RESUMO
BACKGROUND: 3', 5'-cyclic AMP (cAMP) regulates numerous cardiac functions. Various hormones and neurotransmitters elevate intracellular cAMP (i[cAMP]) in cardiomyocytes through activating GsPCRs (stimulatory-G-protein-coupled-receptors) and membrane-bound ACs (adenylyl cyclases). Increasing evidence has indicated that stimulating different GsPCRs and ACs exhibits distinct, even opposite effects, on cardiomyocyte viability. However, the underlying mechanisms are not fully understood. METHODS: We used molecular and pharmacological approaches to investigate how different GsPCR/cAMP signaling differentially regulate cardiomyocyte viability with in vitro, ex vivo, and in vivo models. RESULTS: For prodeath GsPCRs, we explored ß1AR (beta1-adrenergic receptor) and H2R (histamine-H2-receptor). We found that their prodeath effects were similarly dependent on AC5 activation, ATP release to the extracellular space via PANX1 (pannexin-1) channel, and extracellular ATP (e[ATP])-mediated signaling involving in P2X7R (P2X purinoceptor 7) and CaMKII (Ca2+/calmodulin-dependent protein kinase II). PANX1 phosphorylation at Serine 206 by cAMP-dependent-PKA (protein-kinase-A) promoted PANX1 activation, which was critical in ß1AR- or H2R-induced cardiomyocyte death in vitro and in vivo. ß1AR or H2R was localized proximately to PANX1, which permits ATP release. For prosurvival GsPCRs, we explored adenosine-A2-receptor (A2R), CGRPR (calcitonin-gene-related-peptide-receptor), and RXFP1 (relaxin-family peptide-receptor 1). Their prosurvival effects were dependent on AC6 activation, cAMP efflux via MRP4 (multidrug resistance protein 4), extracellular cAMP metabolism to adenosine (e[cAMP]-to-e[ADO]), and e[ADO]-mediated signaling. A2R, CGRPR, or RXFP1 was localized proximately to MRP4, which enables cAMP efflux. Interestingly, exogenously increasing e[cAMP] levels by membrane-impermeable cAMP protected against cardiomyocyte death in vitro and in ex vivo and in vivo mouse hearts with ischemia-reperfusion injuries. CONCLUSIONS: Our findings indicate that the functional diversity of different GsPCRs in cardiomyocyte viability could be achieved by their ability to form unique signaling complexes (signalosomes) that determine the fate of cAMP: either stimulate ATP release by activating PKA or directly efflux to be e[cAMP].
Assuntos
AMP Cíclico , Miócitos Cardíacos , Camundongos , Animais , AMP Cíclico/metabolismo , Miócitos Cardíacos/metabolismo , Adenosina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Peptídeos/metabolismoRESUMO
Of the many multidrug resistance (MDR) mechanisms, the ATP binding cassette (ABC) transporters expelling drug molecules out of cells is a major culprit in limiting the efficacy of present-day anticancer drugs. The present review offers an updated information on structure, function, and regulatory mechanisms of major MDR related ABC transporters such as P-glycoprotein, MRP1, BCRP and effect of modulators on their functions. An attempt has been made to provide a focused information on different modulators of ABC transporters so as utilize them in clinical practice for amelioration of emerging MDR crisis in cancer treatment. Finally, the importance ABC transporters as therapeutic targets has been discussed in the light of future strategic planning for translating the ABC transporter inhibitors in clinical practice.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/uso terapêutico , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Resistencia a Medicamentos Antineoplásicos , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/farmacologia , Proteínas de Neoplasias/uso terapêutico , Resistência a Múltiplos Medicamentos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/química , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Trifosfato de AdenosinaRESUMO
Multidrug resistance is a primary factor in the poor response to chemotherapy and subsequent death in gastric cancer patients. However, the molecular mechanisms involved remain unclear. In this study, the high expression of special AT-rich sequence binding protein 1 (SATB1) in gastric cancer was found to be associated with reduced sensitivity to various chemotherapy drugs. Our results demonstrate that SATB1 can promote chemotherapy resistance in gastric cancer in vitro and in vivo. SATB1 exerts its effect by enhancing the activity of multiple ATP-binding cassette (ABC) transporters (P-glycoprotein, multidrug resistance-associated protein, and breast cancer resistance protein) in gastric cancer cell lines. We also found that SATB1 affects ABC transporters by altering the subcellular localization of the ABC transporter rather than its expression. Subsequently, we confirmed that Ezrin binds to various ABC transporters and affects their subcellular localization. In addition, we found that SATB1 can also bind to the Ezrin promoter and regulate its expression. In the present study, we elucidate the mechanism of SATB1-mediated multidrug resistance in gastric cancer, providing a basis for SATB1 as a potential target for reversal of resistance.
Assuntos
Proteínas de Ligação à Região de Interação com a Matriz , Neoplasias Gástricas , Humanos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Neoplasias/metabolismo , Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologiaRESUMO
Recently, we have isolated and identified several bioactive flavonoids and stilbenoids with potential anticancer activity from Thai orchids. In this study, we further investigated the cytotoxic and chemosensitizing activities of these phytochemicals (namely, pinocembrin, cardamonin, isalpinin, galangin, pinosylvin monomethyl ether, 2,3'-dihydroxy-5'-methoxystilbene, (E)-2,5'-dihydroxy-2'-(4-hydroxybenzyl)-3'-methoxystilbene, 2,3-dihydroxy-3',5'-dimethoxystilbene, 2,3'-dihydroxy-5,5'-dimethoxystilbene, 3,4'-dihydroxy-5-methoxystilbene and batatasin III) against breast cancer MCF7 cells and its two multidrug resistant (MDR) sublines (MCF7/DOX and MCF7/MX). Cytotoxicity was determined with MTT assay for the estimation of the half maximal cytotoxic concentrations (IC50). Effects of the test compounds on activities of efflux transporters (BCRP, P-gp, MRP1, and MRP2) were evaluated with substrate accumulation assays using fluorometry and flow cytometry analysis. Out of these 11 test compounds, the stilbene pinosylvin monomethyl ether displayed its cytotoxicity specifically toward MCF7 cells (IC50 = 6.2 ± 1.2 µM, 72-h incubation) with 4.96 folds higher than normal fibroblast. Its potency decreased in MCF7/DOX and MCF7/MX cells by 3.94 and 7.38 folds, respectively. Our transporter assay indicated that this stilbene significantly reduced the activities of P-gp, MRP1, and MRP2, but not BCRP. After 48-h co-incubation, this stilbene (at 2 µM) synergistically increased doxorubicin- and mitoxantrone-mediated cytotoxicity in MCF7, MCF7/DOX, and MCF7/MX cells potentially by increasing the intracellular level of cytotoxic drug. Pinosylvin monomethyl ether could sensitize breast cancer cells to chemotherapy and overcome MDR, in part, via the inhibition of drug efflux transporters.
Assuntos
Antineoplásicos , Neoplasias da Mama , Estilbenos , Humanos , Feminino , Resistência a Múltiplos Medicamentos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Estilbenos/farmacologia , Éteres/farmacologia , Neoplasias da Mama/tratamento farmacológicoRESUMO
Although there are a lot of chemical drugs to treat breast cancer, increasing drug resistance of cancer cells has strongly hindered the effectiveness of chemotherapy. ATP-binding cassette transporters represented by P-glycoprotein (P-gp), multidrug resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) play an important role in drug resistance. This study aims to investigate the effect of 7-O-geranylquercetin (GQ) combining microRNA-451(miR-451) on reversing drug resistance of breast cancer and reveal the mechanism related to P-gp. Real-time RT-PCR and western blot assays showed that miR-326, miR-328, miR-451 and miR-155 inhibitor down-regulated the expression of genes MRP1, BCRP, MDR1 and the corresponding proteins MRP1, BCRP, P-gp, respectively. Cell counting kit-8 (CCK-8) assay indicated that these miRNAs reversed the resistance of MCF-7/ADR cells to Adriamycin (ADR), and miR-451 showed the greatest reversal effect. Combination of GQ and miR-451 enhanced the inhibitory effects of ADR on the proliferation and migration of MCF-7/ADR cells, and attenuated the expression of MDR1 and P-gp in MCF-7/ADR cells. A xenograft tumor model was used to show that GQ and miR-451 amplified the antitumor effect of ADR in nude mice, while western blot and immunohistochemical assays revealed the decreased expression of P-gp in tumor tissues. These results suggest that GQ and miR-451 have synergistic effect on reversing drug resistance through reducing the expression of MDR1 and P-gp in breast cancer MCF-7/ADR cells.
Assuntos
Neoplasias da Mama , MicroRNAs , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , 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/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Proteínas de Neoplasias/metabolismo , Quercetina/análogos & derivadosRESUMO
MRP-1 is implicated in multidrug resistance and was described as prognostic in high-risk patients with soft-tissue sarcoma (STS) in a previous study. The current research aimed to validate MRP-1 prognostic/predictive value in localized sarcomas treated with anthracyclines plus ifosfamide within the ISG-1001 phase III study. In addition, the inhibitory activity on MRP-1 was investigated in preclinical studies to identify new combinations able to increase the efficacy of standard chemotherapy in STS. MRP-1 expression was assessed by IHC in tissue microarrays from patients with STS and tested for correlation with disease-free survival (DFS) and overall survival (OS). In vitro studies tested the efficacy of MRP-1 inhibitors (nilotinib, ripretinib, selumetinib, and avapritinib) in sarcoma cell lines. The effect of combinations of the most active MRP-1 inhibitors and chemotherapy was measured on the basis of apoptosis. MRP-1 was evaluable in 231 of 264 cases who entered the study. MRP-1 expression (strong intensity) was independently associated with worse DFS [HR, 1.78; 95% confidence interval (CI), 1.11-2.83; P = 0.016], in the multivariate analysis, with a trend for a worse OS (HR, 1.78; 95% CI, 0.97-3.25; P = 0.062). In vitro studies showed that the addition of MRP-1 inhibitors (nilotinib or avapritinib) to doxorubicin plus palifosfamide, significantly increased cell death in SK-UT-1 and CP0024 cell lines. MRP-1 is an adverse predictive factor in localized high-risk patients with STS treated with neoadjuvant anthracyclines plus ifosfamide followed by surgery. In vitro findings support the clinical assessment of the combination of chemotherapy and MRP-1 inhibitors as a promising strategy to overcome the drug ceiling effect for chemotherapy.
Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos/uso terapêutico , Sarcoma/tratamento farmacológico , Pesquisa Translacional Biomédica/métodos , Feminino , Humanos , Masculino , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Valor Preditivo dos Testes , PrognósticoRESUMO
Cancer, a major public health problem, is one of the world's top leading causes of death. Common treatments for cancer include cytotoxic chemotherapy, surgery, targeted drugs, endocrine therapy, and immunotherapy. However, despite the outstanding achievements in cancer therapies during the last years, resistance to conventional chemotherapeutic agents and new targeted drugs is still the major challenge. In the present review, we explain the different mechanisms involved in cancer therapy and the detailed outlines of cancer drug resistance regarding multidrug resistance-associated proteins (MRPs) and their role in treatment failures by common chemotherapeutic agents. Further, different modulators of MRPs are presented. Finally, we outlined the models used to analyze MRP transporters and proposed a future impact that may set up a base or pave the way for many researchers to investigate the cancer MRP further.
Assuntos
Antineoplásicos , Neoplasias , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Previsões , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/uso terapêutico , Neoplasias/tratamento farmacológicoRESUMO
Oxidative stress induced in live HeLa cells by menadione (2-methyl-1,4-napthaquinone) was studied in real time by scanning electrochemical microscopy (SECM). The hydrophobic molecule menadione diffuses through a living cell membrane where it is toxic to the cell. However, in the cell it is conjugated with glutathione to form thiodione. Thiodione is then recognized and transported across the cell membrane via the ATP-driven MRP1 pump. In the extracellular environment, thiodione was detected by the SECM tip at levels of 140, 70, and 35 µM upon exposure of the cells to menadione concentrations of 500, 250, and 125 µM, respectively. With the aid of finite element modeling, the kinetics of thiodione transport was determined to be 1.6 10(-7) m/s, about 10 times faster than menadione uptake. Selective inhibition of these MRP1 pumps inside live HeLa cells by MK571 produced a lower thiodione concentration of 50 µM in presence of 500 µM menadione and 50 µM MK571. A similar reduced (50% drop) thiodione efflux was observed in the presence of monoclonal antibody QCRL-4, a selective blocking agent of the MRP1 pumps. The reduced thiodione flux confirmed that thiodione was transported by MRP1, and that glutathione is an essential substrate for MRP1-mediated transport. This finding demonstrates the usefulness of SECM in quantitative studies of MRP1 inhibitors and suggests that monoclonal antibodies can be a useful tool in inhibiting the transport of these MDR pumps, and thereby aiding in overcoming multidrug resistance.
Assuntos
Glutationa/análogos & derivados , Microscopia de Varredura por Sonda/métodos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Estresse Oxidativo/fisiologia , Vitamina K 3/análogos & derivados , Anticorpos Monoclonais/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Técnicas Eletroquímicas , Análise de Elementos Finitos , Glutationa/metabolismo , Células HeLa , Humanos , Cinética , Simulação de Dinâmica Molecular , Estrutura Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Propionatos/farmacologia , Quinolinas/farmacologia , Vitamina K 3/metabolismoRESUMO
Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[a]pyrene (B[a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or (-)-benzo[a]pyrene-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), the proximate carcinogen of B[a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-2'-deoxyguanosine ((+)-anti-trans-B[a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)-anti-trans-B[a]PDE-dGuo formation in cells treated with (-)-B[a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)-anti-trans-B[a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (-)-B[a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen.
Assuntos
Benzo(a)pireno/antagonistas & inibidores , Benzo(a)pireno/toxicidade , Brônquios/citologia , Carcinógenos/antagonistas & inibidores , Carcinógenos/toxicidade , Adutos de DNA/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Alvéolos Pulmonares/citologia , Brônquios/efeitos dos fármacos , Líquido da Lavagem Broncoalveolar/citologia , Linhagem Celular , DNA/genética , DNA/isolamento & purificação , Dinoprostona/biossíntese , Humanos , Imuno-Histoquímica , Fator 2 Relacionado a NF-E2/metabolismo , Dibenzodioxinas Policloradas/toxicidade , Probenecid/farmacologia , Alvéolos Pulmonares/efeitos dos fármacos , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor Cross-Talk/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/efeitos dos fármacos , Fármacos Renais/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVES: In this study we investigate: 1) the role of multidrug resistance protein-4 (MRP4), an organic anion unidirectional transporter, in modulating aspirin action on human platelet cyclooxygenase (COX)-1; and 2) whether the impairment of aspirin-COX-1 interaction, found in coronary artery bypass grafting (CABG) patients, could be dependent on MRP4-mediated transport. BACKGROUND: Platelets of CABG patients present a reduced sensitivity to aspirin despite in vivo and in vitro drug treatment. Aspirin is an organic anion and could be a substrate for MRP4. METHODS: Intracellular aspirin concentration and drug COX-1 activity, measured by thrombin-induced thromboxane B2 (TxB2) production, were evaluated in platelets obtained from healthy volunteers (HV) and hematopoietic-progenitor cell cultures reducing or not reducing MRP4-mediated transport. Platelet MRP4 expression was evaluated, in platelets from HV and CABG patients, by dot-blot or by immunogold-electromicrographs or immunofluorescence-microscopy analysis. RESULTS: Inhibition of MRP4-mediated transport by dipyridamole or Mk-571 increases aspirin entrapment and its in vitro effect on COX-1 activity (142.7 ± 34.6 pg/10(8) cells vs. 343.7 ± 169.3 pg/108 cells TxB2-production). Platelets derived from megakaryocytes transfected with MRP4 small interfering ribonucleic acid have a higher aspirin entrapment and drug COX-1 activity. Platelets from CABG patients showed a high expression of MRP4 whose in vitro inhibition enhanced aspirin effect on COX-1 (349 ± 141 pg/108 cells vs. 1,670 ± 646 pg/108 cells TxB2-production). CONCLUSIONS: Aspirin is a substrate for MRP4 and can be extruded from platelet through its transportation. Aspirin effect on COX-1 is little-related to MRP4-mediated aspirin transport in HV, but in CABG patients with MRP4 over-expression, its pharmacological inhibition enhances aspirin action in an efficient way.
Assuntos
Aspirina/farmacocinética , Plaquetas/metabolismo , Ponte de Artéria Coronária , Fibrinolíticos/farmacocinética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Inibidores da Agregação Plaquetária/farmacocinética , Adulto , Aspirina/farmacologia , Transporte Biológico/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Células Cultivadas , AMP Cíclico/farmacologia , Ciclo-Oxigenase 1/metabolismo , Dinoprostona/metabolismo , Interações Medicamentosas , Resistência a Medicamentos , Feminino , Fibrinolíticos/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Agregação Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Propionatos/farmacologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Quinolinas/farmacologia , RNA Interferente Pequeno/metabolismo , Ácido Salicílico/farmacocinética , Tromboxano B2/metabolismoAssuntos
Aspirina/farmacocinética , Plaquetas/efeitos dos fármacos , Fibrinolíticos/farmacocinética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Inibidores da Agregação Plaquetária/farmacocinética , Aspirina/farmacologia , Plaquetas/metabolismo , Células Cultivadas , Ponte de Artéria Coronária , Ciclo-Oxigenase 1/metabolismo , Fibrinolíticos/farmacologia , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Inibidores da Agregação Plaquetária/farmacologiaRESUMO
In early studies, it was demonstrated that R-HZ08, S-HZ08 and the racemate had strong reverse efficacy of multidrug resistance in vitro and in vivo (Yan et al., 2008b). The effect was supposed to have direct interaction with multidrug resistance-associated protein (MRP1) in MCF-7/ADM and P-glycoprotein in K562/A02. According to our latest study, we found HZ08 could enhance chemotherapy induced apoptosis by synergistic action on reactive oxygen species generation, GSH depletion, mitochondrial membrane potential depolarization, cytochrome c release and caspase activation. Moreover, the potential selective effect of HZ08 on resistant cells suggested that HZ08 have specific targets for resistance reversal via apoptosis regulation. Therefore, we traced individual influence of HZ08, not only on apoptosis pathway per se but also on apoptosis related intracellular regulation systems. Then we found HZ08 could increase cells in G(0)/G(1) phase and regulate apoptosis related proteins (Bcl-2, Bax) as well as upstream functional molecules (c-Myc and c-Fos), which are usually abnormal in malignancy and responsible for multidrug resistance in MCF-7/ADM. Thereby, chemotherapy induced apoptosis was promoted. R-HZ08 showed better effect than S-HZ08 or the racemate did in most of targets above. Furthermore, HZ08 did not change the concentration of intracellular Ca(2+) which means it would not have side effect as verapamil does. Considering multidrug resistance is multifactorial, HZ08, especially R-HZ08, which could sensitize apoptosis by multiple improvements of upstream malignant characters, will be a promising drug to enhance the effect of chemotherapy in the treatment of multidrug resistant tumor.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Isoquinolinas/farmacologia , Antibióticos Antineoplásicos/farmacologia , Apoptose/fisiologia , Bloqueadores dos Canais de Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Citocromos c/metabolismo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Quimioterapia Combinada , Feminino , Humanos , Isoquinolinas/química , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Verapamil/metabolismo , Verapamil/farmacologiaRESUMO
We present a novel lead for inhibitors of multidrug resistance-associated proteins (MRPs). Compound 1 (4-[(5,6,7,8-tetrahydro-4-oxo-4H-[1]benzothieno[2,3-d][1,3]thiazin-2-yl)amino]benzoic acid) was about six times more potent than the known inhibitor MK571 at MRP1, while at MRP2 its effect was similar to that of MK571. Structural analogs were also evaluated. Among them, compound 2, sharing the 4-aminobenzoic acid substructure with 1, also inhibited MRP1. Both derivatives were inactive against P-gp. It can be concluded that their carboxyl group is needed for inhibition of MRPs and accounts for the selectivity of these compounds.
Assuntos
Ácido 4-Aminobenzoico/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Tiazinas/farmacologia , para-Aminobenzoatos , Ácido 4-Aminobenzoico/química , Animais , Linhagem Celular , Linhagem Celular Tumoral , Cães , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Humanos , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Tiazinas/químicaRESUMO
OBJECTIVE: To study the expression of P-glycoprotein, multi-drug resistance associated protein and major vault protein in pathologic brain specimens, and to investigate their roles in the pathogenesis of refractory epilepsy. METHODS: Immunohistochemical study was performed in pathology specimens from 18 cases of refractory epilepsy (including 5 cases of focal cortical dysplasia, 3 cases of tuberous sclerosis, 5 cases of ganglioglioma and 5 cases of dysembryoplastic neuroepithelial tumor). RESULTS: Both the P-glycoprotein and major vault protein were localized in microvascular endothelium of the lesions. Major vault protein was also seen in balloon cells and some neuronal cells. On the other hand, multi-drug resistance associated protein was mainly localized in the neuronal component of the lesions. In general, the expression of P-glycoprotein and major vault protein in tumoral tissue was higher than that in non-tumoral tissue. The expression of multi-drug resistance associated protein and major vault protein was also different in the neoplastic glial cells of ganglioglioma and dysembryoplastic neuroepithelial tumor. CONCLUSIONS: P-glycoprotein, multi-drug resistance associated protein and major vault protein contribute to the pathogenesis of refractory epilepsy. They may however have different roles, with different cellular localization.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Epilepsia/metabolismo , Ganglioglioma/metabolismo , Malformações do Desenvolvimento Cortical/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Adolescente , Adulto , Encéfalo/metabolismo , Neoplasias Encefálicas/química , Criança , Resistência a Múltiplos Medicamentos , Epilepsia/tratamento farmacológico , Epilepsia/genética , Feminino , Ganglioglioma/genética , Humanos , Masculino , Malformações do Desenvolvimento Cortical/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Esclerose Tuberosa/metabolismo , Adulto JovemRESUMO
OBJECTIVE: To conduct an in vitro study on the effect of recombinant adenovirus microsphere encapusulated antisense MRP (as-mrp) for use in the gene therapy to overcome drug resistance in hepatocellular carcinoma. METHODS: Recombinant adenovirus microsphere encapusulated as-mrp was transfected into hepatocellular carcinoma multidrug resistance cells HepG2/ADM, the fluorescence intensity of transfected cells were observed at 48 hours and 120 hours after transfection. in vitro drug sensitivity was measured by MTT assay; the resistant index of andromycin resistant variants was determined by drawing the cell dosage reaction curves. The levels of MRP mRNA expression were detected by RT-PCR and the ratio of MRP mRNA/beta-actin was detected. Intracelluar rubidomycin (DNR) concertration was examined by flow cytometry (FCM). RESULTS: More than 90% of the HepG2/ADM cells could be transfected when microspheres being 10 mg. Adv microsphere inhibited the expression of mRNA in HepG2/ADM and enhanced the sensitivity of HepG2/ADM to chemotherapeutic drug. CONCLUSION: Recombinant adenovirus microsphere encapusulated as-mrp could effectively reverse HepG2/ADM cells, which would provide an experimental basis for the methods of reversing the multidrug resistance in human hepatocellular carcinoma.
Assuntos
Adenoviridae/genética , Neoplasias Hepáticas/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , RNA Antissenso/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Humanos , Neoplasias Hepáticas/patologia , Microesferas , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , TransfecçãoRESUMO
The human ATP-binding cassette proteins MRP1 (ABCC1), MRP2 (ABCC2) and MRP3 (ABCC3) are active transporters of antineoplastic drugs as well as conjugated metabolites and other organic anions. In addition to being substrates, many glucuronide, glutathione and sulfate conjugates can also inhibit the transport activities of these MRP-related proteins, sometimes in a glutathione (GSH)-dependent manner. Nicotine is the major addictive component of cigarette smoke. Three glucuronide metabolites of this compound have been identified in vivo: nicotine-N-glucuronide, cotinine-N-glucuronide and trans-hydroxycotinine-O-glucuronide. In this study, we first chemically synthesized trans-hydroxycotinine-O-glucuronide and then tested the ability of this compound, nicotine-N-glucuronide and cotinine-N-glucuronide to modulate the vesicular transport of several organic anions by MRP1, MRP2 and MRP3. We observed that none of the three metabolites at concentrations up to 100muM significantly affected organic anion transport by MRP1 or MRP2, either in the absence or presence of GSH. MRP3-mediated transport of 17beta-estradiol 17-(beta-d-glucuronide) and methotrexate were partially inhibited by trans-hydroxycotinine-O-glucuronide (300 microM) (by 70% and 50%, respectively), whereas nicotine-N-glucuronide and cotinine-N-glucuronide had no effect. We conclude that the physiological functions of MRP1, MRP2 and MRP3 are not likely to be substantially affected by nicotine glucuronide metabolites at concentrations achievable in human serum.
Assuntos
Resistência a Múltiplos Medicamentos , Glucuronatos/farmacologia , Proteínas de Membrana Transportadoras/farmacologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Nicotina/análogos & derivados , Nicotina/farmacologia , Transportadores de Cassetes de Ligação de ATP , Técnicas de Cultura de Células , Estimulantes Ganglionares/metabolismo , Glucuronatos/metabolismo , Humanos , Transporte de Íons , Rim/citologia , Proteína 2 Associada à Farmacorresistência Múltipla , Nicotina/metabolismo , Compostos de Piridínio , FumarRESUMO
BACKGROUND: Resistance of cancer cells against anticancer agents is caused partly by multidrug resistance-associated protein 1 (MRP1). The exact mechanism of MRP1-involved multidrug resistance has not yet been clarified, although glutathione (GSH) is likely to have a role for the resistance to occur. N-acetylcysteine (NAC) is a pro-glutathione drug. DL-buthionine (S,R)-sulfoximine (BSO) inhibits GSH synthesis. The aim of our study was to investigate the effect of NAC and BSO on MRP1-mediated doxorubicin resistance in human embryonic kidney (HEK293) and its MRP1-transfected 293MRP cells. MATERIALS AND METHODS: Human embryonic kidney cells were transfected with a plasmid encoding the whole MRP1 gene. Both cells were incubated with doxorubicin in the presence or absence of NAC and/or BSO. The viability of both cells was determined under different incubation conditions. Glutathione, glutathione S-transferase (GST) and glutathione peroxidase (GPx) levels were measured in the cell extracts obtained from both cells incubated with different drugs. RESULTS: N-acetylcysteine increased the resistance of both cells against doxorubicin. DL-buthionine (S,R)-sulfoximine decreased NAC-enhanced MRP1-mediated doxorubicin resistance, indicating that induction of MRP1-mediated doxorubicin resistance depends on GSH synthesis. Doxorubicin decreased the cellular GSH concentration and increased GPx activity. Glutathione S-transferase activity was decreased by NAC. CONCLUSION: Our results demonstrate that NAC enhances MRP1-mediated doxorubicin resistance and this effect depends on GSH synthesis. DL-buthionine (S,R)-sulfoximine seems a promising chemotherapy improving agent in MRP1 overexpressing tumour cells.
Assuntos
Acetilcisteína/farmacologia , Antibióticos Antineoplásicos/uso terapêutico , Doxorrubicina/uso terapêutico , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Transferase/metabolismo , Humanos , TransfecçãoRESUMO
Multidrug resistance associated proteins (MRPs) and P-glycoprotein (P-gp) are involved in hepatobiliary transport of various compounds. Our aim was (1) to define transporter specificity of the cholescintigraphic agents 99mTc-HIDA and 99mTc-MIBI, which are used clinically for myocardial perfusion measurements; and (2) to deduce MRP and P-gp functions in vivo from hepatic 99mTc kinetics. Accumulation of radioactivity was measured in the human tumor cell lines GLC4, GLC4/ADR150x (MRP1-overexpressing/P-gp-negative) and GLC4/P-gp (P-gp-overexpressing). Bile secretion was quantified in untreated and in glutathione-depleted control and MRP2-deficient (GY/TR-) rats. Hepatobiliary transport was measured using a gamma camera in both types of rats. 99mTc-HIDA accumulated 5.8-fold less in GLC4/ADR150x calls than in GLC4 or GLC4/P-gp cells. In GLC4/ADR150x, the cellular 99mTc-HIDA content was increased 3.4-fold by the MRP1,2 inhibitor MK571 (50 microM), while MK571 had no measurable effect in GLC4 and GLC4/P-gp cells. 99mTc-MIBI accumulated less in GLC4/P-gp and GLC4/ADR150x cells than in GLC4 cells. Bile secretion of 99mTc-HIDA was impaired in GY/TR- compared to control rats and not affected by glutathione depletion in GY/TR- rats. Hepatic secretion of 99mTc-HIDA was slower in GY/TR- (t1/2 40 min) than in control rats (t1/2 7 min). Bile secretion of 99mTc-MIBI was similar in both rat strains and impaired by glutathione depletion in control rats only, indicating compensatory activity of additional transporter(s) in GY/TR- rats. 99mTc-HIDA is transported only by MRP1,2 only, while 99mTc-MIBI is transported by P-gp and MRP1,2. The results indicate that hepatic P-gp and MRP1,2 function can be assessed in vivo by sequential use of both radiopharmaceuticals.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/farmacologia , Fígado/diagnóstico por imagem , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Compostos Radiofarmacêuticos/farmacocinética , Lidofenina Tecnécio Tc 99m/farmacocinética , Tecnécio Tc 99m Sestamibi/farmacocinética , Tecnécio/farmacocinética , Animais , Bile/metabolismo , Glutationa/metabolismo , Fígado/fisiologia , Masculino , Cintilografia , Ratos , Ratos WistarRESUMO
New active drugs are needed for the treatment of primary brain tumors in both children and adults. S16020 is a cytotoxic olivacine derivative that inhibits topoisomerase II. The aim of the study was to determine its antitumor activity in athymic mice bearing subcutaneous medulloblastoma (IGRM33, 34, 57) and glioblastoma (IGRG88, 93, 121) xenografts treated at an advanced stage of tumor growth in comparison with that of doxorubicin. Animals were randomly assigned to receive i.v. S16020 or doxorubicin weekly for three consecutive weeks. The optimal dose was 80 mg/kg per week. S16020 demonstrated a significant antitumor activity in two out of three medulloblastoma xenografts. IGRM57 xenografts were highly sensitive with 100% tumor regressions and a tumor growth delay (TGD) of 102 days, while one of eight IGRM34 xenografts showed a partial regression with a TGD of 16 days. Doxorubicin was significantly more active than S16020 in these two models. IGRM33, a model established from a tumor in relapse after chemotherapy and radiotherapy, was refractory to both drugs. S16020 demonstrated a significant antitumor activity in the three glioblastoma xenografts evaluated. The wild-type p53 IGRG93 xenograft was highly sensitive with 100% tumor regressions and a TGD of 54 days. IGRG121 (wt p53) and IGRG88 (mutant p53) were moderately sensitive with TGDs of 33 and 23 days, respectively. Doxorubicin showed greater activity in two of these models. All six xenografts exhibited low expression of mdr1 as quantitated by RT-PCR, and no correlation was found with the activity of either drug. Conversely, a low activity of the two drugs was significantly associated with a high expression of MRP1 in medulloblastomas. Finally, no relationship was observed between drug sensitivity to either drug and expression of their target, topoisomerase IIalpha. In conclusion, S16020 and doxorubicin showed significant antitumor activity in brain tumor xenografts treated at an advanced stage of tumor growth. Their activity was related to MRP1 expression in medulloblastomas.
Assuntos
Carbazóis/farmacologia , Neoplasias Cerebelares/tratamento farmacológico , Doxorrubicina/farmacologia , Glioblastoma/tratamento farmacológico , Meduloblastoma/tratamento farmacológico , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Piridinas/farmacologia , Inibidores da Topoisomerase II , Animais , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/veterinária , Quimioterapia Combinada , Feminino , Regulação da Expressão Gênica , Glioblastoma/patologia , Glioblastoma/veterinária , Meduloblastoma/patologia , Meduloblastoma/veterinária , Camundongos , Camundongos Nus , Proteínas Associadas à Resistência a Múltiplos Medicamentos/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante HeterólogoRESUMO
This study investigated a high-throughput assay to measure multidrug resistance-associated protein (MRP1)-mediated uptake into membrane vesicles. Typically, a rapid filtration technique using a 12-filter vacuum manifold is used. We report here the development of a 96-well microtiter dish assay. MRP1-transfected HeLa cells (HeLa-T5) were used for the membrane vesicle preparations. The uptake of 50nM [3H]leukotriene C(4) (LTC(4)) was measured in a 96-well microtiter dish with rapid filtration onto a Perkin Elmer unifilter GF/B plate using a Perkin Elmer Filtermate 196. Counting of the isotype was conducted with a Perkin Elmer Top Count NXT. Uptake was adenosine 5'-triphosphate-dependent and linear over a 120-s time course. Uptake was inhibited by the leukotriene D(4) antagonist, MK 571, with a k(i) of 0.67 microM, and by the anti-MRP1 monoclonal antibody QCRL-3 but not by QCRL-1. Inhibition by estradiol-17-beta-glucuronide was 35-fold greater than inhibition by estradiol-3-beta-glucuronide. The kinetic parameters for LTC(4) uptake were determined to be a K(m) of 157nM with a V(max) of 344pmol/min/mg protein. The properties of MRP1-mediated transport of LTC(4) are consistent with those previously reported. The microtiter dish assay is a more expedient method for measuring transport into membrane vesicles and will have applications to other transporters.