RESUMO
The diagnosis of renal cell carcinoma is currently based on imaging techniques, mainly because there is no blood marker available for its detection. Thus, there is still the need for the development of novel tumor markers. We examined plasma levels of eight proteins in 15 renal cell carcinoma patients before and after surgery, and in 51 healthy controls using enzyme-linked immunosorbent assay. Plasma levels of α-enolase, calnexin, galectin-1, galectin-3 and lectin mannose-binding 2 were significantly higher in renal cell carcinoma patients than in controls (P < 0.05). Among these proteins, the sensitivities for galectin-1 and galectin-3 were higher than those for calnexin and lectin mannose-binding 2 in the specificity range from 80% to 100%. A combined use of galectin-1 and galectin-3 showed 98% specificity and 47% sensitivity. In addition, the assays showed that plasma α-enolase levels decreased significantly 4 weeks after nephrectomy (P = 0.0034), and this tendency continued until 12 weeks after nephrectomy (P = 0.0156). These findings suggest that α-enolase could be used in the postoperative follow up of renal cell carcinoma patients, whereas the combined use of galectin-1 and galectin-3 might represent a useful tool for primary detection.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma de Células Renais/sangue , Galectina 1/sangue , Galectina 3/sangue , Neoplasias Renais/sangue , Fosfopiruvato Hidratase/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Cuidados Pós-OperatóriosRESUMO
BACKGROUND: In order to clarify the factors causing hyperammonemia and to predict occurrences during treatment with valproic acid (VPA), we investigated the effect of the genetic polymorphism of carbamoyl-phosphate synthase 1 (CPS14217C>A) on susceptibility of hyperammonemia, together with the effect of coadministration of other anticonvulsants. METHODS: Seventy-nine patients with epilepsy were enrolled, and five of them had hyperammonemia. Univariate and multivariate logistic regression analyses were performed. RESULTS: The aspartate aminotransferase level in the patients with hyperammonemia was significantly higher than that in those without hyperammonemia. The risk of hyperammonemia was significantly influenced by the number of anticonvulsants concomitantly administered with VPA. Also, the distribution of the CPS14217C>A genotype differed depending on whether the patients had hyperammonemia or not. No significant effects of CPS14217 genotypes and the number of anticonvulsants coadministered with VPA on the serum concentrations of VPA were observed. The multivariate logistic regression analysis showed that the concomitant administration of two or more anticonvulsants with VPA and the heterozygous or homozygous carrier state of the A allele of the CPS14217C>A polymorphism were independent risk factors for developing hyperammonemia. CONCLUSIONS: These findings suggested that in epileptic patients undergoing VPA therapy, CPS14217A polymorphism and the number of coadministered anticonvulsants would be considered as risk factors for hyperammonemia, even if the serum VPA concentrations were controlled.
Assuntos
Carbamoil-Fosfato Sintase (Amônia)/genética , Epilepsia/tratamento farmacológico , Epilepsia/genética , Hiperamonemia/induzido quimicamente , Hiperamonemia/genética , Polimorfismo Genético , Ácido Valproico/efeitos adversos , Adolescente , Adulto , Distribuição por Idade , Análise de Variância , Anticonvulsivantes/administração & dosagem , Anticonvulsivantes/efeitos adversos , Criança , Estudos de Coortes , Relação Dose-Resposta a Droga , Esquema de Medicação , Feminino , Marcadores Genéticos/efeitos dos fármacos , Genótipo , Humanos , Hiperamonemia/epidemiologia , Incidência , Modelos Logísticos , Masculino , Análise Multivariada , Valores de Referência , Fatores de Risco , Distribuição por Sexo , Ácido Valproico/administração & dosagem , Adulto JovemRESUMO
The objective of this study was to identify novel pharmacogenetic determinants of treatment-related hepatotoxicity during the maintenance phase in children with acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL). Although the authors first determined whether genotypes of drug-metabolizing enzymes and transporters--glutathione S-transferase (GST) genes, GSTM1 positive/null, GSTT1 positive/null and GSTP1 A313G, methylenetetrahydrofolate reductase (MTHFR) C677T, reduced folate carrier 1 (RFC1) G80A, and breast cancer resistant protein (BCRP) C421A--were associated with hepatotoxicity for 24 patients, no significant difference was detected for genotype and allelic frequencies between the patients with and those without severe treatment-related hepatotoxicity. Therefore, the authors explored potential candidate polymorphisms associated with hepatotoxicity using the Illumina Infinium HumanHap300, encompassing more than 318,000 tag single-nucleotide polymorphisms (SNPs), for 8 of 24 patients with or without severe hepatotoxicity. Genome-wide genotyping uncovered a total of 28 candidate SNPs. rs1966862, in Rho GTPase-activating protein 24 (ARHGAP24), was the most significant of the candidates, and the genotypes of rs13424027 (PARD3B), rs1156304 (KCNIP4), rs10255262 (SLC13A1), rs7403531 (RASGRP1), and rs381423 (unidentified gene) were also significantly associated with severe hepatotoxicity. This study suggested rs1966862 (ARHGAP24) and the other SNPs to be predictive factors for drug-induced hepatotoxicity during the maintenance phase in pediatric patients with ALL or LBL.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/genética , Estudos de Associação Genética , Polimorfismo Genético , Leucemia-Linfoma Linfoblástico de Células Precursoras/complicações , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Criança , Pré-Escolar , Feminino , Proteínas Ativadoras de GTPase/genética , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Valor Preditivo dos TestesRESUMO
The purpose of this study is to examine the effects of doxazosin, an alpha-adrenoceptor antagonist, on P-glycoprotein/MDR1-mediated multidrug resistance (MDR) and the transport of anticancer drugs. The effects of doxazosin, prazosin, and terazosin on MDR1-mediated MDR were assessed in human cervical carcinoma HeLa cells and the MDR1-overexpressing derivative Hvrl00-6, established by stepwise increases of the vinblastine concentration in the culture medium. The effects of doxazosin on the transcellular transport and intracellular accumulation of [3H]vinblastine, [3H]daunorubicin, and [3H]digoxin, all MDR1 substrates, were evaluated using LLC-GA5-COL150 cell monolayers, established by transfection of human MDR1 cDNA into porcine kidney epithelial LLC-PK1 cells. The sensitivity to vinblastine and paclitaxel of Hvrl00-6 cells was increased at 3.4- and 17.5-fold, respectively, by the addition of 1 microM doxazosin, whereas prazosin and terazosin had weaker or no such effects. Prazosin at 1 microM had a reversal effect on the sensitivity to vinblastine, whereas terazosin had no effect. In transport experiments, doxazosin concentration dependently increased the apical-to-basal transport of radiolabeled drugs in LLC-GA5-COL150 cells, but did not show remarkable effects on the basal-to-apical transport. In addition, doxazosin restored the intracellular accumulation in a concentration-dependent manner in LLC-GA5-COL150 cells. Doxazosin may partly reverse MDR by inhibiting MDR1-mediated transport, making it a candidate lead compound in the development of a reversing agent for MDR.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Antagonistas Adrenérgicos alfa/farmacologia , Doxazossina/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/fisiologia , Transporte Biológico/efeitos dos fármacos , Daunorrubicina/farmacocinética , Digoxina/farmacocinética , Células HeLa , Humanos , Proteínas de Neoplasias/fisiologia , Prazosina/análogos & derivados , Prazosina/farmacologia , Vimblastina/farmacocinéticaRESUMO
Renal cell carcinoma (RCC) is relatively resistant to chemotherapy and radiotherapy. Recent advances in drug development are providing novel agents for the treatment of RCC, but the effects are still minimal. In addition, there is an urgent need to identify diagnostic markers for RCC. In this report, to discover potential diagnostic markers and therapeutic targets, we subjected RCC samples to a quantitative proteomic analysis utilizing 2-nitrobenzenesulfenyl (NBS) reagent. Proteins were extracted from RCC and adjacent normal tissue, obtained surgically from patients, and labeled with NBS reagent containing six (12)C or (13)C. This was followed by trypsin digestion and the enrichment of labeled peptides. Samples were then subjected to analysis by MALDI-TOF MS. NBS-labeled peptides with a 6 Da difference were identified by MS/MS. Thirty-four proteins were upregulated in more than 60% of the patients of which some were previously known, and some were novel. The identity of a few proteins was confirmed by Western blotting and quantitative real time RT-PCR. The results suggest that NBS-based quantitative proteomic analysis is useful for discovering diagnostic markers and therapeutic targets for RCC.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Proteoma/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/antagonistas & inibidores , Western Blotting , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/tratamento farmacológico , Feminino , Humanos , Neoplasias Renais/diagnóstico , Neoplasias Renais/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Proteoma/genética , Proteoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Previously, MDR1 T-129C polymorphism, encoding multidrug resistant transporter MDR1/P-glycoprotein, was reported to be predictive of poorly-differentiated colorectal adenocarcinomas. Here, VEGF T-1498C, C-634G and C-7T polymorphisms, encoding vascular endothelial growth factor (VEGF), were investigated in terms of their association with differentiation grade. METHODS: VEGF genotypes were determined by TaqMan(R) MGB probe based polymerase chain reaction and evaluated were confirmed by direct sequencing in 36 Japanese patients. RESULTS: VEGF T-1498C, but not C-634G or C-7T, was predictive of poorly-differentiated ones, and thereby a poor prognosis (p = 0.064 for genotype, p = 0.037 for allele), and this effect can be explained by that on VEGF expression. Treatment of a colorectal adenocarcinoma cell line, HCT-15, with sodium butyrate, a typical differentiating agent, resulted in an increase of alkaline phosphatase activity and MDR1 mRNA expression, but in a decrease of VEGF mRNA expression. The transfection of VEGF small interfering RNA (siRNA) induced the expression of MDR1 mRNA to 288-332% of the control level, whereas MDR1 siRNA had no effect on VEGF mRNA expression. CONCLUSIONS: VEGF T-1498C polymorphism is also a candidate marker predictive of poorly-differentiated colorectal adenocarcinomas, but further investigations with a large number of patients should be addressed to draw a conclusion.
Assuntos
Adenocarcinoma/genética , Transformação Celular Neoplásica/genética , Neoplasias Colorretais/genética , Fator A de Crescimento do Endotélio Vascular/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adulto , Idoso , Fosfatase Alcalina/metabolismo , Povo Asiático/genética , Butiratos/farmacologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Feminino , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/metabolismo , RNA Interferente PequenoRESUMO
The intrinsic or acquired resistance to anticancer drugs remains one of the most significant factors impeding the progress of cancer chemotherapy. This phenomenon often involves simultaneous resistance to other anticancer drugs that differ in their chemical structure and mode of action and are not even used in chemotherapy. This phenotype has been called multidrug resistance (MDR). Although the cellular basis underlying MDR is not fully understood, several factors mediating therapy resistance in tumors have been proposed. One of the mechanisms leading to chemoresistance of tumor cells is the increased activity of transporter proteins. The best-characterized transporter protein is MDR1/P-glycoprotein, and a number of clinical investigations have suggested that its intrinsic or acquired overexpression resulted in a poor clinical outcome of chemotherapy. Various types of compounds and techniques for the reversal of MDR1/P-glycoprotein-mediated MDR have been developed, and efforts have concentrated on the inhibition of function and suppression of expression. This review summarizes the current state of knowledge of MDR1/P-glycoprotein and the modulation of MDR by targeting MDR1/P-glycoprotein.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Genes MDR/genética , Genes MDR/fisiologia , Neoplasias/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Animais , Humanos , FarmacogenéticaRESUMO
Recent advances in pharmacogenomics have suggested the association of clinical outcome of glucocorticoid-based anti-inflammatory therapy with a single nucleotide polymorphism at position 3435 in exon 26 (C3435T) of the MDR1 gene. In the present study, the effects of the MDR1 C3435T genotype on the time-dependent profiles of gene expression and function of MDR1/P-glycoprotein were evaluated in peripheral blood mononuclear cells (PBMCs) under lipopolysaccharide (LPS)-induced experimental acute inflammation. LPS treatment resulted in the rapid elevation of IL-1beta and TNF-alpha mRNA levels relative to beta-actin mRNA at 1 h, with a subsequent slight decrease at 3 h after the treatment, while the down-regulation of the relative concentration of MDR1 mRNA was found at 3 h, not at 1 h, after LPS treatment. Here, the C3435T genotype-dependent down-regulations of MDR1 mRNA level were found for CC(3435) and CT(3435), but not for TT(3435), and were 64.1+/-10.1%, 71.4+/-5.9% and 100.0+/-22.5% (+/-S.D.), respectively, of their respective baseline levels, which were independent of C3435T (0.010+/-0.005, 0.011+/-0.013 and 0.009+/-0.006 (+/-S.D.), respectively). The C3435T genotype-dependent down-regulation was supported by the increase of the intracellular accumulation of calcein in PBMCs treated with LPS for 72 h, and the increase was more predominant for CC(3435) than TT(3435). These data suggested that glucocorticoid-based anti-inflammatory therapy might be more effective for C(3435)-allele carriers than non-carriers.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Expressão Gênica/genética , Inflamação/fisiopatologia , Leucócitos Mononucleares/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Doença Aguda , Adulto , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Citometria de Fluxo , Fluoresceínas/química , Fluoresceínas/metabolismo , Fluoresceínas/farmacocinética , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacocinética , Expressão Gênica/efeitos dos fármacos , Genótipo , Humanos , Inflamação/sangue , Inflamação/imunologia , Interleucina-1/genética , Interleucina-1/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Masculino , Polimorfismo Genético , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The genotype frequencies of MDR1 T-129C, C1236T, G2677A,T and C3435T SNPs were compared in 154 healthy Japanese and 100 healthy Caucasians to provide basic information on the inter-ethnic differences of pharmacotherapeutic outcome. The variants were found at allelic frequencies of 5.5%, 65.6%, 16.6%, 40.6% and 40.6%, for T-129C, C1236T, G2677A, G2677T and C3435T, respectively, in Japanese, and at 5.1%, 45.9%, 3.6%, 46.4% and 56.6%, respectively, in Caucasians, with a statistically significant difference for C1236T, G2677A,T and C3435T (p<0.001). G2677A was about 5-fold more frequent in Japanese than Caucasians. These genotype frequencies were also investigated in 95 Japanese patients with colorectal cancer (CRC) and esophageal squamous cell carcinoma (ESCC), but no significant difference was detected, when compared with healthy Japanese subjects. The haplotype frequency reached a total of about 85% in Japanese with the following 4 major haplotypes; T(-129)-T1236-T2677-T3435 (36.1%), T(-129)-T1236-G2677-C3435 (22.5%), T(-129)-C1236-G2677-C3435 (14.2%) and T(-129)-C1236-A2677-C3435 (13.3%). The second and fourth haplotypes were hardly inferred in Caucasian, whereas T(-129)-C1236-G2677-T3435 (12.8%) was found to be Caucasian-specific. There was a tendency for higher frequencies of the T(-129)/C-(129)-C1236-A2677-C3435 haplotype in Japanese CRC patients and T(-129)-T1236-T2677-T3435 haplotype in Japanese ESCC patients, compared with that in healthy Japanese subjects.
Assuntos
Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/genética , Neoplasias Esofágicas/epidemiologia , Neoplasias Esofágicas/genética , Genes MDR/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , DNA/genética , Neoplasias Esofágicas/tratamento farmacológico , Feminino , Frequência do Gene , Genótipo , Haplótipos , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , População BrancaRESUMO
The aim of this work is to develop a highly sensitive assay system for P-gp-mediated transport by using two methods, induction of P-gp and short-term culture of Caco-2 cells. To induce P-gp in Caco-2 cells, cells were cultured in vinblastine-containing medium. The mRNA level of P-gp was approximately 7-fold higher in Caco-2 cells cultured with vinblastine (P-gp-induced Caco-2 cells) than in control cells. Western blot analysis showed a significant increase in P-gp expression. After cell differentiation, the mRNA level of P-gp was downregulated, however, P-gp-induced Caco-2 cells still possessed a 5.6-fold higher mRNA level of P-gp compared to control cells. Polarized transport of substrate drugs was greater in the monolayer of P-gp-induced cells than in that of control cells. Moreover, we found that P-gp expression in Caco-2 cells could be further enhanced by applying the higher concentration of vinblastine. Transport activity of P-gp in Caco-2 cells cultured with higher concentration of vinblastine was markedly higher than that in P-gp-induced Caco-2 cells and was comparable with that in MDR1-MDCKII cells. In conclusion, this study provided a stable and highly sensitive in vitro assay system that can identify compounds that are subject to P-gp-mediated efflux.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Preparações Farmacêuticas/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Animais , Antineoplásicos Fitogênicos/farmacocinética , Antineoplásicos Fitogênicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Western Blotting , Células CACO-2 , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/genética , Processos de Crescimento Celular/fisiologia , Linhagem Celular , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Digoxina/farmacocinética , Digoxina/farmacologia , Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Transportador 1 de Peptídeos , Quinidina/farmacocinética , Quinidina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Simportadores/genética , Simportadores/metabolismo , Simportadores/fisiologia , Verapamil/farmacocinética , Verapamil/farmacologia , Vimblastina/farmacocinética , Vimblastina/farmacologiaRESUMO
Cystatin C (Cys-C) has been recently paid great attention as a better endogenous marker of the glomerular filtration rate than creatinine (Cr). In this study, the usefulness of Cys-C was compared with Cr in terms of the estimation of the steady-state serum trough concentrations of digoxin in Japanese patients. Forty patients treated with digoxin and 56 healthy elderly subjects were participated in this study. The serum levels of Cys-C and Cr in the patients were higher than those in the healthy elderly subjects, but the increase of Cys-C was more predominant in the patients. Their levels were well-correlated for both of the healthy elderly subjects (r=0.691) and patients (r=0.774), but the serum concentrations of digoxin were better correlated with those of the reciprocal values of Cr (r=0.667) than those of Cys-C (r=0.383), presumably due to the fact that digoxin and Cr were excreted via both glomerular filtration and tubular secretion. Cys-C is useful for the substratification of the patients diagnosed to have normal renal function with Cr of < 1.3 mg/dL into those with normal and pseudo-normal renal function, resulting in the corresponding serum concentrations of digoxin.
RESUMO
The involvement of the multidrug resistant transporter MDR1/P-glycoprotein in the penetration of haloperidol into the brain and absorption in the intestine was investigated to examine its role in inter/intra-individual variability, using the porcine kidney epithelial cell line LLC-PK(1) and its MDR1-overexpressing transfectant, LLC-GA5-COL150. The inhibitory effect of haloperidol on other MDR1 substrates was also investigated in terms of the optimization of haloperidol-based pharmacotherapy. The transepithelial transport of [(3)H]haloperidol did not differ between the two cell lines, and vinblastine, a typical MDR1 substrate, had no effect on the transport, suggesting that haloperidol is not a substrate for MDR1, and it is unlikely that MDR function affects haloperidol absorption and brain distribution, and thereby the response to haloperidol. However, haloperidol was found to have an inhibitory effect on the MDR1-mediated transport of [(3)H]digoxin and [(3)H]vinblastine with an IC50 value of 7.84+/-0.76 and 3.60+/-0.64 microM, respectively, suggesting that the intestinal absorption, not distribution into the brain, of MDR1 substrate drugs could be altered by the co-administration of haloperidol in the clinical setting, although further clinical studies are needed.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Haloperidol/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Antipsicóticos/metabolismo , Antipsicóticos/farmacologia , Transporte Biológico/efeitos dos fármacos , Digoxina/metabolismo , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Haloperidol/metabolismo , Humanos , Células LLC-PK1 , Suínos , Fatores de Tempo , Transfecção , Trítio , Vimblastina/metabolismoRESUMO
To clarify the effect of therapeutic moderate hypothermia on drug distribution, transepithelial transport via multi-drug resistance protein 1 (MDR1) (also called P-glycoprotein or ABCB1) was evaluated at various temperatures in vitro using LLC-GA5-COL150 cells, which were established by transfecting human MDR1 complementary deoxyribonucleic acid into kidney epithelial LLC-PK(1) cells and express MDR1 on the apical membrane. MDR1 is expressed in the blood-brain barrier to limit drug distribution to the brain by exporting exogenous substances including calcium blockers and antiarrhythmic drugs. Digoxin was used as a typical substrate, as well as the non-substrate tetracycline and paracellular marker inulin. MDR1-mediated transport of digoxin decreased at lower temperatures. Transport of tetracycline also decreased at lower temperatures, probably due to changes in membrane fluidity. However, no change was found at over 32 degrees C, suggesting that passive diffusion does not change during moderate hypothermia. The distribution of MDR1 substrates should be considered during hypothermic conditions, as the clinical outcome could be affected.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Encéfalo/metabolismo , Digoxina/farmacocinética , Inibidores Enzimáticos/farmacocinética , Hipotermia Induzida/métodos , Inibidores da Síntese de Proteínas/uso terapêutico , Quinidina/farmacocinética , Tetraciclina/farmacocinética , Antiarrítmicos , Barreira Hematoencefálica/metabolismo , Bloqueadores dos Canais de Cálcio/farmacocinética , Epitélio/metabolismo , Genes MDR/genética , HumanosRESUMO
The on-chip genotyping system ("the electrochemical DNA chip") has been developed as a more cost-effective genotyping system and was applied to MDR1 genotyping in the present study, which is required for wide use in clinical application and for personalized medication based on genotype. The electrochemical DNA chip was optimized and applied to simultaneous genotyping of four MDR1 polymorphisms (T-129C, C1236T, G2677(A,T) and C3435T) using synthetic model oligonucleotide DNA and human genomic DNA. The electrochemical DNA chip successfully gave the T-129C, C1236T, G2677(A,T) and C3435T genotypes, which were completely consistent with those determined by direct sequencing. In conclusion, the electrochemical DNA chip is useful for simultaneous determination of some genotypes and haplotypes, and efficient genotyping using this system can support future genotype-phenotype studies at a large scale.
Assuntos
Genes MDR/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Polimorfismo de Nucleotídeo Único/genética , Sequência de Bases , Sondas de DNA , Genótipo , Dados de Sequência MolecularRESUMO
The effects of micafungin on cytochrome P450 3A4 (CYP3A4) metabolic and multidrug resistance protein 1 (MDR1) transport activities were investigated and compared with those of amphotericin B and four azole antifungal drugs (ketoconazole, itraconazole, fluconazole and miconazole). The effects on the metabolic activity of CYP3A4 were examined by measuring nifedipine oxidase activity in human liver microsomes and the effects on MDR1 transport activity were evaluated using [3H]digoxin in MDR1-overexpressing LLC-GA5-COL150 cells. An inhibitory effect on CYP3A4 activity was found for ketoconazole, itraconazole and miconazole, with 50% inhibitory concentrations of 11.7, 32.6 and 74.2 nM, respectively. Fluconazole and micafungin had only slight inhibitory effects and amphotericin B had no effect. The MDR1-mediated transport of [3H]digoxin was inhibited by ketoconazole and itraconazole, and slightly by miconazole. It is suggested that micafungin and amphotericin B would be unlikely to cause drug-drug interactions by inhibition of CYP3A4 and MDR1. A positive correlation between the inhibitory effects on CYP3A4 and MDR1 activities was observed, and the physicochemical mechanisms involved and impact on clinical treatment should be studied further.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Antifúngicos/farmacologia , Azóis/farmacologia , Inibidores das Enzimas do Citocromo P-450 , Lipoproteínas/farmacologia , Peptídeos Cíclicos/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/metabolismo , Equinocandinas , Humanos , Técnicas In Vitro , Concentração Inibidora 50 , Lipopeptídeos , Micafungina , Microssomos Hepáticos/metabolismo , SuínosRESUMO
The effect of the C3435T mutation at exon 26 of the MDR1 gene on the expression levels of MDR1 messenger ribonucleic acid (mRNA) was evaluated by means of real-time polymerase chain reaction in 51 biopsy specimens of duodenum obtained from 13 healthy Japanese subjects. The mRNA levels of MDR1 were 0.38 +/- 0.15, 0.56 +/- 0.14, and 1.13 +/- 0.42 (mean value +/- SE) in the subjects with the homozygote of wild-type allele (C/C), compound heterozygote with mutant T allele (C/T), and the homozygote of the mutant allele (T/T), respectively, reasonably explaining the lower digoxin serum concentration after administration of a single oral dose to subjects harboring a mutant T allele. Good correlation (r =.797; P <.01) was observed between the mRNA concentrations of MDR1 and CYP3A4 in the individual biopsy specimens. This finding suggested a lower plasma concentration of the substrates for CYP3A4 in subjects harboring the C3435T mutation of the MDR1 gene.
Assuntos
Duodeno/metabolismo , Enterócitos/metabolismo , Éxons/genética , Regulação da Expressão Gênica/genética , Genes MDR/genética , Proteínas Mitocondriais , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Mutação/genética , RNA Mensageiro/biossíntese , Proteínas de Saccharomyces cerevisiae , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/biossíntese , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Adulto , Alelos , Citocromo P-450 CYP3A , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Citosina/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Digoxina/sangue , Duodeno/citologia , Duodeno/enzimologia , Enterócitos/enzimologia , Inibidores Enzimáticos/sangue , Humanos , Japão , Masculino , Oxigenases de Função Mista/biossíntese , Oxigenases de Função Mista/genética , Proteína 3 Homóloga a MutS , RNA Mensageiro/genética , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genética , Timina/metabolismoRESUMO
Most drug responses are determined by the interplay of several gene products that influence pharmacokinetics and pharmacodynamics, i.e., drug metabolizing enzymes, drug transporters, and drug targets. With the sequencing of the human genome, it has been estimated that approximately 500-1200 genes code for drug transporters. Concerning the effects of genetic polymorphisms on pharmacotherapy, the best characterized drug transporter is the multidrug resistant transporter P-glycoprotein/MDR1, the gene product of MDR1. Little such information is available on other drug transporters. MDR1 is a glycosylated membrane protein of 170 kDa, belonging to the ATP-binding cassette superfamily, and is expressed mainly in intestines, liver, kidneys and brain. A number of various types of structurally unrelated drugs are substrates for MDR1, and their intestinal absorption, hepatobiliary secretion, renal secretion and brain transport are regulated by MDR1. The first investigation on the effects of MDR1 genotypes on pharmacotherapy was reported in 2000: a silent single nucleotide polymorphism (SNP), C3435T in exon 26, was found to be associated with the duodenal expression of MDR1, and thereby the plasma concentration of digoxin after oral administration. At present, a total of 28 SNPs have been found at 27 positions on the MDR1 gene. Clinical investigations on the association of MDR1 genotypes with the expression and function of MDR1 in tissues, and with pharmacokinetics and pharmacodynamics have mainly focused on C3435T; however, there are still discrepancies in the results, suggesting that the haplotype of the gene should be analyzed instead of a SNP. C3435T is also reported to be a risk factor for a certain class of diseases including the inflammatory bowel diseases, Parkinson's disease and renal epithelial tumor, and this also might be explained by the effects on MDR1 expression and function. In this review, the latest reports on the effects of genetic polymorphisms of MDR1 on pharmacotherapy are summarized, and the pharmacogenetics of other transporters is briefly introduced.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Tratamento Farmacológico , Preparações Farmacêuticas/metabolismo , Farmacogenética/métodos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/farmacocinética , Transporte Biológico/fisiologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Genoma Humano , Genótipo , Humanos , Modelos Biológicos , Polimorfismo GenéticoRESUMO
The multi-drug resistant transporter MDR1/P-glycoprotein, the gene product of MDR1, is a glycosylated membrane protein of 170 kDa, belonging to the ATP-binding cassette (ABC) superfamily of membrane transporters. MDR1 was originally isolated from resistant tumor cells as part of the mechanism of multi-drug resistance, but over the last decade, it has been elucidated that human MDR1 is also expressed throughout the body to confer intrinsic resistance to the tissues by exporting unnecessary or toxic exogeneous substances or metabolites. A number of various types of structurally unrelated drugs are substrates for MDR1, and MDR1 and other transporters are recognized as an important class of proteins for regulating pharmacokinetics and pharmacodynamics. In 2000, Hoffmeyer et al. performed a systemic screening for MDR1 polymorphisms and indicated that a single nucleotide polymorphism (SNP), C3435T in exon 26, which caused no amino acid change, was associated with the duodenal expression of MDR1 and thereby the plasma concentrations of digoxin after oral administration. Interethnic differences in genotype frequencies of C3435T have been clarified, and, at present, a total of 28 SNPs have been found at 27 positions on the MDR1 gene. Clinical studies on the effects of C3435T on MDR1 expression and function in the tissues, and also on the pharmacokinetics and pharmacodynamics have been performed around the world; however, there are still discrepancies in the results, suggesting that the haplotype analysis of the gene should be included instead of SNP detection, and the design of clinical trials must be carefully planned to avoid misinterpretations. A polymorphism of C3435T is also reported to be a risk factor for a certain class of diseases such as the inflammatory bowel diseases, Parkinson's disease and renal epithelial tumor, and this might also be explained by the effects on MDR1 expression and function. In this review, the latest reports are summarized for the future individualization of pharmacotherapy based on MDR1 genotyping.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Preparações Farmacêuticas/metabolismo , Farmacogenética/métodos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/farmacocinética , Animais , HumanosRESUMO
St John's wort (SJW) is Hypericum perforatum L., Hypericaceae, a herbaceous perennial plant native to Europe and Asia, and its various preparations are widely used for the treatment of mild-to-moderately severe depressive disorders. With increasingly prevalent use, the interactions with SJW preparations with co-administered drugs have been reported, presumably via MDR1-mediated processes. In this paper, the effects of SJW extract on antiproliferative effects of anticancer drugs and the expression of MDR1 mRNA were examined using HeLa and its MDR1-overexpressing subline. The effects on MDR1-mediated transport were also evaluated using [(3)H]digoxin and LLC-GA5-COL150 cells, which were established by transfection of human MDR1 cDNA into porcine kidney epithelial LLC-PK(1) cells. The content of hypericin, a presumed active moiety within SJW extract, was determined by HPLC with a photo diode array to be 0.085 (w/w)%, and the effects of hypericin were also evaluated and compared with those of SJW extract. It was concluded that SJW extract reversed the cytotoxicity of paclitaxel and slightly of daunorubicin, down-regulated MDR1 mRNA, and inhibited MDR1-mediated transport, presumably due to other components than hypericin.
RESUMO
The multidrug transporter MDR1 (P-glycoprotein)-mediated interaction between digoxin and 29 antihypertensive drugs of various types was examined by using the MDR1 overexpressing LLC-GA5-COL150 cells, which were established by transfecting MDR1 cDNA into porcine kidney epithelial LLC-PK1 cells. These cells construct monolayers with tight junctions, and enable the evaluation of transcellular transport. The MDR1 was highly expressed on the apical membrane (urine side). The basal-to-apical and apical-to-basal transcellular transport of [3H]digoxin in LLC-GA5-COL150 cells was time- and temperature-dependent. The basal-to-apical transport of [3H]digoxin was markedly increased, whereas the apical-to-basal transport was decreased in LLC-GA5-COL150 cells, compared with the host LLC-PK1 cells, suggesting that [3H]digoxin was a substrate for MDR1. Most of the Ca2+ channel blockers used here markedly inhibited basal-to-apical transport and increased apical-to-basal transport. Exceptions were diltiazem, nifedipine and nitrendipine, which hardly showed inhibitory effects on transcellular transport of [3H]digoxin. Alpha-blocker doxazosin and beta-blocker carvedilol also inhibited transcellular transport of [3H]digoxin, but none of the angiotensin converting enzyme inhibitors and AT1 angiotensin II receptor antagonists used here were active. These observations will promote understanding of the digoxin-drug interactions resulting from their actions on MDR1, and which may aid in avoiding these unexpected effects of digoxin.