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Glioblastoma (GBM), the most commonly occurring primary tumor arising within the central nervous system, is characterized by high invasiveness and poor prognosis. In spite of the improvement in surgical techniques, along with the administration of chemo- and radiation therapy and the incessant investigation in search of prospective therapeutic targets, the local recurrence that frequently occurs within the peritumoral brain tissue makes GBM the most malignant and terminal type of astrocytoma. In the current study, we investigated both GBM and peritumoral tissues obtained from 55 hospitalized patients and the expression of three molecules involved in the onset of resistance/unresponsiveness to chemotherapy: O6-methylguanine methyltransferase (MGMT), breast cancer resistance protein (BCRP1), and A2B5. We propose that the expression of these molecules in the peritumoral tissue might be crucial to promoting the development of early tumorigenic events in the tissue surrounding GBM as well as responsible for the recurrence originating in this apparently normal area and, accordingly, for the resistance to treatment with the standard chemotherapeutic regimen. Notably, the inverse correlation found between MGMT expression in peritumoral tissue and patients' survival suggests a prognostic role for this protein.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Neoplasias Encefálicas/metabolismo , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Glioblastoma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Biomarcadores Tumorais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica , Hospitalização , Humanos , Masculino , Estudos Prospectivos , Microambiente TumoralRESUMO
This study aimed to reveal age-related changes in the expression and activity of seven hepatic drug metabolizing enzymes (DMEs) in male wild-type and breast cancer resistance protein knockout (Bcrp1-/- ) FVB mice. The protein expression of four cytochrome P450 (Cyps) (Cyp3a11, 2d22, 2e1, and 1a2), and three UDP-glucuronosyltransferases (Ugts) (Ugt1a1, 1a6a, and 1a9) in liver microsomes of wild-type and Bcrp1-/- FVB mice at different ages were determined using a validated ultra high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method. The activities and mRNA levels of these DMEs were measured using the probe substrates method and real-time PCR, respectively. In the liver of wild-type FVB mice, Cyp3a11, 2d22, 2e1, 1a2, Ugt1a1, and 1a6a displayed maximum protein levels at 6-9 weeks of age. Cyp1a2, Ugt1a1, 1a6a, and 1a9 showed maximum activities at 6-9 weeks of age, whereas Cyp3a11, 2d22, and 2e1 showed maximum activities in 1-3-week-old mice. Additionally, most of the DMEs showed maximum mRNA levels in 17-week-old mice liver. Compared with wild-type FVB mice, the protein levels of these DMEs showed no significant changes in Bcrp1-/- FVB mice liver. However, the activity of Cyp2e1 was increased and that of Cyp2d22 was decreased. In conclusion, the seven hepatic DMEs in FVB mice liver showed significant alterations in an isoform-specific manner with increased age. Although the protein levels of these DMEs showed no significant changes, the activities of Cyp2e1 and 2d22 were changed in Bcrp1-/- mice.
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Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Envelhecimento/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Glucuronosiltransferase/genética , Masculino , Camundongos Knockout , RNA Mensageiro/metabolismoRESUMO
Mycobacterium tuberculosis (MTB) is notorious for persisting within host macrophages. Efflux pumps decrease intracellular drug levels, thus fostering persistence of MTB during therapy. Isoniazid (INH) and pyrazinamide (PZA) are substrates of the efflux pump breast cancer resistance protein-1 (BCRP-1), which is inhibited by chloroquine (CQ). In this study, BCRP-1 was found to be expressed on macrophages of human origin and on foamy giant cells at the site of MTB infection. In the current in vitro study, interferon-gamma (IFNγ) increased the expression of BCRP-1 in macrophages derived from the human monocytic leukaemia cell line THP-1. Using a BCRP-1-specific fluorescent dye and radioactively labelled INH, it was demonstrated that efflux from macrophages increased upon activation with IFNγ. CQ was able to inhibit active efflux and augmented the intracellular concentrations both of INH and the dye. In agreement, CQ and specific inhibition of BCRP-1 increased the antimycobacterial activity of INH against intracellular MTB. Although PZA behaved differently, CQ had comparable advantageous effects on the intracellular pharmacokinetics and activity of PZA. The adjunctive effects of CQ on intracellular killing of MTB were measurable at concentrations achievable in humans at approved therapeutic doses. Therefore, CQ, a widely used and worldwide available drug, may potentiate the efficacy of standard MTB therapy against bacteria in the intracellular compartment.
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Antituberculosos/farmacologia , Cloroquina/farmacologia , Sinergismo Farmacológico , Isoniazida/farmacologia , Macrófagos/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/farmacocinética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Humanos , Macrófagos/imunologia , Proteínas de Neoplasias/antagonistas & inibidores , Células THP-1RESUMO
AIM: We aimed to evaluate the influence of genetic polymorphisms involved in deferasirox metabolism and transport on its pharmacokinetics and treatment toxicity, in a cohort of ß-thalassaemic children. PATIENTS & METHODS: Drug plasma concentrations were measured by a HPLC-UV method. Allelic discrimination for UGT1A1, UGT1A3, CYP1A1, CYP1A2, CYP2D6, MRP2 and BCRP1 polymorphisms was performed by real-time PCR. RESULTS: CYP1A1 rs2606345AA influenced Ctrough (p = 0.001) and t1/2 (p = 0.042), CYP1A1 rs4646903TC/CC (p = 0.005) and BCRP1 rs2231142GA/AA (p = 0.005) influenced Tmax and CYP2D6 rs1135840CG/GG influenced Cmax (p = 0.044). UGT1A1 rs887829TT (p = 0.002) and CYP1A2 rs762551CC (p = 0.019) resulted as predictive factor of ferritin levels and CYP1A1 rs2606345CA/AA (p = 0.021) and CYP1A2 rs762551AC/CC (p = 0.027) of liver iron concentration. CONCLUSION: Our data suggest the usefulness of deferasirox pharmacogenetics in pediatric treatment optimization.
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Benzoatos , Quelantes de Ferro , Variantes Farmacogenômicos , Polimorfismo de Nucleotídeo Único , Triazóis , Talassemia beta/tratamento farmacológico , Adolescente , Benzoatos/sangue , Benzoatos/uso terapêutico , Benzoatos/toxicidade , Criança , Pré-Escolar , Estudos de Coortes , Deferasirox , Feminino , Humanos , Quelantes de Ferro/farmacocinética , Quelantes de Ferro/uso terapêutico , Quelantes de Ferro/toxicidade , Masculino , Testes Farmacogenômicos , Triazóis/sangue , Triazóis/uso terapêutico , Triazóis/toxicidade , Talassemia beta/genética , Talassemia beta/metabolismoRESUMO
Efflux by the ATP-binding cassette (ABC) transporters affects the pharmacokinetic profile of drugs and it has been implicated in drug-drug interactions as well as its major role in multi-drug resistance in cancer. It is therefore important for the pharmaceutical industry to be able to understand what phenomena rule ABC substrate recognition. Considering a high degree of substrate overlap between various members of ABC transporter family, it is advantageous to employ a multi-label classification approach where predictions made for one transporter can be used for modeling of the other ABC transporters. Here, we present decision tree-based QSAR classification models able to simultaneously predict substrates and non-substrates for BCRP1, P-gp/MDR1 and MRP1 and MRP2, using a dataset of 1493 compounds. To this end, two multi-label classification QSAR modelling approaches were adopted: Binary Relevance (BR) and Classifier Chain (CC). Even though both multi-label models yielded similar predictive performances in terms of overall accuracies (close to 70 %), the CC model overcame the problem of skewed performance towards identifying substrates compared with non-substrates, which is a common problem in the literature. The models were thoroughly validated by using external testing, applicability domain and activity cliffs characterization. In conclusion, a multi-label classification approach is an appropriate alternative for the prediction of ABC efflux.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Ligantes , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , Transportadores de Cassetes de Ligação de ATP/metabolismo , Algoritmos , Estrutura Molecular , Ligação Proteica , Reprodutibilidade dos Testes , Especificidade por SubstratoRESUMO
Temozolomide (TMZ) is an oral alkylating agent used to treat glioblastoma multiforme (GBM) and astrocytomas. However, at least 50% of TMZ treated patients do not respond to TMZ. This is due primarily to the over-expression of O6-methylguanine methyltransferase (MGMT) and/or lack of a DNA repair pathway in GBM cells. Multiple GBM cell lines are known to contain TMZ resistant cells and several acquired TMZ resistant GBM cell lines have been developed for use in experiments designed to define the mechanism of TMZ resistance and the testing of potential therapeutics. However, the characteristics of intrinsic and adaptive TMZ resistant GBM cells have not been systemically compared. This article reviews the characteristics and mechanisms of TMZ resistance in natural and adapted TMZ resistant GBM cell lines. It also summarizes potential treatment options for TMZ resistant GBMs.
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Over the years chemotherapy failure has been a vital research topic as researchers have been striving to discover reasons behind it. The extensive studies carried out on chemotherapeutic agents confirm that resistance to chemotherapy is a major reason for treatment failure. "Resistance to chemotherapy," however, is a comprehensive phrase that refers to a variety of different mechanisms in which ATP-binding cassette (ABC) mediated efflux dominates. The ABC is one of the largest gene superfamily of transporters among both eukaryotes and prokaryotes; it represents a variety of genes that code for proteins, which perform countless functions, including drug efflux - a natural process that protects cells from foreign chemicals. Up to date, chemotherapy failure due to ABC drug efflux is an active research topic that continuously provides further evidence on multiple drug resistance (MDR), aiding scientists in tackling and overcoming this issue. This review focuses on drug resistance by ABC efflux transporters in human, viral, parasitic, fungal and bacterial cells and highlights the importance of the MDR permeability glycoprotein being the mutual ABC transporter among all studied organisms. Current developments and future directions to overcome this problem are also discussed.
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INTRODUCTION: The efflux transporters P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2) are expressed at the blood-brain barrier (BBB), and can limit the access of a wide range of drugs to the brain. In this study we developed a PET-CT imaging method for non-invasive, quantitative analysis of the effect of ABCB1 and ABCG2 on brain penetration of the anti-cancer drug gefitinib, and demonstrated the applicability of this method for identification and quantification of potential modulators of ABCB1 and ABCB2 using the dual inhibitor elacridar. METHODS: In vitro cellular accumulation studies with [(14)C]-gefitinib were conducted in LLC-PK1, MDCKII, and the corresponding ABCB1/Abcb1a and ABCG2/Abcg2 overexpressing cell lines. Subsequently, in vivo brain penetration of [(18)F]-gefitinib was quantified by PET-CT imaging studies in wild-type, Abcg2(-/-), Abcb1a/1b(-/-), and Abcb1a/1b;Abcg2(-/-) mice. RESULTS: In vitro studies showed that [(14)C]-gefitinib is a substrate of the human ABCB1 and ABCG2 transporters. After i.v. administration of [(18)F]-gefitinib (1mg/kg), PET-CT imaging showed 2.3-fold increased brain levels of [(18)F]-gefitinib in Abcb1a/1b;Abcg2(-/-) mice, compared to wild-type. Levels in single knockout animals were not different from wild-type, showing that Abcb1a/1b and Abcg2 together limit access of [(18)F]-gefitinib to the brain. Furthermore, enhanced brain accumulation of [(18)F]-gefitinib after administration of the ABCB1 and ABCG2 inhibitor elacridar (10 mg/kg) could be quantified with PET-CT imaging. CONCLUSIONS: PET-CT imaging with [(18)F]-gefitinib is a powerful tool to non-invasively assess potential ABCB1- and ABCG2-mediated drug-drug interactions (DDIs) in vivo. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: This minimally-invasive, [(18)F]-based PET-CT imaging method shows the interplay of ABCB1 and ABCG2 at the BBB in vivo. The method may be applied in the future to assess ABCB1 and ABCG2 activity at the BBB in humans, and for personalized treatment with drugs that are substrates of ABCB1 and/or ABCG2.
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Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Barreira Hematoencefálica/metabolismo , Radioisótopos de Flúor , Tomografia por Emissão de Pósitrons , Quinazolinas/metabolismo , Tomografia Computadorizada por Raios X , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Acridinas/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Barreira Hematoencefálica/diagnóstico por imagem , Barreira Hematoencefálica/efeitos dos fármacos , Linhagem Celular Tumoral , Interações Medicamentosas , Gefitinibe , Humanos , Masculino , Camundongos , Quinazolinas/farmacocinética , Tetra-Hidroisoquinolinas/farmacologia , Distribuição TecidualRESUMO
Boceprevir (BOC) is a directly-acting antiviral agent for the treatment of hepatitis C virus genotype 1 (HCV-1) infection. It is a mixture of two stereoisomers, the inactive R and the active S isomers. No data have previously been published on BOC intracellular accumulation. In this study, BOC isomer concentrations in peripheral blood mononuclear cells (PBMCs) and plasma were determined. The influence of various single nucleotide polymorphisms (SNPs) on plasma and intracellular drug exposure at Week 4 of triple therapy were also evaluated. Plasma and intracellular BOC concentrations were determined at the end of the dosing interval (C(trough)) using a UPLC-MS/MS validated method. Allelic discrimination was performed through real-time PCR. Median plasma concentrations were 65.97 ng/mL for the S isomer and 36.31 ng/mL for the R isomer; the median S/R plasma concentration ratio was 1.66. The median PBMC concentration was 2285.88 ng/mL for the S isomer; the R isomer was undetectable within PBMCs. The median S isomer PBMC/plasma concentration ratio was 28.59. A significant positive correlation was found between plasma and PBMC S isomer concentrations. ABCB1 1236, SLC28A2 124 and IL28B rs12979860 SNPs were associated with the S isomer PBMC/plasma concentration ratio. In regression models, S isomer plasma levels and FokI polymorphism were able to predict S isomer intracellular exposure, whereas SNPs in AKR1, BCRP1 and SLC28A2 predicted the S isomer PBMC/plasma concentration ratio. No similar data regarding BOC pharmacogenetics and pharmacokinetics have been published previously. This study adds a novel and useful overview of the pharmacological properties of this drug.
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Antivirais/farmacocinética , Citosol/química , Leucócitos Mononucleares/química , Plasma/química , Polimorfismo de Nucleotídeo Único , Prolina/análogos & derivados , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Adulto , Cromatografia Líquida de Alta Pressão , Feminino , Técnicas de Genotipagem , Humanos , Interferons , Interleucinas/genética , Masculino , Proteínas de Membrana Transportadoras/genética , Pessoa de Meia-Idade , Farmacogenética , Prolina/farmacocinética , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas em TandemRESUMO
The side population (SP) phenotype, defined by the ability of a cell to efflux fluorescent dyes such as Hoechst, is common to several stem/progenitor cell types. In avian species, SP phenotype has been identified in pubertal and adult testes, but nothing is known about its expression during prenatal development of a male gonad. In this study, we characterized the Hoechst SP phenotype via the cytofluorimetric analysis of disaggregated testes on different days of chicken embryonic development. Male prenatal gonads contained a fraction of SP cells at each stage analyzed. At least two main SP fractions, named P3 and P4, were identified. The percentage of P3 fraction decreased as development proceeds, whereas P4 cell number was not affected by gonad growth. Functional inhibition of BCRP1 channel membrane using Verapamil and/or Ko143 showed that P3, but not P4 phenotype, was dependent on BCRP1 activity. Molecular analysis of both P3- and P4-sorted fractions revealed a differential RNA expression pattern, indicating that P3 cells mainly contained germinal stem cell markers, whereas P4 was preferentially composed of both Sertoli and Leydig cell progenitor markers. Finally, these findings provided evidence that the SP phenotype is a common feature of both germ and somatic cells detected in chicken developing testis.