RESUMO
Membrane transporters play an important role in the absorption, distribution, metabolism and excretion of drugs. The cellular accumulation of many drugs is the result of the net function of efflux and influx transporters. Efflux transporters such as P-glycoprotein/ABCB1 have been shown to confer multidrug resistance in cancer. Although expression of uptake transporters has been confirmed in cancer cells, their role in chemotherapy response has not been systematically investigated. In the present study we have adapted a fluorescence-based cytotoxic assay to characterize the influence of key drug-transporters on the toxicity of approved anticancer drugs. Co-cultures of fluorescently labeled parental and transporter-expressing cells (expressing ABCB1, ABCG2 or OATP2B1) were screened against 101 FDA-approved anticancer drugs, using a novel, automated, triple fluorescence-based cytotoxicity assay. By measuring the survival of parental and transporter-expressing cells in co-cultures, we identify those FDA-approved anticancer drugs, whose toxicity is influenced by ABCB1, ABCG2 or OATP2B1. In addition to confirming known substrates of ABCB1 and ABCG2, the fluorescence-based cytotoxicity assays identified anticancer agents whose toxicity was increased in OATP2B1 expressing cells. Interaction of these compounds with OATP2B1 was verified in dedicated transport assays using cell-impermeant fluorescent substrates. Understanding drug-transporter interactions is needed to increase the efficacy of chemotherapeutic agents. Our results highlight the potential of the fluorescence-based HT screening system for identifying transporter substrates, opening the way for the design of therapeutic approaches based on the inhibition or even the exploitation of transporters in cancer cells.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Proteínas Luminescentes/genética , Transportadores de Ânions Orgânicos/metabolismo , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Simulação por Computador , Citometria de Fluxo , Corantes Fluorescentes , Proteínas de Fluorescência Verde/genética , Humanos , Transportadores de Ânions Orgânicos/genética , Especificidade por Substrato , Transdução Genética , Proteína Vermelha FluorescenteRESUMO
BACKGROUND & AIMS: Gut bacterial sphingolipids, primarily produced by Bacteroidetes, have dual roles as bacterial virulence factors and regulators of the host mucosal immune system, including regulatory T cells and invariant natural killer T cells. Patients with inflammatory bowel disease display altered sphingolipids profiles in fecal samples. However, how bacterial sphingolipids modulate mucosal homeostasis and regulate intestinal inflammation remains unclear. METHODS: We used dextran sodium sulfate (DSS)-induced colitis in mice monocolonized with Bacteroides fragilis strains expressing or lacking sphingolipids to assess the influence of bacterial sphingolipids on intestinal inflammation using transcriptional, protein, and cellular analyses. Colonic explant and organoid were used to study the function of bacterial sphingolipids. Host mucosal immune cells and cytokines were profiled and characterized using flow cytometry, enzyme-linked immunosorbent assay, and Western blot, and cytokine function in vivo was investigated by monoclonal antibody injection. RESULTS: B fragilis sphingolipids exacerbated intestinal inflammation. Mice monocolonized with B fragilis lacking sphingolipids exhibited less severe DSS-induced colitis. This amelioration of colitis was associated with increased production of interleukin (IL)-22 by ILC3. Mice colonized with B fragilis lacking sphingolipids following DSS treatment showed enhanced epithelial STAT3 activity, intestinal cell proliferation, and antimicrobial peptide production. Protection against DSS colitis associated with B fragilis lacking sphingolipids was reversed on IL22 blockade. Furthermore, bacterial sphingolipids restricted epithelial IL18 production following DSS treatment and interfered with IL22 production by a subset of ILC3 cells expressing both IL18R and major histocompatibility complex class II. CONCLUSIONS: B fragilis-derived sphingolipids exacerbate mucosal inflammation by impeding epithelial IL18 expression and concomitantly suppressing the production of IL22 by ILC3 cells.
Assuntos
Bacteroides fragilis , Colite , Sulfato de Dextrana , Interleucina 22 , Interleucinas , Esfingolipídeos , Animais , Esfingolipídeos/metabolismo , Interleucinas/metabolismo , Camundongos , Colite/imunologia , Colite/patologia , Colite/induzido quimicamente , Colite/microbiologia , Colite/metabolismo , Sulfato de Dextrana/toxicidade , Bacteroides fragilis/imunologia , Modelos Animais de Doenças , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Mucosa Intestinal/microbiologia , Mucosa Intestinal/metabolismo , Fator de Transcrição STAT3/metabolismo , Camundongos Endogâmicos C57BLRESUMO
The loss of IL-10R function leads to severe early onset colitis and, in murine models, is associated with the accumulation of immature inflammatory colonic macrophages. We have shown that IL-10R-deficient colonic macrophages exhibit increased STAT1-dependent gene expression, suggesting that IL-10R-mediated inhibition of STAT1 signaling in newly recruited colonic macrophages might interfere with the development of an inflammatory phenotype. Indeed, STAT1-/- mice exhibit defects in colonic macrophage accumulation after Helicobacter hepaticus infection and IL-10R blockade, and this was phenocopied in mice lacking IFNγR, an inducer of STAT1 activation. Radiation chimeras demonstrated that reduced accumulation of STAT1-deficient macrophages was based on a cell-intrinsic defect. Unexpectedly, mixed radiation chimeras generated with both wild-type and IL-10R-deficient bone marrow indicated that rather than directly interfering with STAT1 function, IL-10R inhibits the generation of cell extrinsic signals that promote the accumulation of immature macrophages. These results define the essential mechanisms controlling the inflammatory macrophage accumulation in inflammatory bowel diseases.
Assuntos
Colite , Camundongos , Animais , Colite/metabolismo , Macrófagos/metabolismo , Receptores de Interleucina-10/genética , Receptores de Interleucina-10/metabolismo , Transdução de Sinais , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Macrophages are a heterogeneous population of mononuclear phagocytes abundantly distributed throughout the intestinal compartments that adapt to microenvironmental specific cues. In adult mice, the majority of intestinal macrophages exhibit a mature phenotype and are derived from blood monocytes. In the steady-state, replenishment of these cells is reduced in the absence of the chemokine receptor CCR2. Within the intestine of mice with colitis, there is a marked increase in the accumulation of immature macrophages that demonstrate an inflammatory phenotype. Here, we asked whether CCR2 is necessary for the development of colitis in mice lacking the receptor for IL10. We compared the development of intestinal inflammation in mice lacking IL10RA or both IL10RA and CCR2. The absence of CCR2 interfered with the accumulation of immature macrophages in IL10R-deficient mice, including a novel population of rounded submucosal Iba1+ cells, and reduced the severity of colitis in these mice. In contrast, the absence of CCR2 did not reduce the augmented inflammatory gene expression observed in mature intestinal macrophages isolated from mice lacking IL10RA. These data suggest that both newly recruited CCR2-dependent immature macrophages and CCR2-independent residual mature macrophages contribute to the development of intestinal inflammation observed in IL10R-deficient mice.
Assuntos
Colite/imunologia , Subunidade alfa de Receptor de Interleucina-10/imunologia , Intestinos/imunologia , Monócitos/imunologia , Receptores CCR2/imunologia , Animais , Colite/genética , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-10/genética , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Knockout , Receptores CCR2/genéticaRESUMO
Organic anion-transporting polypeptide 1A2 (OATP1A2), expressed in the human blood-brain barrier, promotes drug uptake from the blood and hence can be exploited for central nervous system-targeted drug delivery. The thyroid transporter OATP1C1, expressed in the choroid plexus and in astrocytes, is also a potential pharmacological target. Based on their established pharmacological relevance, screening the drug interaction profile of OATP1A2 and OATP1C1 is highly desirable. However, drug interaction screens require suitable model systems and functional assays. In the current study, uptake of a set of cell-impermeable fluorescent dyes was screened in HEK-293 and A431 cell lines overexpressing OATP1A2 and OATP1C1. Based on the uptake of fluorescent dye substrates, a functional assay was developed, which was used to characterize OATP inhibitors/substrates. We identify Live/Dead Green (LDG), Live-or-Dye 488, and sulforhodamines 101, G, and B as novel fluorescent substrates of OATP1A2 and OATP1C1. We show that LDG uptake is proportional to OATP1A2/1C1 expression, allowing the isolation of cells expressing high transporter levels. Additionally, dye uptake can be used to characterize the drug interaction pattern of OATP1A2 and OATP1C1. We demonstrate that third-generation P-glycoprotein inhibitors elacridar, tariquidar, and zosuquidar inhibit OATP1A2 function. Increased toxicity of elacridar in OATP1A2-expressing cells suggests that OATP1A2 may modulate the distribution of this compound. The fluorescence-based assays developed in the current study are a good alternative of radioligand-based tests and pave the way toward high-throughput screens for OATP1A2/1C1 drug interaction studies.
Assuntos
Acridinas/farmacologia , Dibenzocicloeptenos/farmacologia , Corantes Fluorescentes/química , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Quinolinas/farmacologia , Tetra-Hidroisoquinolinas/farmacologia , Acridinas/química , Linhagem Celular , Dibenzocicloeptenos/química , Humanos , Transportadores de Ânions Orgânicos/análise , Transportadores de Ânions Orgânicos/metabolismo , Quinolinas/química , Tetra-Hidroisoquinolinas/químicaRESUMO
Organic anion-transporting polypeptide 3A1 (OATP3A1) is a membrane transporter mediating the cellular uptake of various hormones such as estrone-3-sulfate, prostaglandins E1 and E2 and thyroxine. OATP3A1 is widely expressed in the human body and its presence in tissue-blood barriers, neurons and muscle cells marks it as a potential pharmacological target. Herein we demonstrate that an otherwise membrane impermeant, zwitterionic fluorescent coumarin probe, bearing a sulfonate function is a potent substrate of human OATP3A1, thus readily transported into HEK-293-OATP3A1 cells allowing functional investigation and the screen of drug interactions of the OATP3A1 transporter. At the same time, dyes lacking either the sulfonate motif or the coumarin scaffold showed a dramatic decrease in affinity or even a complete loss of transport. Furthermore, we observed a distinct inhibition/activation pattern in the OATP3A1-mediated uptake of closely related fluorescent coumarin derivatives differing only in the presence of the sulfonate moiety. Additionally, we detected a synergistic effect between one of the probes tested and the endogenous OATP substrate estrone-3-sulfate. These data, together with docking results indicate the presence of at least two cooperative substrate binding sites in OATP3A1. Besides providing the first sensitive probe for testing OATP3A1 substrate/inhibitor interactions, our results also help to understand substrate recognition and transport mechanism of the poorly characterized OATP3A1. Moreover, coumarins are good candidates for OATP3A1-targeted drug delivery and as pharmacological modulators of OATP3A1.
Assuntos
Cumarínicos/metabolismo , Cumarínicos/farmacologia , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacologia , Transportadores de Ânions Orgânicos/metabolismo , Cumarínicos/química , Corantes Fluorescentes/química , Células HEK293 , Humanos , Transportadores de Ânions Orgânicos/química , Estrutura Secundária de Proteína , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologiaRESUMO
Detoxification in hepatocytes is a strictly controlled process, in which the governed action of membrane transporters involved in the uptake and efflux of potentially dangerous molecules has a crucial role. Major transporters of hepatic clearance belong to the ABC (ATP Binding Cassette) and Solute Carrier (SLC) protein families. Organic anion-transporting polypeptide OATP1B1 (encoded by the SLCO1B1 gene) is exclusively expressed in the sinusoidal membrane of hepatocytes, where it mediates the cellular uptake of bile acids, bilirubin, and also that of various drugs. The removal of toxic molecules from hepatocytes to the bile is accomplished by several ABC transporters, including P-glycoprotein (ABCB1), MRP2 (ABCC2) and BCRP (ABCG2). Owing to their pharmacological relevance, monitoring drug interaction with OATP1B1/3 and ABC proteins is recommended. Our aim was to assess the interaction of recently identified fluorescent OATP substrates (various dyes used in cell viability assays, pyranine, Cascade Blue hydrazide (CB) and sulforhodamine 101 (SR101)) (Bakos et al., 2019; Patik et al., 2018) with MRP2 and ABCG2 in order to find fluorescent probes for the simultaneous characterization of both uptake and efflux processes. Transport by MRP2 and ABCG2 was investigated in inside-out membrane vesicles (IOVs) allowing a fast screen of the transport of membrane impermeable substrates by efflux transporters. Next, transcellular transport of shared OATP and ABC transporter substrate dyes was evaluated in MDCKII cells co-expressing OATP1B1 and MRP2 or ABCG2. Our results indicate that pyranine is a general substrate of OATP1B1, OATP1B3 and OATP2B1, and we find that the dye Live/Dead Violet and CB are good tools to investigate ABCG2 function in IOVs. Besides their suitability for MRP2 functional tests in the IOV setup, pyranine, CB and SR101 are the first dual probes that can be used to simultaneously measure OATP1B1 and MRP2 function in polarized cells by a fluorescent method.
Assuntos
Corantes Fluorescentes , Transportadores de Ânions Orgânicos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Interações Medicamentosas , Hepatócitos , Proteínas de Neoplasias , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de SolutoRESUMO
The generation of tissue-resident memory T cells (TRM) is an essential aspect of immunity at mucosal surfaces, and it has been suggested that preferential generation of TRM is one of the principal advantages of mucosally administered vaccines. We have previously shown that antigen-specific, IL-17-producing CD4+ T cells can provide capsular antibody-independent protection against nasal carriage of Streptococcus pneumoniae; but whether pneumococcus-responsive TRM are localized within the nasal mucosa and are sufficient for protection from carriage has not been determined. Here, we show that intranasal administration of live or killed pneumococci to mice generates pneumococcus-responsive IL-17A-producing CD4+ mucosal TRM. Furthermore, we show that these cells are sufficient to mediate long-lived, neutrophil-dependent protection against subsequent pneumococcal nasal challenge. Unexpectedly, and in contrast with the prevailing paradigm, we found that parenteral administration of killed pneumococci also generates protective IL-17A+CD4+ TRM in the nasal mucosa. These results demonstrate a critical and sufficient role of TRM in prevention of pneumococcal colonization, and further that these cells can be generated by parenteral immunization. Our findings therefore have important implications regarding the generation of immune protection at mucosal surfaces by vaccination.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Nariz/imunologia , Infecções Pneumocócicas/imunologia , Vacinas Estreptocócicas/imunologia , Streptococcus pneumoniae/fisiologia , Animais , Células Cultivadas , Resistência à Doença , Humanos , Memória Imunológica , Interleucina-17/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , VacinaçãoRESUMO
Organic Anion-Transporting Polypeptides are multispecific membrane proteins that regulate the passage of crucial endobiotics and drugs across pharmacological barriers. OATP1B1 and OATP1B3 have been described to play a major role in the hepatic uptake of statins, antivirals and various chemotherapeutics; whereas the pharmacological role of the ubiquitously expressed OATP2B1 is less well characterized. According to current industry standards, in vitro testing for susceptibility to OATP1B1 and 1B3 mediated transport is recommended for drug candidates that are eliminated in part via the liver. Here we show that human OATP1B1, 1B3 and 2B1 transport a series of commercially available viability dyes that are generally believed to be impermeable to intact cells. We demonstrate that the intracellular accumulation of Zombie Violet, Live/Dead Green, Cascade Blue and Alexa Fluor 405 is specifically increased by OATPs. Inhibition of Cascade Blue or Alexa Fluor 405 uptake by known OATP substrates/inhibitors yielded IC50 values in agreement with gold-standard radioligand assays. The fluorescence-based assays described in this study provide a new tool for testing OATP1B/2B1 drug interactions.
Assuntos
Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo , Animais , Transporte Biológico , Corantes/metabolismo , Cães , Interações Medicamentosas , Células HEK293 , Humanos , Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado/antagonistas & inibidores , Células Madin Darby de Rim Canino , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Células Sf9 , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/antagonistas & inibidoresRESUMO
To assess the hepatic disposition of erlotinib, we performed positron emission tomography (PET) scans with [11 C]erlotinib in healthy volunteers without and with oral pretreatment with a therapeutic erlotinib dose (300 mg). Erlotinib pretreatment significantly decreased the liver exposure to [11 C]erlotinib with a concomitant increase in blood exposure, pointing to the involvement of a carrier-mediated hepatic uptake mechanism. Using cell lines overexpressing human organic anion-transporting polypeptides (OATPs) 1B1, 1B3, or 2B1, we show that [11 C]erlotinib is selectively transported by OATP2B1. Our data suggest that at PET microdoses hepatic uptake of [11 C]erlotinib is mediated by OATP2B1, whereas at therapeutic doses OATP2B1 transport is saturated and hepatic uptake occurs mainly by passive diffusion. We propose that [11 C]erlotinib may be used as a hepatic OATP2B1 probe substrate and erlotinib as an OATP2B1 inhibitor in clinical drug-drug interaction studies, allowing the contribution of OATP2B1 to the hepatic uptake of drugs to be revealed.
Assuntos
Cloridrato de Erlotinib/farmacocinética , Hepatócitos/metabolismo , Fígado/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Inibidores de Proteínas Quinases/farmacocinética , Adulto , Radioisótopos de Carbono , Difusão , Cloridrato de Erlotinib/metabolismo , Feminino , Humanos , Técnicas In Vitro , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Masculino , Tomografia por Emissão de Pósitrons , Inibidores de Proteínas Quinases/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/metabolismo , Adulto JovemRESUMO
INTRODUCTION: The in vivo fate and effectiveness of a drug depends highly on its absorption, distribution, metabolism, excretion and toxicity (ADME-Tox). Organic anion transporting polypeptides (OATPs) are membrane proteins involved in the cellular uptake of various organic compounds, including clinically used drugs. Since OATPs are significant players in drug absorption and distribution, modulation of OATP function via pharmacotherapy with OATP substrates/inhibitors, or modulation of their expression, affects drug pharmacokinetics. Given their cancer-specific expression, OATPs may also be considered anticancer drug targets. Areas covered: We describe the human OATP family, discussing clinically relevant consequences of altered OATP function. We offer a critical analysis of published data on the role of OATPs in ADME and in drug-drug interactions, especially focusing on OATP1A2, 1B1, 1B3 and 2B1. Expert opinion: Four members of the OATP family, 1A2, 1B1, 1B3 and 2B1, have been characterized in detail. As biochemical and pharmacological knowledge on the other OATPs is lacking, it seems timely to direct research efforts towards developing the experimental framework needed to investigate the transport mechanism and substrate specificity of the poorly described OATPs. In addition, elucidating the role of OATPs in tumor development and therapy response are critical avenues for further research.
Assuntos
Transportadores de Ânions Orgânicos/metabolismo , Peptídeos/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Antineoplásicos/farmacologia , Transporte Biológico , Interações Medicamentosas , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Transportadores de Ânions Orgânicos/genética , Preparações Farmacêuticas/administração & dosagem , Farmacocinética , Especificidade por SubstratoRESUMO
Organic Anion Transporting Polypeptides (OATPs), encoded by genes of the Solute Carrier Organic Anion (SLCO) family, are transmembrane proteins involved in the uptake of various compounds of endogenous or exogenous origin. In addition to their physiological roles, OATPs influence the pharmacokinetics and drug-drug interactions of several clinically relevant compounds. To examine the function and molecular interactions of human OATPs, including several poorly characterized family members, we expressed all 11 human OATPs at high levels in the baculovirus-Sf9 cell system. We measured the temperature- and inhibitor-sensitive cellular accumulation of sodium fluorescein and fluorescein-methotrexate, two fluorescent substrates of the OATPs, OATP1B1 and 1B3. OATP1B1 and 1B3 were functional in Sf9 cells, showing rapid uptake (t1/2(fluorescein-methotrexate) 2.64 and 4.16 min, and t1/2(fluorescein) 6.71 and 5.58 min for OATP1B1 and 1B3, respectively) and high-affinity transport (Km(fluorescein-methotrexate) 0.23 and 0.53 µM, and Km(fluorescein) 25.73 and 38.55 µM for OATP1B1 and 1B3, respectively) of both substrates. We found that sodium fluorescein is a general substrate of all human OATPs: 1A2, 1B1, 1B3, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, 5A1 and 6A1, while fluorescein-methotrexate is only transported by 1B1, 1B3, 1A2 and 2B1. Acidic extracellular pH greatly facilitated fluorescein uptake by all OATPs, and new molecular interactions were detected (between OATP2B1 and Imatinib, OATP3A1, 5A1 and 6A1 and estradiol 17-ß-d-glucuronide, and OATP1C1 and 4C1 and prostaglandin E2). These studies demonstrate, for the first time, that the insect cell system is suitable for the functional analysis of the entire human OATP family, and for drug-OATP interaction screening.