RESUMO
P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancer; it plays important roles in determining the pharmacokinetics of many drugs. Understanding the structural basis of P-gp, substrate polyspecificity has been hampered by its intrinsic flexibility, which is facilitated by a 75-residue linker that connects the two halves of P-gp. Here we constructed a mutant murine P-gp with a shortened linker to facilitate structural determination. Despite dramatic reduction in rhodamine 123 and calcein-AM transport, the linker-shortened mutant P-gp possesses basal ATPase activity and binds ATP only in its N-terminal nucleotide-binding domain. Nine independently determined structures of wild type, the linker mutant, and a methylated P-gp at up to 3.3 Å resolution display significant movements of individual transmembrane domain helices, which correlated with the opening and closing motion of the two halves of P-gp. The open-and-close motion alters the surface topology of P-gp within the drug-binding pocket, providing a mechanistic explanation for the polyspecificity of P-gp in substrate interactions.
Assuntos
Mutação de Sentido Incorreto , Rodamina 123/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Transporte Biológico Ativo/fisiologia , Cristalografia por Raios X , Células HeLa , Humanos , Rodamina 123/metabolismo , Especificidade por Substrato/fisiologiaRESUMO
The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter's broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human-mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-cross-linking with [(125)I]iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Corantes Fluorescentes/metabolismo , Modelos Moleculares , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/química , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Antineoplásicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Transformada , Resistencia a Medicamentos Antineoplásicos , Células HeLa , Humanos , Cinética , Lepidópteros , Moduladores de Transporte de Membrana/farmacologia , Camundongos , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Multidrug resistance (MDR) is a major obstacle to the successful chemotherapy of cancer. MDR is often the result of overexpression of ATP-binding cassette transporters following chemotherapy. A common ATP-binding cassette transporter that is overexpressed in MDR cancer cells is P-glycoprotein, which actively effluxes drugs against a concentration gradient, producing an MDR phenotype. Collateral sensitivity (CS), a phenomenon of drug hypersensitivity, is defined as the ability of certain compounds to selectively target MDR cells, but not the drug-sensitive parent cells from which they were derived. The drug tiopronin has been previously shown to elicit CS. However, unlike other CS agents, the mechanism of action was not dependent on the expression of P-glycoprotein in MDR cells. We have determined that the CS activity of tiopronin is mediated by the generation of reactive oxygen species (ROS) and that CS can be reversed by a variety of ROS-scavenging compounds. Specifically, selective toxicity of tiopronin toward MDR cells is achieved by inhibition of glutathione peroxidase (GPx), and the mode of inhibition of GPx1 by tiopronin is shown in this report. Why MDR cells are particularly sensitive to ROS is discussed, as is the difficulty in exploiting this hypersensitivity to tiopronin in the clinic.
Assuntos
Inibidores Enzimáticos/farmacologia , Glutationa Peroxidase/antagonistas & inibidores , Tiopronina/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Glutationa Peroxidase/química , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Tiomalatos/farmacologiaRESUMO
Acquired drug resistance in cancer continues to be a challenge in cancer therapy, in part due to overexpression of the drug efflux transporter P-glycoprotein (P-gp, MDR1, ABCB1). NSC73306 is a thiosemicarbazone compound that displays greater toxicity against cells expressing functional P-gp than against other cells. Here, we investigate the cellular uptake of NSC73306, and examine its interaction with P-gp and copper transporter 1 (CTR1, SLC31A1). Overexpression of P-gp sensitizes LLC-PK1 cells to NSC73306. Cisplatin (IC50 = 77 µM), cyclosporin A (IC50 = 500 µM), and verapamil (IC50 = 700 µM) inhibited cellular accumulation of [(3)H]NSC73306. Cellular hypertoxicity of NSC73306 to P-gp-expressing cells was inhibited by cisplatin in a dose-dependent manner. Cells transiently expressing the cisplatin uptake transporter CTR1 (SLC31A1) showed increased [(3)H]NSC73306 accumulation. In contrast, CTR1 knockdown decreased [(3)H]NSC73306 accumulation. The presence of NSC73306 reduced CTR1 levels, similar to the negative feedback of CTR1 levels by copper or cisplatin. Surprisingly, although cisplatin is a substrate of CTR1, we found that CTR1 protein was overexpressed in high-level cisplatin-resistant KB-CP20 and BEL7404-CP20 cell lines. We confirmed that the CTR1 protein was functional, as uptake of NSC73306 was increased in KB-CP20 cells compared to their drug-sensitive parental cells, and downregulation of CTR1 in KB-CP20 cells reduced [(3)H]NSC73306 accumulation. These results suggest that NSC73306 is a transport substrate of CTR1.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Indóis/farmacocinética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Células COS , Chlorocebus aethiops , Cisplatino/administração & dosagem , Cisplatino/química , Cobre/química , Transportador de Cobre 1 , Ciclosporina/química , Células HEK293 , Humanos , Concentração Inibidora 50 , Células LLC-PK1 , RNA Interferente Pequeno/metabolismo , Reprodutibilidade dos Testes , Suínos , Tiossemicarbazonas/química , Verapamil/administração & dosagemRESUMO
While chemotherapy remains the most effective treatment for disseminated tumors, acquired or intrinsic drug resistance accounts for approximately 90% of treatment failure. Multidrug resistance (MDR), the simultaneous resistance to drugs that differ both structurally and mechanistically, often results from drug efflux pumps in the cell membrane that reduce intracellular drug levels to less than therapeutic concentrations. Expression of the MDR transporter P-glycoprotein (P-gp, MDR1, ABCB1) has been shown to correlate with overall poor chemotherapy response and prognosis. This review will focus on collateral sensitivity (CS), the ability of compounds to kill MDR cells selectively over the parental cells from which they were derived. Insights into CS may offer an alternative strategy for the clinical resolution of MDR, as highly selective and potent CS agents may lead to drugs that are effective at MDR cell killing and tumor resensitization. Four main mechanistic hypotheses for CS will be reviewed, followed by a discussion on quantitative and experimental evaluation of CS.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transporte Biológico/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/uso terapêutico , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Metabolismo Energético/efeitos dos fármacos , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Prognóstico , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Verapamil/uso terapêuticoRESUMO
CONTEXT: Whether hypofractionated radiation therapy (RT) compared with conventionally fractionated RT provides comparable or possibly improved cancer control without increased toxicity in localized prostate cancer (PC) remains unknown. OBJECTIVE: Realizing from the CHHiP trial that outcomes are highly sensitive to the dose fractionation schedule and number of treatments, we conducted a systematic review and meta-analysis selecting only the randomized noninferiority trials, because the randomized arms closely approximated one another in terms of the dose fractionation schedule, and compared cancer control and toxicity of hypofractionated RT with conventionally fractionated RT for localized PC. EVIDENCE ACQUISITION: Randomized noninferiority trials evaluating hypofractionated (2.4-4Gy daily fractions for 15-30 treatments) versus conventionally fractionated RT (1.8-2Gy daily fractions for 40-45 treatments) in men with localized PC were selected. Studies that were not noninferiority trials, used extreme hypofractionation, or treated metastatic disease were excluded. Three studies were retained for analysis. Data were pooled using a random-effects model to determine hazard ratio (HR) and risk ratio (RR). Heterogeneity was assessed via chi-square test, I2 statistics, and metaregression. The primary outcome was disease-free survival (DFS), defined as death from any cause or biochemical, local, regional, or distant progression. EVIDENCE SYNTHESIS: Of the 5484 men, 3553 (64.8%) had intermediate-risk PC. Hypofractionated RT as compared with conventionally fractionated RT was associated with significantly improved DFS (HR 0.869; 95% confidence interval [CI], 0.757, 0.998; p=0.047), whereas overall survival was not (HR 0.84; 95% CI, 0.66, 1.07; p=0.16). Acute grade 2 or higher gastrointestinal toxicity was significantly increased with hypofractionation (RR 1.42; 95% CI 1.15, 1.77; p=0.002); however, this did not translate into late grade 2 or higher gastrointestinal toxicity. An increase in late grade 2 or higher genitourinary complications was observed (RR 1.18; 95% CI 0.98, 1.43; p=0.08). CONCLUSIONS: Hypofractionated RT as compared with conventionally fractionated RT could improve DFS in men with intermediate-risk PC and, therefore, would be reasonable to consider in men who do not have risk factors for late genitourinary complications. PATIENT SUMMARY: Treatment with a shorter course of radiation, using higher doses per treatment over fewer days, may be the preferred approach in appropriately selected patients with localized prostate cancer.
Assuntos
Neoplasias da Próstata/radioterapia , Hipofracionamento da Dose de Radiação , Estudos de Equivalência como Asunto , Humanos , Masculino , Neoplasias da Próstata/patologia , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Most patients who die of cancer have disseminated disease that has become resistant to multiple therapeutic modalities. Ample evidence suggests that the expression of ATP-binding cassette (ABC) transporters, especially the multidrug resistance protein 1 (MDR1, also known as P-glycoprotein or P-gp), which is encoded by ABC subfamily B member 1 (ABCB1), can confer resistance to cytotoxic and targeted chemotherapy. However, the development of MDR1 as a therapeutic target has been unsuccessful. At the time of its discovery, appropriate tools for the characterization and clinical development of MDR1 as a therapeutic target were lacking. Thirty years after the initial cloning and characterization of MDR1 and the implication of two additional ABC transporters, the multidrug resistance-associated protein 1 (MRP1; encoded by ABCC1)), and ABCG2, in multidrug resistance, interest in investigating these transporters as therapeutic targets has waned. However, with the emergence of new data and advanced techniques, we propose to re-evaluate whether these transporters play a clinical role in multidrug resistance. With this Opinion article, we present recent evidence indicating that it is time to revisit the investigation into the role of ABC transporters in efficient drug delivery in various cancer types and at the blood-brain barrier.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Resistência a Múltiplos Medicamentos/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Barreira Hematoencefálica , Humanos , Terapia de Alvo Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genéticaRESUMO
The efflux transporter P-glycoprotein (P-gp) is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , DNA Complementar/genética , Escherichia coli/crescimento & desenvolvimento , Regiões Promotoras Genéticas/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Sítios de Ligação , Western Blotting , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Citometria de Fluxo , Células HEK293 , Humanos , Camundongos , Microscopia Confocal , Plasmídeos/genética , Conformação Proteica , Fator sigma/metabolismo , Espectrometria de FluorescênciaAssuntos
Automonitorização da Glicemia , Glicemia , Glucose , Hemoglobinas Glicadas/análise , Humanos , HipoglicemiantesRESUMO
Extracts of the sponge genus Candidaspongia showed selective cytotoxicity toward melanoma cells in the NCI 60-cell-line screen. Continued investigation of the Candidaspongia sp. extracts led to the isolation of three new tedanolide analogues, precandidaspongiolides A (1) and B (2) and candidaspongiolide B (4), as well as candidaspongiolide A (3) and tedanolide (5). Semisynthetic derivatives were also generated to develop SAR. Candidaspongiolides A/B were the most potent and showed low nanomolar activity against several melanoma cell lines.
Assuntos
Proliferação de Células/efeitos dos fármacos , Macrolídeos/química , Macrolídeos/síntese química , Melanoma/patologia , Linhagem Celular Tumoral , Humanos , Macrolídeos/farmacologia , Relação Estrutura-AtividadeRESUMO
A major challenge in the treatment of cancer is multidrug resistance (MDR) that develops during chemotherapy. Here we demonstrate that tiopronin (1), a thiol-substituted N-propanoylglycine derivative, was selectively toxic to a series of cell lines expressing the drug efflux pump P-glycoprotein (P-gp, ABCB1) and MRP1 (ABCC1). Treatment of MDR cells with 1 led to instability of the ABCB1 mRNA and consequently a reduction in P-gp protein, despite functional assays demonstrating that tiopronin does not interact with P-gp. Long-term exposure of P-gp-expressing cells to 1 sensitized them to doxorubicin and paclitaxel, both P-gp substrates. Treatment of MRP1-overexpressing cells with tiopronin led to a significant reduction in MRP1 protein. Synthesis and screening of analogues of tiopronin demonstrated that the thiol functional group was essential for collateral sensitivity while substitution of the amino acid backbone altered but did not destroy specificity, pointing to future development of targeted analogues.
Assuntos
Antineoplásicos/farmacologia , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Tiopronina/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Doxorrubicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Produção de Droga sem Interesse Comercial , Paclitaxel/farmacologia , Estabilidade de RNA , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade , Tiopronina/síntese química , Tiopronina/químicaRESUMO
Cancer multidrug resistance (MDR) mediated by ATP-binding cassette (ABC) transporters presents a significant unresolved clinical challenge. One strategy to resolve MDR is to develop compounds that selectively kill cells overexpressing the efflux transporter P-glycoprotein (MDR1, P-gp, ABCB1). We have previously reported structure-activity studies based around the lead compound NSC73306 (1, 1-isatin-4-(4'-methoxyphenyl)-3-thiosemicarbazone, 4.3-fold selective). Here we sought to extend this work on MDR1-selective analogues by establishing whether 1 showed "robust" activity against a range of cell lines expressing P-gp. We further aimed to synthesize and test analogues with varied substitution at the N4-position, and substitution around the N4-phenyl ring of isatin-ß-thiosemicarbazones (IBTs), to identify compounds with increased MDR1-selectivity. Compound 1 demonstrated MDR1-selectivity against all P-gp-expressing cell lines examined. This selectivity was reversed by inhibitors of P-gp ATPase activity. Structural variation at the 4'-phenyl position of 1 yielded compounds of greater MDR1-selectivity. Two of these analogues, 1-isatin-4-(4'-nitrophenyl)-3-thiosemicarbazone (22, 8.3-fold selective) and 1-isatin-4-(4'-tert-butyl phenyl)-3-thiosemicarbazone (32, 14.8-fold selective), were selected for further testing and were found to retain the activity profile of 1. These compounds are the most active IBTs identified to date.