addition, the APOE4 status was analyzed. RESULTS: The novel ABCA7 SNP, rs3752246, tended to be associated with AD in our study. Variants in ABCB1 were significantly less frequent in AD cases older than 65 years of age and among females. This association of ABCB1 2677G>T (rs2032582) was more pronounced in APOE4-negative cases (p = 0.005). However, only ABCC2 3972C>T (rs3740066) was significantly associated with AD risk after logistic regression analysis including all variants. Other transporters showed a lack of association. CONCLUSION: Our results support the hypothesis that ABCB1 and possibly other ABC-transporters are involved in the process of Aß accumulation in the aging brain and may modulate the risk for AD in an allele-specific manner, and thus might represent a new target for prevention and treatment of AD.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Doença de Alzheimer/genética , Estudos de Associação Genética , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Idoso , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Polimorfismo de Nucleotídeo Único , Caracteres SexuaisRESUMO
Chemoresistance of tumors is often reported to be due to overexpression of efflux transporters or genetic alterations of signaling pathways. More recently, there is increasing evidence that epigenetic modification contributes to the phenomenon of drug resistance. Despite alteration of DNA methylation or histone modifications, deregulated miRNA expression patterns of tumor cells have been identified as interfering with drug response. Attempts to modify the expression of selected miRNAs have partly led to intriguing improvements of chemotherapy response. This review focuses on the major epigenetic mechanisms, including the role of miRNA expression contributing to drug resistance and the role of epigenetic drugs to overcome nonresponse arising under conventional chemotherapy.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , MicroRNAs/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , MicroRNAs/genéticaRESUMO
Overexpression of the microsomal enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1, human gene HSD11B1 or SDR26C1) associates with the metabolic syndrome as well as some inflammatory diseases. HSD11B1 expression is known to be highly tissue-specific and driven by two distinct promoters, an aspect which to date has been studied very little. Here, we sought to assess differential promoter usage in various glucocorticoid target tissues and cell lines. As transcription from the distal promoter P1 or the proximal promoter P2 results in transcripts differing in the 5'-UTR, we first used 5'-UTR-specific primers for their detection in semi-quantitative PCR after reverse transcription. We also established a quantitative Real-Time PCR method using 5'-UTR-specific fluorescent probes in combination with 5'-UTR-specific primers for absolute quantification of the two human transcripts in a duplex approach. The combined results demonstrate that transcription from P2 predominated in most tissues and cell lines assessed, including human liver, human lung, human subcutaneous adipose tissue, and the cell lines A549, Caco-2, C2C12 and 3T3-L1. Transcription from P1 predominated in the human tumor cell lines A431 and HT-29 and contributed significantly to overall HSD11B1 expression in human lung. Moreover, HSD11B1 transcription was upregulated in C2C12 and 3T3-L1 cells in the course of differentiation to myotubes and adipocytes, respectively, mostly reflecting increased transcription from P2, although also transcription from P1 gradually increased in the differentiation process. Interestingly, HSD11B1 transcript levels in all tested human tumor cell lines, namely A431, A549, Caco-2, HCT-116, HepG2, HT-29 and PANC-1, were either close to detection limit or undetectable. In conclusion, the results provide the first evidence for tissue- and differentiation state-specific promoter usage in expression of human HSD11B1. Finally, HSD11B1 appears to be frequently downregulated in human tumor cell lines.