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Sample records for multidrug resistance-linked abcg2

  1. Synthetic Analogs of Curcumin Modulate the Function of Multidrug Resistance-Linked ATP-Binding Cassette Transporter ABCG2.

    PubMed

    Murakami, Megumi; Ohnuma, Shinobu; Fukuda, Michihiro; Chufan, Eduardo E; Kudoh, Katsuyoshi; Kanehara, Keigo; Sugisawa, Norihiko; Ishida, Masaharu; Naitoh, Takeshi; Shibata, Hiroyuki; Iwabuchi, Yoshiharu; Ambudkar, Suresh V; Unno, Michiaki

    2017-11-01

    Multidrug resistance (MDR) caused by the overexpression of ATP-binding cassette (ABC) transporters in cancer cells is a major obstacle in cancer chemotherapy. Previous studies have shown that curcumin, a natural product and a dietary constituent of turmeric, inhibits the function of MDR-related ABC transporters, including ABCB1, ABCC1, and especially ABCG2. However, the limited bioavailability of curcumin prevents its use for modulation of the function of these transporters in the clinical setting. In this study, we investigated the effects of 24 synthetic curcumin analogs with increased bioavailability on the transport function of ABCG2. The screening of the 24 synthetic analogs by means of flow cytometry revealed that four of the curcumin analogs (GO-Y030, GO-Y078, GO-Y168, and GO-Y172) significantly inhibited the efflux of the ABCG2 substrates, mitoxantrone and pheophorbide A, from ABCG2-overexpressing K562/breast cancer resistance protein (BCRP) cells. Biochemical analyses showed that GO-Y030, GO-Y078, and GO-Y172 stimulated the ATPase activity of ABCG2 at nanomolar concentrations and inhibited the photolabeling of ABCG2 with iodoarylazidoprazosin, suggesting that these analogs interact with the substrate-binding sites of ABCG2. In addition, when used in cytotoxicity assays, GO-Y030 and GO-Y078 were found to improve the sensitivity of the anticancer drug, SN-38, in K562/BCRP cells. Taken together, these results suggest that nontoxic synthetic curcumin analogs with increased bioavailability, especially GO-Y030 and GO-Y078, inhibit the function of ABCG2 by directly interacting at the substrate-binding site. These synthetic curcumin analogs could therefore be developed as potent modulators to overcome ABCG2-mediated MDR in cancer cells. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  2. The naphthoquinones, vitamin K3 and its structural analog plumbagin, are substrates of the multidrug resistance-linked ABC drug transporter ABCG2

    PubMed Central

    Shukla, Suneet; Wu, Chung-Pu; Nandigama, Krishnamachary; Ambudkar, Suresh V.

    2008-01-01

    Vitamin K3 (Menadione; 2-methyl-1,4-naphthoquinone) is a structural precursor of vitamins K1 and K2 which are essential for blood clotting. The naturally occurring structural analog of this vitamin, plumbagin (5-hydroxy-menadione), is known to modulate cellular proliferation, apoptosis, carcinogenesis, and radioresistance. We, here, report that both vitamin K3 and plumbagin are substrates of the multidrug resistance-linked ATP binding cassette (ABC) drug transporter, ABCG2. Vitamin K3 and plumbagin specifically inhibited the ABCG2-mediated efflux of mitoxantrone, but did not have any effect on the ABCB1-mediated efflux of rhodamine 123. This inhibition of ABCG2 function was due to their interaction at the substrate-binding site(s). They inhibited the binding of [125I]-Iodoarylazidoprazosin (IAAP), a substrate of ABCG2, to this transporter in a concentration-dependent manner with IC50 values of 7.3 and 22.6 μM, respectively, but had no effect on the binding of this photoaffinity analog to ABCB1. Both compounds stimulated ABCG2-mediated ATP hydrolysis and also inhibited the mitoxantrone-stimulated ATPase activity of this transporter, but did not have any significant effect on the ATPase activity of ABCB1. In a cytotoxicity assay, ABCG2-expressing HEK cells were 2.8- and 2.3-fold resistant to plumbagin and vitamin K3, respectively, compared to the control cells, suggesting that they are substrates of this transporter. Collectively, these data demonstrate for the first time that vitamin K3 is a substrate of the ABCG2 transporter. Thus, ABCG2 may have a role in the regulation of vitamin K3 levels in the body. In addition, vitamin K3 and its structural derivative, plumbagin, could potentially be used to modulate ABCG2 function. PMID:18065489

  3. A combination of curcumin with either gramicidin or ouabain selectively kills cells that express the multidrug resistance-linked ABCG2 transporter.

    PubMed

    Rao, Divya K; Liu, Haiyan; Ambudkar, Suresh V; Mayer, Michael

    2014-11-07

    This paper introduces a strategy to kill selectively multidrug-resistant cells that express the ABCG2 transporter (also called breast cancer resistance protein, or BCRP). The approach is based on specific stimulation of ATP hydrolysis by ABCG2 transporters with subtoxic doses of curcumin combined with stimulation of ATP hydrolysis by Na(+),K(+)-ATPase with subtoxic doses of gramicidin A or ouabain. After 72 h of incubation with the drug combinations, the resulting overconsumption of ATP by both pathways inhibits the efflux activity of ABCG2 transporters, leads to depletion of intracellular ATP levels below the viability threshold, and kills resistant cells selectively over cells that lack ABCG2 transporters. This strategy, which was also tested on a clinically relevant human breast adenocarcinoma cell line (MCF-7/FLV1), exploits the overexpression of ABCG2 transporters and induces caspase-dependent apoptotic cell death selectively in resistant cells. This work thus introduces a novel strategy to exploit collateral sensitivity (CS) with a combination of two clinically used compounds that individually do not exert CS. Collectively, this work expands the current knowledge on ABCG2-mediated CS and provides a potential strategy for discovery of CS drugs against drug-resistant cancer cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Evidence for dual mode of action of a thiosemicarbazone, NSC73306: A potent substrate of the multidrug resistance-linked ABCG2 transporter

    PubMed Central

    Wu, Chung-Pu; Shukla, Suneet; Calcagno, Anna Maria; Hall, Matthew D.; Gottesman, Michael M.; Ambudkar, Suresh V.

    2008-01-01

    Multidrug resistance due to reduced drug accumulation is a phenomenon predominantly caused by the overexpression of members of the ATP-binding cassette transporters, including ABCB1 (P-glycoprotein), ABCG2 and several ABCC family members (MRPs). We previously reported that a thiosemicarbazone derivative, NSC73306, is cytotoxic to carcinoma cells that overexpress functional P-glycoprotein and it re-sensitizes these cells to chemotherapeutics. In this study, we investigated the effect of NSC73306 on cells overexpressing other ABC drug transporters, including ABCG2, MRP1, MRP4 and MRP5. Our findings demonstrated that NSC73306 is not more toxic to cells that overexpress these transporters compared to their respective parental cells, and these transporters do not confer resistance to NSC73306 either. In spite of this, we observed that NSC73306 is a transport substrate for ABCG2 that can effectively inhibit ABCG2-mediated drug transport and reverse resistance to both mitoxantrone and topotecan in ABCG2-expressing cells. Interactions between NSC73306 and the ABCG2 drug-binding site(s) were confirmed by its stimulatory effect on ATPase activity (140–150 nM concentration required for 50% stimulation) and by inhibition of [125I]-Iodoarylazidoprazosin photolabeling (50% inhibition at 250–400 nM) of the substrate-binding site(s). Overall, NSC73306 appears to be a potent modulator of ABCG2 that does not interact with MRP1, MRP4 or MRP5. Collectively, these data suggest that NSC73306 can potentially be used, due to its dual mode of action, as an effective agent to overcome drug resistance by eliminating P-glycoprotein-overexpressing cells, and by acting as a potent modulator that re-sensitizes ABCG2-expressing cancer cells to chemotherapeutics. PMID:18089722

  5. The calcium channel blockers, 1,4-dihydropyridines, are substrates of the multidrug resistance-linked ABC drug transporter, ABCG2.

    PubMed

    Shukla, Suneet; Robey, Robert W; Bates, Susan E; Ambudkar, Suresh V

    2006-07-25

    The human ATP-binding cassette transporter, ABCG2, confers resistance to multiple chemotherapeutic agents and also affects the bioavailability of different drugs. [(125)I]Iodoarylazidoprazosin (IAAP) and [(3)H]azidopine were used for photoaffinity labeling of ABCG2 in this study. We show here for the first time that both of these photoaffinity analogues are transport substrates for ABCG2 and that [(3)H]azidopine can also be used to photolabel both wild-type R482-ABCG2 and mutant T482-ABCG2. We further used these assays to screen for potential substrates or modulators of ABCG2 and observed that 1,4-dihydropyridines such as nicardipine and nifedipine, which are clinically used as antihypertensive agents, inhibited the photolabeling of ABCG2 with [(125)I]IAAP and [(3)H]azidopine as well as the transport of these photoaffinity analogues by ABCG2. Furthermore, [(3)H]nitrendipine and bodipy-Fl-dihydropyridine accumulation assays showed that these compounds are transported by ABCG2. These dihydropyridines also inhibited the efflux of the known ABCG2 substrates, mitoxantrone and pheophorbide-a, from ABCG2-overexpressing cells, and nicardipine was more potent in inhibiting this transport. Both nicardipine and nifedipine stimulated the ATPase activity of ABCG2, and the nifedipine-stimulated activity was inhibited by fumitremorgin C, suggesting that these agents might interact at the same site on the transporter. In addition, nontoxic concentrations of dihydropyridines increased the sensitivity of ABCG2-expressing cells to mitoxantrone by 3-5-fold. In aggregate, results from the photoaffinity labeling and efflux assays using [(125)I]IAAP and [(3)H]azidopine demonstrate that 1,4-dihydropyridines are substrates of ABCG2 and that these photolabels can be used to screen new substrates and/or inhibitors of this transporter.

  6. Chemotherapeutic drug-induced ABCG2 promoter demethylation as a novel mechanism of acquired multidrug resistance.

    PubMed

    Bram, Eran E; Stark, Michal; Raz, Shachar; Assaraf, Yehuda G

    2009-12-01

    ABCG2 is an efflux transporter conferring multidrug resistance (MDR) on cancer cells. However, the initial molecular events leading to its up-regulation in MDR tumor cells are poorly understood. Herein, we explored the impact of drug treatment on the methylation status of the ABCG2 promoter and consequent reactivation of ABCG2 gene expression in parental tumor cell lines and their MDR sublines. We demonstrate that ABCG2 promoter methylation is common in T-cell acute lymphoblastic leukemia (T-ALL) lines, also present in primary T-ALL lymphoblast specimens. Furthermore, drug selection with sulfasalazine and topotecan induced a complete demethylation of the ABCG2 promoter in the T-ALL and ovarian carcinoma model cell lines CCRF-CEM and IGROV1, respectively. This resulted in a dramatic induction of ABCG2 messenger RNA levels (235- and 743-fold, respectively) and consequent acquisition of an ABCG2-dependent MDR phenotype. Quantitative genomic polymerase chain reaction and ABCG2 promoter-luciferase reporter assay did not reveal ABCG2 gene amplification or differential transcriptional trans-activation, which could account for ABCG2 up-regulation in these MDR cells. Remarkably, mimicking cytotoxic bolus drug treatment through 12- to 24-hour pulse exposure of ABCG2-silenced leukemia cells, to clinically relevant concentrations of the chemotherapeutic agents daunorubicin and mitoxantrone, resulted in a marked transcriptional up-regulation of ABCG2. Our findings establish that antitumor drug-induced epigenetic reactivation of ABCG2 gene expression in cancer cells is an early molecular event leading to MDR. These findings have important implications for the emergence, clonal selection, and expansion of malignant cells with the MDR phenotype during chemotherapy.

  7. High-affinity interaction of tyrosine kinase inhibitors with the ABCG2 multidrug transporter.

    PubMed

    Ozvegy-Laczka, Csilla; Hegedus, Tamás; Várady, György; Ujhelly, Olga; Schuetz, John D; Váradi, András; Kéri, György; Orfi, László; Német, Katalin; Sarkadi, Balázs

    2004-06-01

    Tyrosine kinase inhibitors (TKIs) are promising new agents for specific inhibition of malignant cell growth and metastasis formation. Because most of the TKIs have to reach an intracellular target, specific membrane transporters may significantly modulate their effectiveness. In addition, the hydrophobic TKIs may interact with so-called multidrug transporters and thus alter the cellular distribution of unrelated pharmacological agents. In the present work, we show that certain TKIs, already in the clinical phase of drug development, directly interact with the ABCG2 multidrug transporter protein with a high affinity. We found that in several in vitro assay systems, STI-571 (Gleevec; imatinib mesylate), ZD1839 (Iressa; gefitinib), and N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (EKI-785) interacted with ABCG2 at submicromolar concentrations, whereas other multidrug transporters, human multidrug resistance protein (P-glycoprotein, ABCB1) and human multidrug resistance protein 1 (ABCC1), showed much lower reactivity toward these agents. Low concentrations of the TKIs examined selectively modulated ABCG2-ATPase activity, inhibited ABCG2-dependent active drug extrusion, and significantly affected drug resistance patterns in cells expressing ABCG2. Our results indicate that multidrug resistance protein modulation by TKIs may be an important factor in the clinical treatment of cancer patients. These data also raise the possibility that an extrusion of TKIs by multidrug transporters, e.g., ABCG2, may be involved in tumor cell TKI resistance.

  8. Membrane cholesterol selectively modulates the activity of the human ABCG2 multidrug transporter.

    PubMed

    Telbisz, Agnes; Müller, Marianna; Ozvegy-Laczka, Csilla; Homolya, László; Szente, Lajos; Váradi, András; Sarkadi, Balázs

    2007-11-01

    The human ABCG2 multidrug transporter provides protection against numerous toxic compounds and causes multidrug resistance in cancer. Here we examined the effects of changes in membrane cholesterol on the function of this protein. Human ABCG2 was expressed in mammalian and in Sf9 insect cells, and membrane cholesterol depletion or enrichment was achieved by preincubation with beta cyclodextrin or its cholesterol-loaded form. We found that mild cholesterol depletion of intact mammalian cells inhibited ABCG2-dependent dye and drug extrusion in a reversible fashion, while the membrane localization of the transporter protein was unchanged. Cholesterol enrichment of cholesterol-poor Sf9 cell membrane vesicles greatly increased ABCG2-driven substrate uptake, substrate-stimulated ATPase activity, as well as the formation of a catalytic cycle intermediate (nucleotide trapping). Interestingly, modulation of membrane cholesterol did not significantly affect the function of the R482G or R482T substrate mutant ABCG2 variants, or that of the MDR1 transporter. The selective, major effect of membrane cholesterol on the wild-type ABCG2 suggests a regulation of the activity of this multidrug transporter during processing or in membrane micro-domain interactions. The experimental recognition of physiological and pharmacological substrates of ABCG2, as well as the fight against cancer multidrug resistance may be facilitated by demonstrating the key role of membrane cholesterol in this transport activity.

  9. OSI-930 analogues as novel reversal agents for ABCG2-mediated multidrug resistance.

    PubMed

    Kuang, Ye-Hong; Patel, Jay P; Sodani, Kamlesh; Wu, Chung-Pu; Liao, Li-Qiu; Patel, Atish; Tiwari, Amit K; Dai, Chun-Ling; Chen, Xiang; Fu, Li-Wu; Ambudkar, Suresh V; Korlipara, Vijaya L; Chen, Zhe-Sheng

    2012-09-15

    OSI-930, a dual c-Kit and KDR tyrosine kinase inhibitor, is reported to have undergone a Phase I dose escalation study in patients with advanced solid tumors. A series of fifteen pyridyl and phenyl analogues of OSI-930 were designed and synthesized. Extensive screening of these compounds led to the discovery that nitropyridyl and ortho-nitrophenyl analogues, VKJP1 and VKJP3, were effective in reversing ABC subfamily G member 2 (ABCG2) transporter-mediated multidrug resistance (MDR). VKJP1 and VKJP3 significantly sensitized ABCG2-expressing cells to established substrates of ABCG2 including mitoxantrone, SN-38, and doxorubicin in a concentration-dependent manner, but not to the non-ABCG2 substrate cisplatin. However, they were unable to reverse ABCB1- or ABCC1-mediated MDR indicating their selectivity for ABCG2. Western blotting analysis was performed to evaluate ABCG2 expression and it was found that neither VKJP1 nor VKJP3 significantly altered ABCG2 protein expression for up to 72 h. [(3)H]-mitoxantrone accumulation study demonstrated that VKJP1 and VKJP3 increased the intracellular accumulation of [(3)H]-mitoxantrone, a substrate of ABCG2. VKJP1 and VKJP3 also remarkably inhibited the transport of [(3)H]-methotrexate by ABCG2 membrane vesicles. Importantly, both VKJP1 and VKJP3 were efficacious in stimulating the activity of ATPase of ABCG2 and inhibited the photoaffinity labeling of this transporter by its substrate [(125)I]-iodoarylazidoprazosin. The results suggested that VKJP1 and VKJP3, specifically inhibit the function of ABCG2 through direct interaction with its substrate binding site(s). Thus VKJP1 and VKJP3 represent a new class of drugs for reducing MDR in ABCG2 over-expressing tumors.

  10. High level functional expression of the ABCG2 multidrug transporter in undifferentiated human embryonic stem cells.

    PubMed

    Apáti, Agota; Orbán, Tamás I; Varga, Nóra; Németh, Andrea; Schamberger, Anita; Krizsik, Virág; Erdélyi-Belle, Boglárka; Homolya, László; Várady, György; Padányi, Rita; Karászi, Eva; Kemna, Evelien W M; Német, Katalin; Sarkadi, Balázs

    2008-12-01

    Expression of multidrug resistance ABC transporters has been suggested as a functional marker and chemoprotective element in early human progenitor cell types. In this study we examined the expression and function of the key multidrug-ABC transporters, ABCB1, ABCC1 and ABCG2 in two human embryonic stem (HuES) cell lines. We detected a high level ABCG2 expression in the undifferentiated HuES cells, while the expression of this protein significantly decreased during early cell differentiation. ABCG2 in HuES cells provided protection against mitoxantrone toxicity, with a drug-stimulated overexpression of the transporter. No significant expression of ABCB1/ABCC1 was found either in the undifferentiated or partially differentiated HuES cells. Examination of the ABCG2 mRNA in HuES cells indicated the use of selected promoter sites and a truncated 3' untranslated region, suggesting a functionally distinct regulation of this transporter in undifferentiated stem cells. The selective expression of the ABCG2 multidrug transporter indicates that ABCG2 can be applied as a marker for undifferentiated HuES cells. Moreover, protection of embryonic stem cells against xenobiotics and endobiotics may depend on ABCG2 expression and regulation.

  11. Xanthines Down-Regulate the Drug Transporter ABCG2 and Reverse Multidrug Resistance

    PubMed Central

    Ding, Rui; Shi, Jia; Pabon, Kirk

    2012-01-01

    ABCG2 is an ATP-binding-cassette (ABC) transporter that confers multidrug resistance (MDR) to tumor cells by extruding a broad variety of chemotherapeutic agents, ultimately leading to failure of cancer therapy. Thus, the down-regulation of ABCG2 expression and/or function has been proposed as part of a regimen to improve cancer therapeutic efficacy. In this study, we found that a group of xanthines including caffeine, theophylline, and dyphylline can dramatically decrease ABCG2 protein in cells that have either moderate (BeWo, a placental choriocarcinoma cell line) or high (MCF-7/MX100, a breast cancer drug-resistant cell subline) levels of ABCG2 expression. This down-regulation is time-dependent, dose-dependent, and reversible. Using lysosomal inhibitors, we found that xanthines decreased ABCG2 by inducing its rapid internalization and lysosome-mediated degradation. As a consequence, caffeine treatment significantly increased the retention of an established ABCG2 substrate in MCF-7/MX100 cells but not in parental MCF-7 cells and sensitized the MDR cells to the chemotherapeutic agent mitoxantrone (MX); combination treatment with MX and caffeine decreased the IC50 of MX ∼10-fold and induced a greater degree of apoptotic cell death than MX treatment alone. Taken together, our results describe a novel function for this large class of therapeutically relevant compounds and suggest that a subset of xanthines could be developed as combination therapy to improve the efficacy of anticancer drugs that are ABCG2 substrates. PMID:22113078

  12. Ins and outs of the ABCG2 multidrug transporter: an update on in vitro functional assays.

    PubMed

    Hegedus, Csilla; Szakács, Gergely; Homolya, László; Orbán, Tamás I; Telbisz, Agnes; Jani, Márton; Sarkadi, Balázs

    2009-01-31

    The major aim of this chapter is to provide a critical overview of the in vitro methods available for studying the function of the ABCG2 multidrug transporter protein. When describing the most applicable assay systems, in each case we present a short overview relevant to ABC multidrug transporters in general, and then we concentrate on the tools applicable to analysis of substrate-drug interactions, the effects of potential activators and inhibitors, and the role of polymorphisms of the ABCG2 transporter. Throughout this chapter we focus on recently developed assay systems, which may provide new possibilities for analyzing the pharmacological aspects of this medically important protein.

  13. Lipid regulation of the ABCB1 and ABCG2 multidrug transporters.

    PubMed

    Hegedüs, Csilla; Telbisz, Ágnes; Hegedűs, Tamás; Sarkadi, Balázs; Özvegy-Laczka, Csilla

    2015-01-01

    This chapter deals with the interactions of two medically important multidrug ABC transporters (MDR-ABC), ABCB1 and ABCG2, with lipid molecules. Both ABCB1 and ABCG2 are capable of transporting a wide range of hydrophobic drugs and xenobiotics and are involved in cancer chemotherapy resistance. Therefore, the exploration of their mechanism of action has major therapeutic consequences. As discussed here in detail, both ABCB1 and ABCG2 are significantly affected by various lipid compounds especially those residing in their close proximity in the plasma membrane. ABCB1 is capable of transporting lipids and lipid derivatives, and thus may alter the general membrane composition by "flopping" membrane lipid constituents, while there is no such information regarding ABCG2. Still, both ABCB1 and ABCG2 show complex interactions with a variety of lipid molecules, and the transporters are significantly modulated by cholesterol and cholesterol derivatives at the posttranslational level. In this chapter, we explore the molecular details of the direct transporter-lipid interactions, the potential role of lipid-sensor domains within the proteins, as well as the application of experimental site-directed mutagenesis, detailed structural studies, and in silico modeling for examining these interactions. We also discuss the regulation of ABCB1 and ABCG2 expression at the transcriptional level, occurring through nuclear receptors involved in lipid sensing. The better understanding of lipid interactions with these medically important MDR-ABC transporters may significantly improve further drug development and clinical treatment options.

  14. Targeting the ABCG2-overexpressing multidrug resistant (MDR) cancer cells by PPARγ agonists

    PubMed Central

    To, Kenneth K W; Tomlinson, Brian

    2013-01-01

    Background and Purpose Multidrug resistance (MDR), usually mediated by overexpression of efflux transporters such as P-gp, ABCG2 and/or MRP1, remains a major obstacle hindering successful cancer chemotherapy. There has been great interest in the development of inhibitors towards these transporters to circumvent resistance. However, since the inhibition of transporter is not specific to cancer cells, a decrease in the cytotoxic drug dosing may be needed to prevent excess toxicity, thus undermining the potential benefit brought about by a drug efflux inhibitor. The design of potent MDR modulators specific towards resistant cancer cells and devoid of drug-drug interactions will be needed to effect MDR reversal. Experimental Approach Recent evidence suggests that the PTEN/PI3K/Akt pathway may be exploited to alter ABCG2 subcellular localization, thereby circumventing MDR. Three PPARγ agonists (telmisartan, pioglitazone and rosiglitazone) that have been used in the clinics were tested for their effect on the PTEN/PI3K/Akt pathway and possible reversal of ABCG2-mediated drug resistance. Key Results The PPARγ agonists were found to be weak ABCG2 inhibitors by drug efflux assay. They were also shown to elevate the reduced PTEN expression in a resistant and ABCG2-overexpressing cell model, which inhibit the PI3K-Akt pathway and lead to the relocalization of ABCG2 from the plasma membrane to the cytoplasma, thus apparently circumventing the ABCG2-mediated MDR. Conclusions and Implications Since this PPARγ/PTEN/PI3K/Akt pathway regulating ABCG2 is only functional in drug-resistant cancer cells with PTEN loss, the PPARγ agonists identified may represent promising agents targeting resistant cells for MDR reversal. PMID:24032744

  15. Mitoxantrone is expelled by the ABCG2 multidrug transporter directly from the plasma membrane.

    PubMed

    Homolya, László; Orbán, Tamás I; Csanády, László; Sarkadi, Balázs

    2011-01-01

    ABC multidrug transporter proteins expel a wide variety of structurally unrelated, mostly hydrophobic compounds from cells. The special role of these transporters both at the physiological barriers and in cancer cells is based on their extremely broad substrate recognition. Since hydrophobic compounds are known to partition into the lipid bilayer and accumulate in membranes, the "classical pump" model for the mechanism of multidrug transporter proteins has been challenged, and alternative models suggesting substrate recognition within the lipid bilayer have been proposed. Although much effort has been made to validate this concept, unambiguous evidence for direct drug extrusion from the plasma membrane has not been provided yet. Here we show a detailed on-line microscopic analysis of cellular extrusion of fluorescent anti-cancer drugs, mitoxantrone and pheophorbide A, by a key human multidrug transporter, ABCG2. Using the fully active GFP-tagged ABCG2 and exploiting the special character of mitoxantrone that gains fluorescence in the lipid environment, we were able to determine transporter-modulated drug concentrations separately in the plasma membrane and the intracellular compartments. Different kinetic models describing the various transport mechanisms were generated and the experimental data were analyzed using these models. On the basis of the kinetic analysis, drug extrusion from the cytoplasm can be excluded, thus, our results indicate that ABCG2 extrudes mitoxantrone directly from the plasma membrane.

  16. Constitutive AhR activation leads to concomitant ABCG2-mediated multidrug resistance in cisplatin-resistant esophageal carcinoma cells.

    PubMed

    To, Kenneth K W; Yu, Le; Liu, Shuwen; Fu, Jianhua; Cho, Chi Hin

    2012-06-01

    Esophageal squamous cell carcinoma (ESCC) is a highly malignant disease that is generally not responding to chemotherapy. It is particularly predominant in China. Although ESCC is significantly associated with cigarette smoking, the relationship between its molecular pathogenesis and responsiveness to chemotherapy and cigarette smoke remains elusive. This study reported the constitutive activation of aryl hydrocarbon receptor (AhR), leading to ABCG2 upregulation and the multidrug resistance (MDR) phenotype, in ESCC cell lines with acquired cisplatin resistance. Reporter gene assay, chromatin immunoprecipitation analysis and specific gene knockdown confirmed that the enhanced AhR binding to a xenobiotic response element (XRE) within the ABCG2 promoter is responsible for ABCG2 overexpression. A HSP90 inhibitor (17-AAG) and two AhR antagonists (kaempferol and salicylamide) were shown to inhibit ABCG2 upregulation, thereby reversing the ABCG2-mediated MDR. Our data therefore advocate the use of these inhibitors as novel chemosensitizers for the treatment of esophageal cancer.

  17. PI3-kinase and mTOR inhibitors differently modulate the function of the ABCG2 multidrug transporter.

    PubMed

    Hegedüs, Csilla; Truta-Feles, Krisztina; Antalffy, Géza; Brózik, Anna; Kasza, Ildikó; Német, Katalin; Orbán, Tamás I; Özvegy-Laczka, Csilla; Váradi, András; Sarkadi, Balázs

    2012-04-20

    The ATP-binding cassette (ABC) transporter ABCG2 plays an important role in tissue detoxification and confers multidrug resistance to cancer cells. Identification of expressional and functional cellular regulators of this multidrug transporter is therefore intensively pursued. The PI3-kinase/Akt signaling axis has been implicated as a key element in regulating various cellular functions, including the expression and plasma membrane localization of ABCG2. Here we demonstrate that besides inhibiting their respective target kinases, the pharmacological PI3-kinase inhibitor LY294002 and the downstream mTOR kinase inhibitor rapamycin also directly inhibit ABCG2 function. In contrast, wortmannin, another commonly used pharmacological inhibitor of PI3-kinase does not interact with the transporter. We suggest that direct functional modulation of ABCG2 should be taken into consideration when pharmacological agents are applied to dissect the specific role of PI3-kinase/Akt/mTOR signaling in cellular functions.

  18. Flavonoids from Eight Tropical Plant Species That Inhibit the Multidrug Resistance Transporter ABCG2

    PubMed Central

    Versiani, Muhammad Ali; Diyabalanage, Thushara; Ratnayake, Ranjala; Henrich, Curtis J.; Bates, Susan E.; McMahon, James B.; Gustafson, Kirk R.

    2013-01-01

    Overexpression of ABCG2, a membrane-bound multidrug transporter, can make tumor cells resistant to treatment with conventional chemotherapeutic agents. A high-throughput screening effort with the NCI repository of natural product extracts revealed that eight tropical plant extracts significantly inhibited the function of ABCG2. This activity was tracked throughout the extract fractionation process to a series of ABCG2 inhibitory flavonoids (1–13). Their structures were identified by a combination of NMR, mass spectrometry, and circular dichroism studies, and this resulted in the elucidation of (2S)-5,7,3′-trihydroxy-4′-methoxy-8-(3″-methylbut-2″-enyl)-flavonone (1), (2S)-5,7,3′,5′-tetrahydroxy-8-[3″,8″ -dimethylocta-2″(E),7″-dienyl]flavonone (3), and 5,7,3′-trihydroxy-3,5′-dimethoxy-2′-(3′-methylbut-2-enyl)flavone (12) as new compounds. PMID:21275386

  19. Alectinib (CH5424802) antagonizes ABCB1- and ABCG2-mediated multidrug resistance in vitro, in vivo and ex vivo

    PubMed Central

    Yang, Ke; Chen, Yifan; To, Kenneth Kin Wah; Wang, Fang; Li, Delan; Chen, Likun; Fu, Liwu

    2017-01-01

    Alectinib, an inhibitor of anaplastic lymphoma kinase (ALK), was approved by the Food and Drug Administration (FDA) for the treatment of patients with ALK-positive non-small cell lung cancer (NSCLC). Here we investigated the reversal effect of alectinib on multidrug resistance (MDR) induced by ATP-binding cassette (ABC) transporters, which is the primary cause of chemotherapy failure. We provide the first evidence that alectinib increases the sensitivity of ABCB1- and ABCG2-overexpressing cells to chemotherapeutic agents in vitro and in vivo. Mechanistically, alectinib increased the intracellular accumulation of ABCB1/ABCG2 substrates such as doxorubicin (DOX) and Rhodamine 123 (Rho 123) by inhibiting the efflux function of the transporters in ABCB1- or ABCG2-overexpressing cells but not in their parental sensitive cells. Furthermore, alectinib stimulated ATPase activity and competed with substrates of ABCB1 or ABCG2 and competed with [125I] iodoarylazidoprazosin (IAAP) photolabeling bound to ABCB1 or ABCG2 but neither altered the expression and localization of ABCB1 or ABCG2 nor the phosphorylation levels of AKT and ERK. Alectinib also enhanced the cytotoxicity of DOX and the intracellular accumulation of Rho 123 in ABCB1-overexpressing primary leukemia cells. These findings suggest that alectinib combined with traditional chemotherapy may be beneficial to patients with ABCB1- or ABCG2-mediated MDR. PMID:28303028

  20. Overcoming Multidrug Resistance via Photodestruction of ABCG2-Rich Extracellular Vesicles Sequestering Photosensitive Chemotherapeutics

    PubMed Central

    Goler-Baron, Vicky; Assaraf, Yehuda G.

    2012-01-01

    Multidrug resistance (MDR) remains a dominant impediment to curative cancer chemotherapy. Efflux transporters of the ATP-binding cassette (ABC) superfamily including ABCG2, ABCB1 and ABCC1 mediate MDR to multiple structurally and functionally distinct antitumor agents. Recently we identified a novel mechanism of MDR in which ABCG2-rich extracellular vesicles (EVs) form in between attached neighbor breast cancer cells and highly concentrate various chemotherapeutics in an ABCG2-dependent manner, thereby sequestering them away from their intracellular targets. Hence, development of novel strategies to overcome MDR modalities is a major goal of cancer research. Towards this end, we here developed a novel approach to selectively target and kill MDR cancer cells. We show that illumination of EVs that accumulated photosensitive cytotoxic drugs including imidazoacridinones (IAs) and topotecan resulted in intravesicular formation of reactive oxygen species (ROS) and severe damage to the EVs membrane that is shared by EVs-forming cells, thereby leading to tumor cell lysis and the overcoming of MDR. Furthermore, consistent with the weak base nature of IAs, MDR cells that are devoid of EVs but contained an increased number of lysosomes, highly accumulated IAs in lysosomes and upon photosensitization were efficiently killed via ROS-dependent lysosomal rupture. Combining targeted lysis of IAs-loaded EVs and lysosomes elicited a synergistic cytotoxic effect resulting in MDR reversal. In contrast, topotecan, a bona fide transport substrate of ABCG2, accumulated exclusively in EVs of MDR cells but was neither detected in lysosomes of normal breast epithelial cells nor in non-MDR breast cancer cells. This exclusive accumulation in EVs enhanced the selectivity of the cytotoxic effect exerted by photodynamic therapy to MDR cells without harming normal cells. Moreover, lysosomal alkalinization with bafilomycin A1 abrogated lysosomal accumulation of IAs, consequently preventing

  1. Vandetanib (Zactima, ZD6474) Antagonizes ABCC1- and ABCG2-Mediated Multidrug Resistance by Inhibition of Their Transport Function

    PubMed Central

    Zheng, Li-sheng; Wang, Fang; Li, Yu-hong; Zhang, Xu; Chen, Li-ming; Liang, Yong-ju; Dai, Chun-ling; Yan, Yan-yan; Tao, Li-yang; Mi, Yan-jun; Yang, An-kui; To, Kenneth Kin Wah; Fu, Li-wu

    2009-01-01

    Background ABCC1 and ABCG2 are ubiquitous ATP-binding cassette transmembrane proteins that play an important role in multidrug resistance (MDR). In this study, we evaluated the possible interaction of vandetanib, an orally administered drug inhibiting multiple receptor tyrosine kinases, with ABCC1 and ABCG2 in vitro. Methodology and Principal Findings MDR cancer cells overexpressing ABCC1 or ABCG2 and their sensitive parental cell lines were used. MTT assay showed that vandetanib had moderate and almost equal-potent anti-proliferative activity in both sensitive parental and MDR cancer cells. Concomitant treatment of MDR cells with vandetanib and specific inhibitors of ABCC1 or ABCG2 did not alter their sensitivity to the former drug. On the other hand, clinically attainable but non-toxic doses of vandetanib were found to significantly enhance the sensitivity of MDR cancer cells to ABCC1 or ABCG2 substrate antitumor drugs. Flow cytometric analysis showed that vandetanib treatment significantly increase the intracellular accumulation of doxorubicin and rhodamine 123, substrates of ABCC1 and ABCG2 respectively, in a dose-dependent manner (P<0.05). However, no significant effect was shown in sensitive parental cell lines. Reverse transcription-PCR and Western blot analysis showed that vandetanib did not change the expression of ABCC1 and ABCG2 at both mRNA and protein levels. Furthermore, total and phosphorylated forms of AKT and ERK1/2 remained unchanged after vandetanib treatment in both sensitive and MDR cancer cells. Conclusions Vandetanib is unlikely to be a substrate of ABCC1 or ABCG2. It overcomes ABCC1- and ABCG2-mediated drug resistance by inhibiting the transporter activity, independent of the blockade of AKT and ERK1/2 signal transduction pathways. PMID:19390592

  2. Structure and Function of ABCG2-Rich Extracellular Vesicles Mediating Multidrug Resistance

    PubMed Central

    Goler-Baron, Vicky; Assaraf, Yehuda G.

    2011-01-01

    Multidrug resistance (MDR) is a major impediment to curative cancer chemotherapy. The ATP-Binding Cassette transporters ABCG2, ABCB1 and ABCC2 form a unique defense network against multiple structurally and functionally distinct chemotherapeutics, thereby resulting in MDR. Thus, deciphering novel mechanisms of MDR and their overcoming is a major goal of cancer research. Recently we have shown that overexpression of ABCG2 in the membrane of novel extracellular vesicles (EVs) in breast cancer cells results in mitoxantrone resistance due to its dramatic sequestration in EVs. However, nothing is known about EVs structure, biogenesis and their ability to concentrate multiple antitumor agents. To this end, we here found that EVs are structural and functional homologues of bile canaliculi, are apically localized, sealed structures reinforced by an actin-based cytoskeleton and secluded from the extracellular milieu by the tight junction proteins occludin and ZO-1. Apart from ABCG2, ABCB1 and ABCC2 were also selectively targeted to the membrane of EVs. Moreover, Ezrin-Radixin-Moesin protein complex selectively localized to the border of the EVs membrane, suggesting a key role for the tethering of MDR pumps to the actin cytoskeleton. The ability of EVs to concentrate and sequester different antitumor drugs was also explored. Taking advantage of the endogenous fluorescence of anticancer drugs, we found that EVs-forming breast cancer cells display high level resistance to topotecan, imidazoacridinones and methotrexate via efficient intravesicular drug concentration hence sequestering them away from their cellular targets. Thus, we identified a new modality of anticancer drug compartmentalization and resistance in which multiple chemotherapeutics are actively pumped from the cytoplasm and highly concentrated within the lumen of EVs via a network of MDR transporters differentially targeted to the EVs membrane. We propose a composite model for the structure and function of MDR

  3. Antibody binding shift assay for rapid screening of drug interactions with the human ABCG2 multidrug transporter.

    PubMed

    Telbisz, Ágnes; Hegedüs, Csilla; Özvegy-Laczka, Csilla; Goda, Katalin; Várady, György; Takáts, Zoltán; Szabó, Eszter; Sorrentino, Brian P; Váradi, András; Sarkadi, Balázs

    2012-01-23

    The ABCG2 multidrug transporter protein has been identified as a key player in cancer drug resistance and xenobiotic elimination, as its actively transported substrates include anticancer drugs, intermediates of heme metabolism, xenobiotics, and also drug conjugates. Several transported substrates at higher concentrations, and some anticancer agents even at low concentrations directly inhibit the ABCG2 transporter, thus it is difficult to provide estimation for pharmacologically important ABCG2-dependent interactions. In addition, as documented here, in mutant variants of the transporter, inhibitors of the wild-type ABCG2 may become actively transported substrates. In this paper we describe a rapid in vitro assay to identify transport modulation by measuring the cell surface interaction of a conformation sensitive monoclonal antibody (5D3) with ABCG2 in intact cells. As documented, in conjunction with membrane ATPase, transport and cytotoxicity measurements, this assay provides a reliable estimate of concentration-dependent modulation of ABCG2 by newly emerging pharmacophores. A high-throughput, 96-well plate assay platform is also provided.

  4. Bafetinib (INNO-406) reverses multidrug resistance by inhibiting the efflux function of ABCB1 and ABCG2 transporters

    NASA Astrophysics Data System (ADS)

    Zhang, Yun-Kai; Zhang, Guan-Nan; Wang, Yi-Jun; Patel, Bhargav A.; Talele, Tanaji T.; Yang, Dong-Hua; Chen, Zhe-Sheng

    2016-05-01

    ATP-Binding Cassette transporters are involved in the efflux of xenobiotic compounds and are responsible for decreasing drug accumulation in multidrug resistant (MDR) cells. Discovered by structure-based virtual screening algorithms, bafetinib, a Bcr-Abl/Lyn tyrosine kinase inhibitor, was found to have inhibitory effects on both ABCB1- and ABCG2-mediated MDR in this in-vitro investigation. Bafetinib significantly sensitized ABCB1 and ABCG2 overexpressing MDR cells to their anticancer substrates and increased the intracellular accumulation of anticancer drugs, particularly doxorubicin and [3H]-paclitaxel in ABCB1 overexpressing cells; mitoxantrone and [3H]-mitoxantrone in ABCG2 overexpressing cells, respectively. Bafetinib stimulated ABCB1 ATPase activities while inhibited ABCG2 ATPase activities. There were no significant changes in the expression level or the subcellular distribution of ABCB1 and ABCG2 in the cells exposed to 3 μM of bafetinib. Overall, our study indicated that bafetinib reversed ABCB1- and ABCG2-mediated MDR by blocking the drug efflux function of these transporters. These findings might be useful in developing combination therapy for MDR cancer treatment.

  5. Interaction of the EGFR inhibitors gefitinib, vandetanib, pelitinib and neratinib with the ABCG2 multidrug transporter: implications for the emergence and reversal of cancer drug resistance.

    PubMed

    Hegedüs, Csilla; Truta-Feles, Krisztina; Antalffy, Géza; Várady, György; Német, Katalin; Ozvegy-Laczka, Csilla; Kéri, György; Orfi, László; Szakács, Gergely; Settleman, Jeffrey; Váradi, András; Sarkadi, Balázs

    2012-08-01

    Human ABCG2 is a plasma membrane glycoprotein that provides physiological protection against xenobiotics. ABCG2 also significantly influences biodistribution of drugs through pharmacological tissue barriers and confers multidrug resistance to cancer cells. Moreover, ABCG2 is the molecular determinant of the side population that is characteristically enriched in normal and cancer stem cells. Numerous tumors depend on unregulated EGFR signaling, thus inhibition of this receptor by small molecular weight inhibitors such as gefitinib, and the novel second generation agents vandetanib, pelitinib and neratinib, is a promising therapeutic option. In the present study, we provide detailed biochemical characterization regarding the interaction of these EGFR inhibitors with ABCG2. We show that ABCG2 confers resistance to gefitinib and pelitinib, whereas the intracellular action of vandetanib and neratinib is unaltered by the presence of the transporter. At higher concentrations, however, all these EGFR inhibitors inhibit ABCG2 function, thereby promoting accumulation of ABCG2 substrate drugs. We also report enhanced expression of ABCG2 in gefitinib-resistant non-small cell lung cancer cells, suggesting potential clinical relevance of ABCG2 in acquired drug resistance. Since ABCG2 has important impact on both the pharmacological properties and anti-cancer efficiencies of drugs, our results regarding the novel EGFR inhibitors should provide useful information about their therapeutic applicability against ABCG2-expressing cancer cells depending on EGFR signaling. In addition, the finding that these EGFR inhibitors efficiently block ABCG2 function may help to design novel drug-combination therapeutic strategies.

  6. The role of the human ABCG2 multidrug transporter and its variants in cancer therapy and toxicology.

    PubMed

    Cervenak, Judit; Andrikovics, Hajnalka; Ozvegy-Laczka, Csilla; Tordai, Attila; Német, Katalin; Váradi, András; Sarkadi, Balázs

    2006-03-08

    The human multidrug resistance ABC transporters provide a protective function in our body against a large number of toxic compounds. These proteins, residing in the plasma membrane, perform an active, ATP-dependent extrusion of such xenobiotics. However, the same proteins are also used by the tumor cells to fight various anticancer agents. ABCG2 is an important member of the multidrug resistance proteins, an 'ABC half transporter', which functions as a homodimer in the cell membrane. In this review, we provide a basic overview of ABCG2 function in physiology and drug metabolism, but concentrate on the discussion of mutations and polymorphisms discovered in this protein. Interestingly, a single nucleotide mutation, changing amino acid 482 from arginine to threonine or glycine in ABCG2, results in a major increase in the catalytic activity and a wider drug recognition by this protein. Still, this mutation proved to be an in vitro artifact, produced only in heavily drug-selected cell lines. In contrast, at least two, but possibly more polymorphic variants of ABCG2 were found to be present in large human populations with different ethnic background. However, currently available experimental data regarding the cellular expression, localization and function of these ABCG2 variants are strongly contradictory. Since, the proteins produced by these variant alleles may differently modulate cancer treatment, general drug absorption and toxicity, may represent risk factors in fetal toxicity, or alter the differentiation of stem cells, their exact characterization is a major challenge in this field.

  7. Jump into a New Fold-A Homology Based Model for the ABCG2/BCRP Multidrug Transporter.

    PubMed

    László, Laura; Sarkadi, Balázs; Hegedűs, Tamás

    2016-01-01

    ABCG2/BCRP is a membrane protein, involved in xenobiotic and endobiotic transport in key pharmacological barriers and drug metabolizing organs, in the protection of stem cells, and in multidrug resistance of cancer. Pharmacogenetic studies implicated the role of ABCG2 in response to widely used medicines and anticancer agents, as well as in gout. Its Q141K variant exhibits decreased functional expression thus increased drug accumulation and decreased urate secretion. Still, there has been no reliable molecular model available for this protein, as the published structures of other ABC transporters could not be properly fitted to the ABCG2 topology and experimental data. The recently published high resolution structure of a close homologue, the ABCG5-ABCG8 heterodimer, revealed a new ABC transporter fold, unique for ABCG proteins. Here we present a structural model of the ABCG2 homodimer based on this fold and detail the experimental results supporting this model. In order to describe the effect of mutations on structure and dynamics, and characterize substrate recognition and cholesterol regulation we performed molecular dynamics simulations using full length ABCG2 protein embedded in a membrane bilayer and in silico docking simulations. Our results show that in the Q141K variant the introduced positive charge diminishes the interaction between the nucleotide binding and transmembrane domains and the R482G variation alters the orientation of transmembrane helices. Moreover, the R482 position, which plays a role the substrate specificity of the transporter, is located in one of the substrate binding pockets identified by the in silico docking calculations. In summary, the ABCG2 model and in silico simulations presented here may have significant impact on understanding drug distribution and toxicity, as well as drug development against cancer chemotherapy resistance or gout.

  8. Jump into a New Fold—A Homology Based Model for the ABCG2/BCRP Multidrug Transporter

    PubMed Central

    László, Laura; Sarkadi, Balázs

    2016-01-01

    ABCG2/BCRP is a membrane protein, involved in xenobiotic and endobiotic transport in key pharmacological barriers and drug metabolizing organs, in the protection of stem cells, and in multidrug resistance of cancer. Pharmacogenetic studies implicated the role of ABCG2 in response to widely used medicines and anticancer agents, as well as in gout. Its Q141K variant exhibits decreased functional expression thus increased drug accumulation and decreased urate secretion. Still, there has been no reliable molecular model available for this protein, as the published structures of other ABC transporters could not be properly fitted to the ABCG2 topology and experimental data. The recently published high resolution structure of a close homologue, the ABCG5-ABCG8 heterodimer, revealed a new ABC transporter fold, unique for ABCG proteins. Here we present a structural model of the ABCG2 homodimer based on this fold and detail the experimental results supporting this model. In order to describe the effect of mutations on structure and dynamics, and characterize substrate recognition and cholesterol regulation we performed molecular dynamics simulations using full length ABCG2 protein embedded in a membrane bilayer and in silico docking simulations. Our results show that in the Q141K variant the introduced positive charge diminishes the interaction between the nucleotide binding and transmembrane domains and the R482G variation alters the orientation of transmembrane helices. Moreover, the R482 position, which plays a role the substrate specificity of the transporter, is located in one of the substrate binding pockets identified by the in silico docking calculations. In summary, the ABCG2 model and in silico simulations presented here may have significant impact on understanding drug distribution and toxicity, as well as drug development against cancer chemotherapy resistance or gout. PMID:27741279

  9. Multidrug transporter ABCG2/breast cancer resistance protein secretes riboflavin (vitamin B2) into milk.

    PubMed

    van Herwaarden, Antonius E; Wagenaar, Els; Merino, Gracia; Jonker, Johan W; Rosing, Hilde; Beijnen, Jos H; Schinkel, Alfred H

    2007-02-01

    The multidrug transporter breast cancer resistance protein (BCRP/ABCG2) is strongly induced in the mammary gland during pregnancy and lactation. We here demonstrate that BCRP is responsible for pumping riboflavin (vitamin B(2)) into milk, thus supplying the young with this important nutrient. In Bcrp1(-/-) mice, milk secretion of riboflavin was reduced >60-fold compared to that in wild-type mice. Yet, under laboratory conditions, Bcrp1(-/-) pups showed no riboflavin deficiency due to concomitant milk secretion of its cofactor flavin adenine dinucleotide, which was not affected. Thus, two independent secretion mechanisms supply vitamin B(2) equivalents to milk. BCRP is the first active riboflavin efflux transporter identified in mammals and the first transporter shown to concentrate a vitamin into milk. BCRP activity elsewhere in the body protects against xenotoxins by reducing their absorption and mediating their excretion. Indeed, Bcrp1 activity increased excretion of riboflavin into the intestine and decreased its systemic availability in adult mice. Surprisingly, the paradoxical dual utilization of BCRP as a xenotoxin and a riboflavin pump is evolutionarily conserved among mammals as diverse as mice and humans. This study establishes the principle that an ABC transporter can transport a vitamin into milk and raises the possibility that other vitamins and nutrients are likewise secreted into milk by ABC transporters.

  10. Multidrug Transporter ABCG2/Breast Cancer Resistance Protein Secretes Riboflavin (Vitamin B2) into Milk▿

    PubMed Central

    van Herwaarden, Antonius E.; Wagenaar, Els; Merino, Gracia; Jonker, Johan W.; Rosing, Hilde; Beijnen, Jos H.; Schinkel, Alfred H.

    2007-01-01

    The multidrug transporter breast cancer resistance protein (BCRP/ABCG2) is strongly induced in the mammary gland during pregnancy and lactation. We here demonstrate that BCRP is responsible for pumping riboflavin (vitamin B2) into milk, thus supplying the young with this important nutrient. In Bcrp1−/− mice, milk secretion of riboflavin was reduced >60-fold compared to that in wild-type mice. Yet, under laboratory conditions, Bcrp1−/− pups showed no riboflavin deficiency due to concomitant milk secretion of its cofactor flavin adenine dinucleotide, which was not affected. Thus, two independent secretion mechanisms supply vitamin B2 equivalents to milk. BCRP is the first active riboflavin efflux transporter identified in mammals and the first transporter shown to concentrate a vitamin into milk. BCRP activity elsewhere in the body protects against xenotoxins by reducing their absorption and mediating their excretion. Indeed, Bcrp1 activity increased excretion of riboflavin into the intestine and decreased its systemic availability in adult mice. Surprisingly, the paradoxical dual utilization of BCRP as a xenotoxin and a riboflavin pump is evolutionarily conserved among mammals as diverse as mice and humans. This study establishes the principle that an ABC transporter can transport a vitamin into milk and raises the possibility that other vitamins and nutrients are likewise secreted into milk by ABC transporters. PMID:17145775

  11. Regulation of the Function of the Human ABCG2 Multidrug Transporter by Cholesterol and Bile Acids: Effects of Mutations in Potential Substrate and Steroid Binding Sites

    PubMed Central

    Telbisz, Ágnes; Hegedüs, Csilla; Váradi, András; Sarkadi, Balázs

    2014-01-01

    ABCG2 (ATP-binding cassette, subfamily G, member 2) is a plasma membrane glycoprotein that actively extrudes xenobiotics and endobiotics from the cells and causes multidrug resistance in cancer. In the liver, ABCG2 is expressed in the canalicular membrane of hepatocytes and excretes its substrates into the bile. ABCG2 is known to require high membrane cholesterol content for maximal activity, and by examining purified ABCG2 reconstituted in proteoliposomes we have recently shown that cholesterol is an essential activator, while bile acids significantly modify the activity of this protein. In the present work, by using isolated insect cell membrane preparations expressing human ABCG2 and its mutant variants, we have analyzed whether certain regions in this protein are involved in sterol recognition. We found that replacing ABCG2-R482 with large amino acids does not affect cholesterol dependence, but changes to small amino acids cause altered cholesterol sensitivity. When leucines in the potential steroid-binding element (SBE, aa 555–558) of ABCG2 were replaced by alanines, cholesterol dependence of ABCG2 activity was strongly reduced, although the L558A mutant variant when purified and reconstituted still required cholesterol for full activity. Regarding the effect of bile acids in isolated membranes, we found that these compounds decreased ABCG2-ATPase in the absence of drug substrates, which did not significantly affect substrate-stimulated ATPase activity. These ABCG2 mutant variants also altered bile acid sensitivity, although cholic acid and glycocholate were not transported by the protein. We suggest that the aforementioned two regions in ABCG2 are important for sterol sensing and may represent potential targets for pharmacologic modulation of ABCG2 function. PMID:24384916

  12. Function-dependent conformational changes of the ABCG2 multidrug transporter modify its interaction with a monoclonal antibody on the cell surface.

    PubMed

    Ozvegy-Laczka, Csilla; Várady, György; Köblös, Gabriella; Ujhelly, Olga; Cervenak, Judit; Schuetz, John D; Sorrentino, Brian P; Koomen, Gerrit-Jan; Váradi, András; Német, Katalin; Sarkadi, Balázs

    2005-02-11

    The human ABCG2 protein is an important primary active transporter for hydrophobic compounds in several cell types, and its overexpression causes multidrug resistance in tumors. A monoclonal antibody (5D3) recognizes this protein on the cell surface. In ABCG2-expressing cells 5D3 antibody showed a saturable labeling and inhibited ABCG2 transport and ATPase function. However, at low antibody concentrations 5D3 binding to intact cells depended on the actual conformation of the ABCG2 protein. ATP depletion or the addition of the ABCG2 inhibitor Ko143 significantly increased, whereas the vanadate-induced arrest of ABCG2 strongly decreased 5D3 binding. The binding of the 5D3 antibody to a non-functional ABCG2 catalytic center mutant (K86M) in intact cells was not affected by the addition of vanadate but still increased with the addition of Ko143. In isolated membrane fragments the ligand modulation of 5D3 binding to ABCG2 could be analyzed in detail. In this case 5D3 binding was maximum in the presence of ATP, ADP, or Ko143, whereas the non-hydrolysable ATP analog, adenosine 5'-(beta,gamma-imido)triphosphate (AMP-PNP), and nucleotide trapping by vanadate decreased antibody binding. In membranes expressing the ABCG2-K86M mutant, ATP, ADP, and AMP-PNP decreased, whereas Ko143 increased 5D3 binding. Based on these data we suggest that the 5D3 antibody can be used as a sensitive tool to reveal intramolecular changes, reflecting ATP binding, the formation of a catalytic intermediate, or substrate inhibition within the transport cycle of the ABCG2 protein.

  13. Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter.

    PubMed

    Ozvegy-Laczka, Csilla; Laczkó, Rozália; Hegedus, Csilla; Litman, Thomas; Várady, György; Goda, Katalin; Hegedus, Tamás; Dokholyan, Nikolay V; Sorrentino, Brian P; Váradi, András; Sarkadi, Balázs

    2008-09-19

    Human ABCG2 is a plasma membrane glycoprotein working as a homodimer or homo-oligomer. The protein plays an important role in the protection/detoxification of various tissues and may also be responsible for the multidrug-resistant phenotype of cancer cells. In our previous study we found that the 5D3 monoclonal antibody shows a function-dependent reactivity to an extracellular epitope of the ABCG2 transporter. In the current experiments we have further characterized the 5D3-ABCG2 interaction. The effect of chemical cross-linking and the modulation of extracellular S-S bridges on the transporter function and 5D3 reactivity of ABCG2 were investigated in depth. We found that several protein cross-linkers greatly increased 5D3 labeling in ABCG2 expressing HEK cells; however, there was no correlation between covalent dimer formation, the inhibition of transport activity, and the increase in 5D3 binding. Dithiothreitol treatment, which reduced the extracellular S-S bridge-forming cysteines of ABCG2, had no effect on transport function but caused a significant decrease in 5D3 binding. When analyzing ABCG2 mutants carrying Cys-to-Ala changes in the extracellular loop, we found that the mutant C603A (lacking the intermolecular S-S bond) showed comparable transport activity and 5D3 reactivity to the wild-type ABCG2. However, disruption of the intramolecular S-S bridge (in C592A, C608A, or C592A/C608A mutants) in this loop abolished 5D3 binding, whereas the function of the protein was preserved. Based on these results and ab initio folding simulations, we propose a model for the large extracellular loop of the ABCG2 protein.

  14. Enhanced therapeutic effect of Adriamycin on multidrug resistant breast cancer by the ABCG2-siRNA loaded polymeric nanoparticles assisted with ultrasound

    PubMed Central

    Teng, Yanwei; Sun, Ying; Li, Fan; Zhang, Xiangyu; Xu, Yuanyuan; Duan, Yourong; Du, Lianfang

    2015-01-01

    The overexpression of the breast cancer resistance protein (ABCG2) confers resistance to Adriamycin (ADR) in breast cancer. The silencing of ABCG2 using small interfering RNA (siRNA) could be a promising approach to overcome multidrug resistance (MDR) in cancer cells. To deliver ABCG2-siRNA effectively into breast cancer cells, we used mPEG-PLGA-PLL (PEAL) nanoparticles (NPs) with ultrasound-targeted microbubble destruction (UTMD). PEAL NPs were prepared with an emulsion-solvent evaporation method. The NPs size was about 131.5 ± 6.5 nm. The siRNA stability in serum was enhanced. The intracellular ADR concentration increased after the introduction of siRNA-loaded NPs. After intravenous injection of PEAL NPs in tumor-bearing mice, the ABCG2-siRNA-loaded NPs with UTMD efficiently silenced the ABCG2 gene and enhanced the ADR susceptibility of MCF-7/ADR (ADR resistant human breast cancer cells). The siRNA-loaded NPs with UTMD + ADR showed better tumor inhibition effect and good safety in vivo. These results indicate that ADR-chemotherapy in combination with ABCG2-siRNA is an attractive strategy to treat breast cancer. PMID:26575421

  15. Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense

    SciTech Connect

    Leslie, Elaine M.; Deeley, Roger G.; Cole, Susan P.C. . E-mail: coles@post.queensu.ca

    2005-05-01

    In tumor cell lines, multidrug resistance is often associated with an ATP-dependent decrease in cellular drug accumulation which is attributed to the overexpression of certain ATP-binding cassette (ABC) transporter proteins. ABC proteins that confer drug resistance include (but are not limited to) P-glycoprotein (gene symbol ABCB1), the multidrug resistance protein 1 (MRP1, gene symbol ABCC1), MRP2 (gene symbol ABCC2), and the breast cancer resistance protein (BCRP, gene symbol ABCG2). In addition to their role in drug resistance, there is substantial evidence that these efflux pumps have overlapping functions in tissue defense. Collectively, these proteins are capable of transporting a vast and chemically diverse array of toxicants including bulky lipophilic cationic, anionic, and neutrally charged drugs and toxins as well as conjugated organic anions that encompass dietary and environmental carcinogens, pesticides, metals, metalloids, and lipid peroxidation products. P-glycoprotein, MRP1, MRP2, and BCRP/ABCG2 are expressed in tissues important for absorption (e.g., lung and gut) and metabolism and elimination (liver and kidney). In addition, these transporters have an important role in maintaining the barrier function of sanctuary site tissues (e.g., blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier or placenta). Thus, these ABC transporters are increasingly recognized for their ability to modulate the absorption, distribution, metabolism, excretion, and toxicity of xenobiotics. In this review, the role of these four ABC transporter proteins in protecting tissues from a variety of toxicants is discussed. Species variations in substrate specificity and tissue distribution of these transporters are also addressed since these properties have implications for in vivo models of toxicity used for drug discovery and development.

  16. Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter.

    PubMed

    Gál, Zita; Hegedüs, Csilla; Szakács, Gergely; Váradi, András; Sarkadi, Balázs; Özvegy-Laczka, Csilla

    2015-02-01

    Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter.

  17. Estrogen Enhances the Expression of the Multidrug Transporter Gene ABCG2—Increasing Drug Resistance of Breast Cancer Cells through Estrogen Receptors

    PubMed Central

    Chang, Fung-Wei; Fan, Hueng-Chuen; Liu, Jui-Ming; Fan, Tai-Ping; Jing, Jin; Yang, Chia-Ling; Hsu, Ren-Jun

    2017-01-01

    Background: Multidrug resistance is a major obstacle in the successful therapy of breast cancer. Studies have proved that this kind of drug resistance happens in both human cancers and cultured cancer cell lines. Understanding the molecular mechanisms of drug resistance is important for the reasonable design and use of new treatment strategies to effectively confront cancers. Results: In our study, ATP-binding cassette sub-family G member 2 (ABCG2), adenosine triphosphate (ATP) synthase and cytochrome c oxidase subunit VIc (COX6C) were over-expressed more in the MCF-7/MX cell line than in the normal MCF7 cell line. Therefore, we believe that these three genes increase the tolerance of MCF7 to mitoxantrone (MX). The data showed that the high expression of COX6C made MCF-7/MX have more stable on mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) expression than normal MCF7 cells under hypoxic conditions. The accumulation of MX was greater in the ATP-depleted treatment MCF7/MX cells than in normal MCF7/MX cells. Furthermore, E2 increased the tolerance of MCF7 cells to MX through inducing the expression of ABCG2. However, E2 could not increase the expression of ABCG2 after the inhibition of estrogen receptor α (ERα) in MCF7 cells. According to the above data, under the E2 treatment, MDA-MB231, which lacks ER, had a higher sensitivity to MX than MCF7 cells. Conclusions: E2 induced the expression of ABCG2 through ERα and the over-expressed ABCG2 made MCF7 more tolerant to MX. Moreover, the over-expressed ATP synthase and COX6c affected mitochondrial genes and function causing the over-expressed ABCG2 cells pumped out MX in a concentration gradient from the cell matrix. Finally lead to chemoresistance. PMID:28098816

  18. Multidrug transporter ABCG2 prevents tumor cell death induced by the epidermal growth factor receptor inhibitor Iressa (ZD1839, Gefitinib).

    PubMed

    Elkind, N Barry; Szentpétery, Zsófia; Apáti, Agota; Ozvegy-Laczka, Csilla; Várady, György; Ujhelly, Olga; Szabó, Katalin; Homolya, László; Váradi, András; Buday, László; Kéri, György; Német, Katalin; Sarkadi, Balázs

    2005-03-01

    Iressa (ZD1839, Gefitinib), used in clinics to treat non-small cell lung cancer patients, is a tyrosine kinase receptor inhibitor that leads to specific decoupling of epidermal growth factor receptor (EGFR) signaling. Recent data indicate that Iressa is especially effective in tumors with certain EGFR mutations; however, a subset of these tumors does not respond to Iressa. In addition, certain populations have an elevated risk of side effects during Iressa treatment. The human ABCG2 (BCRP/MXR/ABCP) transporter causes cancer drug resistance by actively extruding a variety of cytotoxic drugs, and it functions physiologically to protect our tissues from xenobiotics. Importantly, ABCG2 modifies absorption, distribution, and toxicity of several pharmacologic agents. Previously, we showed that ABCG2 displays a high-affinity interaction with several tyrosine kinase receptor inhibitors, including Iressa. Here, we show that the expression of ABCG2, but not its nonfunctional mutant, protects the EGFR signaling-dependent A431 tumor cells from death on exposure to Iressa. This protection is reversed by the ABCG2-specific inhibitor, Ko143. These data, reinforced with cell biology and biochemical experiments, strongly suggest that ABCG2 can actively pump Iressa. Therefore, variable expression and polymorphisms of ABCG2 may significantly modify the antitumor effect as well as the absorption and tissue distribution of Iressa.

  19. Development of sulfasalazine resistance in human T cells induces expression of the multidrug resistance transporter ABCG2 (BCRP) and augmented production of TNFα

    PubMed Central

    van der Heijden, J; de Jong, M C; Dijkmans, B; Lems, W; Oerlemans, R; Kathmann, I; Schalkwijk, C; Scheffer, G; Scheper, R; Jansen, G

    2004-01-01

    Objective: To determine whether overexpression of cell membrane associated drug efflux pumps belonging to the family of ATP binding cassette (ABC) proteins contributes to a diminished efficacy of sulfasalazine (SSZ) after prolonged cellular exposure to this disease modifying antirheumatic drug (DMARD). Methods: A model system of human T cells (CEM) was used to expose cells in vitro to increasing concentrations of SSZ for a period of six months. Cells were then characterised for the expression of drug efflux pumps: P-glycoprotein (Pgp, ABCB1), multidrug resistance protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2). Results: Prolonged exposure of CEM cells to SSZ provoked resistance to SSZ as manifested by a 6.4-fold diminished antiproliferative effect of SSZ compared with parental CEM cells. CEM cells resistant to SSZ (CEM/SSZ) showed a marked induction of ABCG2/BCRP, Pgp expression was not detectable, while MRP1 expression was even down regulated. A functional role of ABCG2 in SSZ resistance was demonstrated by 60% reversal of SSZ resistance by the ABCG2 blocker Ko143. Release of the proinflammatory cytokine tumour necrosis factor α (TNFα) was threefold higher in CEM/SSZ cells than in CEM cells. Moreover, twofold higher concentrations of SSZ were required to inhibit TNFα release from CEM/SSZ cells compared with CEM cells. Conclusion: Collectively, ABCG2 induction, augmented TNFα release, and less efficient inhibition of TNFα production by SSZ may contribute to diminished efficacy after prolonged exposure to SSZ. These results warrant further clinical studies to verify whether drug efflux pumps, originally identified for their roles in cytostatic drug resistance, can also be induced by SSZ or other DMARDs. PMID:14722201

  20. Expression levels of the ABCG2 multidrug transporter in human erythrocytes correspond to pharmacologically relevant genetic variations.

    PubMed

    Kasza, Ildikó; Várady, György; Andrikovics, Hajnalka; Koszarska, Magdalena; Tordai, Attila; Scheffer, George L; Németh, Adrienn; Szakács, Gergely; Sarkadi, Balázs

    2012-01-01

    We have developed a rapid, simple and reliable, antibody-based flow cytometry assay for the quantitative determination of membrane proteins in human erythrocytes. Our method reveals significant differences between the expression levels of the wild-type ABCG2 protein and the heterozygous Q141K polymorphic variant. Moreover, we find that nonsense mutations on one allele result in a 50% reduction in the erythrocyte expression of this protein. Since ABCG2 polymorphisms are known to modify essential pharmacokinetic parameters, uric acid metabolism and cancer drug resistance, a direct determination of the erythrocyte membrane ABCG2 protein expression may provide valuable information for assessing these conditions or for devising drug treatments. Our findings suggest that erythrocyte membrane protein levels may reflect genotype-dependent tissue expression patterns. Extension of this methodology to other disease-related or pharmacologically important membrane proteins may yield new protein biomarkers for personalized diagnostics.

  1. Expression Levels of the ABCG2 Multidrug Transporter in Human Erythrocytes Correspond to Pharmacologically Relevant Genetic Variations

    PubMed Central

    Kasza, Ildikó; Várady, György; Andrikovics, Hajnalka; Koszarska, Magdalena; Tordai, Attila; Scheffer, George L.; Németh, Adrienn; Szakács, Gergely; Sarkadi, Balázs

    2012-01-01

    We have developed a rapid, simple and reliable, antibody-based flow cytometry assay for the quantitative determination of membrane proteins in human erythrocytes. Our method reveals significant differences between the expression levels of the wild-type ABCG2 protein and the heterozygous Q141K polymorphic variant. Moreover, we find that nonsense mutations on one allele result in a 50% reduction in the erythrocyte expression of this protein. Since ABCG2 polymorphisms are known to modify essential pharmacokinetic parameters, uric acid metabolism and cancer drug resistance, a direct determination of the erythrocyte membrane ABCG2 protein expression may provide valuable information for assessing these conditions or for devising drug treatments. Our findings suggest that erythrocyte membrane protein levels may reflect genotype-dependent tissue expression patterns. Extension of this methodology to other disease-related or pharmacologically important membrane proteins may yield new protein biomarkers for personalized diagnostics. PMID:23166586

  2. Breast cancer resistance protein (BCRP/ABCG2): its role in multidrug resistance and regulation of its gene expression

    PubMed Central

    Nakanishi, Takeo; Ross, Douglas D.

    2012-01-01

    Breast cancer resistance protein (BCRP)/ATP-binding cassette subfamily G member 2 (ABCG2) is an ATP-binding cassette (ABC) transporter identified as a molecular cause of multidrug resistance (MDR) in diverse cancer cells. BCRP physiologically functions as a part of a self-defense mechanism for the organism; it enhances elimination of toxic xenobiotic substances and harmful agents in the gut and biliary tract, as well as through the blood-brain, placental, and possibly blood-testis barriers. BCRP recognizes and transports numerous anticancer drugs including conventional chemotherapeutic and targeted small therapeutic molecules relatively new in clinical use. Thus, BCRP expression in cancer cells directly causes MDR by active efflux of anticancer drugs. Because BCRP is also known to be a stem cell marker, its expression in cancer cells could be a manifestation of metabolic and signaling pathways that confer multiple mechanisms of drug resistance, self-renewal (sternness), and invasiveness (aggressiveness), and thereby impart a poor prognosis. Therefore, blocking BCRP-mediated active efflux may provide a therapeutic benefit for cancers. Delineating the precise molecular mechanisms for BCRP gene expression may lead to identification of a novel molecular target to modulate BCRP-mediated MDR. Current evidence suggests that BCRP gene transcription is regulated by a number of trans-acting elements including hypoxia inducible factor 1α, estrogen receptor, and peroxisome proliferator-activated receptor. Furthermore, alternative promoter usage, demethylation of the BCRP promoter, and histone modification are likely associated with drug-induced BCRP overexpression in cancer cells. Finally, PI3K/AKT signaling may play a critical role in modulating BCRP function under a variety of conditions. These biological events seem involved in a complicated manner. Untangling the events would be an essential first step to developing a method to modulate BCRP function to aid patients with

  3. Role of Abcg2 During Mouse Embroyonic Stem Cell Diffferentiation

    EPA Science Inventory

    Role of Abcg2 During Mouse Embryonic Stem Cell Differentiation. Abcg2 is a multidrug resistance ATP-binding cassette (ABC) transporter whose activity may be considered a hallmark of stem cell plasticity. The role of Abcg2 during early embryogenesis, however, is unclear. Studies...

  4. Role of Abcg2 During Mouse Embroyonic Stem Cell Diffferentiation

    EPA Science Inventory

    Role of Abcg2 During Mouse Embryonic Stem Cell Differentiation. Abcg2 is a multidrug resistance ATP-binding cassette (ABC) transporter whose activity may be considered a hallmark of stem cell plasticity. The role of Abcg2 during early embryogenesis, however, is unclear. Studies...

  5. ABCG2 -- a transporter for all seasons.

    PubMed

    Sarkadi, Balázs; Ozvegy-Laczka, Csilla; Német, Katalin; Váradi, András

    2004-06-01

    The human ABCG2 (ABCP/MXR/BCRP) protein is a recently recognized ABC half-transporter, which forms homodimers in the plasma membrane and actively extrudes a wide variety of chemically unrelated compounds from the cells. This protein protects our cells and tissues against various xenobiotics, with a crucial role in the intestine, liver, placenta, and the blood-brain barrier. Moreover, ABCG2 seems to have a key function in stem cell protection/regulation, and also in hypoxic defense mechanisms. Widely occurring single nucleotide polymorphisms in ABCG2 may affect absorption and distribution, altering the effectiveness and toxicity of drugs in large populations. At the clinics, overexpression of ABCG2 in tumor cells confers cancer multidrug resistance to a variety of newly developed anticancer agents. On the other hand, specific substrate mutants of ABCG2 are advocated for use as selectable markers in stem-cell based gene therapy.

  6. BCRP/ABCG2 inhibitors: a patent review (2009-present).

    PubMed

    Wiese, Michael

    2015-01-01

    BCRP/ABCG2 is a polyspecific ATP binding cassette transporter involved in multidrug resistance of tumors. Compared with P-glycoprotein/ABCB1 only a few inhibitors are known. Identification of ABCG2 inhibitors in compound libraries and efforts made to develop new ABCG2 inhibitors are discussed in the review. Additionally, development of new test systems for testing ABCG2 activity and medical applications of ABCG2 inhibitors and activators are reviewed. ABCG2 is highly expressed in side-population cells, which possess stem cell properties and are resistant to chemotherapy and radiotherapy. These cells are thought to lead to a relapse after chemotherapy. Therefore, inhibition of ABCG2 could have an additional benefit besides counteracting multidrug resistance. Among the new chemical entities, the bivalent flavonoids seem promising, as selective and broad spectrum inhibitors were found within this class of compounds. However, the available pharmacological data are rather preliminary.

  7. Promoter methylation patterns of ABCB1, ABCC1 and ABCG2 in human cancer cell lines, multidrug-resistant cell models and tumor, tumor-adjacent and tumor-distant tissues from breast cancer patients

    PubMed Central

    Spitzwieser, Melanie; Pirker, Christine; Koblmüller, Bettina; Pfeiler, Georg; Hacker, Stefan; Berger, Walter; Heffeter, Petra; Cichna-Markl, Margit

    2016-01-01

    Overexpression of ABCB1, ABCC1 and ABCG2 in tumor tissues is considered a major cause of limited efficacy of anticancer drugs. Gene expression of ABC transporters is regulated by multiple mechanisms, including changes in the DNA methylation status. Most of the studies published so far only report promoter methylation levels for either ABCB1 or ABCG2, and data on the methylation status for ABCC1 are scarce. Thus, we determined the promoter methylation patterns of ABCB1, ABCC1 and ABCG2 in 19 human cancer cell lines. In order to contribute to the elucidation of the role of DNA methylation changes in acquisition of a multidrug resistant (MDR) phenotype, we also analyzed the promoter methylation patterns in drug-resistant sublines of the cancer cell lines GLC-4, SW1573, KB-3-1 and HL-60. In addition, we investigated if aberrant promoter methylation levels of ABCB1, ABCC1 and ABCG2 occur in tumor and tumor-surrounding tissues from breast cancer patients. Our data indicates that hypomethylation of the ABCC1 promoter is not cancer type-specific but occurs in cancer cell lines of different origins. Promoter methylation was found to be an important mechanism in gene regulation of ABCB1 in parental cancer cell lines and their drug-resistant sublines. Overexpression of ABCC1 in MDR cell models turned out to be mediated by gene amplification, not by changes in the promoter methylation status of ABCC1. In contrast to the promoters of ABCC1 and ABCG2, the promoter of ABCB1 was significantly higher methylated in tumor tissues than in tumor-adjacent and tumor-distant tissues from breast cancer patients. PMID:27689338

  8. ABCG2/BCRP: Specific and Nonspecific Modulators.

    PubMed

    Peña-Solórzano, Diana; Stark, Simone Alexandra; König, Burkhard; Sierra, Cesar Augusto; Ochoa-Puentes, Cristian

    2017-09-01

    Multidrug resistance (MDR) in cancer cells is the development of resistance to a variety of structurally and functionally nonrelated anticancer drugs. This phenomenon has become a major obstacle to cancer chemotherapy seriously affecting the clinical outcome. MDR is associated with increased drug efflux from cells mediated by an energy-dependent mechanism involving the ATP-binding cassette (ABC) transporters, mainly P-glycoprotein (ABCB1), the MDR-associated protein-1 (ABCC1), and the breast cancer resistance protein (ABCG2). The first two transporters have been widely studied already and reviews summarized the results. The ABCG2 protein has been a subject of intense study since its discovery as its overexpression has been detected in resistant cell lines in numerous types of human cancers. To date, a long list of modulators of ABCG2 exists and continues to increase. However, little is known about the clinical consequences of ABCG2 modulation. This makes the design of novel, potent, and nontoxic inhibitors of this efflux protein a major challenge to reverse MDR and thereby increase the success of chemotherapy. The aim of the present review is to describe and highlight specific and nonspecific modulators of ABCG2 reported to date based on the selectivity of the compounds, as many of them are effective against one or more ABC transport proteins. © 2016 Wiley Periodicals, Inc.

  9. Physiological and pharmacological roles of ABCG2 (BCRP): recent findings in Abcg2 knockout mice.

    PubMed

    Vlaming, Maria L H; Lagas, Jurjen S; Schinkel, Alfred H

    2009-01-31

    The multidrug transporter ABCG2 (BCRP/MXR/ABCP) can actively extrude a broad range of endogenous and exogenous substrates across biological membranes. ABCG2 limits oral availability and mediates hepatobiliary and renal excretion of its substrates, and thus influences the pharmacokinetics of many drugs. Recent work, relying mainly on the use of Abcg2(-/-) mice, has revealed important contributions of ABCG2 to the blood-brain, blood-testis and blood-fetal barriers. Together, these functions indicate a primary biological role of ABCG2 in protecting the organism from a range of xenobiotics. In addition, several other physiological functions of ABCG2 have been observed, including extrusion of porphyrins and/or porphyrin conjugates from hematopoietic cells, liver and harderian gland, as well as secretion of vitamin B(2) (riboflavin) and possibly other vitamins (biotin, vitamin K) into breast milk. However, the physiological significance of these processes has been difficult to establish, indicating that there is still a lot to learn about this intriguing protein.

  10. Modulating the function of ATP-binding cassette subfamily G member 2 (ABCG2) with inhibitor cabozantinib.

    PubMed

    Zhang, Guan-Nan; Zhang, Yun-Kai; Wang, Yi-Jun; Barbuti, Anna Maria; Zhu, Xi-Jun; Yu, Xin-Yue; Wen, Ai-Wen; Wurpel, John N D; Chen, Zhe-Sheng

    2017-01-25

    Cabozantinib (XL184) is a small molecule tyrosine kinase receptor inhibitor, which targets c-Met and VEGFR2. Cabozantinib has been approved by the Food and Drug Administration to treat advanced medullary thyroid cancer and renal cell carcinoma. In the present study, we evaluated the ability of cabozantinib to modulate the function of the ATP-binding cassette subfamily G member 2 (ABCG2) by sensitizing cells that are resistant to ABCG2 substrate antineoplastic drugs. We used a drug-selected resistant cell line H460/MX20 and three ABCG2 stable transfected cell lines ABCG2-482-R2, ABCG2-482-G2, and ABCG2-482-T7, which overexpress ABCG2. Cabozantinib, at non-toxic concentrations (3 or 5μM), sensitized the ABCG2-overexpressing cells to mitoxantrone, SN-38, and topotecan. Our results indicate that cabozantinib reverses ABCG2-mediated multidrug resistance by antagonizing the drug efflux function of the ABCG2 transporter instead of downregulating its expression. The molecular docking analysis indicates that cabozantinib binds to the drug-binding site of the ABCG2 transporter. Overall, our findings demonstrate that cabozantinib inhibits the ABCG2 transporter function and consequently enhances the effect of the antineoplastic agents that are substrates of ABCG2. Cabozantinib may be a useful agent in anticancer treatment regimens for patients who are resistant to ABCG2 substrate drugs.

  11. Impact of genetic variability in the ABCG2 gene on ABCG2 expression, function, and interaction with AT1 receptor antagonist telmisartan.

    PubMed

    Deppe, Sylvia; Ripperger, Anne; Weiss, Johanna; Ergün, Süleyman; Benndorf, Ralf A

    2014-01-24

    The ATP-binding cassette transporter ABCG2 plays a prominent role in cardiovascular and cancer pathophysiology, is involved in the pathogenesis of gout, and affects pharmacokinetics of numerous drugs. Telmisartan, a widely used AT1 receptor antagonist, inhibits the transport capacity of ABCG2 and may cause drug-drug interactions, especially in individuals carrying polymorphism that facilitate the telmisartan-ABCG2 interaction. Thus, the aim of this study was to identify ABCG2 polymorphisms and somatic mutations with relevance for the telmisartan-ABCG2 interaction. For this purpose, a cellular system for the conditional expression of ABCG2 was established. ABCG2 variants were generated via site-directed mutagenesis. Interaction of telmisartan with these ABCG2 variants was investigated in HEK293-Tet-On cells using the pheophorbide A efflux assay. Moreover, expression of ABCG2 variants was studied in these cells. Importantly, protein levels of the Q141K and F489L variant were significantly reduced, a phenomenon that was partly reversed by pharmacological proteasome inhibition. Moreover, basal pheophorbide A efflux capacity of S248P, F431L, and F489L variants was significantly impaired. Interestingly, inhibition of ABCG2-mediated pheophorbide A transport by telmisartan was almost abolished in cells expressing the R482G variant, whereas it was largely increased in cells expressing the F489L variant. We conclude that the arginine residue at position 482 of the ABCG2 molecule is of major importance for the interaction of telmisartan with this ABC transporter. Furthermore, individuals carrying the F489L polymorphism may be at increased risk of developing adverse drug reactions in multi-drug regimens involving ABCG2 substrates and telmisartan. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. ABCG2 is not able to catalyze glutathione efflux and does not contribute to GSH-dependent collateral sensitivity

    PubMed Central

    Gauthier, Charlotte; Ozvegy-Laczka, Csilla; Szakacs, Gergely; Sarkadi, Balazs; Di Pietro, Attilio

    2013-01-01

    ABCG2 is a key human ATP-binding cassette (ABC) transporter mediating cancer cell chemoresistance. In the case of ABCC1, another multidrug transporter, earlier findings documented that certain modulators greatly increase ABCC1-mediated glutathione (GSH) efflux and, upon depletion of intracellular GSH, induce “collateral sensitivity” leading to the apoptosis of multidrug resistant cells. Recently, it has been suggested that ABCG2 may mediate an active GSH transport. In order to explore if ABCG2-overexpressing cells may be similarly targeted, we first looked for the effects of ABCG2 expression on cellular GSH levels, and for an ABCG2-dependent GSH transport in HEK293 and MCF7 cells. We found that, while ABCG2 overexpression altered intracellular GSH levels in these transfected or drug-selected cells, ABCG2 inhibitors or transport modulators did not influence GSH efflux. We then performed direct measurements of drug-stimulated ATPase activity and 3H-GSH transport in inside-out membrane vesicles of human ABC transporter-overexpressing Sf9 insect cells. Our results indicate that ABCG2-ATPase is not modulated by GSH and, in contrast to ABCC1, ABCG2 does not catalyze any significant GSH transport. Our data suggest no direct interaction between the ABCG2 transporter and GSH, although a long-term modulation of cellular GSH by ABCG2 cannot be excluded. PMID:24312054

  13. ABCG2 is not able to catalyze glutathione efflux and does not contribute to GSH-dependent collateral sensitivity.

    PubMed

    Gauthier, Charlotte; Ozvegy-Laczka, Csilla; Szakacs, Gergely; Sarkadi, Balazs; Di Pietro, Attilio

    2013-01-01

    ABCG2 is a key human ATP-binding cassette (ABC) transporter mediating cancer cell chemoresistance. In the case of ABCC1, another multidrug transporter, earlier findings documented that certain modulators greatly increase ABCC1-mediated glutathione (GSH) efflux and, upon depletion of intracellular GSH, induce "collateral sensitivity" leading to the apoptosis of multidrug resistant cells. Recently, it has been suggested that ABCG2 may mediate an active GSH transport. In order to explore if ABCG2-overexpressing cells may be similarly targeted, we first looked for the effects of ABCG2 expression on cellular GSH levels, and for an ABCG2-dependent GSH transport in HEK293 and MCF7 cells. We found that, while ABCG2 overexpression altered intracellular GSH levels in these transfected or drug-selected cells, ABCG2 inhibitors or transport modulators did not influence GSH efflux. We then performed direct measurements of drug-stimulated ATPase activity and (3)H-GSH transport in inside-out membrane vesicles of human ABC transporter-overexpressing Sf9 insect cells. Our results indicate that ABCG2-ATPase is not modulated by GSH and, in contrast to ABCC1, ABCG2 does not catalyze any significant GSH transport. Our data suggest no direct interaction between the ABCG2 transporter and GSH, although a long-term modulation of cellular GSH by ABCG2 cannot be excluded.

  14. Dietary polyacetylenes of the falcarinol type are inhibitors of breast cancer resistance protein (BCRP/ABCG2).

    PubMed

    Tan, Kee W; Killeen, Daniel P; Li, Yan; Paxton, James W; Birch, Nigel P; Scheepens, Arjan

    2014-01-15

    Polyacetylenes of the falcarinol type are present in vegetables such as carrots and parsley. They display interesting bioactivities and hold potential as health-promoting and therapeutic agents. In this study, falcarinol, falcarindiol, falcarindiol 3-acetate and falcarindiol 3,8-diacetate were examined for their modulation on breast cancer resistance protein (BCRP/ABCG2), an efflux transporter important for xenobiotic absorption and disposition, and multidrug resistance in cancer. Falcarinol, falcarindiol, and falcarindiol 3-acetate were extracted from carrots and falcarindiol 3,8-diacetate prepared from falcarindiol. Their modulatory effects on ABCG2 were studied using three methods-mitoxantrone accumulation, vesicular transport, and ATPase assay. The polyacetylenes inhibited mitoxantrone (an ABCG2 substrate) efflux in ABCG2-overexpressing HEK293 cells. The inhibitory effect was confirmed in the vesicular transport assay, in which concentration-dependent inhibition of methotrexate (an ABCG2 substrate) uptake into ABCG2-overexpressing Sf9 membrane vesicles was observed (IC50=19.7-41.7µM). The polyacetylenes also inhibited baseline and sulfasalazine-stimulated vanadate-sensitive ATPase activities in ABCG2-overexpressing Sf9 membrane vesicles (IC50=19.3-79.3µM). This is the first report of an inhibitory effect of polyacetylenes on ABCG2. These results indicate a prospective use of polyacetylenes as multidrug resistance reversal agents, a possible role of ABCG2 in the absorption and disposition of polyacetylenes, and potential food-drug interactions between polyacetylene-rich foods and ABCG2 substrate drugs.

  15. Structural determinants of imidazoacridinones facilitating antitumor activity are crucial for substrate recognition by ABCG2.

    PubMed

    Bram, Eran E; Adar, Yamit; Mesika, Nufar; Sabisz, Michal; Skladanowski, Andrzej; Assaraf, Yehuda G

    2009-05-01

    Symadex is the lead acridine compound of a novel class of imidazoacridinones (IAs) currently undergoing phase II clinical trials for the treatment of various cancers. Recently, we have shown that Symadex is extruded by ABCG2-overexpressing lung cancer A549/K1.5 cells, thereby resulting in a marked resistance to certain IAs. To identify the IA residues essential for substrate recognition by ABCG2, we here explored the ability of ABCG2 to extrude and confer resistance to a series of 23 IAs differing at defined residue(s) surrounding their common 10-azaanthracene structure. Taking advantage of the inherent fluorescent properties of IAs, ABCG2-dependent efflux and drug resistance were determined in A549/K1.5 cells using flow cytometry in the presence or absence of fumitremorgin C, a specific ABCG2 transport inhibitor. We find that a hydroxyl group at one of the R1, R2, or R3 positions in the proximal IA ring was essential for ABCG2-mediated efflux and consequent IA resistance. Moreover, elongation of the common distal aliphatic side chain attenuated ABCG2-dependent efflux, thereby resulting in the retention of parental cell sensitivity. Hence, the current study offers novel molecular insight into the structural determinants that facilitate ABCG2-mediated drug efflux and consequent drug resistance using a unique platform of fluorescent IAs. Moreover, these results establish that the IA determinants mediating cytotoxicity are precisely those that facilitate ABCG2-dependent drug efflux and IA resistance. The possible clinical implications for the future design of novel acridines that overcome ABCG2-dependent multidrug resistance are discussed.

  16. A-803467, a tetrodotoxin-resistant sodium channel blocker, modulates ABCG2-mediated MDR in vitro and in vivo

    PubMed Central

    Patel, Atish; Zhang, Yun-Kai; Wang, Yi-Jun; Shukla, Suneet; Kathawala, Rishil J.; Kumar, Priyank; Gupta, Pranav; Ambudkar, Suresh V.; Wurpel, John N. D.; Chen, Zhe-Sheng

    2015-01-01

    ATP-binding cassette subfamily G member 2 (ABCG2) is a member of the ABC transporter superfamily proteins, which has been implicated in the development of multidrug resistance (MDR) in cancer, apart from its physiological role to remove toxic substances out of the cells. The diverse range of substrates of ABCG2 includes many antineoplastic agents such as topotecan, doxorubicin and mitoxantrone. ABCG2 expression has been reported to be significantly increased in some solid tumors and hematologic malignancies, correlated to poor clinical outcomes. In addition, ABCG2 expression is a distinguishing feature of cancer stem cells, whereby this membrane transporter facilitates resistance to the chemotherapeutic drugs. To enhance the chemosensitivity of cancer cells, attention has been focused on MDR modulators. In this study, we investigated the effect of a tetrodotoxin-resistant sodium channel blocker, A-803467 on ABCG2-overexpressing drug selected and transfected cell lines. We found that at non-toxic concentrations, A-803467 could significantly increase the cellular sensitivity to ABCG2 substrates in drug-resistant cells overexpressing either wild-type or mutant ABCG2. Mechanistic studies demonstrated that A-803467 (7.5 μM) significantly increased the intracellular accumulation of [3H]-mitoxantrone by inhibiting the transport activity of ABCG2, without altering its expression levels. In addition, A-803467 stimulated the ATPase activity in membranes overexpressed with ABCG2. In a murine model system, combination treatment of A-803467 (35 mg/kg) and topotecan (3 mg/kg) significantly inhibited the tumor growth in mice xenografted with ABCG2-overexpressing cancer cells. Our findings indicate that a combination of A-803467 and ABCG2 substrates may potentially be a novel therapeutic treatment in ABCG2-positive drug resistant cancers. PMID:26515463

  17. Synthesis and Investigation of Tetrahydro-β-carboline Derivatives as Inhibitors of the Breast Cancer Resistance Protein (ABCG2).

    PubMed

    Spindler, Anna; Stefan, Katja; Wiese, Michael

    2016-07-14

    The breast cancer resistance protein (ABCG2) transports chemotherapeutic drugs out of cells, which makes it a major player in mediating multidrug resistance (MDR) of cancer cells. To overcome this mechanism, inhibitors of ABCG2 can be used. Only a few potent and selective ABCG2 inhibitors have been discovered, i.e., fumitremorgin C (FTC), Ko143, and the alkaloid harmine, which contain a tetrahydro-β-carboline or β-carboline backbone, respectively. However, toxicity and or instability prevent their use in vivo. Therefore, there is a need for further potent inhibitors. We synthesized and pharmacologically investigated 37 tetrahydro-β-carboline derivatives. The inhibitory activity of two compounds (51, 52) is comparable to that of Ko143, and they are selective for ABCG2 over ABCB1. Furthermore, they are able to reverse the ABCG2-mediated resistance toward SN-38 and inhibit the ATPase activity. The cytotoxicity data show that their inhibitory effect is substantially higher than their toxicity.

  18. Hop-derived prenylflavonoids are substrates and inhibitors of the efflux transporter breast cancer resistance protein (BCRP/ABCG2).

    PubMed

    Tan, Kee W; Cooney, Janine; Jensen, Dwayne; Li, Yan; Paxton, James W; Birch, Nigel P; Scheepens, Arjan

    2014-11-01

    Hops (Humulus lupulus L.) produce unique prenylflavonoids that exhibit interesting bioactivities. This study investigates the interactions between selected prenylflavonoids and breast cancer resistance protein (BCRP/ABCG2), an efflux transporter important for xenobiotic bioavailability and multidrug resistance (MDR). ABCG2-inhibitory activity of xanthohumol (XN), isoxanthohumol (IX), 6-prenylnaringenin (6-PN), 8-prenylnaringenin (8-PN), and 6,8-diprenylnarigenin (6,8-diPN) was evaluated using mitoxantrone accumulation and vesicular transport assays. XN, IX, and 8-PN were tested for a substrate-type relationship with ABCG2 using ATPase and bidirectional transport assays. The prenylflavonoids exhibited significant ABCG2-inhibitory activities in mitoxantrone accumulation and vesicular transport assays. In the ATPase assay, XN, IX, and 8-PN inhibited baseline and sulfasalazine-stimulated ATPase activities with IC50 of 2.16-27.0 μM. IX and 8-PNalso displayed bell-shaped activation curves in Ko143-suppressed membranes, indicating a substrate-type relationship. For IX, efflux ratios of 1.25 ± 0.21 and 9.18 ± 0.56 were observed in wild type and ABCG2-overexpressing MDCKII cell monolayers, respectively. The latter was reduced to 1.25 ± 0.15 in the presence of the ABCG2-specific inhibitor Ko143, demonstrating an ABCG2-mediated efflux of IX. Additionally, evidence was shown for the involvement of ABCG2 in the efflux of 8-PN and/or its sulfate conjugate. Prenylflavonoids are potent inhibitors of ABCG2 and therefore implicated in ABCG2-mediated food/herb-drug interactions and MDR. ABCG2-mediated efflux of prenylflavonoids may represent one mechanism that regulates prenylflavonoid bioavailability. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Acryloylphenylcarboxamides: A New Class of Breast Cancer Resistance Protein (ABCG2) Modulators.

    PubMed

    Kraege, Stefanie; Köhler, Sebastian C; Wiese, Michael

    2016-10-13

    Chalcones are easily synthesized natural precursors of secondary plant metabolites, and their derivatives show various biological activities including inhibition of ABC transporters. Especially, their role as inhibitors of ABCG2, the most recently discovered ABC transporter involved in multidrug resistance, inspired the synthesis of new structurally diverse derivatives. Therefore, we combined the typical chalcone moiety with several acid chlorides by using an amide linker at position 2', 3', or 4' on ring A of the chalcone. The resulting 35 compounds covered a wide spectrum of substitution patterns, which allowed development of structure-activity relationships and to find the optimal structural features for further investigations. Synthesized acryloylphenylcarboxamides were investigated for their inhibitory activity against ABCG2 and their behavior toward ABCB1 and ABCC1. Furthermore, for the most promising compounds, their intrinsic cytotoxicity and their ability to reverse ABCG2-mediated multidrug resistance were determined. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Specific inhibition of the ABCG2 transporter could improve the efficacy of photodynamic therapy.

    PubMed

    Bebes, Attila; Nagy, Tünde; Bata-Csörgo, Zsuzsanna; Kemény, Lajos; Dobozy, Attila; Széll, Márta

    2011-11-03

    Photodynamic therapy is based on the selective accumulation of a photosensitizer in tumors, followed by destruction of the target tissue by a light source. Protoporphyrin IX, a well-known photosensitizer, was recently reported as an endogenous substrate for the multidrug transporter ABCG2. We investigated the role of ABCG2 protein in the porphyrin extrusion ability of keratinocytes, with regard to the impact of the specific inhibition of ABCG2 by a non-toxic fumitremorgin C analog, Ko-134, on photodynamic therapy efficacy. We studied the level of porphyrin accumulation in response to delta-aminolevulinic acid pretreatment in proliferating and highly differentiated HaCaT keratinocytes. An in vitro model of photodynamic therapy on HaCaT cells was established with a therapeutically approved narrow-bandwidth red-light source. The porphyrin extrusion ability of HaCaT cells proved to correlate with their ABCG2 expression which was higher in proliferating cells than in differentiated cells. Moreover, the specific inhibition of ABCG2 by Ko-134 enhanced the sensitivity of keratinocytes to photodynamic therapy in vitro. These results suggest that ABCG2 may serve as a target molecule via which to improve the photodynamic therapy of skin lesions: its inhibition by the non-toxic Ko-134 is a promising therapeutic modality.

  1. Regulation and expression of the ATP-binding cassette transporter ABCG2 in human embryonic stem cells.

    PubMed

    Padmanabhan, Raji; Chen, Kevin G; Gillet, Jean-Pierre; Handley, Misty; Mallon, Barbara S; Hamilton, Rebecca S; Park, Kyeyoon; Varma, Sudhir; Mehaffey, Michele G; Robey, Pamela G; McKay, Ronald D G; Gottesman, Michael M

    2012-10-01

    The expression and function of several multidrug transporters (including ABCB1 and ABCG2) have been studied in human cancer cells and in mouse and human adult stem cells. However, the expression of ABCG2 in human embryonic stem cells (hESCs) remains unclear. Limited and contradictory results in the literature from two research groups have raised questions regarding its expression and function. In this study, we used quantitative real-time PCR, Northern blots, whole genome RNA sequencing, Western blots, and immunofluorescence microscopy to study ABCG2 expression in hESCs. We found that full-length ABCG2 mRNA transcripts are expressed in undifferentiated hESC lines. However, ABCG2 protein was undetectable even under embryoid body differentiation or cytotoxic drug induction. Moreover, surface ABCG2 protein was coexpressed with the differentiation marker stage-specific embryonic antigen-1 of hESCs, following constant BMP-4 signaling at days 4 and 6. This expression was tightly correlated with the downregulation of two microRNAs (miRNAs) (i.e., hsa-miR-519c and hsa-miR-520h). Transfection of miRNA mimics and inhibitors of these two miRNAs confirmed their direct involvement in the regulation ABCG2 translation. Our findings clarify the controversy regarding the expression of the ABCG2 gene and also provide new insights into translational control of the expression of membrane transporter mRNAs by miRNAs in hESCs.

  2. Regulation and Expression of the ATP-binding Cassette Transporter ABCG2 in Human Embryonic Stem Cells

    PubMed Central

    Padmanabhan, Raji; Chen, Kevin G.; Gillet, Jean-Pierre; Handley, Misty; Mallon, Barbara S.; Hamilton, Rebecca S.; Park, Kyeyoon; Varma, Sudhir; Mehaffey, Michele G.; Robey, Pamela G.; McKay, Ronald D.G.; Gottesman, Michael M.

    2012-01-01

    The expression and function of several multidrug transporters (including ABCB1 and ABCG2) have been studied in human cancer cells and in mouse and human adult stem cells. However, the expression of ABCG2 in human embryonic stem cells (hESCs) remains unclear. Limited and contradictory results in the literature from two research groups have raised questions regarding its expression and function. In this study, we used quantitative real-time PCR, Northern blots, whole genome RNA sequencing, Western blots, and immunofluorescence microscopy to study ABCG2 expression in hESCs. We found that full-length ABCG2 mRNA transcripts are expressed in undifferentiated hESC lines. However, ABCG2 protein was undetectable even under embryoid body differentiation or cytotoxic drug induction. Moreover, surface ABCG2 protein was coexpressed with the differentiation marker SSEA-1 of hESCs, following constant BMP-4 signaling at days 4 and 6. This expression was tightly correlated with the down-regulation of two microRNAs (i.e., hsa-miR-519c and hsa-miR-520h). Transfection of microRNA mimics and inhibitors of these two microRNAs confirmed their direct involvement in the regulation ABCG2 translation. Our findings clarify the controversy regarding the expression of the ABCG2 gene and also provide new insights into translational control of the expression of membrane transporter mRNAs by microRNAs in hESCs. PMID:22887864

  3. Effect of ABCG2/BCRP Expression on Efflux and Uptake of Gefitinib in NSCLC Cell Lines

    PubMed Central

    Galetti, Maricla; Petronini, Pier Giorgio; Fumarola, Claudia; Cretella, Daniele; La Monica, Silvia; Bonelli, Mara; Cavazzoni, Andrea; Saccani, Francesca; Caffarra, Cristina; Andreoli, Roberta; Mutti, Antonio; Tiseo, Marcello; Ardizzoni, Andrea; Alfieri, Roberta R.

    2015-01-01

    Background BCRP/ABCG2 emerged as an important multidrug resistance protein, because it confers resistance to several classes of cancer chemotherapeutic agents and to a number of novel molecularly-targeted therapeutics such as tyrosine kinase inhibitors. Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor used in the treatment of patients with advanced non small cell lung cancer (NSCLC) carrying activating EGFR mutations. Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism. Aim The present study, performed in a panel of NSCLC cell lines expressing different ABCG2 plasma membrane levels, was designed to investigate the effect of the efflux transporter ABCG2 on intracellular gefitinib accumulation, by dissecting the contribution of uptake and efflux processes. Methods and Results Our findings indicate that gefitinib, in lung cancer cells, inhibits ABCG2 activity, as previously reported. In addition, we suggest that ABCG2 silencing or overexpression affects intracellular gefitinib content by modulating the uptake rather than the efflux. Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake. Conclusions Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells. PMID:26536031

  4. Single-step doxorubicin-selected cancer cells overexpress the ABCG2 drug transporter through epigenetic changes

    PubMed Central

    Calcagno, A M; Fostel, J M; To, K K W; Salcido, C D; Martin, S E; Chewning, K J; Wu, C-P; Varticovski, L; Bates, S E; Caplen, N J; Ambudkar, S V

    2008-01-01

    Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines. PMID:18382425

  5. Effects of the lipid environment, cholesterol and bile acids on the function of the purified and reconstituted human ABCG2 protein.

    PubMed

    Telbisz, Ágnes; Özvegy-Laczka, Csilla; Hegedűs, Tamás; Váradi, András; Sarkadi, Balázs

    2013-03-01

    The human ABCG2 multidrug transporter actively extrudes a wide range of hydrophobic drugs and xenobiotics recognized by the transporter in the membrane phase. In order to examine the molecular nature of the transporter and its effects on the lipid environment, we have established an efficient protocol for the purification and reconstitution of the functional protein. We found that the drug-stimulated ATPase and the transport activity of ABCG2 are fully preserved by applying excess lipids and mild detergents during solubilization, whereas a detergent-induced dissociation of the ABCG2 dimer causes an irreversible inactivation. By using the purified and reconstituted protein we demonstrate that cholesterol is an essential activator, whereas bile acids are important modulators of ABCG2 activity. Both wild-type ABCG2 and its R482G mutant variant require cholesterol for full activity, although they exhibit different cholesterol sensitivities. Bile acids strongly decrease the basal ABCG2-ATPase activity both in the wild-type ABCG2 and in the mutant variant. These data reinforce the results for the modulatory effects of cholesterol and bile acids of ABCG2 investigated in a complex cell membrane environment. Moreover, these experiments open the possibility to perform functional and structural studies with a purified, reconstituted and highly active ABCG2 multidrug transporter.

  6. Inhibition of breast cancer resistance protein (ABCG2) in human myeloid dendritic cells induces potent tolerogenic functions during LPS stimulation.

    PubMed

    Jin, Jun-O; Zhang, Wei; Wong, Ka-Wing; Kwak, Minseok; van Driel, Ian R; Yu, Qing

    2014-01-01

    Breast cancer resistance protein (ABCG2), a member of the ATP-binding cassette transporters has been identified as a major determinant of multidrug resistance (MDR) in cancer cells, but ABC transporter inhibition has limited therapeutic value in vivo. In this research, we demonstrated that inhibition of efflux transporters ABCG2 induced the generation of tolerogenic DCs from human peripheral blood myeloid DCs (mDCs). ABCG2 expression was present in mDCs and was further increased by LPS stimulation. Treatment of CD1c+ mDCs with an ABCG2 inhibitor, Ko143, during LPS stimulation caused increased production of IL-10 and decreased production of pro-inflammatory cytokines and decreased expression of CD83 and CD86. Moreover, inhibition of ABCG2 in monocyte-derived DCs (MDDCs) abrogated the up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in these cells in response to LPS. Furthermore, CD1c+ mDCs stimulated with LPS plus Ko143 inhibited the proliferation of allogeneic and superantigen-specific syngenic CD4+ T cells and promoted expansion of CD25+FOXP3+ regulatory T (Treg) cells in an IL-10-dependent fashion. These tolerogenic effects of ABCG2 inhibition could be abolished by ERK inhibition. Thus, we demonstrated that inhibition of ABCG2 in LPS-stimulated mDCs can potently induce tolerogenic potentials in these cells, providing crucial new information that could lead to development of better strategies to combat MDR cancer.

  7. CD147 mediates chemoresistance in breast cancer via ABCG2 by affecting its cellular localization and dimerization.

    PubMed

    Zhou, Shuangyuan; Liao, Liqiu; Chen, Chen; Zeng, Weiqi; Liu, Shuang; Su, Juan; Zhao, Shuang; Chen, Mingliang; Kuang, Yehong; Chen, Xiang; Li, Jie

    2013-09-01

    CD147 and ABCG2 both have been reported to mediate Multidrug resistance (MDR) in breast cancer. Recent study demonstrates that CD147 could form a complex with ABCG2 on the cell membrane in primary effusion lymphoma. However, whether these two molecules regulate each other in breast cancer and result in MDR is not clear. We established four MCF-7 cell lines transfected with CD147 and/or ABCG2 and found that CD147 could increase the expression and dimerization of ABCG2, affect its cellular localization and regulate its drug transporter function. The findings derived from cells were confirmed subsequently in clinic samples of chemotherapy-sensitive/resistant breast cancer.

  8. Osimertinib (AZD9291) Attenuates the Function of Multidrug Resistance-Linked ATP-Binding Cassette Transporter ABCB1 in Vitro.

    PubMed

    Hsiao, Sung-Han; Lu, Yu-Jen; Li, Yan-Qing; Huang, Yang-Hui; Hsieh, Chia-Hung; Wu, Chung-Pu

    2016-06-06

    The effectiveness of cancer chemotherapy is often circumvented by multidrug resistance (MDR) caused by the overexpression of ATP-binding cassette (ABC) drug transporter ABCB1 (MDR1, P-glycoprotein). Several epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been shown previously capable of modulating the function of ABCB1 and reversing ABCB1-mediated MDR in human cancer cells. Furthermore, some TKIs are transported by ABCB1, which results in low oral bioavailability, reduced distribution, and the development of acquired resistance to these TKIs. In this study, we investigated the interaction between ABCB1 and osimertinib, a novel selective, irreversible third-generation EGFR TKI that has recently been approved by the U.S. Food and Drug Administration. We also evaluated the potential impact of ABCB1 on the efficacy of osimertinib in cancer cells, which can present a therapeutic challenge to clinicians in the future. We revealed that although osimertinib stimulates the ATPase activity of ABCB1, overexpression of ABCB1 does not confer resistance to osimertinib. Our results suggest that it is unlikely that the overexpression of ABCB1 can be a major contributor to the development of osimertinib resistance in cancer patients. More significantly, we revealed an additional action of osimertinib that directly inhibits the function of ABCB1 without affecting the expression level of ABCB1, enhances drug-induced apoptosis, and reverses the MDR phenotype in ABCB1-overexpressing cancer cells. Considering that osimertinib is a clinically approved third-generation EGFR TKI, our findings suggest that a combination therapy with osimertinib and conventional anticancer drugs may be beneficial to patients with MDR tumors.

  9. In vitro and in vivo evidence for the importance of breast cancer resistance protein transporters (BCRP/MXR/ABCP/ABCG2).

    PubMed

    Meyer zu Schwabedissen, Henriette E; Kroemer, Heyo K

    2011-01-01

    The breast cancer resistance protein (BCRP/ABCG2) is a member of the G-subfamiliy of the ATP-binding cassette (ABC)-transporter superfamily. This half-transporter is assumed to function as an important mechanism limiting cellular accumulation of various compounds. In context of its tissue distribution with localization in the sinusoidal membrane of hepatocytes, and in the apical membrane of enterocytes ABCG2 is assumed to function as an important mechanism facilitating hepatobiliary excretion and limiting oral bioavailability, respectively. Indeed functional assessment performing mouse studies with genetic deletion or chemical inhibition of the transporter, or performing pharmacogenetic studies in humans support this assumption. Furthermore the efflux function of ABCG2 has been linked to sanctuary blood tissue barriers as described for placenta and the central nervous system. However, in lactating mammary glands ABCG2 increases the transfer of substrates into milk thereby increasing the exposure to potential noxes of a breastfed newborn. With regard to its broad substrate spectrum including various anticancer drugs and environmental carcinogens the function of ABCG2 has been associated with multidrug resistance and tumor development/progression. In terms of cancer biology current research is focusing on the expression and function of ABCG2 in immature stem cells. Recent findings support the notion that the physiological function of ABCG2 is involved in the elimination of uric acid resulting in higher risk for developing gout in male patients harboring genetic variants. Taken together ABCG2 is implicated in various pathophysiological and pharmacological processes.

  10. The breast cancer resistance protein BCRP (ABCG2) concentrates drugs and carcinogenic xenotoxins into milk.

    PubMed

    Jonker, Johan W; Merino, Gracia; Musters, Sandra; van Herwaarden, Antonius E; Bolscher, Ellen; Wagenaar, Els; Mesman, Elly; Dale, Trevor C; Schinkel, Alfred H

    2005-02-01

    Contamination of milk with drugs, pesticides and other xenotoxins can pose a major health risk to breast-fed infants and dairy consumers. Here we show that the multidrug transporter BCRP (encoded by ABCG2) is strongly induced in the mammary gland of mice, cows and humans during lactation and that it is responsible for the active secretion of clinically and toxicologically important substrates such as the dietary carcinogen PhIP, the anticancer drug topotecan and the antiulcerative cimetidine into mouse milk.

  11. Generation of an ABCG2{sup GFPn-puro} transgenic line - A tool to study ABCG2 expression in mice

    SciTech Connect

    Orford, Michael; Mean, Richard; Lapathitis, George; Genethliou, Nicholas; Panayiotou, Elena; Panayi, Helen; Malas, Stavros

    2009-06-26

    The ATP-binding cassette (ABC) transporter 2 (ABCG2) is expressed by stem cells in many organs and in stem cells of solid tumors. These cells are isolated based on the side population (SP) phenotype, a Hoechst 3342 dye efflux property believed to be conferred by ABCG2. Because of the limitations of this approach we generated transgenic mice that express Nuclear GFP (GFPn) coupled to the Puromycin-resistance gene, under the control of ABCG2 promoter/enhancer sequences. We show that ABCG2 is expressed in neural progenitors of the developing forebrain and spinal cord and in embryonic and adult endothelial cells of the brain. Using the neurosphere assay, we isolated tripotent ABCG2-expressing neural stem cells from embryonic mouse brain. This transgenic line is a powerful tool for studying the expression of ABCG2 in many tissues and for performing functional studies in different experimental settings.

  12. MBL-II-141, a chromone derivative, enhances irinotecan (CPT-11) anticancer efficiency in ABCG2-positive xenografts

    PubMed Central

    Honorat, Mylène; Guitton, Jérôme; Gauthier, Charlotte; Bouard, Charlotte; Lecerf-Schmidt, Florine; Peres, Basile; Terreux, Raphaël; Gervot, Héloïse; Rioufol, Catherine; Boumendjel, Ahcène; Puisieux, Alain; Di Pietro, Attilio; Payen, Léa

    2014-01-01

    ABCG2 is responsible for the multidrug resistance (MDR) phenotype, and strongly modulates cancer outcomes. Its high expression at a number of physiological barriers, including blood-brain and intestinal barriers, impacts on drug pharmacokinetics parameters. We characterized MBL-II-141, a specific and potent ABCG2 inhibitor. Combination of 10 mg/kg MBL-II-141 with the anticancer agent CPT-11 completely blocked the growth of 90% freshly implanted ABCG2-positive tumors. Moreover, the same combination slowed the growth of already established tumors. As required for preclinical development, we defined the main pharmacokinetics parameters of MBL-II-141 and its influence on the kinetics of CPT-11 and its active metabolite SN-38 in mice. MBL-II-141 distribution into the brain occurred at a low, but detectable, level. Interestingly, preliminary data suggested that MBL-II-141 is well tolerated (at 50 mg/kg) and absorbed upon force-feeding. MBL-II-141 induced a potent sensitization of ABCG2-positive xenografts to CPT-11 through in vivo ABCG2 inhibition. MBL-II-141 strongly increased CPT-11 levels in the brain, and therefore would be a valuable agent to improve drug distribution into the brain to efficiently treat aggressive gliomas. Safety and other pharmacological data strongly support the reglementary preclinical development of MBL-II-141. PMID:25474134

  13. 4-Anilino-2-pyridylquinazolines and -pyrimidines as Highly Potent and Nontoxic Inhibitors of Breast Cancer Resistance Protein (ABCG2).

    PubMed

    Krapf, Michael K; Gallus, Jennifer; Wiese, Michael

    2017-05-25

    Multidrug resistance (MDR) mediated by ATP-binding cassette (ABC) transport proteins remains a major problem in the chemotherapeutic treatment of cancer and might be overcome by inhibition of the transporter. Because of the lack of understanding, the complex mechanisms involved in the transport process, in particular for breast cancer resistance protein (BCRP/ABCG2), there is a persistent need for studies of inhibitors of ABCG2. In this study, we investigated a systematic series of 4-substituted-2-pyridylquinazolines in terms of their inhibitory potency as well as selectivity toward ABCG2. For comparison, the quinazoline scaffold was reduced to the significantly smaller 4-methylpyrimidine basic structure. Furthermore, the cytotoxicity and the ability to reverse MDR was tested with the chemotherapeutic agents SN-38 and mitoxantrone (MX). Interaction of the compounds with ABCG2 was investigated by a colorimetric ATPase assay. Enzyme kinetic studies were carried out with Hoechst 33342 as fluorescent dye and substrate of ABCG2 to elucidate the compounds binding modes.

  14. New, highly potent and non-toxic, chromone inhibitors of the human breast cancer resistance protein ABCG2.

    PubMed

    Pires, Amanda do Rocio Andrade; Lecerf-Schmidt, Florine; Guragossian, Nathalie; Pazinato, Jaqueline; Gozzi, Gustavo Jabor; Winter, Evelyn; Valdameri, Glaucio; Veale, Alexander; Boumendjel, Ahcène; Di Pietro, Attilio; Pérès, Basile

    2016-10-21

    Breast cancer resistance protein (BCRP/ABCG2) is one of the major transporters involved in the efflux of anticancer compounds, contributing to multidrug resistance (MDR). Inhibition of ABCG2-mediated transport is then considered a promising strategy for overcoming MDR in tumors. We recently identified a chromone derivative, namely MBL-II-141 as a selective ABCG2 inhibitor, with relevant in vivo activity. Here, we report the pharmacomodulation of MBL-II-141, with the aim of identifying key pharmacophoric elements to design more potent selective and non-toxic inhibitors. Through rational structural modifications of MBL-II-141, using simple and affordable chemistry, we obtained highly active and easily-made inhibitors of ABCG2. Among the investigated compounds, derivative 4a, was found to be 3-fold more potent than MBL-II-141. It was similarly efficient as the reference inhibitor Ko143 but with the advantage of a lower intrinsic cytotoxicity, and therefore constitutes the best ABCG2 inhibitor ever reported displaying a very high therapeutic ratio. Copyright © 2016. Published by Elsevier Masson SAS.

  15. Optimization of Acryloylphenylcarboxamides as Inhibitors of ABCG2 and Comparison with Acryloylphenylcarboxylates.

    PubMed

    Kraege, Stefanie; Stefan, Katja; Köhler, Sebastian C; Wiese, Michael

    2016-11-21

    ABCG2 belongs to the superfamily of ATP binding cassette (ABC) proteins and is associated with the limited success of anticancer chemotherapy, given its responsibility for the cross-resistance of tumor cells, known as multidrug resistance (MDR). Several classes of ABCG2 inhibitors were developed for increasing the efficacy of chemotherapy. A series of chalcones coupled to an additional aromatic residue was synthesized and investigated for their inhibition of ABC transporters. In our previous work we determined the preferred position of the linker on the A-ring to be ortho, and found several substitution patterns at the additional ring that improved potency. In this study we investigated whether a methoxy group that improved the inhibitory activity of chalcones would also be beneficial for the acryloylphenylcarboxamide scaffold. Indeed, this modification led to highly potent ABCG2 inhibitors. To support the hypothesis of a beneficial effect of the amide linker, six acryloylphenylcarboxylates were synthesized and investigated for their inhibitory activity. Replacement of the amide linker with an ester group resulted in decreased inhibition. Molecular modeling showed that the conformational preference of both series differs, thereby explaining the positive effect of the amide linker. Several compounds were characterized in detail by investigating their intrinsic cytotoxicity and capacity to reverse MDR in MTT assays and their effect on vanadate-sensitive ATPase activity. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Modulation of ABCC1 and ABCG2 proteins by ouabain in human breast cancer cells.

    PubMed

    DA Silva, Vanessa Amil; DA Silva, Karla Andreza Elizeu Pereira; Delou, João Marcos Azevedo; DA Fonseca, Leonardo Marques; Lopes, Anibal Gil; Capella, Márcia Alves Marques

    2014-03-01

    ABCC1 and ABCG2 are two transporters associated with multi-drug resistance to cancer chemotherapy. Ouabain is a cardiotonic steroid, currently considered as a hormone associated with arterial hypertension. Previous studies have suggested that ouabain can modulate ABCB1 and ABCC1 expression in cancer and renal cell lines. The present study investigated the effects of physiological concentrations of ouabain on the expression and activity of ABCC1 and ABCG2 in two human breast cancer cell lines, MCF7 and MDA-MB-231, the first known to be responsive to estrogens. Cell viability and proliferation assays showed that 1 μM ouabain reduced proliferation of MCF7, but not if MDA-MB-231 cells. On the other hand, 10 nM ouabain increased proliferation of MDA-MB-231, but not of MCF7 cells. Ouabain (10 nM) prevented the cytotoxic effects of doxorubicin in MCF7 cells, but not in MDA-MB-231 cells. Treatment of cells under different ouabain concentrations for 24 h did not cause any significant effects in the expression of ABCG2 or ABCC1 in either cell line. However, the activity of ABCC1 was increased when MCF7 and MDA-MB-231 cells were treated with 10 mM and 1 nM ouabain respectively. These results claim attention to the possibility that breast cancer patients with high levels of endogenous ouabain may have different responses to chemotherapy.

  17. Hedgehog pathway inhibitor HhAntag691 is a potent inhibitor of ABCG2/BCRP and ABCB1/Pgp.

    PubMed

    Zhang, Yimao; Laterra, John; Pomper, Martin G

    2009-01-01

    HhAntag691 (GDC-0449), a low-molecular weight inhibitor of the tumor-promoting hedgehog (Hh) signaling pathway, has been used to treat medulloblastoma in animal models and has recently entered clinical trials for a variety of solid tumors. Here, we show that HhAntag691 inhibits multiple ATP-binding cassette (ABC) transporters. ATP-binding cassette transporters are within a family of membrane proteins, the overexpression of which is associated with multidrug resistance, a major impediment to successful cancer treatment. HhAntag691 is a potent inhibitor of two ABC transporters, ABCG2/BCRP and ABCB1/Pgp, and is a mild inhibitor of ABCC1/MRP1. In ABCG2-overexpressing HEK293 cells, HhAntag691 increased retention of the fluorescent ABCG2 substrate BODIPY-prazosin and resensitized these cells to mitoxantrone, an antineoplastic ABCG2 substrate. In Madin-Darby canine kidney II cells engineered to overexpress Pgp or MRP1, HhAntag691 increased the retention of calcein-AM and resensitized them to colchicine. HhAntag691 also resensitized human non-small cell lung carcinoma cells NCI-H460/par and NCI-H460/MX20, which overexpress ABCG2 in response to mitoxantrone, to mitoxantrone, and to topotecan or SN-38. The IC(50) values of HhAntag691 for inhibition of ABCG2 and Pgp were approximately 1.4 and approximately 3.0 microM, respectively. Because ABC transporters are highly expressed at the blood-brain barrier and on many tumor cells, they contribute significantly to treatment failure of many types of cancer, particularly of those within the neuraxis. In addition to its effect on Hh signaling, the ability of HhAntag691 and related compounds to inhibit two key ABC transporters could contribute to their effectiveness in treating malignancies.

  18. Phenolic indeno[1,2-b]indoles as ABCG2-selective potent and non-toxic inhibitors stimulating basal ATPase activity

    PubMed Central

    Gozzi, Gustavo Jabor; Bouaziz, Zouhair; Winter, Evelyn; Daflon-Yunes, Nathalia; Honorat, Mylène; Guragossian, Nathalie; Marminon, Christelle; Valdameri, Glaucio; Bollacke, Andre; Guillon, Jean; Pinaud, Noël; Marchivie, Mathieu; Cadena, Silvia M; Jose, Joachim; Le Borgne, Marc; Di Pietro, Attilio

    2015-01-01

    Ketonic indeno[1,2-b]indole-9,10-dione derivatives, initially designed as human casein kinase II (CK2) inhibitors, were recently shown to be converted into efficient inhibitors of drug efflux by the breast cancer resistance protein ABCG2 upon suited substitutions including a N5-phenethyl on C-ring and hydrophobic groups on D-ring. A series of ten phenolic and seven p-quinonic derivatives were synthesized and screened for inhibition of both CK2 and ABCG2 activities. The best phenolic inhibitors were about threefold more potent against ABCG2 than the corresponding ketonic derivatives, and showed low cytotoxicity. They were selective for ABCG2 over both P-glycoprotein and MRP1 (multidrug resistance protein 1), whereas the ketonic derivatives also interacted with MRP1, and they additionally displayed a lower interaction with CK2. Quite interestingly, they strongly stimulated ABCG2 ATPase activity, in contrast to ketonic derivatives, suggesting distinct binding sites. In contrast, the p-quinonic indenoindoles were cytotoxic and poor ABCG2 inhibitors, whereas a partial inhibition recovery could be reached upon hydrophobic substitutions on D-ring, similarly to the ketonic derivatives. PMID:26170632

  19. ABCG2-overexpressing S1-M1-80 cell xenografts in nude mice keep original biochemistry and cell biological properties.

    PubMed

    Wang, Fang; Liang, Yong-Ju; Wu, Xing-Ping; Su, Xiao-Dong; Fu, Li-Wu

    2012-03-01

    S1-M1-80 cells, derived from human colon carcinoma S1 cells, are mitoxantrone-selected ABCG2-overexpressing cells and are widely used in in vitro studies of multidrug resistance(MDR). In this study, S1-M1-80 cell xenografts were established to investigate whether the MDR phenotype and cell biological properties were maintained in vivo. Our results showed that the proliferation, cell cycle, and ABCG2 expression level in S1-M1-80 cells were similar to those in cells isolated from S1-M1-80 cell xenografts (named xS1-M1-80 cells). Consistently, xS1-M1-80 cells exhibited high levels of resistance to ABCG2 substrates such as mitoxantrone and topotecan, but remained sensitive to the non-ABCG2 substrate cisplatin. Furthermore, the specific ABCG2 inhibitor Ko143 potently sensitized xS1-M1-80 cells to mitoxantrone and topotecan. These results suggest that S1-M1-80 cell xenografts in nude mice retain their original cytological characteristics at 9 weeks. Thus, this model could serve as a good system for further investigation of ABCG2-mediated MDR.

  20. Osimertinib (AZD9291) Enhanced the Efficacy of Chemotherapeutic Agents in ABCB1- and ABCG2-Overexpressing Cells In Vitro, In Vivo, and Ex Vivo.

    PubMed

    Chen, Zhen; Chen, Yifan; Xu, Meng; Chen, Likun; Zhang, Xu; To, Kenneth Kin Wah; Zhao, Hongyun; Wang, Fang; Xia, Zhongjun; Chen, Xiaoqin; Fu, Liwu

    2016-08-01

    The overexpression of ATP-binding cassette (ABC) transporters has been proved to be a major trigger for multidrug resistance (MDR) in certain types of cancer. In our study, we investigated whether osimertinib (AZD9291), a third-generation irreversible tyrosine kinase inhibitor of both activating EGFR mutations and resistance-associated T790M point mutation, could reverse MDR induced by ABCB1 and ABCG2 in vitro, in vivo, and ex vivo Our results showed that osimertinib significantly increased the sensitivity of ABCB1- and ABCG2-overexpressing cells to their substrate chemotherapeutic agents in vitro and in the model of ABCB1-overexpressing KBv200 cell xenograft in nude mice. Mechanistically, osimertinib increased the intracellular accumulations of doxorubicin (DOX) and Rhodamine 123 (Rho 123) by inhibiting the efflux function of the transporters in ABCB1- or ABCG2-overexpressing cells but not in their parental sensitive cells. Furthermore, osimertinib stimulated the ATPase activity of both ABCB1 and ABCG2 and competed with the [(125)I] iodoarylazidoprazosin photolabeling bound to ABCB1 or ABCG2, but did not alter the localization and expression of ABCB1 or ABCG2 in mRNA and protein levels nor the phosphorylations of EGFR, AKT, and ERK. Importantly, osimertinib also enhanced the cytotoxicity of DOX and intracellular accumulation of Rho 123 in ABCB1-overexpressing primary leukemia cells. Overall, these findings suggest osimertinib reverses ABCB1- and ABCG2-mediated MDR via inhibiting ABCB1 and ABCG2 from pumping out chemotherapeutic agents and provide possibility for cancer combinational therapy with osimertinib in the clinic. Mol Cancer Ther; 15(8); 1845-58. ©2016 AACR.

  1. Synthesis and biological investigation of 2,4-substituted quinazolines as highly potent inhibitors of breast cancer resistance protein (ABCG2).

    PubMed

    Krapf, Michael K; Gallus, Jennifer; Wiese, Michael

    2017-08-10

    Expression of ABCG2, a member of the ABC transporter superfamily, has been correlated to the clinical outcome of multiple cancers and is often associated with the occurrence of multidrug resistance (MDR) in chemotherapy. Inhibition of the transport protein by potent and selective inhibitors might be a way to treat cancer more efficiently and improve the therapy of cancer patients. Recently we reported the synthesis of new inhibitors based on a quinazoline scaffold. In the present study more structural variations were explored. Compounds with 3,4-dimethoxy groups and meta or para nitro substituents were found to be highly potent inhibitors of ABCG2. The most potent compound was more than five-fold more potent than Ko143, one of the best inhibitors of ABCG2. To determine the new compounds selectivity toward ABCG2 their inhibitory effects on ABCB1 and ABCC1 were also investigated identifying selective as well as broadspectrum inhibitors. Furthermore, intrinsic cytotoxicity and efficacy regarding the reversal of multidrug resistance toward SN-38 and mitoxantrone were explored. The most potent compounds were able to reverse the resistance toward the cytostatic agents with EC50 values below 20 nM. Additionally, the type of interaction between inhibitors and the ABCG2 substrate Hoechst 33342 was investigated yielding competitive and non-competitive interactions suggesting different modes of binding. Finally the effect of the derivatives on vanadate-sensitive ATPase activity of ABCG2 was determined. According to the different effects on ATPase activity we conclude the existence of different binding sites. This study provides the structural requirements for high potency inhibition and elucidates the interaction with ABCG2 setting the basis for further studies. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. [Expression and significance of ABCG2 in human malignant glioma].

    PubMed

    Chu, Liang; Huang, Qiang; Zhai, De-Zhong; Zhu, Qing; Huo, Hong-Mei; Dong, Jun; Qian, Zhi-Yuan; Wang, Ai-Dong; Lan, Qing; Gao, Yi-Lu

    2007-10-01

    ATP-binding cassette transporter protein ABCG2 is a marker derived from hematopoietic stem cells. However, its role in tumorigenesis and malignant progression of glioma is unclear. This study was to investigate the expression and significance of ABCG2 in gliomas of different malignant grades. A microarray chip containing glioma tissues of different malignant grades, implanted glioma xenografts in nude mice, spheroids of glioma cell lines and glioma stem cells was prepared and examined for the expression of ABCG2 with immunohistochemical staining. The positive rate of ABCG2 was 26.8% in the 71 specimens of human glioma tissues, with 11.1% in grade I gliomas, 8% in grade II gliomas, 43.5% in grade III gliomas, and 42.9% in grade IV gliomas; it was significantly higher in grade III-IV gliomas than in grade I-II gliomas (chi2=10.710, P=0.001). The positive rate of ABCG2 was 100% in implanted glioma xenografts in nude mice, gliomas stem cells, and neural stem cells. It was also expressed in some normal tissues. The positive cells surrounded and invaded into vessels in glioma tissues. ABCG2 is overexpressed in glioma stem cells, glioma tissues of higher grades, and implanted glioma xenografts. The positive cells distribute around vessels in glioma tissues.

  3. Synthesis and Biological Evaluation of 4-Anilino-quinazolines and -quinolines as Inhibitors of Breast Cancer Resistance Protein (ABCG2).

    PubMed

    Krapf, Michael K; Wiese, Michael

    2016-06-09

    Chemotherapeutic treatment of cancer often fails due to overexpression of the ATP-binding cassette (ABC) transport proteins, like ABCG2, triggering active efflux of various structurally unrelated drugs. This so-called multidrug resistance (MDR) may be reversed by selective, potent, and nontoxic inhibitors of ABCG2. As only a few potent inhibitors are known, new compounds based on a 4-substituted-2-phenylquinazoline scaffold were investigated. Substitution with hydroxy, cyano, nitro, acetamido, and fluoro led to high inhibitory activities toward ABCG2. The ability to reverse MDR of the most active compounds was confirmed in a MTT efficacy assay. Moreover, a negligibly low intrinsic cytotoxicity was found resulting in a high therapeutic ratio. Investigations of the inhibitory activity toward ABCB1 and ABCC1 yielded a high selectivity toward ABCG2 for the quinazoline compounds. Quinoline-based analogues showed lower inhibitory activity and selectivity. The study yielded a variety of promising compounds, some with superior properties compared to those of the standard inhibitor Ko143.

  4. ABCG2-overexpressing H460/MX20 cell xenografts in athymic nude mice maintained original biochemical and cytological characteristics

    PubMed Central

    Zhang, Wei; Chen, Zhen; Chen, Likun; Wang, Fang; Li, Furong; Wang, Xiaokun; Fu, Liwu

    2017-01-01

    H460/MX20 are derived from large cell lung cancer H460 cell line and then transformed into ABCG2-overexpressing cells by mitoxantrone’s induction, which are widely used in study of multidrug resistance (MDR) in vitro. To establish and spread the model of H460/MX20 cell xenografts, we investigated whether cell biological characteristics and the MDR phenotype were maintained in vivo model. Our results demonstrated that the cell proliferation, cell cycle, and ABCG2 expression level in xH460/MX20 cells isolated from H460/MX20 cell xenografts were similar to H460/MX20 cells in vitro. Importantly, xH460/MX20 cells exhibited high levels of resistance to ABCG2 substrates such as mitoxantrone and topotecan as H460/MX20 cells did. Furthermore, lapatinib, the inhibitor of ABCG2, potently reversed mitoxantrone- and topotecan-resistance of xH460/MX20 cells. Taken together, these results suggest that H460/MX20 cell xenografts in athymic nude mice still retain their original cytological characteristics and MDR phenotype. Thus, the H460/MX20 cell xenografts model could serve as a sound model in vivo for study on reversal MDR. PMID:28059154

  5. ABCG2-overexpressing H460/MX20 cell xenografts in athymic nude mice maintained original biochemical and cytological characteristics.

    PubMed

    Zhang, Wei; Chen, Zhen; Chen, Likun; Wang, Fang; Li, Furong; Wang, Xiaokun; Fu, Liwu

    2017-01-06

    H460/MX20 are derived from large cell lung cancer H460 cell line and then transformed into ABCG2-overexpressing cells by mitoxantrone's induction, which are widely used in study of multidrug resistance (MDR) in vitro. To establish and spread the model of H460/MX20 cell xenografts, we investigated whether cell biological characteristics and the MDR phenotype were maintained in vivo model. Our results demonstrated that the cell proliferation, cell cycle, and ABCG2 expression level in xH460/MX20 cells isolated from H460/MX20 cell xenografts were similar to H460/MX20 cells in vitro. Importantly, xH460/MX20 cells exhibited high levels of resistance to ABCG2 substrates such as mitoxantrone and topotecan as H460/MX20 cells did. Furthermore, lapatinib, the inhibitor of ABCG2, potently reversed mitoxantrone- and topotecan-resistance of xH460/MX20 cells. Taken together, these results suggest that H460/MX20 cell xenografts in athymic nude mice still retain their original cytological characteristics and MDR phenotype. Thus, the H460/MX20 cell xenografts model could serve as a sound model in vivo for study on reversal MDR.

  6. Camptothecin and cisplatin upregulate ABCG2 and MRP2 expression by activating the ATM/NF-κB pathway in lung cancer cells.

    PubMed

    Ke, Shi Zhong; Ni, Xiao Yan; Zhang, Yue Hua; Wang, Yi Nan; Wu, Bin; Gao, Feng Guang

    2013-04-01

    Multidrug resistance (MDR) formation is an important problem in lung cancer chemotherapy. Our study showed that both camptothecin and cisplatin could not only induce ATM and NF-κB activation but also upregulate expression of the MDR-related genes ABCG2, MRP2 in NCI-H446 cells. Moreover, camptothecin and cisplatin-induced ABCG2 and MRP2 upregulation could be impaired by ATM and NF-κB inhibitors, indicating a relationship between ATM, NF-κB activation and MDR formation in lung cancer chemo-therapy. Our study indicates that ATM may serve as a potential molecular target for MDR formation in lung cancer chemotherapy.

  7. Targeting BCRP/ABCG2 by RNA interference enhances the chemotherapy sensitivity of human colon cancer side population cells.

    PubMed

    Hu, Jun; Li, Jian; Yue, Xin; Wang, Jia-Cang; Wang, Jun-Feng; Liu, Jian-Zhong; Kong, Da-Lu

    2017-04-01

    Relapse and metastasis are frequent in colon cancer and may be linked to stem cell characteristics. This study isolated side population (SP) cells from a colon cancer cell line (Colo-320) and examined their self-renewal and differentiation abilities. Compared to non-SP (NSP) cells, SP colon cancer cells were more tumorigenic in vivo and exhibited more invasive characteristics and a greater ability to form colonies. Additionally, more cells were in G0/G1 phase and more highly expressed the multidrug resistance protein BCRP/ABCG2. We achieved enhanced chemotherapy sensitivity by transfecting SP cells with a hairpin-like, small interfering RNA (siRNA) eukaryotic expression plasmid targeting BCRP/ABCG2.

  8. ABCG2 in peptic ulcer: gene expression and mutation analysis.

    PubMed

    Salagacka-Kubiak, Aleksandra; Żebrowska, Marta; Wosiak, Agnieszka; Balcerczak, Mariusz; Mirowski, Marek; Balcerczak, Ewa

    2016-08-01

    The aim of this study was to evaluate the participation of polymorphism at position C421A and mRNA expression of the ABCG2 gene in the development of peptic ulcers, which is a very common and severe disease. ABCG2, encoded by the ABCG2 gene, has been found inter alia in the gastrointestinal tract, where it plays a protective role eliminating xenobiotics from cells into the extracellular environment. The materials for the study were biopsies of gastric mucosa taken during a routine endoscopy. For genotyping by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) at position C421A, DNA was isolated from 201 samples, while for the mRNA expression level by real-time PCR, RNA was isolated from 60 patients. The control group of healthy individuals consisted of 97 blood donors. The dominant genotype in the group of peptic ulcer patients and healthy individuals was homozygous CC. No statistically significant differences between healthy individuals and the whole group of peptic ulcer patients and, likewise, between the subgroups of peptic ulcer patients (infected and uninfected with Helicobacter pylori) were found. ABCG2 expression relative to GAPDH expression was found in 38 of the 60 gastric mucosa samples. The expression level of the gene varies greatly among cases. The statistically significant differences between the intensity (p = 0.0375) of H. pylori infection and ABCG2 gene expression have been shown. It was observed that the more intense the infection, the higher the level of ABCG2 expression.

  9. Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein

    SciTech Connect

    Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V.; Xia, Di

    2016-07-27

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space groupP1), with unit-cell parametersa= 40.67,b= 44.91,c= 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.

  10. Crystal structure of the antigen-binding fragment of a monoclonal antibody specific for the multidrug-resistance-linked ABC transporter human P-glycoprotein.

    PubMed

    Esser, Lothar; Shukla, Suneet; Zhou, Fei; Ambudkar, Suresh V; Xia, Di

    2016-08-01

    P-glycoprotein (P-gp) is a polyspecific ATP-dependent transporter linked to multidrug resistance in cancers that plays important roles in the pharmacokinetics of a large number of drugs. The drug-resistance phenotype of P-gp can be modulated by the monoclonal antibody UIC2, which specifically recognizes human P-gp in a conformation-dependent manner. Here, the purification, sequence determination and high-resolution structure of the Fab fragment of UIC2 (UIC2/Fab) are reported. Purified UIC2/Fab binds human P-gp with a 1:1 stoichiometry. Crystals of UIC2/Fab are triclinic (space group P1), with unit-cell parameters a = 40.67, b = 44.91, c = 58.09 Å, α = 97.62, β = 99.10, γ = 94.09°, and diffracted X-rays to 1.6 Å resolution. The structure was determined by molecular replacement and refined to 1.65 Å resolution. The asymmetric unit contains one molecule of UIC2/Fab, which exhibits a positively charged antigen-binding surface, suggesting that it might recognize an oppositely charged extracellular epitope of P-gp.

  11. The ABCG2 efflux transporter from rabbit placenta: Cloning and functional characterization.

    PubMed

    Halwachs, Sandra; Kneuer, Carsten; Gohlsch, Katrin; Müller, Marian; Ritz, Vera; Honscha, Walther

    2016-02-01

    In human placenta, the ATP-binding cassette efflux transporter ABCG2 is highly expressed in syncytiotrophoblast cells and mediates cellular excretion of various drugs and toxins. Hence, physiological ABCG2 activity substantially contributes to the fetoprotective placenta barrier function during gestation. Developmental toxicity studies are often performed in rabbit. However, despite its toxicological relevance, there is no data so far on functional ABCG2 expression in this species. Therefore, we cloned ABCG2 from placenta tissues of chinchilla rabbit. Sequencing showed 84-86% amino acid sequence identity to the orthologues from man, rat and mouse. We transduced the rabbit ABCG2 clone (rbABCG2) in MDCKII cells and stable rbABCG2 gene and protein expression was shown by RT-PCR and Western blot analysis. The rbABCG2 efflux activity was demonstrated with the Hoechst H33342 assay using the specific ABCG2 inhibitor Ko143. We further tested the effect of established human ABCG2 (hABCG2) drug substrates including the antibiotic danofloxacin or the histamine H2-receptor antagonist cimetidine on H33342 accumulation in MDCKII-rbABCG2 or -hABCG2 cells. Human therapeutic plasma concentrations of all tested drugs caused a comparable competitive inhibition of H33342 excretion in both ABCG2 clones. Altogether, we first showed functional expression of the ABCG2 efflux transporter in rabbit placenta. Moreover, our data suggest a similar drug substrate spectrum of the rabbit and the human ABCG2 efflux transporter. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. Hedgehog signaling regulates drug sensitivity by targeting ABC transporters ABCB1 and ABCG2 in epithelial ovarian cancer.

    PubMed

    Chen, Yi; Bieber, Marcia M; Teng, Nelson N H

    2014-08-01

    A major challenge of successful chemotherapy in ovarian cancer is overcoming intrinsic or acquired multi-drug resistance caused by active drug efflux mediated by ATP-binding cassette (ABC) transporters. Regulation of these transporters in ovarian cancer is poorly understood. We have found that abnormal expression of the hedgehog (Hh) signaling pathway transcription factor Gli1 is involved in the regulation of ABC transporters ABCB1 and ABCG2 in ovarian cancer. Hh is a known regulator of cancer cell proliferation and differentiation in several other types of invasive and metastatic malignancies. Our work has demonstrated that Gli1 is abnormally activated in a portion of ovarian cancers. Inhibition of Gli1 expression decreases ABCB1 and ABCG2 gene expression levels and enhances the response of ovarian cancer cells to certain chemotherapeutic drugs. The underlying mechanism is a direct association of Gli1 with a specific consensus sequence located in the promoter region of ABCB1 and ABCG2 genes. This study provides new understanding of ABC gene regulation by Hh signaling pathway, which may lead to the identification of new markers to detect and to anticipate ovarian cancer chemotherapy drug sensitivity.

  13. Gemcitabine upregulates ABCG2/BCRP and modulates the intracellular pharmacokinetic profiles of bioluminescence in pancreatic cancer cells.

    PubMed

    Sun, Yue; Gu, Mancang; Zhu, Lixin; Liu, Junying; Xiong, Yang; Wei, Yinghui; Li, Fanzhu

    2016-03-01

    A lack of methods capable of exploring real-time intracellular drug deposition has since limited the investigation of gemcitabine-induced multidrug resistance in vitro and in vivo. Specifically, resistance induced by D-luciferin, a substrate of the breast cancer resistance protein (ABCG2/BCRP), has recently attracted clinical attention, but further investigation has been limited. Herein, the intracellular pharmacokinetic behavior of D-luciferin was investigated in pancreatic cancer cell lines in real time by using bioluminescence imaging. To achieve this feat, BxPC3 and Panc1 pancreatic cancer cells overexpressing firefly luciferase were treated with gemcitabine in a dose and time gradient manner in vitro. The intracellular pharmacokinetic profiles of each group were then determined through the acquisition of bioluminescent signal intensity of D-luciferin in cells. Simultaneously, key pharmacokinetic parameters including area under the curve, elimination rate constant (K), and mean resident time were calculated according to the noncompartment model. ABCG2 protein levels following gemcitabine treatment were detected through western blot, and gemcitabine showed no significant effect on luciferase activity over dimethyl sulfoxide (DMSO) as a control (P>0.05). However, gemcitabine significantly increased K values while suppressing area under the curve and mean resident time compared with DMSO (P<0.05) and increased ABCG2 expression over DMSO-treated cells. In addition, gemcitabine increased the elimination rate of the ABCG2 substrate, D-luciferin, and decreased D-luciferin accumulation in BxPC3 and Panc1 cells in a dose-response manner. Advances made herein illustrate the versatility of the in-vitro bioluminescent model and its capability to observe the onset of chemoresistance in real time.

  14. ABCG2 expression, function, and promoter methylation in human multiple myeloma

    PubMed Central

    Turner, Joel G.; Gump, Jana L.; Zhang, Chunchun; Cook, James M.; Marchion, Douglas; Hazlehurst, Lori; Munster, Pamela; Schell, Michael J.; Dalton, William S.; Sullivan, Daniel M.

    2006-01-01

    We investigated the role of the breast cancer resistance protein (BCRP/ABCG2) in drug resistance in multiple myeloma (MM). Human MM cell lines, and MM patient plasma cells isolated from bone marrow, were evaluated for ABCG2 mRNA expression by quantitative polymerase chain reaction (PCR) and ABCG2 protein, by Western blot analysis, immunofluorescence microscopy, and flow cytometry. ABCG2 function was determined by measuring topotecan and doxorubicin efflux using flow cytometry, in the presence and absence of the specific ABCG2 inhibitor, tryprostatin A. The methylation of the ABCG2 promoter was determined using bisulfite sequencing. We found that ABCG2 expression in myeloma cell lines increased after exposure to topotecan and doxorubicin, and was greater in logphase cells when compared with quiescent cells. Myeloma patients treated with topotecan had an increase in ABCG2 mRNA and protein expression after treatment with topotecan, and at relapse. Expression of ABCG2 is regulated, at least in part, by promoter methylation both in cell lines and in patient plasma cells. Demethylation of the promoter increased ABCG2 mRNA and protein expression. These findings suggest that ABCG2 is expressed and functional in human myeloma cells, regulated by promoter methylation, affected by cell density, up-regulated in response to chemotherapy, and may contribute to intrinsic drug resistance. PMID:16917002

  15. Functional characterization of the ABCG2 5' non-coding exon variants: Stem cell specificity, translation efficiency and the influence of drug selection.

    PubMed

    Sándor, Sára; Jordanidisz, Theodora; Schamberger, Anita; Várady, György; Erdei, Zsuzsa; Apáti, Ágota; Sarkadi, Balázs; Orbán, Tamás I

    2016-07-01

    ABCG2 is a multidrug transporter with wide substrate specificity, and is believed to protect several cell types from various xenobiotics and endobiotics. This "guardian" function is important in numerous cell types and tissue barriers but becomes disadvantageous by being responsible for the multidrug resistance phenotype in certain tumor cells. ABCG2 regulation at the protein level has already been extensively studied, however, regulation at the mRNA level, especially the functional role of the various 5' untranslated exon variants (5' UTRs) has been elusive. In the present work, we describe a comprehensive characterization of four ABCG2 mRNA variants with different exon 1 sequences, investigate drug inducibility, stem cell specificity, mRNA stability, and translation efficiency. Although certain variants (E1B and E1C) are considered as "constitutive" mRNA isoforms, we show that chemotoxic drugs significantly alter the expression pattern of distinct ABCG2 mRNA isoforms. When examining human embryonic stem cell lines, we provide evidence that variant E1A has an expression pattern coupled to undifferentiated stem cell stage, as its transcript level is regulated parallel to mRNAs of Oct4 and Nanog pluripotency marker genes. When characterizing the four exon 1 variants we found no significant differences in terms of mRNA stabilities and half-lives of the isoforms. In contrast, variant E1U showed markedly lower translation efficiency both at the total protein level or regarding the functional presence in the plasma membrane. Taken together, these results indicate that the different 5' UTR variants play an important role in cell type specific regulation and fine tuning of ABCG2 expression.

  16. ABCG2 deficiency in skin impairs re-epithelialization in cutaneous wound healing.

    PubMed

    Chang, Hsiao-Min; Huang, Wen-Yen; Lin, Sung-Jan; Huang, Wei-Chao; Shen, Chia-Rui; Mao, Wan-Yu; Shen, Chia-Ning

    2016-05-01

    The ATP-binding cassette transporter ABCG2 is expressed in the interfollicular epidermis and mediates the side-population phenotype in skin cells. However, the role of ABCG2 in skin is unclear. Increased expression levels of ABCG2 were found at the basal layer of transitional epidermis adjacent to cutaneous wounds in human patients, indicating that ABCG2 may be involved in regulating the wound healing process. To investigate the role of ABCG2 in cutaneous wound healing, full-thickness skin wounds were created in ABCG2 knockout (ABCG2-KO) and wild-type mice. The healing process was analysed and revealed that ABCG2 deficiency in skin results in delays in wound closure and impairments in re-epithelialization, as evidenced by reductions in both suprabasal differentiation and in p63-expressing keratinocytes migrating from transitional epidermis to epithelial tongues. The reduction in p63-expressing cells may be due to elevated levels of reactive oxygen species in ABCG2-KO epidermis, which can cause DNA damage and lead to proliferation arrest. To determine whether ABCG2 deficiency affects the potency of epidermal stem/progenitor cells (EPCs), transplantation studies were carried out, which demonstrated that ABCG2-KO EPCs display higher levels of γH2AX and lose the capacity to differentiate into suprabasal keratinocytes. A competitive repopulation assay confirmed that ABCG2 expression is critical for the proper expansion and differentiation of EPCs in cutaneous wounds. As EPCs are known to contribute to the healing of larger wounds, the current findings imply a functional role for ABCG2 in the expansion and differentiation of p63-expressing EPCs. Thus, ABCG2 deficiency in skin impairs re-epithelialization in cutaneous wound healing.

  17. Functional characterization of rainbow trout (Oncorhynchus mykiss) Abcg2a (Bcrp) transporter.

    PubMed

    Zaja, Roko; Popović, Marta; Lončar, Jovica; Smital, Tvrtko

    2016-12-01

    ABCG2 (BCRP - breast cancer resistance protein) belongs to the ATP-binding cassette (ABC) superfamily. It plays an important role in the disposition and elimination of xeno- and endobiotics and/or their metabolites in mammals. Likewise, the protective role of ABC transporters, including Abcg2, has been reported for aquatic organisms. In our previous study we have cloned the full gene sequence of rainbow trout (Oncorhynchus mykiss) Abcg2a and showed its high expression in liver and primary hepatocytes. Based on those insights, the main goal of this study was to perform a detailed functional characterization of trout Abcg2a using insect ovary cells (Spodoptera frugiperda, Sf9) as a heterologous expression system. Membrane vesicles preparations from Sf9 cells were used for the ATPase assay determinations and basic biochemical properties of fish Abcg2a versus human ABCG2 have been compared. A series of 39 physiologically and/or environmentally relevant substances was then tested on interaction with trout Abcg2a and human ABCG2. Correlation analysis reveals highly similar pattern of activation and inhibition. Significant activation of trout Abcg2a ATPase was observed for prazosin, doxorubicine, sildenafil, furosemid, propranolol, fenofibrate and pheophorbide. Pesticides showed either a weak activation (malathione) or strong (endosulfan) to weak (chlorpyrifos, fenoxycarb, DDE) inhibition of trout Abcg2a ATPase while the highest activation was obtained for benzo(a)pyrene, curcumine and testosterone. In conclusion, data from this study offer the first characterization of fish Abcg2a, reveal potent interactors among physiologically or environmentally relevant substances and point to similarities regarding strengths and interactor preferences between human ABCG2 and fish Abcg2a. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Downregulation of mdr1 and abcg2 genes is a mechanism of inhibition of efflux pumps mediated by polymeric amphiphiles.

    PubMed

    Cuestas, María L; Castillo, Amalia I; Sosnik, Alejandro; Mathet, Verónica L

    2012-11-01

    The ability of cells to acquire resistance to multiple pharmaceuticals, namely multidrug resistance (MDR), is often mediated by the over-expression of efflux transporters of the ATP-binding cassette (ABC) superfamily; for example P-glycoprotein (P-gp or MDR1), breast cancer resistance protein (BCRP or ABCG2), and multidrug resistance-associated protein MRP1. ABCs pump drug molecules out of cells against a concentration gradient, reducing their intracellular concentration. The ability of polymeric amphiphiles to inhibit ABCs as well as the cellular pathways involved in the inhibition has been extensively investigated. This work investigated for the first time the effect of branched poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamines) on the levels of mRNA encoding for MDR1, BCRP and MRP1, in a human hepatoma cell line (Huh7). Copolymers with a broad range of molecular weights and hydrophilic-lipophilic balances were assayed. Results confirmed the down-regulation of mdr1 and abcg2 genes. Conversely, the mrp1 gene was not affected. These findings further support the versatility of these temperature- and pH-responsive copolymers to overcome drug resistance in cancer and infectious diseases.

  19. Tyrosine kinase inhibitors enhance ciprofloxacin-induced phototoxicity by inhibiting ABCG2.

    PubMed

    Mealey, Katrina L; Dassanayake, Sandamali; Burke, Neal S

    2014-01-01

    The tyrosine kinase inhibitor (TKI) class of anticancer agents inhibits ABCG2-mediated drug efflux. ABCG2 is an important component of the blood-retinal barrier, where it limits retinal exposure to phototoxic compounds such as fluoroquinolone antibiotics. Patients treated with TKIs would be expected to be at greater risk for retinal phototoxicity. Using an in vitro system, our results indicate that the TKIs gefitinib and imatinib abrogate the ability of ABCG2 to protect cells against ciprofloxacin-induced phototoxicity. We conclude that the concurrent administration of ABCG2 inhibitors with photoreactive fluoroquinolone antibiotics may result in retinal damage.

  20. Abcg2-Labeled Cells Contribute to Different Cell Populations in the Embryonic and Adult Heart.

    PubMed

    Doyle, Michelle J; Maher, Travis J; Li, Qinglu; Garry, Mary G; Sorrentino, Brian P; Martin, Cindy M

    2016-02-01

    ATP-binding cassette transporter subfamily G member 2 (Abcg2)-expressing cardiac-side population cells have been identified in the developing and adult heart, although the role they play in mammalian heart growth and regeneration remains unclear. In this study, we use genetic lineage tracing to follow the cell fate of Abcg2-expressing cells in the embryonic and adult heart. During cardiac embryogenesis, the Abcg2 lineage gives rise to multiple cardiovascular cell types, including cardiomyocytes, endothelial cells, and vascular smooth muscle cells. This capacity for Abcg2-expressing cells to contribute to cardiomyocytes decreases rapidly during the postnatal period. We further tested the role of the Abcg2 lineage following myocardial injury. One month following ischemia reperfusion injury, Abcg2-expressing cells contributed significantly to the endothelial cell lineage, however, there was no contribution to regenerated cardiomyocytes. Furthermore, consistent with previous results showing that Abcg2 plays an important cytoprotective role during oxidative stress, we show an increase in Abcg2 labeling of the vasculature, a decrease in the scar area, and a moderate improvement in cardiac function following myocardial injury. We have uncovered a difference in the capacity of Abcg2-expressing cells to generate the cardiovascular lineages during embryogenesis, postnatal growth, and cardiac regeneration.

  1. Enhanced efficacy of photodynamic therapy by inhibiting ABCG2 in colon cancers.

    PubMed

    Kim, Ju Hee; Park, Jae Myung; Roh, Yoon Jin; Kim, In-Wook; Hasan, Tayyaba; Choi, Myung-Gyu

    2015-07-07

    Photodynamic therapy (PDT) contains a photosensitizing process, which includes cellular uptake of photosensitizer and delivery of light to the target. ATP-binding cassette subfamily G2 (ABCG2) regulates endogenous protoporphyrin levels. In human colon cancers, it is not fully examined the role of ABCG2 in porphyrin-based photodynamic therapy. SW480 and HT29 cells were selected because they showed low and high ABCG2 expression levels, respectively. Pyropheophorbid-a (PPa) was used as a photosensitizer. Cells were exposed to a 670 nm diod laser. Cell viability and necrosi apoptosis was examined. Production level of singlet oxygen was detected with the photomultiplier-tube s/ -based singlet oxygen detection system. SW480 cells, which expressed lower level of ABCG2, showed the higher uptake of PPa than HT-29 cells. The uptake level of PPa was significantly correlated with the decreased cell viability after PDT. Pretreatment with a ABCG2 inhibitor, Ko-143, significantly enhanced the PDT efficacy in HT29 cells compared to vehicle-pretreated cells. To confirm the ABCG2 effect on PDT, we established ABCG2 over-expressing stable cells in SW480 cells (SW480/ABCG2). Furthermore, SW480/ABCG2 cells showed significantly decreased PDT effect compared to the control cells. The increased or decreased cell survival was significantly correlated with the production level of singlet oxygen after PDT. ABCG2 plays an important role in determining the PDT efficacy by controlling the photosensitizer efflux rate. This implies the control of ABCG2 expression may be a potential solution to enhance photosensitivity.

  2. Gene and functional up-regulation of the BCRP/ABCG2 transporter in hepatocellular carcinoma

    PubMed Central

    2012-01-01

    Background The Breast Cancer Resistance Protein (BCRP/ABCG2) is one member of ABC transporters proteins super family responsible of drug resistance. Since data on ABCG2 expression in liver malignances are scanty, here we report the expression of ABCG2 in adult human hepatocellular carcinoma (HCC) in both in vivo and in vitro models with different degree of malignancy. Methods In cell lines derived from human hepatocellular carcinoma, ABCG2 gene expression was assessed by reverse transcription quantitative real time PCR and function by Hoechst 33342 efflux assay; protein content was assessed by SDS-PAGE Western blot. Results ABCG2 expression was found to be highest in the most undifferentiated cell lines, and this was related with a higher functional activity. ABCG2 expression was sensitive to antineoplastic drugs since exposure to 5 μM doxorubicin for 24 hours resulted in significant up-regulations of ABCG2 in all cell lines, particularly in those lines with low basal ABCG2 expression (p<0.01). The gene expression was also investigated in 51 adult liver tissues with HCC and related cirrhosis; normal liver tissue was used as control. ABCG2 gene expression was higher in HCC than both cirrhotic paired tissue and normal tissue. This up-regulation was greater (p<0.05) in pathological poorly differentiated grade G3/G4 than in well-differentiated G1/G2 HCC. Conclusions Our results suggest a correlation of ABCG2 gene expression and differentiation stage both in human and HCC derived cell lines. The rapid up-regulation of ABCG2 to exposure to doxorubicin emphasizes the importance of this transporter in accounting for drug resistance in liver tumors. PMID:23153066

  3. Brain Accumulation of Ponatinib and Its Active Metabolite, N-Desmethyl Ponatinib, Is Limited by P-Glycoprotein (P-GP/ABCB1) and Breast Cancer Resistance Protein (BCRP/ABCG2).

    PubMed

    Kort, Anita; van Hoppe, Stéphanie; Sparidans, Rolf W; Wagenaar, Els; Beijnen, Jos H; Schinkel, Alfred H

    2017-10-02

    Ponatinib is an oral BCR-ABL1 inhibitor for treatment of advanced leukemic diseases that carry the Philadelphia chromosome, specifically containing the T315I mutation yielding resistance to previously approved BCR-ABL1 inhibitors. Using in vitro transport assays and knockout mouse models, we investigated whether the multidrug efflux transporters ABCB1 and ABCG2 transport ponatinib and whether they, or the drug-metabolizing enzyme CYP3A, affect the oral availability and brain accumulation of ponatinib and its active N-desmethyl metabolite (DMP). In vitro, mouse Abcg2 and human ABCB1 modestly transported ponatinib. In mice, both Abcb1 and Abcg2 markedly restricted brain accumulation of ponatinib and DMP, but not ponatinib oral availability. Abcg2 deficiency increased DMP plasma levels ∼3-fold. Cyp3a deficiency increased the ponatinib plasma AUC 1.4-fold. Our results suggest that pharmacological inhibition of ABCG2 and ABCB1 during ponatinib therapy might benefit patients with brain (micro)metastases positioned behind an intact blood-brain barrier, or with substantial expression of these transporters in the malignant cells. CYP3A inhibitors might increase ponatinib oral availability, enhancing efficacy but possibly also toxicity of this drug.

  4. Abcg2 labels multiple cell types in skeletal muscle and participates in muscle regeneration

    PubMed Central

    Doyle, Michelle J.; Zhou, Sheng; Tanaka, Kathleen Kelly; Pisconti, Addolorata; Farina, Nicholas H.; Sorrentino, Brian P.

    2011-01-01

    Skeletal muscle contains progenitor cells (satellite cells) that maintain and repair muscle. It also contains muscle side population (SP) cells, which express Abcg2 and may participate in muscle regeneration or may represent a source of satellite cell replenishment. In Abcg2-null mice, the SP fraction is lost in skeletal muscle, although the significance of this loss was previously unknown. We show that cells expressing Abcg2 increased upon injury and that muscle regeneration was impaired in Abcg2-null mice, resulting in fewer centrally nucleated myofibers, reduced myofiber size, and fewer satellite cells. Additionally, using genetic lineage tracing, we demonstrate that the progeny of Abcg2-expressing cells contributed to multiple cell types within the muscle interstitium, primarily endothelial cells. After injury, Abcg2 progeny made a minor contribution to regenerated myofibers. Furthermore, Abcg2-labeled cells increased significantly upon injury and appeared to traffic to muscle from peripheral blood. Together, these data suggest an important role for Abcg2 in positively regulating skeletal muscle regeneration. PMID:21949413

  5. Hyperuricemia in acute gastroenteritis is caused by decreased urate excretion via ABCG2

    PubMed Central

    Matsuo, Hirotaka; Tsunoda, Tomoyuki; Ooyama, Keiko; Sakiyama, Masayuki; Sogo, Tsuyoshi; Takada, Tappei; Nakashima, Akio; Nakayama, Akiyoshi; Kawaguchi, Makoto; Higashino, Toshihide; Wakai, Kenji; Ooyama, Hiroshi; Hokari, Ryota; Suzuki, Hiroshi; Ichida, Kimiyoshi; Inui, Ayano; Fujimori, Shin; Shinomiya, Nariyoshi

    2016-01-01

    To clarify the physiological and pathophysiological roles of intestinal urate excretion via ABCG2 in humans, we genotyped ABCG2 dysfunctional common variants, Q126X (rs72552713) and Q141K (rs2231142), in end-stage renal disease (hemodialysis) and acute gastroenteritis patients, respectively. ABCG2 dysfunction markedly increased serum uric acid (SUA) levels in 106 hemodialysis patients (P = 1.1 × 10−4), which demonstrated the physiological role of ABCG2 for intestinal urate excretion because their urate excretion almost depends on intestinal excretion via ABCG2. Also, ABCG2 dysfunction significantly elevated SUA in 67 acute gastroenteritis patients (P = 6.3 × 10−3) regardless of the degree of dehydration, which demonstrated the pathophysiological role of ABCG2 in acute gastroenteritis. These findings for the first time show ABCG2-mediated intestinal urate excretion in humans, and indicates the physiological and pathophysiological importance of intestinal epithelium as an excretion pathway besides an absorption pathway. Furthermore, increased SUA could be a useful marker not only for dehydration but also epithelial impairment of intestine. PMID:27571712

  6. ABCG2 membrane transporter in mature human erythrocytes is exclusively homodimer.

    PubMed

    Leimanis, Mara L; Georges, Elias

    2007-03-09

    The human ABCG2 protein, a member of ABC transporter family, was shown to transport anti-cancer drugs and normal cell metabolites. Earlier studies have demonstrated the expression of ABCG2 in hematopoietic stem cells and erythroid cells; however little is known about the expression and activity of ABCG2 in mature erythrocytes. In this report, we show that ABCG2 in mature human erythrocytes migrates with an apparent molecular mass of 140 kDa, under reducing conditions, on Fairbanks SDS gel system. In contrast, tumor cells expressing higher levels of ABCG2 show no detectable homodimers, when resolved under identical reducing conditions. Analysis of the same membrane extracts from tumor cells and human erythrocytes on Laemmli SDS gel system, where samples are boiled in the presence of increasing concentrations of disulfide reducing conditions and then analyzed, migrate with an apparent molecular mass of 70 kDa or a monomer. Drug transport studies using Pheophorbide A, a substrate of ABCG2, show the protein to be active in erythrocytes. Furthermore, Fumitremorgin C, a specific inhibitor of ABCG2 increases the accumulation of Pheophorbide A in erythrocytes and drug-resistant cells but not in the parental drug-sensitive cells. Given the ability of ABCG2 to transport protoprophyrin IX or heme, these findings may have implications on the normal function of erythrocytes.

  7. Role of ABCB1, ABCG2, ABCC2 and ABCC5 transporters in placental passage of zidovudine.

    PubMed

    Neumanova, Zuzana; Cerveny, Lukas; Ceckova, Martina; Staud, Frantisek

    2016-01-01

    Zidovudine (AZT) is one of the most frequently used antiretroviral drugs in prevention of perinatal transmission of HIV. However, safety concerns on AZT use in pregnancy still persist as severe side effects are associated with AZT exposure in children. In our study we aimed to contribute to current knowledge on AZT transplacental transport and to evaluate potential involvement of the main human drug efflux ATP-binding cassette (ABC) transporters, p-glycoprotein (ABCB1), breast cancer resistance protein (ABCG2) and multidrug resistance-associated proteins 2 and 5 (ABCC2 and ABCC5) in the disposition of AZT between mother and fetus. In order to elucidate this issue we investigated the effect of selected ABC transporters on AZT transepithelial transport across MDCKII cell monolayers. In addition we used the in situ method of dually perfused rat term placenta to further study the role of ABC transporters in AZT transplacental transport. In vitro studies revealed significant effect of ABCB1 and ABCG2 on AZT transport which was subsequently confirmed also on organ level. Lamivudine, an antiretroviral agent commonly co-administered with AZT, did not affect ABC transporter-mediated AZT transfer.

  8. ABCG2 dysfunction causes hyperuricemia due to both renal urate underexcretion and renal urate overload

    PubMed Central

    Matsuo, Hirotaka; Nakayama, Akiyoshi; Sakiyama, Masayuki; Chiba, Toshinori; Shimizu, Seiko; Kawamura, Yusuke; Nakashima, Hiroshi; Nakamura, Takahiro; Takada, Yuzo; Oikawa, Yuji; Takada, Tappei; Nakaoka, Hirofumi; Abe, Junko; Inoue, Hiroki; Wakai, Kenji; Kawai, Sayo; Guang, Yin; Nakagawa, Hiroko; Ito, Toshimitsu; Niwa, Kazuki; Yamamoto, Ken; Sakurai, Yutaka; Suzuki, Hiroshi; Hosoya, Tatsuo; Ichida, Kimiyoshi; Shimizu, Toru; Shinomiya, Nariyoshi

    2014-01-01

    Gout is a common disease which results from hyperuricemia. We have reported that the dysfunction of urate exporter ABCG2 is the major cause of renal overload (ROL) hyperuricemia, but its involvement in renal underexcretion (RUE) hyperuricemia, the most prevalent subtype, is not clearly explained so far. In this study, the association analysis with 644 hyperuricemia patients and 1,623 controls in male Japanese revealed that ABCG2 dysfunction significantly increased the risk of RUE hyperuricemia as well as overall and ROL hyperuricemia, according to the severity of impairment. ABCG2 dysfunction caused renal urate underexcretion and induced hyperuricemia even if the renal urate overload was not remarkable. These results show that ABCG2 plays physiologically important roles in both renal and extra-renal urate excretion mechanisms. Our findings indicate the importance of ABCG2 as a promising therapeutic and screening target of hyperuricemia and gout. PMID:24441388

  9. Structure and Function of the Human Breast Cancer Resistance Protein (BCRP/ABCG2)

    PubMed Central

    Ni, Zhanglin; Bikadi, Zsolt; Rosenberg, Mark F.; Mao, Qingcheng

    2010-01-01

    The human breast cancer resistance protein (BCRP/ABCG2) is the second member of the G subfamily of the large ATP-binding cassette (ABC) transporter superfamily. BCRP was initially discovered in multidrug resistant breast cancer cell lines where it confers resistance to chemotherapeutic agents such as mitoxantrone, topotecan and methotrexate by extruding these compounds out of the cell. BCRP is capable of transporting non-chemotherapy drugs and xenobiotiocs as well, including nitrofurantoin, prazosin, glyburide, and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine. BCRP is frequently detected at high levels in stem cells, likely providing xenobiotic protection. BCRP is also highly expressed in normal human tissues including the small intestine, liver, brain endothelium, and placenta. Therefore, BCRP has been increasingly recognized for its important role in the absorption, elimination, and tissue distribution of drugs and xenobiotics. At present, little is known about the transport mechanism of BCRP, particularly how it recognizes and transports a large number of structurally and chemically unrelated drugs and xenobiotics. Here, we review current knowledge of structure and function of this medically important ABC efflux drug transporter. PMID:20812902

  10. Pharmacogenomics of the human ABC transporter ABCG2: from functional evaluation to drug molecular design

    NASA Astrophysics Data System (ADS)

    Ishikawa, Toshihisa; Tamura, Ai; Saito, Hikaru; Wakabayashi, Kanako; Nakagawa, Hiroshi

    2005-10-01

    In the post-genome-sequencing era, emerging genomic technologies are shifting the paradigm for drug discovery and development. Nevertheless, drug discovery and development still remain high-risk and high-stakes ventures with long and costly timelines. Indeed, the attrition of drug candidates in preclinical and development stages is a major problem in drug design. For at least 30% of the candidates, this attrition is due to poor pharmacokinetics and toxicity. Thus, pharmaceutical companies have begun to seriously re-evaluate their current strategies of drug discovery and development. In that light, we propose that a transport mechanism-based design might help to create new, pharmacokinetically advantageous drugs, and as such should be considered an important component of drug design strategy. Performing enzyme- and/or cell-based drug transporter, interaction tests may greatly facilitate drug development and allow the prediction of drug-drug interactions. We recently developed methods for high-speed functional screening and quantitative structure-activity relationship analysis to study the substrate specificity of ABC transporters and to evaluate the effect of genetic polymorphisms on their function. These methods would provide a practical tool to screen synthetic and natural compounds, and these data can be applied to the molecular design of new drugs. In this review article, we present an overview on the genetic polymorphisms of human ABC transporter ABCG2 and new camptothecin analogues that can circumvent AGCG2-associated multidrug resistance of cancer.

  11. Role of Breast Cancer Resistance Protein (BCRP/ABCG2) in Cancer Drug Resistance

    PubMed Central

    Natarajan, Karthika; Xie, Yi; Baer, Maria R.; Ross, Douglas D.

    2012-01-01

    Since cloning of the ATP-binding cassette (ABC) family member breast cancer resistance protein (BCRP/ABCG2) and its characterization as a multidrug resistance efflux transporter in 1998, BCRP has been the subject of more than two thousand scholarly articles. In normal tissues, BCRP functions as a defense mechanism against toxins and xenobiotics, with expression in the gut, bile canaliculi, placenta, blood-testis and blood-brain barriers facilitating excretion and limiting absorption of potentially toxic substrate molecules, including many cancer chemotherapeutic drugs. BCRP also plays a key role in heme and folate homeostasis, which may help normal cells survive under conditions of hypoxia. BCRP expression appears to be a characteristic of certain normal tissue stem cells termed “side population cells,” which are identified on flow cytometric analysis by their ability to exclude Hoechst 33342, a BCRP substrate fluorescent dye. Hence, BCRP expression may contribute to the natural resistance and longevity of these normal stem cells. Malignant tissues can exploit the properties of BCRP to survive hypoxia and to evade exposure to chemotherapeutic drugs. Evidence is mounting that many cancers display subpopulations of stem cells that are responsible for tumor self-renewal. Such stem cells frequently manifest the “side population” phenotype characterized by expression of BCRP and other ABC transporters. Along with other factors, these transporters may contribute to the inherent resistance of these neoplasms and their failure to be cured. PMID:22248732

  12. Porphyrin Homeostasis Maintained by ABCG2 Regulates Self-Renewal of Embryonic Stem Cells

    PubMed Central

    Chen, Yun-Nan; Shen, Chia-Rui; Yan, Yu-Ting; Tsai, Sheng-Ta; Chen, Chung-Hsuan; Shen, Chia-Ning

    2008-01-01

    Background Under appropriate culture conditions, undifferentiated embryonic stem (ES) cells can undergo multiple self-renewal cycles without loss of pluripotency suggesting they must be equipped with specific defense mechanisms to ensure sufficient genetic stability during self-renewal expansion. The ATP binding cassette transporter ABCG2 is expressed in a wide variety of somatic and embryonic stem cells. However, whether it plays an important role in stem cell maintenance remains to be defined. Methodology/Principal Findings Here we provide evidence to show that an increase in the level of ABCG2 was observed accompanied by ES colony expansion and then were followed by decreases in the level of protoporphyrin IX (PPIX) indicating that ABCG2 plays a role in maintaining porphyrin homoeostasis. RNA-interference mediated inhibition of ABCG2 as well as functional blockage of ABCG2 transporter with fumitremorgin C (FTC), a specific and potent inhibitor of ABCG2, not only elevated the cellular level of PPIX, but also arrest the cell cycle and reduced expression of the pluripotent gene Nanog. Overexpression of ABCG2 in ES cells was able to counteract the increase of endogenous PPIX induced by treatment with 5-Aminolevulinic acid suggesting ABCG2 played a direct role in removal of PPIX from ES cells. We also found that excess PPIX in ES cells led to elevated levels of reactive oxygen species which in turn triggered DNA damage signals as indicated by increased levels of γH2AX and phosphorylated p53. The increased level of p53 reduced Nanog expression because RNA- interference mediated inhibition of p53 was able to prevent the downregulation of Nanog induced by FTC treatment. Conclusions/Significance The present work demonstrated that ABCG2 protects ES cells from PPIX accumulation during colony expansion, and that p53 and γH2AX acts as a downstream checkpoint of ABCG2-dependent defense machinery in order to maintain the self-renewal of ES cells. PMID:19107196

  13. ABCG2 regulatory single-nucleotide polymorphisms alter in-vivo enhancer activity and expression.

    PubMed

    Eclov, Rachel J; Kim, Mee J; Chhibber, Aparna; Smith, Robin P; Ahituv, Nadav; Kroetz, Deanna L

    2017-09-18

    The expression and activity of the breast cancer resistance protein (ABCG2) contributes toward the pharmacokinetics of endogenous and xenobiotic substrates. The effect of genetic variation on the activity of cis-regulatory elements and nuclear response elements in the ABCG2 locus and their contribution toward ABCG2 expression have not been investigated systematically. In this study, the effect of genetic variation on the in-vitro and in-vivo enhancer activity of six previously identified liver enhancers in the ABCG2 locus was examined. Reference and variant liver enhancers were tested for their ability to alter luciferase activity in vitro in HepG2 and HEK293T cell lines and in vivo using a hydrodynamic tail vein assay. Positive in-vivo single-nucleotide polymorphisms (SNPs) were tested for association with gene expression and for altered protein binding in electrophoretic mobility shift assays. Multiple SNPs were found to alter enhancer activity in vitro. Four of these variants (rs9999111, rs12508471, ABCG2RE1*2, and rs149713212) decreased and one (rs2725263) increased enhancer activity in vivo. In addition, rs9999111 and rs12508471 were associated with ABCG2 expression in lymphoblastoid cell lines, lymphocytes, and T cells, and showed increased HepG2 nuclear protein binding. This study identifies SNPs within regulatory regions of the ABCG2 locus that alter enhancer activity in vitro and in vivo. Several of these SNPs correlate with tissue-specific ABCG2 expression and alter DNA/protein binding. These SNPs could contribute toward reported tissue-specific variability in ABCG2 expression and may influence the correlation between ABCG2 expression and disease risk or the pharmacokinetics and pharmacodynamics of breast cancer resistance protein substrates.

  14. Synthesis and biological evaluation of flavones and benzoflavones as inhibitors of BCRP/ABCG2.

    PubMed

    Juvale, Kapil; Stefan, Katja; Wiese, Michael

    2013-09-01

    Multidrug resistance (MDR) often leads to a failure of cancer chemotherapy. Breast Cancer Resistance Protein (BCRP/ABCG2), a member of the superfamily of ATP binding cassette proteins has been found to confer MDR in cancer cells by transporting molecules with amphiphilic character out of the cells using energy from ATP hydrolysis. Inhibiting BCRP can be a solution to overcome MDR. We synthesized a series of flavones, 7,8-benzoflavones and 5,6-benzoflavones with varying substituents at positions 3, 3' and 4' of the (benzo)flavone structure. All synthesized compounds were tested for BCRP inhibition in Hoechst 33342 and pheophorbide A accumulation assays using MDCK cells expressing BCRP. All the compounds were further screened for their P-glycoprotein (P-gp) and Multidrug resistance-associated protein 1 (MRP1) inhibitory activity by calcein AM accumulation assay to check the selectivity towards BCRP. In addition most active compounds were investigated for their cytotoxicity. It was observed that in most cases 7,8-benzoflavones are more potent in comparison to the 5,6-benzoflavones. In general it was found that presence of a 3-OCH3 substituent leads to increase in activity in comparison to presence of OH or no substitution at position 3. Also, it was found that presence of 3',4'-OCH3 on phenyl ring lead to increase in activity as compared to other substituents. Compound 24, a 7,8-benzoflavone derivative was found to be most potent being 50 times selective for BCRP and showing very low cytotoxicity at higher concentrations.

  15. Expression of Potential Cancer Stem Cell Marker ABCG2 is Associated with Malignant Behaviors of Hepatocellular Carcinoma

    PubMed Central

    Luo, Weihuan; Jiao, Hongbo; Jiang, Chunping

    2013-01-01

    Background. Despite improvement in treatment, the prognosis of hepatocellular carcinoma (HCC) remains disastrous. Cancer stem cells (CSCs) may be responsible for cancer malignant behaviors. ATP-binding cassette, subfamily G, member 2 (ABCG2) is widely expressed in both normal and cancer stem cells and may play an important role in cancer malignant behaviors. Methods. The expression of ABCG2 in HCC tissues and SMMC-7721 cells was examined, and the relevance of ABCG2 expression with clinical characteristics was analyzed. ABCG2+ and ABCG2− cells were sorted, and the potential of tumorigenicity was determined. Expression level of ABCG2 was manipulated by RNA interference and overexpression. Malignant behaviors including proliferation, drug resistance, migration, and invasion were studied in vitro. Results. Expression of ABCG2 was found in a minor group of cells in HCC tissues and cell lines. ABCG2 expression showed tendencies of association with unfavorable prognosis factors. ABCG2 positive cells showed a superior tumorigenicity. Upregulation of ABCG2 enhanced the capacity of proliferation, doxorubicin resistance, migration, and invasion potential, while downregulation of ABCG2 significantly decreased these malignant behaviors. Conclusion. Our results indicate that ABCG2 is a potential CSC marker for HCC. Its expression level has a close relationship with tumorigenicity, proliferation, drug resistance, and metastasis ability. PMID:24194752

  16. Association of the ABCG2 C421A polymorphism with prostate cancer risk and survival

    PubMed Central

    Gardner, Erin R.; Ahlers, Christoph M.; Shukla, Suneet; Sissung, Tristan M.; Ockers, Sandra B.; Price, Douglas K.; Hamada, Akinobu; Robey, Robert W.; Steinberg, Seth M.; Ambudkar, Suresh V.; Dahut, William L.; Figg, William D.

    2009-01-01

    Objective To determine if the C421A single nucleotide polymorphism (SNP) in the ATP-binding cassette transporter ABCG2 increases prostate cancer risk or affects survival. Patients, subjects and methods Numerous studies have suggested that dietary, hormonal and environmental factors all play a role in the initiation in prostate cancer; among these, the carcinogenic heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a known substrate of the ABCG2. A SNP of ABCG2, C421A, resulting in a glutamine to lysine change at amino acid 141, has been shown to result in decreased function of the protein. Due to the expression of ABCG2 in the prostate, together with the purported role of dietary carcinogens and steroids in the development and progression of prostate cancer, 311 individuals were genotyped for the ABCG2 C421A SNP, 170 patients with androgen-independent prostate cancer (AIPC) and 141 ‘healthy’ controls. We also evaluated the effect of this SNP on the intracellular accumulation of PhIP and testosterone in vitro. Results There were no significant differences in the prevalence of prostate cancer based on ABCG2 genetic variation in this population. However, survival was significantly longer for individuals with wild-type ABCG2, as compared with those hetero- or homozygous for the C421A SNP (7.4 years vs 5.3 years, P = 0.044). Conclusion Intracellular accumulation of PhIP was 80% higher in HEK293 cells transfected with Q141K ABCG2 than in wild-type cells, confirming that this SNP decreases transport of PhIP. In contrast, testosterone was not transported by either wild-type or variant transfected cells, nor did it act as in inhibitor of ABCG2 in subsequent transport assays. PMID:18710444

  17. ABCG2 polymorphisms in gout: insights into disease susceptibility and treatment approaches

    PubMed Central

    Cleophas, MC; Joosten, LA; Stamp, LK; Dalbeth, N; Woodward, OM; Merriman, Tony R

    2017-01-01

    As a result of the association of a common polymorphism (rs2231142, Q141K) in the ATP-binding cassette G2 (ABCG2) transporter with serum urate concentration in a genome-wide association study, it was revealed that ABCG2 is an important uric acid transporter. This review discusses the relevance of ABCG2 polymorphisms in gout, possible etiological mechanisms, and treatment approaches. The 141K ABCG2 urate-increasing variant causes instability in the nucleotide-binding domain, leading to decreased surface expression and function. Trafficking of the protein to the cell membrane is altered, and instead, there is an increased ubiquitin-mediated proteasomal degradation of the variant protein as well as sequestration into aggresomes. In humans, this leads to decreased uric acid excretion through both the kidney and the gut with the potential for a subsequent compensatory increase in renal urinary excretion. Not only does the 141K polymorphism in ABCG2 lead to hyperuricemia through renal overload and renal underexcretion, but emerging evidence indicates that it also increases the risk of acute gout in the presence of hyperuricemia, early onset of gout, tophi formation, and a poor response to allopurinol. In addition, there is some evidence that ABCG2 dysfunction may promote renal dysfunction in chronic kidney disease patients, increase systemic inflammatory responses, and decrease cellular autophagic responses to stress. These results suggest multiple benefits in restoring ABCG2 function. It has been shown that decreased ABCG2 141K surface expression and function can be restored with colchicine and other small molecule correctors. However, caution should be exercised in any application of these approaches given the role of surface ABCG2 in drug resistance. PMID:28461764

  18. Sildenafil reverses ABCB1- and ABCG2-mediated chemotherapeutic drug resistance.

    PubMed

    Shi, Zhi; Tiwari, Amit K; Shukla, Suneet; Robey, Robert W; Singh, Satyakam; Kim, In-Wha; Bates, Susan E; Peng, Xingxiang; Abraham, Ioana; Ambudkar, Suresh V; Talele, Tanaji T; Fu, Li-Wu; Chen, Zhe-Sheng

    2011-04-15

    Sildenafil is a potent and selective inhibitor of the type 5 cGMP (cyclic guanosine 3',5'-monophosphate)-specific phosphodiesterase that is used clinically to treat erectile dysfunction and pulmonary arterial hypertension. Here, we report that sildenafil has differential effects on cell surface ABC transporters such as ABCB1, ABCC1, and ABCG2 that modulate intracompartmental and intracellular concentrations of chemotherapeutic drugs. In ABCB1-overexpressing cells, nontoxic doses of sildenafil inhibited resistance and increased the effective intracellular concentration of ABCB1 substrate drugs such as paclitaxel. Similarly, in ABCG2-overexpressing cells, sildenafil inhibited resistance to ABCG2 substrate anticancer drugs, for example, increasing the effective intracellular concentration of mitoxantrone or the fluorescent compound BODIPY-prazosin. Sildenafil also moderately inhibited the transport of E(2)17βG and methotrexate by the ABCG2 transporter. Mechanistic investigations revealed that sildenafil stimulated ABCB1 ATPase activity and inhibited photolabeling of ABCB1 with [(125)I]-iodoarylazidoprazosin (IAAP), whereas it only slightly stimulated ABCG2 ATPase activity and inhibited photolabeling of ABCG2 with [(125)I]-IAAP. In contrast, sildenafil did not alter the sensitivity of parental, ABCB1-, or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drugs, nor did sildenafil affect the function of another ABC drug transporter, ABCC1. Homology modeling predicted the binding conformation of sildenafil within the large cavity of the transmembrane region of ABCB1. Overall, we found that sildenafil inhibits the transporter function of ABCB1 and ABCG2, with a stronger effect on ABCB1. Our findings suggest a possible strategy to enhance the distribution and potentially the activity of anticancer drugs by jointly using a clinically approved drug with known side effects and drug-drug interactions. ©2011 AACR.

  19. Sildenafil reverses ABCB1- and ABCG2-mediated chemotherapeutic drug resistance

    PubMed Central

    Shi, Zhi; Tiwari, Amit K; Shukla, Suneet; Robey, Robert W.; Singh, Satyakam; Kim, In-Wha; Bates, Susan E.; Peng, Xingxiang; Abraham, Ioana; Ambudkar, Suresh V.; Talele, Tanaji T.; Fu, Li-Wu; Chen, Zhe-Sheng

    2011-01-01

    Sildenafil is a potent and selective inhibitor of the type 5 cGMP-specific phosphodiesterase that is used clinically to treat erectile dysfunction and pulmonary arterial hypertension. Here we report that sildenafil has differential effects on cell surface ABC transporters such as ABCB1, ABCC1 and ABCG2 that modulate intracompartmental and intracellular concentrations of chemotherapeutic drugs. In ABCB1-overexpressing cells, non-toxic doses of sildenafil inhibited resistance and increased the effective intracellular concentration of ABCB1 substrate drugs, such as paclitaxel. Similarly, in ABCG2-overexpressing cells, sildenafil inhibited resistance to ABCG2 substrate anticancer drugs, for example, increasing the effective intracellular concentration of mitoxantrone or the fluorescent compound BODIPY-prazosin. Sildenafil also moderately inhibited the transport of E217βG and methotrexate by the ABCG2 transporter. Mechanistic investigations revealed that sildenafil stimulated ABCB1 ATPase activity and inhibited photolabeling of ABCB1 with [125I]-IAAP, whereas it only slightly stimulated ABCG2 ATPase activity and inhibited photolabeling of ABCG2 with [125I]-IAAP. In contrast, Sildenafil did not alter the sensitivity of parental, ABCB1- or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drugs, nor did sildenafil affect the function of another ABC drug transporter ABCC1. Homology modeling predicted the binding conformation of sildenafil within the large cavity of the transmembrane region of ABCB1. Overall, we found that sildenafil inhibits the transporter function of ABCB1 and ABCG2, with a stronger effect on ABCB1. Our findings suggest a possible strategy to enhance the distribution and potentially the activity of anti-cancer drugs by jointly using a clinically approved drug with known side effects and drug-drug interactions. PMID:21402712

  20. ABCG2/BCRP gene expression is related to epithelial-mesenchymal transition inducer genes in a papillary thyroid carcinoma cell line (TPC-1).

    PubMed

    Mato, E; González, C; Moral, A; Pérez, J I; Bell, O; Lerma, E; de Leiva, A

    2014-06-01

    Tumor malignancy is associated with the epithelial-mesenchymal transition (EMT) process and resistance to chemotherapy. However, little is known about the relationship between the EMT and the multidrug-resistance gene in thyroid tumor progression. We investigated whether the expression of the ABCG2/BCRP gene is associated with ZEB1 and other EMT inducer genes involved in tumor dedifferentiation. We established a subpopulation of cells that express the ABCG2/BCRP gene derived from the thyroid papillary carcinoma cell line (TPC-1), the so-called TPC-1 MITO-resistant subline. The most relevant findings in these TPC-1 selected cells were a statistically significant upregulation of ZEB1 and TWIST1 (35- and 15-fold change respectively), no changes in the relative expression of vimentin and SNAIL1, and no expression of E-cadherin. The TPC-1 MITO-resistant subline displayed a faster migration and greater invasive ability than parental cells in correlation with a significant upregulation of the survivin (BIRC5) gene (twofold change, P<0.05). The knockdown of ZEB1 promoted nuclear re-expression of E-cadherin, reduced expression of vimentin, N-cadherin, and BIRC5 genes, and reduced cell migration (P<0.05). Analysis of human thyroid carcinoma showed a slight overexpression of the ABCG2/BCRP at stages I and II (P<0.01), and a higher overexpression at stages III and IV (P<0.01). SNAIL1, TWIST1, and ZEB1 genes showed higher expression at stages III and IV than at stages I and II. E- and N-cadherin genes were upregulated at stages I and II of the disease (ninefold and tenfold change, respectively, P<0.01) but downregulated at stages III and IV (fourfold lower, P<0.01). These results could be a promising starting point for further study of the role of the ABCG2/BCRP gene in the progression of thyroid tumor.

  1. A novel MDCKII in vitro model for assessing ABCG2-drug interactions and regulation of ABCG2 transport activity in the caprine mammary gland by environmental pollutants and pesticides.

    PubMed

    Halwachs, Sandra; Wassermann, Louise; Honscha, Walther

    2014-04-01

    The ABC efflux transporter ABCG2 represents the main route for active secretion of xenobiotics into milk. Thus, ABCG2 regulation by aryl hydrocarbon receptor (AhR) ligands including ubiquitously environmental pollutants is of great toxicological relevance. However, no adequate in vitro model is as yet available to study AhR-dependent ABCG2 regulation in dairy animals. In this study, we therefore systematically investigated the effect of various environmental contaminants and pesticides on ABCG2 efflux activity in MDCKII cells stably expressing mammary ABCG2 from dairy goats. The AhR-agonists TCDD, Aroclor 1254, prochloraz, and iprodione caused a dose- and time-dependent increase in EROD activity. Moreover, TCDD and prochloraz significantly stimulated ABCG2 transport activity through a dose- and time-dependent induction of transporter gene expression. AhR inhibitors like CH223191 significantly reversed TCDD- and prochloraz-induced stimulation of ABCG2 efflux activity. In contrast, non-AhR activators such as PCB 101 had no significant effect on EROD activity, ABCG2 gene expression or transporter activity. As we identified various anthelmintics including monepantel as potential ABCG2 substrates this regulatory mechanism may result in increased milk residues of potentially harmful xenobiotics. Thus, MDCKII-cABCG2 cells may represent a suitable in vitro model to study mammary ABCG2 secretory activity and its potential regulation by AhR-activating contaminants. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. High plasma concentrations of dolutegravir in patients with ABCG2 genetic variants.

    PubMed

    Tsuchiya, Kiyoto; Hayashida, Tsunefusa; Hamada, Akinobu; Oki, Sakurako; Oka, Shinichi; Gatanaga, Hiroyuki

    2017-08-29

    The ATP-binding cassette transporters B1 (ABCB1) and G2 (ABCG2) are both expressed in the intestine and known as efflux transporters of drugs. Dolutegravir was identified recently as a substrate of both ABCB1 and ABCG2. This study aimed to determine the relations between single-nucleotide polymorphisms of ABCB1 and ABCG2 genes and plasma dolutegravir concentrations. Plasma samples were obtained from 42 HIV-1-infected patients treated with dolutegravir-containing regimens 0.5-4 h after dolutegravir dosing. Plasma dolutegravir concentrations were measured by liquid chromatography-mass spectrometry. Genomic DNA was isolated from peripheral blood mononuclear cells. Genotyping of allelic variants of ABCB1 1236 C>T (rs1128503), 2677 G>T/A (rs2032582), 3435 C>T (rs1045642), 4036 A>G (rs3842), and ABCG2 421 C>A (rs2231142) was performed using the TaqMan drug metabolism assays. None of the genotypes in ABCB1 1236 C>T, 2677 G>T/A, 3435 C>T, and 4036 A>G correlated with plasma dolutegravir concentration. In contrast, the mean peak plasma concentration of dolutegravir was significantly higher in the genotypes of ABCG2 421 AA (5002 ng/ml, n=3) compared with the genotypes of ABCG2 421 CC (2569 ng/ml, n=22) and ABCG2 421 CA (2479 ng/ml, n=17) (P=0.0005). The speculated peak level of plasma dolutegravir concentration was significantly higher in ABCG2 genetic variant holders, probably, at least in part, because of low expression levels of efflux transporters in the intestines associated with these genetic variants.

  3. Expression of ABCG2 (BCRP) in mouse models with enhanced erythropoiesis

    PubMed Central

    Latunde-Dada, Gladys O.; Laftah, Abas H.; Masaratana, Patarabutr; McKie, Andrew T.; Simpson, Robert J.

    2014-01-01

    Haem is a structural component of numerous cellular proteins which contributes significantly to iron metabolic processes in mammals but its toxicity demands that cellular levels must be tightly regulated. Breast Cancer Resistance Protein (BCRP/ABCG2), an ATP Binding Cassette G-member protein has been shown to possess porphyrin/haem efflux function. The current study evaluated the expression and regulation of Abcg2 mRNA and protein levels in mouse tissues involved in erythropoiesis. Abcg2 mRNA expression was enhanced in bone marrow hemopoietic progenitor cells from mice that were treated with phenylhydrazine (PHZ). Abcg2 mRNA expression was increased particularly in the extramedullary haematopoietic tissues from all the mice models with enhanced erythropoiesis. Haem oxygenase (ho1) levels tended to increase in the liver of mice with enhanced erythropoiesis and gene expression patterns differed from those observed in the spleen. Efflux of haem biosynthetic metabolites might be dependent on the relative abundance of Abcg2 or ho1 during erythropoiesis. Abcg2 appears to act principally as a safety valve regulating porphyrin levels during the early stages of erythropoiesis and its role in systemic haem metabolism and erythrophagocytosis, in particular, awaits further clarification. PMID:25028581

  4. Interaction of nilotinib, dasatinib and bosutinib with ABCB1 and ABCG2: implications for altered anti-cancer effects and pharmacological properties

    PubMed Central

    Hegedűs, C; Özvegy-Laczka, C; Apáti, Á; Magócsi, M; Német, K; Őrfi, L; Kéri, G; Katona, M; Takáts, Z; Váradi, A; Szakács, G; Sarkadi, B

    2009-01-01

    Background and purpose: ABC multidrug transporters (MDR-ABC proteins) cause multiple drug resistance in cancer and may be involved in the decreased anti-cancer efficiency and modified pharmacological properties of novel specifically targeted agents. It has been documented that ABCB1 and ABCG2 interact with several first-generation, small-molecule, tyrosine kinase inhibitors (TKIs), including the Bcr-Abl fusion kinase inhibitor imatinib, used for the treatment of chronic myeloid leukaemia. Here, we have investigated the specific interaction of these transporters with nilotinib, dasatinib and bosutinib, three clinically used, second-generation inhibitors of the Bcr-Abl tyrosine kinase activity. Experimental approach: MDR-ABC transporter function was screened in both membrane- and cell-based (K562 cells) systems. Cytotoxicity measurements in Bcr-Abl-positive model cells were coupled with direct determination of intracellular TKI concentrations by high-pressure liquid chromatography-mass spectrometry and analysis of the pattern of Bcr-Abl phosphorylation. Transporter function in membranes was assessed by ATPase activity. Key results: Nilotinib and dasatinib were high-affinity substrates of ABCG2, and this protein mediated an effective resistance in cancer cells against these compounds. Nilotinib and dasatinib also interacted with ABCB1, but this transporter provided resistance only against dasatinib. Neither ABCB1 nor ABCG2 induced resistance to bosutinib. At relatively higher concentrations, however, each TKI inhibited both transporters. Conclusions and implications: A combination of in vitro assays may provide valuable preclinical information for the applicability of novel targeted anti-cancer TKIs, even in multidrug-resistant cancer. The pattern of MDR-ABC transporter–TKI interactions may also help to understand the general pharmacokinetics and toxicities of new TKIs. PMID:19785662

  5. Interaction of nilotinib, dasatinib and bosutinib with ABCB1 and ABCG2: implications for altered anti-cancer effects and pharmacological properties.

    PubMed

    Hegedus, C; Ozvegy-Laczka, C; Apáti, A; Magócsi, M; Német, K; Orfi, L; Kéri, G; Katona, M; Takáts, Z; Váradi, A; Szakács, G; Sarkadi, B

    2009-10-01

    ABC multidrug transporters (MDR-ABC proteins) cause multiple drug resistance in cancer and may be involved in the decreased anti-cancer efficiency and modified pharmacological properties of novel specifically targeted agents. It has been documented that ABCB1 and ABCG2 interact with several first-generation, small-molecule, tyrosine kinase inhibitors (TKIs), including the Bcr-Abl fusion kinase inhibitor imatinib, used for the treatment of chronic myeloid leukaemia. Here, we have investigated the specific interaction of these transporters with nilotinib, dasatinib and bosutinib, three clinically used, second-generation inhibitors of the Bcr-Abl tyrosine kinase activity. MDR-ABC transporter function was screened in both membrane- and cell-based (K562 cells) systems. Cytotoxicity measurements in Bcr-Abl-positive model cells were coupled with direct determination of intracellular TKI concentrations by high-pressure liquid chromatography-mass spectrometry and analysis of the pattern of Bcr-Abl phosphorylation. Transporter function in membranes was assessed by ATPase activity. Nilotinib and dasatinib were high-affinity substrates of ABCG2, and this protein mediated an effective resistance in cancer cells against these compounds. Nilotinib and dasatinib also interacted with ABCB1, but this transporter provided resistance only against dasatinib. Neither ABCB1 nor ABCG2 induced resistance to bosutinib. At relatively higher concentrations, however, each TKI inhibited both transporters. A combination of in vitro assays may provide valuable preclinical information for the applicability of novel targeted anti-cancer TKIs, even in multidrug-resistant cancer. The pattern of MDR-ABC transporter-TKI interactions may also help to understand the general pharmacokinetics and toxicities of new TKIs.

  6. Increased expression of the Abcg2 transporter during erythroid maturation plays a role in decreasing cellular protoporphyrin IX levels.

    PubMed

    Zhou, Sheng; Zong, Yang; Ney, Paul A; Nair, Geeta; Stewart, Clinton F; Sorrentino, Brian P

    2005-03-15

    ABCG2/BCRP is a member of the adenosine triphosphate-binding cassette (ABC) transporter family and is expressed in intestine, kidney, and liver, where it modulates the absorption and excretion of xenobiotic compounds. ABCG2 is also expressed in hematopoietic stem cells and erythroid cells; however, little is known regarding its role in hematopoiesis. Abcg2 null mice have increased levels of protoporphyrin IX (PPIX) in erythroid cells, yet the mechanism for this remains uncertain. We have found that Abcg2 mRNA expression was up-regulated in differentiating erythroid cells, coinciding with increased expression of other erythroid-specific genes. This expression pattern was associated with significant amounts of ABCG2 protein on the membrane of mature peripheral blood erythrocytes. Erythroid cells engineered to express ABCG2 had significantly lower intracellular levels of PPIX, suggesting the modulation of PPIX level by ABCG2. This modulating activity was abrogated by treatment with a specific ABCG2 inhibitor, Ko143, implying that PPIX may be a direct substrate for the transporter. Taken together, our results demonstrate that ABCG2 plays a role in regulating PPIX levels during erythroid differentiation and suggest a potential role for ABCG2 as a genetic determinant in erythropoietic protoporphyria.

  7. Induction of ABCG2/BCRP restricts the distribution of zidovudine to the fetal brain in rats.

    PubMed

    Filia, María Fernanda; Marchini, Timoteo; Minoia, Juan Mauricio; Roma, Martín Ignacio; De Fino, Fernanda Teresa; Rubio, Modesto Carlos; Copello, Guillermo J; Evelson, Pablo A; Peroni, Roxana Noemí

    2017-09-01

    Safety concerns for fetus development of zidovudine (AZT) administration as prophylaxis of vertical transmission of HIV persist. We evaluated the participation of the ATP-binding cassette efflux transporter ABCG2 in the penetration of AZT into the fetal brain and the relevance for drug safety. Oral daily doses of AZT (60mg/kg body weight) or its vehicle were administered between post gestational days 11 (E11) and 20 (E20) to Sprague-Dawley pregnant rats. At E21, animals received an intravenous bolus of 60mg AZT/kg body weight in the presence or absence of the ABCG2 inhibitor gefitinib (20mg/kg body weight, ip) and AZT in maternal plasma and fetal brain were measured by HPLC-UV. ABCG2 protein expression in placenta and fetal brain, as well as mitochondrial function and ultrastructure in fetal brain were also analyzed. In utero chronic exposure to AZT markedly induced ABCG2 expression in placenta and fetal brain whereas did not significantly alter mitochondrial functionality in the fetal brain. The area-under-the-concentration-time-curve of AZT significantly decreased in fetal brains isolated from AZT-exposed fetuses compared to control group, but this effect was abolished by ABCG2 inhibition. Our results suggest that the absence of mitochondrial toxicity in the fetal brain after chronic in utero administration of AZT could be attributed to its low accumulation in the tissue caused, at least in part, by ABCG2 overexpression. We propose that any interference with ABCG2 activity due to genetic, pathological or iatrogenic factors would increase the amount of AZT reaching the fetal brain, which could increase the risk of toxicity of this drug on the tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Mutant Gly482 and Thr482 ABCG2 mediate high-level resistance to lipophilic antifolates.

    PubMed

    Bram, Eran; Ifergan, Ilan; Shafran, Assaf; Berman, Bluma; Jansen, Gerrit; Assaraf, Yehuda G

    2006-12-01

    Cellular uptake of hydrophilic antifolates proceeds via the reduced folate carrier whereas lipophilic antifolates enter cells by diffusion. Recently we have shown that transfectant cells overexpressing the mutant G482 ABCG2 displayed 120-6,250-fold resistance to hydrophilic antifolates than untransfected cells upon 4 h drug exposure, but lost almost all their antifolate resistance upon 72 h drug exposure (Shafran et al. in Cancer Res 65:8414-8422, 2005). Here we explored the ability of the wild type (WT) R482-as well as the mutant G482-and T482 ABCG2 to confer resistance to lipophilic antifolate inhibitors of dihydrofolate reductase (trimetrexate, piritrexim, metoprine and pyrimethamine) and thymidylate synthase (AG337, AG377 and AG331). Lipophilic antifolate resistance was determined using growth inhibition assays upon 72 h drug exposure. Cells overexpressing these mutant efflux transporters displayed up to 106-fold resistance to lipophilic antifolates relative to untransfected cells; this resistance was reversed by the specific and potent ABCG2 efflux inhibitor Ko143. In contrast, cells overexpressing the WT R482 ABCG2 exhibited either no or only a low-level of lipophilic antifolate resistance. These results provide the first evidence that overexpression of the mutant G482- and T482 but not the WT R482 ABCG2 confers a high-level of resistance to lipophilic antifolates. The high membrane partitioning of lipophilic antifolates along with the large confinement of ABCG2 to the plasma membrane suggest that these mutant ABCG2 transporters may possibly recognize and extrude lipophilic antifolates from the lipid bilayer. The potential implications to cancer chemotherapy as well as the mechanism of anticancer drug extrusion by these mutant exporters are discussed.

  9. Epigenetic modulation of the drug resistance genes MGMT, ABCB1 and ABCG2 in glioblastoma multiforme

    PubMed Central

    2013-01-01

    Background Resistance of the highly aggressive glioblastoma multiforme (GBM) to drug therapy is a major clinical problem resulting in a poor patient’s prognosis. Beside promoter methylation of the O 6 -methylguanine-DNA-methyltransferase (MGMT) gene the efflux transporters ABCB1 and ABCG2 have been suggested as pivotal factors contributing to drug resistance, but the methylation of ABCB1 and ABCG2 has not been assessed before in GBM. Methods Therefore, we evaluated the proportion and prognostic significance of promoter methylation of MGMT, ABCB1 and ABCG2 in 64 GBM patient samples using pyrosequencing technology. Further, the single nucleotide polymorphisms MGMT C-56 T (rs16906252), ABCB1 C3435T (rs1045642) and ABCG2 C421A (rs2231142) were determined using the restriction fragment length polymorphism method (RFLP). To study a correlation between promoter methylation and gene expression, we analyzed MGMT, ABCB1 and ABCG2 expression in 20 glioblastoma and 7 non-neoplastic brain samples. Results Despite a significantly increased MGMT and ABCB1 promoter methylation in GBM tissue, multivariate regression analysis revealed no significant association between overall survival of glioblastoma patients and MGMT or ABCB1 promoter methylation. However, a significant negative correlation between promoter methylation and expression could be identified for MGMT but not for ABCB1 and ABCG2. Furthermore, MGMT promoter methylation was significantly associated with the genotypes of the MGMT C-56 T polymorphism showing a higher methylation level in the T allele bearing GBM. Conclusions In summary, the data of this study confirm the previous published relation of MGMT promoter methylation and gene expression, but argue for no pivotal role of MGMT, ABCB1 and ABCG2 promoter methylation in GBM patients’ survival. PMID:24380367

  10. Identification of Febuxostat as a New Strong ABCG2 Inhibitor: Potential Applications and Risks in Clinical Situations

    PubMed Central

    Miyata, Hiroshi; Takada, Tappei; Toyoda, Yu; Matsuo, Hirotaka; Ichida, Kimiyoshi; Suzuki, Hiroshi

    2016-01-01

    ATP-binding cassette transporter G2 (ABCG2) is a plasma membrane protein that regulates the pharmacokinetics of a variety of drugs and serum uric acid (SUA) levels in humans. Despite the pharmacological and physiological importance of this transporter, there is no clinically available drug that modulates ABCG2 function. Therefore, to identify such drugs, we investigated the effect of drugs that affect SUA levels on ABCG2 function. This strategy was based on the hypothesis that the changes of SUA levels might caused by interaction with ABCG2 since it is a physiologically important urate transporter. The results of the in vitro screening showed that 10 of 25 drugs investigated strongly inhibited the urate transport activity of ABCG2. Moreover, febuxostat was revealed to be the most promising candidate of all the potential ABCG2 inhibitors based on its potent inhibition at clinical concentrations; the half-maximal inhibitory concentration of febuxostat was lower than its maximum plasma unbound concentrations reported. Indeed, our in vivo study demonstrated that orally administered febuxostat inhibited the intestinal Abcg2 and, thereby, increased the intestinal absorption of an ABCG2 substrate sulfasalazine in wild-type mice, but not in Abcg2 knockout mice. These results suggest that febuxostat might inhibit human ABCG2 at a clinical dose. Furthermore, the results of this study lead to a proposed new application of febuxostat for enhancing the bioavailability of ABCG2 substrate drugs, named febuxostat-boosted therapy, and also imply the potential risk of adverse effects by drug-drug interactions that could occur between febuxostat and ABCG2 substrate drugs. PMID:28082903

  11. Assessment of ABCG2-mediated transport of xenobiotics across the blood-milk barrier of dairy animals using a new MDCKII in vitro model.

    PubMed

    Wassermann, Louise; Halwachs, Sandra; Baumann, Daniela; Schaefer, Ingo; Seibel, Peter; Honscha, Walther

    2013-09-01

    The ATP-binding cassette (ABC) efflux transporter ABCG2 represents the main route for active secretion of drugs and toxins across the blood-milk barrier, thereby producing a potential health risk for dairy consumers through formation of relevant residues in milk. However, no suitable in vitro model is as yet available to systematically investigate ABCG2-mediated transport of xenobiotics into milk of dairy animals. We recently cloned ABCG2 from the lactating mammary gland of dairy cows (bABCG2) and goats (cABCG2). Thus, the objective of this study was to generate a suitable blood-milk barrier in vitro model using polarized MDCKII monolayers stably expressing mammary bABCG2 or cABCG2. ABCG2 protein was localized by confocal microscopy to the apical and lateral plasma membrane of polarized MDCKII cells. Intact barrier function of MDCKII-bABCG2 and MDCKII-cABCG2 monolayers was confirmed by determination of cell permeability of transcellular marker propranolol and paracellular marker atenolol which was ≤1 %. In flux assays, ABCG2 substrate 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) showed preferential basolateral to apical (B > A) transport in ABCG2-MDCKII cells. This apically directed PhIP transport was significantly inhibited by ABCG2 inhibitor fumitremorgin C (FTC) or the flavonoid equol. PhIP B > A transport in MDCKII-bABCG2 monolayers was additionally decreased by ABCG2 inhibitor Ko143. The fluoroquinolone antibiotic enrofloxacin was identified as a substrate of ruminant mammary ABCG2. The analgesic drug sodium salicylate was shown to be substrate of bABCG2 but not of cABCG2. Thus, the generated mammary ABCG2-expressing MDCKII cells represent a valuable tool to study active secretion of drugs and toxins into milk.

  12. Recombinant synthesis of human ABCG2 expressed in the yeast Saccharomyces cerevisiae: an experimental methodological study.

    PubMed

    Jacobs, Anna; Emmert, Dana; Wieschrath, Svenja; Hrycyna, Christine A; Wiese, Michael

    2011-03-01

    Human ABCG2 is an efflux protein belonging to the ATP-binding cassette transporter superfamily. It is expressed in the plasma membrane of different cell types performing various physiological functions. It is the most recently discovered MDR transporter and its structure and function are still not well understood. Thus, expression and functional reconstitution of the protein in different variants and from different sources are important steps for its further investigation. In this work we describe a recombinant synthesis of human ABCG2 R482G from S. cerevisiae. We expressed the human ABCG2 R482G variant in S. cerevisiae and purified the protein from total yeast membranes. Using a panel of sixteen detergents, we analyzed the efficiency of extraction of ABCG2 from membranes by SDS-PAGE and immunoblot analysis. Based on these results, three detergents were selected for further purification studies and two of them, n-octyl-β-D-glucopyranoside and n-dodecyl-β-D-maltopyranoside, yielded functional protein after reconstitution into liposomes. We show here the first example of purified and reconstituted ABCG2 expressed in S. cerevisiae retaining drug-stimulated ATPase activity.

  13. [Construction and identification of eukaryotic plasmid pGC-silencer-U6/Neo/GFP/ABCG2].

    PubMed

    Yu, Yanping; Zhang, Song; Kong, Weijia

    2010-09-01

    To construct three short hairpin RNA (shRNA) interference expression plasmid vectors of human ABCG2 gene, to assay the expression of ABCG2 in a human nasopharyngeal carcinoma (NPC) cell line, CEN-2 cell line, and to detect the RNAi effect of shRNA. Targeting ABCG2 gene sequence, three plasmid expression vectors coding for shRNA and a control vector containing random DNA fragment were constructed. The recombinant plasmids were amplified in Ecoli. DH5 and then identified by restriction digestion, PCR and sequencing. The recombinant plasmids were transfected into CEN-2 cells. ABCG2 expression was assayed by real-time quantitative PCR and Western blot. The construction of pGC-silencer-U6/Neo/GFP/ABCG2 was succeed. The shRNA plasmids significantly down-regulated the ABCG2 expression in CEN-2 cells, at both mRNA level and protein level. Recombinant plasmid 1 had the strongest effect compared with plasmids 2 and 3 (P < 0.05), with an inhibition ratio of 75% at the mRNA level and 68% at the protein level. pGC-silencer-U6/Neo/GFP/ABCG2 has been successfully constructed and it can down-regulate ABCG2 expression after transfected into CEN-2 cells, which could help further studies of ABCG2 functions CEN-2 cell line and contribute to the NPC gene therapy.

  14. ABCG2-mediated dyecycle violet efflux defined side population in benign and malignant prostate

    PubMed Central

    Mathew, Grinu; Timm, Earl A.; Sotomayor, Paula; Godoy, Alejandro; Montecinos, Viviana P.; Smith, Gary J.; Huss, Wendy J.

    2010-01-01

    The efflux of Hoechst 33342 by ATP-binding cassette protein G2 (ABCG2) membrane pump allows reproducible identification of a subpopulation of cells by flow cytometric analysis termed the “side population” (SP). The SP identified by constitutive Hoechst efflux contains the stem/progenitor cell population from bone marrow and many solid organs, including prostate. DyeCycle Violet (DCV) is a cell membrane permeable, fluorescent vital dye that intercalates into DNA and is a substrate for ABCG2-mediated efflux. Therefore, DCV was evaluated in this study as a tool for identification of the SP from prostate cancer cell lines and from freshly harvested human prostate tissue. SPs that demonstrated ABCG2-mediated efflux of DCV were identified in the human prostate cancer cell lines CWR-R1, DU-145 and RWPE-1, but not in the BPH-1, LAPC-4 or PC-3 cell lines. Additionally, a SP was identified in enzymatically disaggregated prostate tumors from Transgenic Adenocarcinoma of Mouse Prostate (TRAMP), human benign prostate tissue and human prostate cancer tissue. The causal role of ABCG2-mediated efflux of DCV in the identification of the SP was confirmed by loss of the SP by incubation with the specific inhibitor of ABCG2, Fumitremorgin C. Expression of ABCG2 in the SP cells was confirmed by qRT-PCR and immunofluorescence analysis. Consequently, DCV represents an important new tool for isolation of viable candidate stem cells/cancer stem cells as a SP from cultured prostate cell lines, and prostate tissue specimens, without the requirement for instrumentation with ultra-violet excitation capability and minimizing the risk of damage to DNA in the sorted population. PMID:19270533

  15. Role of ABCG2 expression driven by cisplatin in platinum-containing chemotherapy for gastric cancer

    PubMed Central

    Zhang, Qiang; Li, Kun; Xu, Jian-Hua; Zhao, Cheng-Gen; Gao, Qi; Wu, Bin; Liu, Xiao-Yan

    2013-01-01

    AIM: To investigate the relationship between increases in expression time of ABCG2 mRNA driven by cisplatin and efficacy of platinum-containing chemotherapy for gastric cancer. METHODS: Tumor specimens and normal control tissues were collected from 78 patients with gastric cancer treated from January 2008 to December 2011. Fresh tumor tissue obtained from the surgically resected specimens was tested within 6 h. Polymerase chain reaction products were run on 2% agarose gels and analyzed under ultraviolet light after ethidium bromide staining. Increases in ABCG2 mRNA expression time were assessed after cancer cells were incubated with cisplatin, and were divided into terciles and compared in relation to clinical outcomes. RESULTS: Among groups classified by expression time of ABCG2 mRNA, no significant differences in baseline clinical characteristics and pathological findings were detected. The median overall time was 14.2 (95%CI: 9.7-18.6), 11.4 (95%CI: 6.3-16.5) and 8.1 (95%CI: 5.4-10.8) in patients with low, intermediate and high increases in ABCG2 mRNA expression times (P < 0.05), respectively. Median survival associated with performance status and tumor node metastasis (TNM) stage showed a similar trend, with longer survival and higher risk for mortality associated with lower performance status score and TNM stage. In a multivariate analysis for survival with Cox proportional-hazards model, increased ABCG2 mRNA expression time was an independent predictor for overall survival. Overall survival was longer with increased ABCG2 mRNA expression times ≤ 0.71 than increased ABCG2 mRNA expression times > 0.71, with a hazard ratio for death of 0.855 (95%CI: 0.615-0.962, P = 0.038). CONCLUSION: Increased ABCG2 mRNA expression time driven by cisplatin is associated with survival of gastric cancer patients, and this may help modify the therapeutic strategies. PMID:24151392

  16. Lack of ABCG2 Leads to Biventricular Dysfunction and Remodeling in Response to Hypoxia

    PubMed Central

    Nagy, Bence M.; Nagaraj, Chandran; Egemnazarov, Bakytbek; Kwapiszewska, Grazyna; Stauber, Rudolf E.; Avian, Alexander; Olschewski, Horst; Olschewski, Andrea

    2017-01-01

    Aims: The ATP-binding cassette (ABC)G2 transporter protects the heart from pressure overload-induced ventricular dysfunction but also protects cancer cells from chemotherapeutic agents. It is upregulated in the myocardium of heart failure patients and clears hypoxia-induced intracellular metabolites. This study employs ABCG2 knockout (KO) mice to elucidate the relevance of ABCG2 for cardiac and pulmonary vascular structure and function in chronic hypoxia, and uses human primary cardiac fibroblasts to investigate the potential role of ABCG2 in cardiac fibrosis. Methods and results: ABCG2 KO and control mice (n = 10) were subjected to 4 weeks normoxia or hypoxia. This allowed for investigation of the interaction between genotype and hypoxia (GxH). In hypoxia, KO mice showed pronounced right (RV) and left (LV) ventricular diastolic dysfunction. Compared to normoxia, end-diastolic pressure (EDP) was increased in control vs. KO mice by +1.1 ± 0.3 mmHg vs. +4.8 ± 0.3 mmHg, p for GxH < 0.001 (RV) and +3.9 ± 0.5 mmHg vs. +11.5 ± 1.6 mmHg, p for GxH = 0.110 (LV). The same applied for myocardial fibrosis with +0.3 ± 0.1% vs. 1.3 ± 0.2%, p for GxH = 0.036 (RV) and +0.06 ± 0.03% vs. +0.36 ± 0.08%, p for GxH = 0.002 (LV), whereas systolic function and capillary density was unaffected. ABCG2 deficiency did not influence hypoxia-induced pulmonary hypertension or vascular remodeling. In line with these observations, human cardiac fibroblasts showed increased collagen production upon ABCG2 silencing in hypoxia (p for GxH = 0.04). Conclusion: Here we provide evidence for the first time that ABCG2 membrane transporter can play a crucial role in ventricular dysfunction and fibrosis in hypoxia-induced pulmonary hypertension. PMID:28270772

  17. ABCG2 is a high-capacity urate transporter and its genetic impairment increases serum uric acid levels in humans.

    PubMed

    Nakayama, Akiyoshi; Matsuo, Hirotaka; Takada, Tappei; Ichida, Kimiyoshi; Nakamura, Takahiro; Ikebuchi, Yuki; Ito, Kousei; Hosoya, Tatsuo; Kanai, Yoshikatsu; Suzuki, Hiroshi; Shinomiya, Nariyoshi

    2011-12-01

    The ATP-binding cassette, subfamily G, member 2 (ABCG2/BCRP) gene encodes a well-known transporter, which exports various substrates including nucleotide analogs such as 3'-azido-3'-deoxythymidine (AZT). ABCG2 is also located in a gout-susceptibility locus (MIM 138900) on chromosome 4q, and has recently been identified by genome-wide association studies to relate to serum uric acid (SUA) and gout. Becuase urate is structurally similar to nucleotide analogs, we hypothesized that ABCG2 might be a urate exporter. To demonstrate our hypothesis, transport assays were performed with membrane vesicles prepared from ABCG2-overexpressing cells. Transport of estrone-3-sulfate (ES), a typical substrate of ABCG2, is inhibited by urate as well as AZT and ES. ATP-dependent transport of urate was then detected in ABCG2-expressing vesicles but not in control vesicles. Kinetic analysis revealed that ABCG2 is a high-capacity urate transporter that maintained its function even under high-urate concentration. The calculated parameters of ABCG2-mediated transport of urate were a Km of 8.24 ± 1.44 mM and a Vmax of 6.96 ± 0.89 nmol/min per mg of protein. Moreover, the quantitative trait locus (QTL) analysis performed in 739 Japanese individuals revealed that a dysfunctional variant of ABCG2 increased SUA as the number of minor alleles of the variant increased (p = 6.60 × 10(-5)). Because ABCG2 is expressed on the apical membrane in several tissues, including kidney, intestine, and liver, these findings indicate that ABCG2, a high-capacity urate exporter, has a physiological role of urate homeostasis in the human body through both renal and extrarenal urate excretion.

  18. ROLE OF ATP BINDING CASSETTE SUB-FAMILY MEMBER 2 (ABCG2) IN MOUSE EMBRYONIC STEM CELL DEVELOPMENT.

    EPA Science Inventory

    ATP binding cassette sub-family member 2 (ABCG2), is a member of the ABC transporter superfamily and a principal xenobiotic transporter. ABCG2 is also highly expressed in certain stem cell populations where it is thought to be related to stem cell plasticity, although the role o...

  19. ROLE OF ATP BINDING CASSETTE SUB-FAMILY MEMBER 2 (ABCG2) IN MOUSE EMBRYONIC STEM CELL DEVELOPMENT.

    EPA Science Inventory

    ATP binding cassette sub-family member 2 (ABCG2), is a member of the ABC transporter superfamily and a principal xenobiotic transporter. ABCG2 is also highly expressed in certain stem cell populations where it is thought to be related to stem cell plasticity, although the role o...

  20. High imatinib dose overcomes insufficient response associated with ABCG2 haplotype in chronic myelogenous leukemia patients

    PubMed Central

    Delord, Marc; Rousselot, Philippe; Cayuela, Jean Michel; Sigaux, François; Guilhot, Joëlle; Preudhomme, Claude; Guilhot, François; Loiseau, Pascale; Raffoux, Emmanuel; Geromin, Daniela; Génin, Emmanuelle; Calvo, Fabien; Bruzzoni-Giovanelli, Heriberto

    2013-01-01

    Pharmacogenetic studies in chronic myelogenous leukemia (CML) typically use a candidate gene approach. In an alternative strategy, we analyzed the impact of single nucleotide polymorphisms (SNPs) in drug transporter genes on the molecular response to imatinib, using a DNA chip containing 857 SNPs covering 94 drug transporter genes. Two cohorts of CML patients treated with imatinib were evaluated: an exploratory cohort including 105 patients treated at 400 mg/d and a validation cohort including patients sampled from the 400 mg/d and 600 mg/d arms of the prospective SPIRIT trial (n=239). Twelve SNPs discriminating patients according to cumulative incidence of major molecular response (CI-MMR) were identified within the exploratory cohort. Three of them, all located within the ABCG2 gene, were validated in patients included in the 400 mg/d arm of the SPIRIT trial. We identified an ABCG2 haplotype (define as G-G, rs12505410 and rs2725252) as associated with significantly higher CI-MMR in patients treated at 400 mg/d. Interestingly, we found that patients carrying this ABCG2 “favorable” haplotype in the 400 mg arm reached similar CI-MMR rates that patients randomized in the imatinib 600 mg/d arm. Our results suggest that response to imatinib may be influenced by constitutive haplotypes in drug transporter genes. Lower response rates associated with “non-favorable” ABCG2 haplotypes may be overcome by increasing the imatinib daily dose up to 600 mg/d. PMID:24123600

  1. Bioluminescent imaging of ABCG2 efflux activity at the blood-placenta barrier

    PubMed Central

    Kumar, Jeyan S.; Wei, Bih-Rong; Madigan, James P.; Simpson, R. Mark; Hall, Matthew D.; Gottesman, Michael M.

    2016-01-01

    Physiologic barriers such as the blood placenta barrier (BPB) and the blood brain barrier protect the underlying parenchyma from pathogens and toxins. ATP-binding cassette (ABC) transporters are transmembrane proteins found at these barriers, and function to efflux xenobiotics and maintain chemical homeostasis. Despite the plethora of ex vivo and in vitro data showing the function and expression of ABC transporters, no imaging modality exists to study ABC transporter activity in vivo at the BPB. In the present study, we show that in vitro models of the placenta possess ABCG2 activity and can specifically transport D-luciferin, the endogenous substrate of firefly luciferase. To test ABCG2 transport activity at the BPB, we devised a breeding strategy to generate a bioluminescent pregnant mouse model to demonstrate transporter function in vivo. We found that coadministering the ABCG2 inhibitors Ko143 and gefitinib with D-luciferin increased bioluminescent signal from fetuses and placentae, whereas the control P-gp inhibitor DCPQ had no effect. We believe that our bioluminescent pregnant mouse model will facilitate greater understanding of the BPB and ABCG2 activity in health and disease. PMID:26853103

  2. SIRT1 prevents hyperuricemia via the PGC-1α/PPARγ-ABCG2 pathway.

    PubMed

    Wang, Juan; Zhu, Xiao-Xia; Liu, Lei; Xue, Yu; Yang, Xue; Zou, He-Jian

    2016-08-01

    Silent information regulator T1 (SIRT1) plays several key roles in the regulation of lipid and glucose homoeostasis. In this study, we investigated the potential role of SIRT1 in hyperuricemia and explored possible mechanisms. Significant hyperuricemia was detected in C57BL/6 mice treated with oxonate and yeast polysaccharide. Resveratrol (RSV), a specific SIRT1 activator, was administered to the mice. SIRT1 suppressed the increased serum uric acid level but up-regulated the expression of urate transporter ATP-binding cassette subfamily G member 2 (ABCG2) in the ileum of hyperuricemic mice. In a human colon carcinoma cell line, SIRT1 promoted ABCG2 production through the deacetylation of peroxisome proliferator-activated receptor (PPAR) γ co-activator 1α (PGC-1α), which then activated the effectors of PPARγ. Interestingly, the SIRT1-induced up-regulation of ABCG2 was significantly inhibited when PGC-1α or PPARγ was blocked by siRNA transfection. Our data demonstrated that SIRT1 and its activator, RSV, have clear anti-hyperuricemia functions in this mouse model. One possible mechanism is the activation of ABCG2 in the ileum through the PGC-1α/PPARγ pathway.

  3. Cholesterol reduces the sensitivity to platinum-based chemotherapy via upregulating ABCG2 in lung adenocarcinoma.

    PubMed

    Wu, Yufeng; Si, Ruirui; Tang, Hong; He, Zhen; Zhu, Hui; Wang, Lili; Fan, Yingchao; Xia, Suhua; He, Zelai; Wang, Qiming

    2015-02-20

    Inoperable lung adenocarcinoma is currently treated with platinum-based chemotherapy. However, the effectiveness of these chemotherapeutic agents is not the same for all patients. Patients either show quick chemoresistance (QCR) or delayed chemoresistance (DCR), which are defined by 87 and 242 days of progression-free survival (PFS) after initial platinum-based treatment, respectively. We found that QCR patients displayed an elevated level of serum cholesterol and that their tumors showed upregulated ABCG2 expression. We propose that chemoresistance may be attributed to cholesterol-induced ABCG2 expression and hypothesize that blocking ABCG2 may increase the efficacy of platinum-based chemotherapeutic agents. Using the MTT cell viability assay, we observed that cotreatment with ABCG2 blocker Nicardipine and platinum-based drugs Cisplatin, Oxaliplatin or Carboplatin significantly decreased cell viability of tumor cells. Importantly, our results also showed that incubating cells with cholesterol prior to chemotherapy treatment or cotreatment increased cell viability of tumor cells relative to the controls. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Activity of ABCG2 Is Regulated by Its Expression and Localization in DHT and Cyclopamine-Treated Breast Cancer Cells.

    PubMed

    Chua, Vivian Y L; Larma, Irma; Harvey, Jennet; Thomas, Marc A; Bentel, Jacqueline M

    2016-10-01

    Elevated expression of the efflux transporter, ATP-binding cassette subfamily G isoform 2 (ABCG2) on the plasma membrane of cancer cells contributes to the development of drug resistance and is a key characteristic of cancer stem cells. In this study, gene expression analysis identified that treatment of the MCF-7 and T-47D breast cancer cell lines with the androgen, 5α-dihydrotestosterone (DHT), and the Hedgehog signaling inhibitor, cyclopamine downregulated ABCG2 mRNA levels. In MCF-7 cells, and in Hoechst 33342(lo) /CD44(hi) /CD24(lo) breast cancer stem-like cells isolated from MCF-7 cultures, ABCG2 was accumulated in cell-to-cell junction complexes and in large cytoplasmic aggresome-like vesicles. DHT treatments, which decreased cellular ABCG2 protein levels, led to diminished ABCG2 localization in both cell-to-cell junction complexes and in cytoplasmic vesicles. In contrast, cyclopamine, which did not alter ABCG2 protein levels, induced accumulation of ABCG2 in cytoplasmic vesicles, reducing its localization in cell-to-cell junction complexes. The reduced localization of ABCG2 at the plasma membrane of MCF-7 cells was associated with decreased efflux of the ABCG2 substrate, mitoxantrone, and increased sensitivity of cyclopamine-treated cultures to the cytotoxic effects of mitoxantrone. Together, these findings indicate that DHT and cyclopamine reduce ABCG2 activity in breast cancer cells by distinct mechanisms, providing evidence to advocate the adjunct use of analogous pharmaceutics to increase or prolong the efficacy of breast cancer treatments. J. Cell. Biochem. 117: 2249-2259, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  5. Masitinib antagonizes ATP-binding cassette subfamily G member 2-mediated multidrug resistance

    PubMed Central

    KATHAWALA, RISHIL J.; CHEN, JUN-JIANG; ZHANG, YUN-KAI; WANG, YI-JUN; PATEL, ATISH; WANG, DE-SHEN; TALELE, TANAJI T.; ASHBY, CHARLES R.; CHEN, ZHE-SHENG

    2014-01-01

    In this in vitro study, we determined whether masitinib could reverse multidrug resistance (MDR) in cells overexpressing the ATP binding cassette subfamily G member 2 (ABCG2) transporter. Masitinib (1.25 and 2.5 μM) significantly decreases the resistance to mitoxantrone (MX), SN38 and doxorubicin in HEK293 and H460 cells overexpressing the ABCG2 transporter. In addition, masitinib (2.5 μM) significantly increased the intracellular accumulation of [3H]-MX, a substrate for ABCG2, by inhibiting the function of ABCG2 and significantly decreased the efflux of [3H]-MX. However, masitinib (2.5 μM) did not significantly alter the expression of the ABCG2 protein. In addition, a docking model suggested that masitinib binds within the transmembrane region of a homology-modeled human ABCG2 transporter. Overall, our in vitro findings suggest that masitinib reverses MDR to various anti-neoplastic drugs in HEK293 and H460 cells overexpressing ABCG2 by inhibiting their transport activity as opposed to altering their levels of expression. PMID:24626598

  6. In vitro evaluation of photosensitivity risk related to genetic polymorphisms of human ABC transporter ABCG2 and inhibition by drugs.

    PubMed

    Tamura, Ai; Onishi, Yuko; An, Ran; Koshiba, Shoko; Wakabayashi, Kanako; Hoshijima, Kazuyuki; Priebe, Waldemar; Yoshida, Takashi; Kometani, Satoshi; Matsubara, Takayoshi; Mikuriya, Kenta; Ishikawa, Toshihisa

    2007-12-01

    Since porphyrins are regarded as endogenous substrates for the ATP-binding cassette (ABC) transporter ABCG2, it is hypothesized that functional impairment owing to genetic polymorphisms or inhibition of ABCG2 by drugs may result in a disruption of cellular porphyrin homeostasis. In the present study, we expressed ABCG2 genetic variants, i.e., V12M, Q141K, S441N, and F489L, as well as the wild type (WT) in Flp-In-293 cells to examine the hypothesis. Cells expressing S441N and F489L variants exhibited high levels of both cellularly accumulated pheophorbide a and photosensitivity, when those cells were incubated with pheophorbide a and irradiated with visible light. To further elucidate the significance of ABCG2 in cellular porphyrin homeostasis, we observed cellular accumulation and compartmentation of porphyrin and pheophorbide a by means of a new fluorescence microscopy technology, and found that accumulation of porphyrin and pheophorbide a in the cytoplasm compartment was maintained at low levels in Flp-In-293 cells expressing ABCG2 WT, V12M, or Q141K. When ABCG2 was inhibited by imatinib or novobiocin, however, those cells became sensitive to light. Based on these results, it is strongly suggested that certain genetic polymorphisms and/or inhibition of ABCG2 by drugs can enhance the potential risk of photosensitivity.

  7. Characterization of ABCG2 gene amplification manifesting as extrachromosomal DNA in mitoxantrone-selected SF295 human glioblastoma cells.

    PubMed

    Rao, V Koneti; Wangsa, Darawalee; Robey, Robert W; Huff, Lyn; Honjo, Yasumasa; Hung, Jeffrey; Knutsen, Turid; Ried, Thomas; Bates, Susan E

    2005-07-15

    The human ABCG2 gene, located on chromosome 4, encodes an ATP-binding cassette half-transporter that has been shown to confer resistance to chemotherapeutic agents. Relatively little is known about the mechanisms controlling expression of ABCG2. In previous studies, we had shown that overexpression of ABCG2 can result from rearrangement or gene amplification involving chromosome 4. To better characterize the mechanisms of ABCG2 overexpression, SF295 glioblastoma cells were exposed to increasing amounts of mitoxantrone to generate the SF295 MX50, MX100, MX250, and MX500 sublines, maintained in mitoxantrone concentrations ranging from 50 to 500 nmol/L. Northern blot analysis confirmed overexpression of ABCG2 mRNA, and immunoblot analysis demonstrated increased protein expression in the selected cell lines. Efflux of BODIPY-prazosin confirmed a functional protein. ABCG2 gene amplification was observed in all resistant sublines, as determined by Southern blot analysis. Fluorescence in situ hybridization (FISH) revealed amplification of ABCG2 via double minute chromosomes (dmins) detected in metaphase chromosome spreads in the SF295 MX50 and MX100 sublines. At higher levels of drug selection, in the MX250 and MX500 sublines, fewer dmins were observed but homogeneously staining regions (hsr) were visible with FISH analysis, revealing reintegration of the ABCG2 gene into multiple chromosomes. Spectral karyotyping (SKY) demonstrated multiple clonal and nonclonal rearrangements of chromosome 4, including hsrs. These results suggest that amplification of ABCG2 occurred initially in the form of dmins, followed by chromosomal reintegration of the amplicon at multiple sites. This occurred with increasing drug-selection pressure, generating a more stable genotype.

  8. Modulating Drug Resistance by Targeting BCRP/ABCG2 Using Retrovirus-Mediated RNA Interference

    PubMed Central

    Yuan, Jianhui; Liu, Wenlan; Deng, Tingting; Li, Zigang; Jin, Yi; Hu, Zhangli

    2014-01-01

    Background The BCRP/ABCG2 transporter, which mediates drug resistance in many types of cells, depends on energy provided by ATP hydrolysis. Here, a retrovirus encoding a shRNA targeting the ATP-binding domain of this protein was used to screen for highly efficient agents that could reverse drug resistance and improve cell sensitivity to drugs, thus laying the foundation for further studies and applications. Methodology/Principal Findings To target the ATP-binding domain of BCRP/ABCG2, pLenti6/BCRPsi shRNA recombinant retroviruses, with 20 bp target sequences starting from the 270th, 745th and 939th bps of the 6th exon, were constructed and packaged. The pLenti6/BCRPsi retroviruses (V-BCRPi) that conferred significant knockdown effects were screened using a drug-sensitivity experiment and flow cytometry. The human choriocarcinoma cell line JAR, which highly expresses endogenous BCRP/ABCG2, was injected under the dorsal skin of a hairless mouse to initiate a JAR cytoma. After injecting V-BCRPi-infected JAR tumor cells into the dorsal skin of hairless mice, BCRP/ABCG2 expression in the tumor tissue was determined using immunohistochemistry, fluorescent quantitative RT-PCR and Western blot analyses. After intraperitoneal injection of BCRP/ABCG2-tolerant 5-FU, the tumor volume, weight change, and apoptosis rate of the tumor tissue were determined using in situ hybridization. V-BCRPi increased the sensitivity of the tumor histiocytes to 5-FU and improved the cell apoptosis-promoting effects of 5-FU in the tumor. Conclusions/Significance The goal of the in vivo and in vitro studies was to screen for an RNA interference recombinant retrovirus capable of stably targeting the ATP-binding domain of BCRP/ABCG2 (V-BCRPi) to inhibit its function. A new method to improve the chemo-sensitivity of breast cancer and other tumor cells was discovered, and this method could be used for gene therapy and functional studies of malignant tumors. PMID:25076217

  9. Modulating drug resistance by targeting BCRP/ABCG2 using retrovirus-mediated RNA interference.

    PubMed

    Xie, Ni; Mou, Lisha; Yuan, Jianhui; Liu, Wenlan; Deng, Tingting; Li, Zigang; Jing, Yi; Jin, Yi; Hu, Zhangli

    2014-01-01

    The BCRP/ABCG2 transporter, which mediates drug resistance in many types of cells, depends on energy provided by ATP hydrolysis. Here, a retrovirus encoding a shRNA targeting the ATP-binding domain of this protein was used to screen for highly efficient agents that could reverse drug resistance and improve cell sensitivity to drugs, thus laying the foundation for further studies and applications. To target the ATP-binding domain of BCRP/ABCG2, pLenti6/BCRPsi shRNA recombinant retroviruses, with 20 bp target sequences starting from the 270th, 745th and 939th bps of the 6th exon, were constructed and packaged. The pLenti6/BCRPsi retroviruses (V-BCRPi) that conferred significant knockdown effects were screened using a drug-sensitivity experiment and flow cytometry. The human choriocarcinoma cell line JAR, which highly expresses endogenous BCRP/ABCG2, was injected under the dorsal skin of a hairless mouse to initiate a JAR cytoma. After injecting V-BCRPi-infected JAR tumor cells into the dorsal skin of hairless mice, BCRP/ABCG2 expression in the tumor tissue was determined using immunohistochemistry, fluorescent quantitative RT-PCR and Western blot analyses. After intraperitoneal injection of BCRP/ABCG2-tolerant 5-FU, the tumor volume, weight change, and apoptosis rate of the tumor tissue were determined using in situ hybridization. V-BCRPi increased the sensitivity of the tumor histiocytes to 5-FU and improved the cell apoptosis-promoting effects of 5-FU in the tumor. The goal of the in vivo and in vitro studies was to screen for an RNA interference recombinant retrovirus capable of stably targeting the ATP-binding domain of BCRP/ABCG2 (V-BCRPi) to inhibit its function. A new method to improve the chemo-sensitivity of breast cancer and other tumor cells was discovered, and this method could be used for gene therapy and functional studies of malignant tumors.

  10. Becatecarin (rebeccamycin analog, NSC 655649) is a transport substrate and induces expression of the ATP-binding cassette transporter, ABCG2, in lung carcinoma cells

    PubMed Central

    Robey, Robert W.; Obrzut, Tomasz; Shukla, Suneet; Polgar, Orsolya; Macalou, Sira; Bahr, Julian C.; Di Pietro, Attilio; Ambudkar, Suresh V.; Bates, Susan E.

    2008-01-01

    Purpose ABCG2 overexpression has been linked to resistance to topoisomerase inhibitors, leading us to examine the potential interaction between ABCG2 and becatecarin. Methods Interaction with ABCG2 was determined by ATPase assay, competition of [125I]iodoarylazidoprazosin (IAAP) photolabeling and flow cytometry. Cellular resistance was measured in four-day cytotoxicity assays. ABCG2 expression was measured by fluorescent-substrate transport assays and immunoblot. Results Becatecarin competed [125I]-IAAP labeling of ABCG2, stimulated ATPase activity and, at concentrations greater than 10 μM, inhibited ABCG2-mediated transport. Becatecarin-selected A549 Bec150 lung carcinoma cells were 3.1-fold, 15-fold, 8-fold, and 6.8-fold resistant to becatecarin, mitoxantrone, SN-38 and topotecan, respectively. A549 Bec150 cells transported the ABCG2 substrates pheophorbide a, mitoxantrone and BODIPY-prazosin and displayed increased staining with the anti-ABCG2 antibody 5D3 compared to parental cells. Increased ABCG2 expression was confirmed by immunoblot. Conclusions Our results suggest that becatecarin is transported by ABCG2 and can induce ABCG2 expression in cancer cells. PMID:19132374

  11. Cichorium intybus L. promotes intestinal uric acid excretion by modulating ABCG2 in experimental hyperuricemia.

    PubMed

    Wang, Yu; Lin, Zhijian; Zhang, Bing; Nie, Anzheng; Bian, Meng

    2017-01-01

    Excessive production and/or reduced excretion of uric acid could lead to hyperuricemia, which could be a major cause of disability. Hyperuricemia has received increasing attention in the last few decades due to its global prevalence. Cichorium intybus L., commonly known as chicory, is a perennial herb of the asteraceae family. It was previously shown to exert potent hypouricemic effects linked with decreasing uric acid formation in the liver by down-regulating the activity of xanthine oxidase, and increasing uric acid excretion by up-regulating the renal OAT3 mRNA expression. The present study aimed to evaluate its extra-renal excretion and possible molecular mechanism underlying the transporter responsible for intestinal uric acid excretion in vivo. Chicory was administered intragastrically to hyperuricemic rats induced by drinking 10% fructose water. The uricosuric effect was evaluated by determining the serum uric acid level as well as the intestinal uric acid excretion by HPLC. The location and expression levels of ATP-binding cassette transporter, sub-family G, member 2 (ABCG2) in jejunum and ileum were analyzed. The administration of chicory decreased the serum uric acid level significantly and increased the intestinal uric acid excretion obviously in hyperuricemic rats induced by 10% fructose drinking. Staining showed that ABCG2 was expressed in the apical membrane of the epithelium and glands of the jejunum and ileum in rats. Further examination showed that chicory enhanced the mRNA and protein expressions of ABCG2 markedly in a dose-dependent manner in jejunum and ileum. These findings indicate that chicory increases uric acid excretion by intestines, which may be related to the stimulation of intestinal uric acid excretion via down-regulating the mRNA and protein expressions of ABCG2.

  12. Application of fluorescent dye substrates for functional characterization of ABC multidrug transporters at a single cell level.

    PubMed

    Nerada, Zsuzsanna; Hegyi, Zoltán; Szepesi, Áron; Tóth, Szilárd; Hegedüs, Csilla; Várady, György; Matula, Zsolt; Homolya, László; Sarkadi, Balázs; Telbisz, Ágnes

    2016-09-01

    ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2- and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. © 2016 International Society for Advancement of Cytometry.

  13. ABCG2 is a selectable marker for enhanced multilineage differentiation potential in periodontal ligament stem cells.

    PubMed

    Szepesi, Áron; Matula, Zsolt; Szigeti, Anna; Várady, György; Szabó, Gyula; Uher, Ferenc; Sarkadi, Balázs; Német, Katalin

    2015-01-15

    Periodontal ligament stem cells (PDLSCs) provide an important source for tissue regeneration and may become especially useful in the formation of osteogenic seeds. PDLSCs can be cultured, expanded, and differentiated in vitro; thus, they may be applied in the long-term treatment of the defects in the dental regions. Here we studied numerous potential markers allowing the selection of human PDLSCs with a maximum differentiation potential. We followed the expression of the ATP-binding cassette subfamily G member 2 (ABCG2) membrane transporter protein and isolated ABCG2-expressing cells by using a monoclonal antibody, recognizing the transporter at the cell surface in intact cells. The expression of the ABCG2 protein, corresponding to the so-called side-population phenotype in various tissue-derived stem cells, was found to be a useful marker for the selection of PDLSCs with enhanced osteogenic, chondrogenic, and adipogenic differentiation. These findings may have important applications in achieving efficient dental tissue regeneration by using stem cells from extracted teeth.

  14. ABCG2 Is a Selectable Marker for Enhanced Multilineage Differentiation Potential in Periodontal Ligament Stem Cells

    PubMed Central

    Szepesi, Áron; Matula, Zsolt; Szigeti, Anna; Várady, György; Szabó, Gyula; Uher, Ferenc; Sarkadi, Balázs

    2015-01-01

    Periodontal ligament stem cells (PDLSCs) provide an important source for tissue regeneration and may become especially useful in the formation of osteogenic seeds. PDLSCs can be cultured, expanded, and differentiated in vitro; thus, they may be applied in the long-term treatment of the defects in the dental regions. Here we studied numerous potential markers allowing the selection of human PDLSCs with a maximum differentiation potential. We followed the expression of the ATP-binding cassette subfamily G member 2 (ABCG2) membrane transporter protein and isolated ABCG2-expressing cells by using a monoclonal antibody, recognizing the transporter at the cell surface in intact cells. The expression of the ABCG2 protein, corresponding to the so-called side-population phenotype in various tissue-derived stem cells, was found to be a useful marker for the selection of PDLSCs with enhanced osteogenic, chondrogenic, and adipogenic differentiation. These findings may have important applications in achieving efficient dental tissue regeneration by using stem cells from extracted teeth. PMID:25101689

  15. DGAT1 and ABCG2 polymorphism in Indian cattle (Bos indicus) and buffalo (Bubalus bubalis) breeds.

    PubMed

    Tantia, Madhu S; Vijh, Ramesh K; Mishra, Bishnu P; Mishra, Bina; Kumar, S T Bharani; Sodhi, Monika

    2006-11-07

    Indian cattle (Bos indicus) and riverine buffalo (Bubalus bubalis) give a poor yield of milk but it has a high fat and protein percentage compared to taurine cattle. The identification of QTLs (Quantitative Trait Loci) on BTA14 and BTA6 and its subsequent fine mapping has led to identification of two non conservative mutations affecting milk production and composition. Our objective was to estimate the frequency of K232A (DGAT1--diacylglycerol-acyltransferase 1) and Y581S (ABCG2--ATP binding cassette sub family G member 2) polymorphisms in diverse cattle and buffalo breeds of India having large variation in terms of milk production. We screened the reported missense mutations in six cattle and five buffalo breeds. The DGAT1K and ABCG2Y alleles were found to be fixed in Indian cattle and buffalo breeds studied. This study provides an indirect evidence that all the Indian cattle and buffalo breeds have fixed alleles with respect to DGAT1 and ABCG2 genes reported to be responsible for higher milk fat yield, higher fat and protein percent.

  16. Abcg2 expression marks tissue-specific stem cells in multiple organs in a mouse progeny tracking model.

    PubMed

    Fatima, Soghra; Zhou, Sheng; Sorrentino, Brian P

    2012-02-01

    The side population phenotype is associated with the Hoechst dye efflux activity of the Abcg2 transporter and identifies hematopoietic stem cells (HSCs) in the bone marrow. This association suggests the direct use of Abcg2 expression to identify adult stem cells in various other organs. We have generated a lineage tracing mouse model based on an allele that coexpresses both Abcg2 and a CreERT2 expression cassette. By crossing these mice with lox-STOP-lox reporter lines (LacZ or YFP), cells that express Abcg2 and their progeny were identified following treatment with tamoxifen (Tam). In the liver and kidney, in which mature cells express Abcg2, reporter gene expression verified the expected physiologic expression pattern of the recombinant allele. Long-term marking of HSCs was seen in multiple peripheral blood lineages from adult mice, demonstrating that Abcg2(+) bone marrow HSCs contribute to steady-state hematopoiesis. Stem cell tracing patterns were seen in the small intestine and in seminiferous tubules in the testis 20 months after Tam treatment, proving that stem cells from these organs express Abcg2. Interstitial cells from skeletal and cardiac muscle were labeled, and some cells were costained with endothelial markers, raising the possibility that these cells may function in the repair response to muscle injury. Altogether, these studies prove that Abcg2 is a stem cell marker for blood, small intestine, testicular germ cells, and possibly for injured skeletal and/or cardiac muscle and provide a new model for studying stem cell activity that does not require transplant-based assays. Copyright © 2011 AlphaMed Press.

  17. Abcg2 expression marks tissue-specific stem cells in multiple organs in a mouse progeny tracking model

    PubMed Central

    Fatima, Soghra; Zhou, Sheng; Sorrentino, Brian P.

    2016-01-01

    The side population (SP) phenotype is associated with the Hoechst dye efflux activity of the Abcg2 transporter and identifies hematopoietic stem cells (HSCs) in the bone marrow. This association suggests the direct use of Abcg2 expression to identify adult stem cells in various other organs. We have generated a lineage tracing mouse model based on an allele that co-expresses both Abcg2 and a CreERT2 expression cassette. By crossing these mice with lox-STOP-lox reporter lines (LacZ or YFP), cells that express Abcg2 and their progeny were identified following treatment with tamoxifen (Tam). In the liver and kidney, in which mature cells express Abcg2, reporter gene expression verified the expected physiologic expression pattern of the recombinant allele. Long term marking of HSCs was seen in multiple peripheral blood lineages from adult mice, demonstrating that Abcg2+ bone marrow HSCs contribute to steady state hematopoiesis. Stem cell tracing patterns were seen in the small intestine and in seminiferous tubules in the testis twenty months after Tam treatment, proving that stem cells from these organs express Abcg2. Interstitial cells from skeletal and cardiac muscle were labeled and some cells co-stained with endothelial markers, raising the question as to whether these may function as tissue-specific muscle stem cells. Altogether, these studies prove that Abcg2 is a stem cell marker for blood, small intestine, and testicular germ cells and provide a new model for studying stem cell activity that does not require transplant-based assays. PMID:22134889

  18. Bioluminescent imaging of drug efflux at the blood–brain barrier mediated by the transporter ABCG2

    PubMed Central

    Bakhsheshian, Joshua; Wei, Bih-Rong; Chang, Ki-Eun; Shukla, Suneet; Ambudkar, Suresh V.; Simpson, R. Mark; Gottesman, Michael M.; Hall, Matthew D.

    2013-01-01

    ATP-binding cassette (ABC) transporters are a group of transmembrane proteins that maintain chemical homeostasis through efflux of compounds out of organelles and cells. Among other functions, ABC transporters play a key role in protecting the brain parenchyma by efflux of xenobiotics from capillary endothelial cells at the blood–brain barrier (BBB). They also prevent the entry of therapeutic drugs at the BBB, thereby limiting their efficacy. One of the key transporters playing this role is ABCG2. Although other ABC transporters can be studied through various imaging modalities, no specific probe exists for imaging ABCG2 function in vivo. Here we show that d-luciferin, the endogenous substrate of firefly luciferase, is a specific substrate for ABCG2. We hypothesized that ABCG2 function at the BBB could be evaluated by using bioluminescence imaging in transgenic mice expressing firefly luciferase in the brain. Bioluminescence signal in the brain of mice increased with coadministration of the ABCG2 inhibitors Ko143, gefitinib, and nilotinib, but not an ABCB1 inhibitor. This method for imaging ABCG2 function at the BBB will facilitate understanding of the function and pharmacokinetic inhibition of this transporter. PMID:24297888

  19. Novel understanding of ABC transporters ABCB1/MDR/P-glycoprotein, ABCC2/MRP2, and ABCG2/BCRP in colorectal pathophysiology.

    PubMed

    Andersen, Vibeke; Svenningsen, Katrine; Knudsen, Lina Almind; Hansen, Axel Kornerup; Holmskov, Uffe; Stensballe, Allan; Vogel, Ulla

    2015-11-07

    To evaluate ATP-binding cassette (ABC) transporters in colonic pathophysiology as they had recently been related to colorectal cancer (CRC) development. Literature search was conducted on PubMed using combinations of the following terms: ABC transporters, ATP binding cassette transporter proteins, inflammatory bowel disease, ulcerative, colitis, Crohn's disease, colorectal cancer, colitis, intestinal inflammation, intestinal carcinogenesis, ABCB1/P-glycoprotein (P-gp/CD243/MDR1), ABCC2/multidrug resistance protein 2 (MRP2) and ABCG2/breast cancer resistance protein (BCRP), Abcb1/Mdr1a, abcc2/Mrp2, abcg2/Bcrp, knock-out mice, tight junction, membrane lipid function. Recently, human studies reported that changes in the levels of ABC transporters were early events in the adenoma-carcinoma sequence leading to CRC. A link between ABCB1, high fat diet and gut microbes in relation to colitis was suggested by the animal studies. The finding that colitis was preceded by altered gut bacterial composition suggests that deletion of Abcb1 leads to fundamental changes of host-microbiota interaction. Also, high fat diet increases the frequency and severity of colitis in specific pathogen-free Abcb1 KO mice. The Abcb1 KO mice might thus serve as a model in which diet/environmental factors and microbes may be controlled and investigated in relation to intestinal inflammation. Potential molecular mechanisms include defective transport of inflammatory mediators and/or phospholipid translocation from one side to the other of the cell membrane lipid bilayer by ABC transporters affecting inflammatory response and/or function of tight junctions, phagocytosis and vesicle trafficking. Also, diet and microbes give rise to molecules which are potential substrates for the ABC transporters and which may additionally affect ABC transporter function through nuclear receptors and transcriptional regulation. Another critical role of ABCB1 was suggested by the finding that ABCB1 expression

  20. Novel understanding of ABC transporters ABCB1/MDR/P-glycoprotein, ABCC2/MRP2, and ABCG2/BCRP in colorectal pathophysiology

    PubMed Central

    Andersen, Vibeke; Svenningsen, Katrine; Knudsen, Lina Almind; Hansen, Axel Kornerup; Holmskov, Uffe; Stensballe, Allan; Vogel, Ulla

    2015-01-01

    AIM: To evaluate ATP-binding cassette (ABC) transporters in colonic pathophysiology as they had recently been related to colorectal cancer (CRC) development. METHODS: Literature search was conducted on PubMed using combinations of the following terms: ABC transporters, ATP binding cassette transporter proteins, inflammatory bowel disease, ulcerative, colitis, Crohns disease, colorectal cancer, colitis, intestinal inflammation, intestinal carcinogenesis, ABCB1/P-glycoprotein (P-gp/CD243/MDR1), ABCC2/multidrug resistance protein 2 (MRP2) and ABCG2/breast cancer resistance protein (BCRP), Abcb1/Mdr1a, abcc2/Mrp2, abcg2/Bcrp, knock-out mice, tight junction, membrane lipid function. RESULTS: Recently, human studies reported that changes in the levels of ABC transporters were early events in the adenoma-carcinoma sequence leading to CRC. A link between ABCB1, high fat diet and gut microbes in relation to colitis was suggested by the animal studies. The finding that colitis was preceded by altered gut bacterial composition suggests that deletion of Abcb1 leads to fundamental changes of host-microbiota interaction. Also, high fat diet increases the frequency and severity of colitis in specific pathogen-free Abcb1 KO mice. The Abcb1 KO mice might thus serve as a model in which diet/environmental factors and microbes may be controlled and investigated in relation to intestinal inflammation. Potential molecular mechanisms include defective transport of inflammatory mediators and/or phospholipid translocation from one side to the other of the cell membrane lipid bilayer by ABC transporters affecting inflammatory response and/or function of tight junctions, phagocytosis and vesicle trafficking. Also, diet and microbes give rise to molecules which are potential substrates for the ABC transporters and which may additionally affect ABC transporter function through nuclear receptors and transcriptional regulation. Another critical role of ABCB1 was suggested by the finding that

  1. The ATP-binding cassette transporter ABCG2 protects against pressure overload-induced cardiac hypertrophy and heart failure by promoting angiogenesis and antioxidant response.

    PubMed

    Higashikuni, Yasutomi; Sainz, Julie; Nakamura, Kazuto; Takaoka, Minoru; Enomoto, Soichiro; Iwata, Hiroshi; Tanaka, Kimie; Sahara, Makoto; Hirata, Yasunobu; Nagai, Ryozo; Sata, Masataka

    2012-03-01

    ATP-binding cassette transporter subfamily G member 2 (ABCG2), expressed in microvascular endothelial cells in the heart, has been suggested to regulate several tissue defense mechanisms. This study was performed to elucidate its role in pressure overload-induced cardiac hypertrophy. Pressure overload was induced in 8- to 12-week-old wild-type and Abcg2-/- mice by transverse aortic constriction (TAC). Abcg2-/- mice showed exaggerated cardiac hypertrophy and ventricular remodeling after TAC compared with wild-type mice. In the early phase after TAC, functional impairment in angiogenesis and antioxidant response in myocardium was found in Abcg2-/- mice. In vitro experiments demonstrated that ABCG2 regulates transport of glutathione, an important endogenous antioxidant, from microvascular endothelial cells. Besides, glutathione transported from microvascular endothelial cells in ABCG2-dependent manner ameliorated oxidative stress-induced cardiomyocyte hypertrophy. In vivo, glutathione levels in plasma and the heart were increased in wild-type mice but not in Abcg2-/- mice after TAC. Treatment with the superoxide dismutase mimetic ameliorated cardiac hypertrophy in Abcg2-/- mice after TAC to the same extent as that in wild-type mice, although cardiac dysfunction with impaired angiogenesis was observed in Abcg2-/- mice. ABCG2 protects against pressure overload-induced cardiac hypertrophy and heart failure by promoting angiogenesis and antioxidant response.

  2. Existence of small slow-cycling Langerhans cells in the limbal basal epithelium that express ABCG2.

    PubMed

    Chen, Wensheng; Hara, Koji; Tian, Qing; Zhao, Kanxing; Yoshitomi, Takeshi

    2007-04-01

    Despite the obvious importance of limbal stem cells in corneal homeostasis and tumorigenesis, little is known about their specific biological characteristics. The purpose of this study was to characterize limbal slow-cycling cells based on the expression of ABCG2 and major histocompatibility complex (MHC) class II and the cell size. Wistar rats were daily injected with 5-bromo-2-deoxyuridine (BrdU) at a dose of 5 mg/100 g for 2 weeks. After 4-week BrdU-free period, corneal tissues were excised, and immunofluorescence staining for ABCG2, BrdU, and MHC class II was performed by confocal microscopy. In another series, corneal tissues of normal rat were double immunostained for ABCG2, keratin 14, keratin 3, CD11c, and MHC class II. In addition, limbal, peripheral and central corneal epithelial sheets were isolated by Dispase II digestion and dissociated into single cell by trypsin digestion and cytospin preparations were double immunostained for ABCG2 and MHC class II. The cell size and nucleus-to-cytoplasm (N/C) ratio of limbal ABCG2+ cells were analyzed and compared with those of cells from other zones. BrdU label-retaining cells (LRCs) with expression of ABCG2 were found in the limbal epithelial basal layer, but not in other parts of the cornea. Approximately 20% of these cells were MHC class II positive. All MHC class II+ cells in the corneal epithelium were positive for CD11c, a marker for dendritic cells (DCs). Double labeling with ABCG2 and keratin 14 showed that nearly four-fifth of limbal ABCG2+ cells were positive for keratin 14 but negative for keratin 3, exhibiting an undifferentiated epithelial cell lineage. Cytospin sample analysis revealed the presence of a distinct population of smaller ABCG2+ cells with expression of MHC class II with a larger N/C ratio in the limbal epithelium. A new population of small slow-cycling cells with large N/C ratio has been found to express ABCG2 in the limbal epithelial basal layer. Some of these cells normally express MHC

  3. Natural allelic variants of bovine ATP-binding cassette transporter ABCG2: increased activity of the Ser581 variant and development of tools for the discovery of new ABCG2 inhibitors.

    PubMed

    Merino, Gracia; Real, Rebeca; Baro, Marta F; Gonzalez-Lobato, Lucia; Prieto, Julio G; Alvarez, Ana I; Marques, Margarita M

    2009-01-01

    ATP-binding cassette transporter ABCG2 [breast cancer resistance protein (BCRP)] is a member of the ABC transporter superfamily that actively extrudes xenotoxins from cells and is a major determinant of the bioavailability of many compounds. ABCG2 expression is strongly induced during lactation in the mammary gland and is related to the active secretion of drugs into the milk. The presence of drug residues and environmental pollutants in milk is an outstanding problem for human milk consumption and milk industrial processes, involving important risks to public health and the dairy industry. In cows, a single nucleotide polymorphism (SNP) in this protein has been described previously (Tyr581) and is associated with higher fat and protein percentages and lower milk yield. However, whether this amino acid substitution affects ABCG2-mediated drug transport in cows, including milk secretion, required further exploration. We cloned the two variants of bovine ABCG2 and evaluated the effect of this SNP on mitoxantrone accumulation assays performed in ovine primary fibroblasts transiently expressing either of the variants. It is interesting to note that statistically significant differences in activity between both variants were observed, and the Ser581 variant was related with an increased efflux activity. In addition, we demonstrated that genistein is a very good inhibitor of bovine ABCG2 and identified new inhibitors of the transporter, such as the macrocyclic lactones, ivermectin, and selamectin. Moreover, the inhibitory effect of these compounds on human and murine ABCG2 homologs was confirmed using transduced Marbin-Dabin canine kidney II cells. These findings may have important implications regarding the presence of drug residues in milk and drug interactions affecting the pharmacological behavior of ABCG2 substrates.

  4. A High-Content Assay Strategy for the Identification and Profiling of ABCG2 Modulators in Live Cells

    PubMed Central

    Antczak, Christophe; Wee, Boyoung; Radu, Constantin; Bhinder, Bhavneet; Holland, Eric C.

    2014-01-01

    Abstract ABCG2 is a member of the ATP-binding cassette (ABC) family of transporters, the overexpression of which has been implicated in resistance to various chemotherapeutic agents. Though a number of cell-based assays to screen for inhibitors have been reported, they do not provide a content-rich platform to discriminate toxic and autofluorescent compounds. To fill this gap, we developed a live high-content cell-based assay to identify inhibitors of ABCG2-mediated transport and, at the same time, assess their cytotoxic effect and potential optical interference. We used a pair of isogenic U87MG human glioblastoma cell lines, with one stably overexpressing the ABCG2 transporter. JC-1 (J-aggregate–forming lipophilic cation 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol carbocyanine iodide) was selected as the optimal reporter substrate for ABCG2 activity, and the resulting assay was characterized by a Z′ value of 0.50 and a signal-to-noise (S/N) ratio of 14 in a pilot screen of ∼7,000 diverse chemicals. The screen led to the identification of 64 unique nontoxic positives, yielding an initial hit rate of 1%, with 58 of them being confirmed activity. In addition, treatment with two selected confirmed positives suppressed the side population of U87MG-ABCG2 cells that was able to efflux the Hoechst dye as measured by flow cytometry, confirming that they constitute potent new ABCG2 transporter inhibitors. Our results demonstrate that our live cell and content-rich platform enables the rapid identification and profiling of ABCG2 modulators, and this new strategy opens the door to the discovery of compounds targeting the expression and/or trafficking of ABC transporters as an alternative to functional inhibitors that failed in the clinic. PMID:23992118

  5. Mutational Analysis of Threonine 402 Adjacent to the GXXXG Dimerization Motif in TM1 of ABCG2

    PubMed Central

    Polgar, Orsolya; Ierano, Caterina; Tamaki, Akina; Stanley, Bradford; Ward, Yvona; Xia, Di; Tarasova, Nadya; Robey, Robert W.; Bates, Susan E.

    2010-01-01

    ABCG2 is an ATP-binding cassette half-transporter important in normal tissue protection, drug distribution and excretion. ABCG2 requires homodimerization for function, though the mechanism for dimerization has not been elucidated. We carried out mutational analysis of threonine 402, three residues away from the GXXXG motif in TM1, to study its potential role in ABCG2 dimerization (TXXXGXXXG). Single mutations to leucine (T402L) or arginine (T402R) did not have significant impact on the ABCG2 protein. On the other hand, combining the T402 mutations with the GXXXG glycine to leucine mutations (T402L/G406L/G410L and T402R/G406L/G410L) resulted in substantially reduced expression, altered glycosylation, degradation by a proteosome independent pathway and partial retention in the ER as suggested by immunostaining, Endo H sensitivity and MG132 and bafilomycin failed effect. The T402L/G406L/G410L mutant when incubated with the ABCG2-substrate MX showed a shift on immunoblot analysis to the band representing the fully matured glycoprotein. The T402R/G406L/G410L mutant carrying the more drastic substitution was found to primarily localize intracellularly. The same set of mutations also displayed impaired dimerization in the TOXCAT assay for TM1 compared to the wild-type. Homology modeling of ABCG2 places the TXXXGXXXG motif at the dimer interface. These studies are consistent with a role for the extended TXXXGXXXG motif in ABCG2 folding, processing, and/or dimerization. PMID:20088606

  6. Common dysfunctional variants of ABCG2 have stronger impact on hyperuricemia progression than typical environmental risk factors

    PubMed Central

    Nakayama, Akiyoshi; Matsuo, Hirotaka; Nakaoka, Hirofumi; Nakamura, Takahiro; Nakashima, Hiroshi; Takada, Yuzo; Oikawa, Yuji; Takada, Tappei; Sakiyama, Masayuki; Shimizu, Seiko; Kawamura, Yusuke; Chiba, Toshinori; Abe, Junko; Wakai, Kenji; Kawai, Sayo; Okada, Rieko; Tamura, Takashi; Shichijo, Yuka; Akashi, Airi; Suzuki, Hiroshi; Hosoya, Tatsuo; Sakurai, Yutaka; Ichida, Kimiyoshi; Shinomiya, Nariyoshi

    2014-01-01

    Gout/hyperuricemia is a common multifactorial disease having typical environmental risks. Recently, common dysfunctional variants of ABCG2, a urate exporter gene also known as BCRP, are revealed to be a major cause of gout/hyperuricemia. Here, we compared the influence of ABCG2 dysfunction on serum uric acid (SUA) levels with other typical risk factors in a cohort of 5,005 Japanese participants. ABCG2 dysfunction was observed in 53.3% of the population investigated, and its population-attributable risk percent (PAR%) for hyperuricemia was 29.2%, much higher than those of the other typical environmental risks, i.e. overweight/obesity (BMI ≥ 25.0; PAR% = 18.7%), heavy drinking (>196 g/week (male) or >98 g/week (female) of pure alcohol; PAR% = 15.4%), and aging (≥60 years old; PAR% = 5.74%). SUA significantly increased as the ABCG2 function decreased (P = 5.99 × 10−19). A regression analysis revealed that ABCG2 dysfunction had a stronger effect than other factors; a 25% decrease in ABCG2 function was equivalent to “an increase of BMI by 1.97-point” or “552.1 g/week alcohol intake as pure ethanol” in terms of ability to increase SUA. Therefore, ABCG2 dysfunction originating from common genetic variants has a much stronger impact on the progression of hyperuricemia than other familiar risks. Our study provides a better understanding of common genetic factors for common diseases. PMID:24909660

  7. Genetic variation in the ABCG2 gene is associated with gout risk in the Chinese Han population.

    PubMed

    Jiri, Mutu; Zhang, Le; Lan, Bing; He, Na; Feng, Tian; Liu, Kai; Jin, Tianbo; Kang, Longli

    2016-01-01

    Gout is a common type of arthritis that is characterized by hyperuricemia, tophi, and joint inflammation. Current evidence suggests that heredity contributes to the progression of gout. Previous studies have shown that regulation of the ATP-binding cassette subfamily G member 2 (ABCG2) pathways plays a role in gout occurrence. To investigate and validate potential genetic associations with the risk of gout, we conducted a case-control study. We conducted 143 cases and 310 controls and genotyped seven single-nucleotide polymorphisms (SNPs) in ABCG2 gene. ABCG2 SNP association analyses were performed using SPSS 17.0 Statistical Package, PLINK Software, HaploView software package, and SHEsis software platform. We identified that four susceptibility SNPs were potentially associated with occurrence of gout. Rs2622621 and rs3114018 in ABCG2 can actually increase the risk of gout in log-additive model (rs2622621, odds ratio (OR) = 1.90, 95% confidence interval (CI) 1.39-2.61, p < 0.001; rs3114018, OR = 1.55, 95% CI 1.13-2.13, p = 0.006). We found that rs17731799G/T-G/G and rs3114020 T/C-T/T in ABCG2 can actually increase the risk of gout in dominant model (rs17731799, OR = 1.67, 95% CI 1.05-2.66, p = 0.028; rs3114020, OR = 1.58, 95% CI 1.00-2.51, p = 0.048). The ABCG2 haplotype "GGCTCTC" (OR = 0.46, 95% CI 0.28-0.75, p = 0.0019) decreased the gout risk. Our results, combined with those from previous studies, suggest that genetic variation in ABCG2 may influence gout susceptibility in the Han population.

  8. Effect of bovine ABCG2 polymorphism Y581S SNP on secretion into milk of enterolactone, riboflavin and uric acid.

    PubMed

    Otero, J A; Miguel, V; González-Lobato, L; García-Villalba, R; Espín, J C; Prieto, J G; Merino, G; Álvarez, A I

    2016-02-01

    The ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP) is an efflux protein involved in the bioavailability and milk secretion of endogenous and exogenous compounds, actively affecting milk composition. A limited number of physiological substrates have been identified. However, no studies have reported the specific effect of this polymorphism on the secretion into milk of compounds implicated in milk quality such as vitamins or endogenous compounds. The bovine ABCG2 Y581S polymorphism is described as a gain-of-function polymorphism that increases milk secretion and decreases plasma levels of its substrates. This work aims to study the impact of Y581S polymorphism on plasma disposition and milk secretion of compounds such as riboflavin (vitamin B2), enterolactone, a microbiota-derived metabolite from the dietary lignan secoisolariciresinol and uric acid. In vitro transport of these compounds was assessed in MDCK-II cells overexpressing the bovine ABCG2 (WT-bABCG2) and its Y581S variant (Y581S-bABCG2). Plasma and milk levels were obtained from Y/Y homozygous and Y/S heterozygous cows. The results show that riboflavin was more efficiently transported in vitro by the Y581S variant, although no differences were noted in vivo. Both uric acid and enterolactone were substrates in vitro of the bovine ABCG2 variants and were actively secreted into milk with a two-fold increase in the milk/plasma ratio for Y/S with respect to Y/Y cows. The in vitro ABCG2-mediated transport of the drug mitoxantrone, as a model substrate, was inhibited by enterolactone in both variants, suggesting the possible in vivo use of this enterolignan to reduce ABCG2-mediated milk drug transfer in cows. The Y581S variant was inhibited to a lesser extent probably due to its higher transport capacity. All these findings point to a significant role of the ABCG2 Y581S polymorphism in the milk disposition of enterolactone and the endogenous molecules riboflavin and uric acid

  9. A new strategy of high-speed screening and quantitative structure-activity relationship analysis to evaluate human ATP-binding cassette transporter ABCG2-drug interactions.

    PubMed

    Saito, Hikaru; Hirano, Hiroyuki; Nakagawa, Hiroshi; Fukami, Takeaki; Oosumi, Keisuke; Murakami, Kaori; Kimura, Hiroko; Kouchi, Takayuki; Konomi, Mami; Tao, Eriko; Tsujikawa, Noboru; Tarui, Shigeki; Nagakura, Makoto; Osumi, Masako; Ishikawa, Toshihisa

    2006-06-01

    The human ATP-binding cassette (ABC) transporter ABCG2 (BCRP/MXR1/ABCP) plays a critical role in cellular protection against xenobiotics as well as pharmacokinetics of drugs in our body. In the present study, we aimed to analyze the quantitative structure-activity relationship (QSAR) latently residing in ABCG2-drug interactions. We first established standard methods for expression of human ABCG2 in insect cells, quality control of plasma membrane samples by using electron microscopy techniques, and high-speed screening of ABCG2 inhibition with test compounds. Plasma membrane vesicles prepared from ABCG2-expressing Sf9 cells were used as a model system to measure the ATP-dependent transport of [3H]methotrexate (MTX). Forty-nine different therapeutic drugs and natural compounds were tested for their ability to inhibit ABCG2-mediated MTX transport. Based on their inhibition profiles, we performed QSAR analysis using chemical fragmentation codes deduced from the structures of test compounds. Multiple linear regression analysis delineated a relationship between the structural components and the extent of ABCG2 inhibition, allowing us to identify one set of structure-specific chemical fragmentation codes that are closely correlated with the inhibition of ABCG2 transport activity. Based on the QSAR analysis data, we predicted the potency of gefitinib to inhibit ABCG2. The validity of our QSAR-based prediction for gefitinib was examined by actual experiments. Our kinetic analysis experiments suggest that the ABCG2-ATP complex binds gefitinib. The present study provides a new strategy for analyzing ABCG2-drug interactions. This strategy is considered to be practical and useful for the molecular designing of new ABCG2 modulators.

  10. C421 allele-specific ABCG2 gene amplification confers resistance to the antitumor triazoloacridone C-1305 in human lung cancer cells.

    PubMed

    Bram, Eran E; Ifergan, Ilan; Grimberg, Michal; Lemke, Krzysztof; Skladanowski, Andrzej; Assaraf, Yehuda G

    2007-06-30

    The A421 ABCG2 genotype is a frequent polymorphism encoding the K141 transporter, which is associated with a significant decrease in transporter expression and function when compared to the wild type (wt) C421 allele encoding the Q141 ABCG2. Here we show that during the acquisition of resistance to the novel triazoloacridone antitumor agent C-1305 in lung cancer cells harboring a heterozygous C421A genotype, a marked C421 allele-specific ABCG2 gene amplification occurred. This monoallelic C421 ABCG2 gene amplification brought about the overexpression of both C421 ABCG2 mRNA and the transporter at the plasma membrane. This resulted in the lack of cellular drug accumulation due to increased efflux of both C1305 and C-1311, a fluorescent imidazoacridone homologue of C-1305, as well as marked resistance to these antitumor agents and to established ABCG2 substrates including mitoxantrone and SN-38. Consistently, the accumulation and sensitivity to these drugs were restored upon incubation with the potent and specific ABCG2 transport inhibitors Ko143 and fumitremorgin C. Moreover, upon transfection into HEK293 cells, the wt Q141 ABCG2 allele displayed a significantly decreased accumulation of C-1311 and increased resistance to C-1305, C-1311 and mitoxantrone, when compared to the K141 ABCG2 transfectant. Hence, the current study provides the first evidence that during the exposure to anticancer drugs, an allele-specific Q141 ABCG2 gene amplification occurs that confers a drug resistance advantage when compared to the K141 ABCG2. These findings have important implications for the selection and expansion of malignant anticancer drug resistant clones during chemotherapy with ABCG2 drugs.

  11. Down-regulation of ABCG2, a urate exporter, by parathyroid hormone enhances urate accumulation in secondary hyperparathyroidism.

    PubMed

    Sugimoto, Ryusei; Watanabe, Hiroshi; Ikegami, Komei; Enoki, Yuki; Imafuku, Tadashi; Sakaguchi, Yoshiaki; Murata, Michiya; Nishida, Kento; Miyamura, Shigeyuki; Ishima, Yu; Tanaka, Motoko; Matsushita, Kazutaka; Komaba, Hirotaka; Fukagawa, Masafumi; Otagiri, Masaki; Maruyama, Toru

    2017-03-01

    Hyperuricemia occurs with increasing frequency among patients with hyperparathyroidism. However, the molecular mechanism by which the serum parathyroid hormone (PTH) affects serum urate levels remains unknown. This was studied in uremic rats with secondary hyperparathyroidism where serum urate levels were found to be increased and urate excretion in the intestine and kidney decreased, presumably due to down-regulation of the expression of the urate exporter ABCG2 in intestinal and renal epithelial membranes. These effects were prevented by administration of the calcimimetic cinacalcet, a PTH suppressor, suggesting that PTH may down-regulate ABCG2 expression. This was directly tested in intestinal Caco-2 cells where the expression of ABCG2 on the plasma membrane was down-regulated by PTH (1-34) while its mRNA level remained unchanged. Interestingly, an inactive PTH derivative (13-34) had no effect, suggesting that a posttranscriptional regulatory system acts through the PTH receptor to regulate ABCG2 plasma membrane expression. As found in an animal study, additional clinical investigations showed that treatment with cinacalcet resulted in significant reductions in serum urate levels together with decreases in PTH levels in patients with secondary hyperparathyroidism undergoing dialysis. Thus, PTH down-regulates ABCG2 expression on the plasma membrane to suppress intestinal and renal urate excretion, and the effects of PTH can be prevented by cinacalcet treatment.

  12. Effects of the ABCG2 and ABCB1 drug transporter polymorphisms on the pharmacokinetics of bicalutamide in humans.

    PubMed

    Kim, Kyoung-Ah; Cha, Yu-Jung; Lee, Hae-Mi; Joo, Hyun-Jin; Park, Ji-Young

    2015-01-01

    Bicalutamide is an oral non-steroidal anti-androgen used in the treatment of prostate cancer. Drug transporters P-glycoprotein encoded by ABCB1 and breast cancer resistance protein (BCRP) encoded by ABCG2 are involved in the transportation of bicalutamide and its treatment failure. We evaluated the roles of ABCB1 and ABCG2 genetic polymorphisms in the pharmacokinetics of bicalutamide in humans. After a single oral dose of 150mg bicalutamide was administered, plasma concentrations of bicalutamide were measured, and pharmacokinetic analyses were performed in 27 healthy subjects according to ABCB1 (c.1236C>T, c.2677G>T/A, and c.3435C>T) and ABCG2 (c.34G>A and c.421C>A). ABCB1 polymorphisms did not affect the plasma levels of bicalutamide and the pharmacokinetic parameters did not differ among ABCB1 genotype groups. However, the ABCG2 c.421C>A polymorphism significantly influenced the plasma levels and pharmacokinetics of bicalutamide gene dose-dependently. The ABCB1 genetic polymorphisms did not influence the pharmacokinetics of bicalutamide. However, ABCG2 c.421C>A significantly and gene dose-dependently influenced its pharmacokinetics, but c.34G>A did not. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Pilot PET Study to Assess the Functional Interplay Between ABCB1 and ABCG2 at the Human Blood–Brain Barrier

    PubMed Central

    Bauer, M; Römermann, K; Karch, R; Wulkersdorfer, B; Stanek, J; Philippe, C; Maier‐Salamon, A; Haslacher, H; Jungbauer, C; Wadsak, W; Jäger, W; Löscher, W; Hacker, M; Zeitlinger, M

    2016-01-01

    ABCB1 and ABCG2 work together at the blood–brain barrier (BBB) to limit brain distribution of dual ABCB1/ABCG2 substrates. In this pilot study we used positron emission tomography (PET) to assess brain distribution of two model ABCB1/ABCG2 substrates ([11C]elacridar and [11C]tariquidar) in healthy subjects without (c.421CC) or with (c.421CA) the ABCG2 single‐nucleotide polymorphism (SNP) c.421C>A. Subjects underwent PET scans under conditions when ABCB1 and ABCG2 were functional and during ABCB1 inhibition with high‐dose tariquidar. In contrast to the ABCB1‐selective substrate (R)‐[11C]verapamil, [11C]elacridar and [11C]tariquidar showed only moderate increases in brain distribution during ABCB1 inhibition. This provides evidence for a functional interplay between ABCB1 and ABCG2 at the human BBB and suggests that both ABCB1 and ABCG2 need to be inhibited to achieve substantial increases in brain distribution of dual ABCB1/ABCG2 substrates. During ABCB1 inhibition c.421CA subjects had significantly higher increases in [11C]tariquidar brain distribution than c.421CC subjects, pointing to impaired cerebral ABCG2 function. PMID:26940368

  14. Pilot PET Study to Assess the Functional Interplay Between ABCB1 and ABCG2 at the Human Blood-Brain Barrier.

    PubMed

    Bauer, M; Römermann, K; Karch, R; Wulkersdorfer, B; Stanek, J; Philippe, C; Maier-Salamon, A; Haslacher, H; Jungbauer, C; Wadsak, W; Jäger, W; Löscher, W; Hacker, M; Zeitlinger, M; Langer, O

    2016-08-01

    ABCB1 and ABCG2 work together at the blood-brain barrier (BBB) to limit brain distribution of dual ABCB1/ABCG2 substrates. In this pilot study we used positron emission tomography (PET) to assess brain distribution of two model ABCB1/ABCG2 substrates ([(11) C]elacridar and [(11) C]tariquidar) in healthy subjects without (c.421CC) or with (c.421CA) the ABCG2 single-nucleotide polymorphism (SNP) c.421C>A. Subjects underwent PET scans under conditions when ABCB1 and ABCG2 were functional and during ABCB1 inhibition with high-dose tariquidar. In contrast to the ABCB1-selective substrate (R)-[(11) C]verapamil, [(11) C]elacridar and [(11) C]tariquidar showed only moderate increases in brain distribution during ABCB1 inhibition. This provides evidence for a functional interplay between ABCB1 and ABCG2 at the human BBB and suggests that both ABCB1 and ABCG2 need to be inhibited to achieve substantial increases in brain distribution of dual ABCB1/ABCG2 substrates. During ABCB1 inhibition c.421CA subjects had significantly higher increases in [(11) C]tariquidar brain distribution than c.421CC subjects, pointing to impaired cerebral ABCG2 function. © 2016, The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

  15. Inhibition of c-Kit, VEGFR-2 (KDR), and ABCG2 by analogues of OSI-930.

    PubMed

    Patel, Jay P; Kuang, Ye-Hong; Chen, Zhe-Sheng; Korlipara, Vijaya L

    2011-11-01

    The quinoline domain of OSI-930, a dual inhibitor of receptor tyrosine kinases (RTKs) c-Kit and KDR, was modified in an effort to further understand the SAR of OSI-930, and the binding site characteristics of c-Kit and KDR. A series of 16 compounds with heteroatom substituted pyridyl and phenyl ring systems was synthesized and evaluated against a panel of kinases including c-Kit and KDR. Aminopyridyl derivative 6 was found to be the most active member of the series with 91% and 57% inhibition of c-Kit at 10μM and 1μM, respectively and 88% and 50% inhibition of KDR at 10μM and 1μM, respectively. The target compounds were also tested for their ability to inhibit efflux of mitoxantrone through inhibition of ATP dependent ABCG2 pump. Nitropyridyl derivative 5 and o-nitrophenyl derivative 7 exhibited complete inhibition of the ABCG2 pump with IC(50) values of 13.67μM and 16.67μM, respectively.

  16. The expressions of ABCC4 and ABCG2 xenobiotic transporters in human keratinocytes are proliferation-related.

    PubMed

    Bebes, Attila; Kis, Kornélia; Nagy, Tünde; Kurunczi, Anita; Polyánka, Hilda; Bata-Csörgo, Zsuzsanna; Kemény, Lajos; Dobozy, Attila; Széll, Márta

    2012-01-01

    Xenobiotic transporters of the ATP-binding cassette (ABC) protein superfamily play important roles in maintaining the biochemical barrier of various tissues, but their precise functions in the skin are not yet known. Screening of the expressions of the known xenobiotic transporter genes in two in vitro keratinocyte differentiation models revealed that the ABCC4 and ABCG2 transporters are highly expressed in proliferating keratinocytes, their expressions decreasing along with differentiation. Abrogation of the ABCC4 and ABCG2 protein functions by siRNA-mediated silencing and chemical inhibition did not affect the proliferation of HaCaT cells. In contrast, disruption of the ABCG2 function had no effect on normal human epidermal keratinocyte proliferation, while the inhibition of ABCC-type transporters by probenecid resulted in a striking decrease in the proliferation of the cells. These results indicate that, besides their possible therapy-modulating effects, xenobiotic transporters may contribute significantly to other keratinocyte functions, such as cell proliferation.

  17. [The effect of MDR1 (P-gp) and ABCG2 on the drug resistance in Hep 2 cells].

    PubMed

    Sun, Zhenfeng; Shen, Bin; Zhang, Jia; Su, Tiantian; Dong, Pin

    2015-06-01

    To study the effect of MDR1 (P-gp) and ABCG2 on the drug resistance in Hep 2 cells. Flow cytometry was used to detect the variations of the antitumor drugs accumulation and discharging, and activity variations when MDR1 and ABCG2 inhibitors were used in Hep-2. The accumulation and discharging of mitoxantrone was significantly higher than the control group when ABCG2 inhibitor FTC was used in Hep-2 (P<0. 05). In contrast, P-gp did not appear similar case; To the mitoxantrone and cisplatin, there was no statistical correlation about activity of Hep-2 between P-gp or ABCG2 antagonist and the control; To the doxorubicin, combining FTC and P-gp, the activity of Hep-2 was higher than the control and difference was significant (P<. 05), In contrast, FTC and P-gp did not appear similar case when used alone; To the 5-FU, when PGP used, the activity of Hep-2 was higher than that in the control and difference was significant (P<0. 05), In con- trast, FTC and FTC+P-gp did not appear similar case; To the paclitaxel, when P-gp or FTC+P-gp used, the activity of Hep-2 was higher than that in the control and difference was significant(P<0. 05). ABCG2 may lead to drug resistance mainly by changing the ability of cell in accumulating and discharging chemotherapy drugs. P-gp has other way. P-gp and ABCG2 play different roles in different drug resistance.

  18. Sildenafil is not a useful modulator of ABCB1 and ABCG2 mediated drug resistance in vivo.

    PubMed

    Lin, Fan; Hoogendijk, Lisette; Buil, Levi; Beijnen, Jos H; van Tellingen, Olaf

    2013-05-01

    Recently, sildenafil was reported to be an inhibitor of P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) in vitro. We have now investigated the in vivo potency of sildenafil. By using wild-type and Abcb1; Abcg2 knockout mice we have investigated the effect of sildenafil on the brain penetration of two substrate drugs (docetaxel and topotecan). Next we have investigated if sildenafil was able to improve the efficacy of doxorubicin against P-glycoprotein expressing CT26 colon cancer cells in syngeneic Balb/c mice. Sildenafil administered orally at a dose of 50mg/kg did not improve the brain penetration of docetaxel and topotecan, although the plasma level of sildenafil was already much higher than can be achieved in humans. On the other hand, sildenafil increased the plasma levels of the cytotoxic drugs, but not by inhibition of Abcb1 or Abcg2, since this effect was also seen in Abcb1;Abcg2 knockout mice. The brain penetration of sildenafil was more than 20-fold higher in Abcb1;Abcg2 mice versus wild-type mice, indicating that sildenafil is a good substrate of the two transporters. Sildenafil was also not able to improve the efficacy of doxorubicin against subcutaneous CT26 tumours. The doxorubicin level in tumour tissue did increase, but so did the concentration of doxorubicin in plasma and heart. These results demonstrate that the potency and specificity of sildenafil as an inhibitor of ABCB1 and ABCG2 is not sufficient to warrant further clinical testing of this agent in combination with anticancer drugs. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. ABCG2pos lung mesenchymal stem cells are a novel pericyte subpopulation that contributes to fibrotic remodeling.

    PubMed

    Marriott, Shennea; Baskir, Rubin S; Gaskill, Christa; Menon, Swapna; Carrier, Erica J; Williams, Janice; Talati, Megha; Helm, Karen; Alford, Catherine E; Kropski, Jonathan A; Loyd, James; Wheeler, Lisa; Johnson, Joyce; Austin, Eric; Nozik-Grayck, Eva; Meyrick, Barbara; West, James D; Klemm, Dwight J; Majka, Susan M

    2014-10-15

    Genesis of myofibroblasts is obligatory for the development of pathology in many adult lung diseases. Adult lung tissue contains a population of perivascular ABCG2(pos) mesenchymal stem cells (MSC) that are precursors of myofibroblasts and distinct from NG2 pericytes. We hypothesized that these MSC participate in deleterious remodeling associated with pulmonary fibrosis (PF) and associated hypertension (PH). To test this hypothesis, resident lung MSC were quantified in lung samples from control subjects and PF patients. ABCG2(pos) cell numbers were decreased in human PF and interstitial lung disease compared with control samples. Genetic labeling of lung MSC in mice enabled determination of terminal lineage and localization of ABCG2 cells following intratracheal administration of bleomycin to elicit fibrotic lung injury. Fourteen days following bleomycin injury enhanced green fluorescent protein (eGFP)-labeled lung MSC-derived cells were increased in number and localized to interstitial areas of fibrotic and microvessel remodeling. Finally, gene expression analysis was evaluated to define the response of MSC to bleomycin injury in vivo using ABCG2(pos) MSC isolated during the inflammatory phase postinjury and in vitro bleomycin or transforming growth factor-β1 (TGF-β1)-treated cells. MSC responded to bleomycin treatment in vivo with a profibrotic gene program that was not recapitulated in vitro with bleomycin treatment. However, TGF-β1 treatment induced the appearance of a profibrotic myofibroblast phenotype in vitro. Additionally, when exposed to the profibrotic stimulus, TGF-β1, ABCG2, and NG2 pericytes demonstrated distinct responses. Our data highlight ABCG2(pos) lung MSC as a novel cell population that contributes to detrimental myofibroblast-mediated remodeling during PF.

  20. Rheumatoid Arthritis Disease Activity Is Determinant for ABCB1 and ABCG2 Drug-Efflux Transporters Function

    PubMed Central

    Atisha-Fregoso, Yemil; Lima, Guadalupe; Pascual-Ramos, Virginia; Baños-Peláez, Miguel; Fragoso-Loyo, Hilda; Jakez-Ocampo, Juan; Contreras-Yáñez, Irazú; Llorente, Luis

    2016-01-01

    Objective To compare drug efflux function of ABCB1 and ABCG2 transporters in rheumatoid arthritis (RA) patients with active disease and in remission. Methods Twenty two active RA patients (DAS28 ≥3.2) and 22 patients in remission (DAS28<2.6) were selected from an early RA clinic. All patients were evaluated at study inclusion and six months later. ABCB1 and ABCG2 functional activity was measured in peripheral lymphocytes by flow cytometry. The percentage of cells able to extrude substrates for ABCB1 and ABCG2 was recorded. Results Active patients had higher ABCB1 and ABCG2 activity compared with patients in remission (median [interquartile range]): 3.9% (1.4–22.2) vs (1.3% (0.6–3.2), p = 0.003 and 3.9% (1.1–13.3) vs 0.9% (0.5–1.9) p = 0.006 respectively. Both transporters correlated with disease activity assessed by DAS28, rho = 0.45, p = 0.002 and rho = 0.47, p = 0.001 respectively. Correlation was observed between the time from the beginning of treatment and transporter activity: rho = 0.34, p = 0.025 for ABCB1 and rho = 0.35, p = 0.018 for ABCG2. The linear regression model showed that DAS28 and the time from the onset of treatment are predictors of ABCB1 and ABCG2 functional activity, even after adjustment for treatment. After six months we calculated the correlation between change in DAS28 and change in the functional activity in both transporters and found a moderate and significant correlation for ABCG2 (rho = 0.28, p = 0.04) and a non-significant correlation for ABCB1 (rho = 0.22, p = 0.11). Conclusions Patients with active RA have an increased function of ABCB1 and ABCG2, and disease activity is the main determinant of this phenomena. PMID:27442114

  1. Effect of oxygen on multidrug resistance in the first trimester human placenta.

    PubMed

    Lye, P; Bloise, E; Dunk, C; Javam, M; Gibb, W; Lye, S J; Matthews, S G

    2013-09-01

    The multidrug resistance proteins, P-glycoprotein (P-gp, encoded by the ABCB1 gene) and breast cancer resistance protein (BCRP, encoded by ABCG2) are highly expressed in the first trimester placenta. These transporters protect the fetus from exposure to maternally derived toxins and xenobiotics. Since oxygen is a regulator of multidrug resistance in various tissues, we hypothesized that changes in oxygen tension alter placental ABCB1/P-gp and ABCG2/BCRP expression in the first trimester. Placental specimens were collected from first (n = 7), second (n = 5) and term pregnancies (n = 5). First trimester placental villous explants were incubated (24 or 48 h) in different oxygen tension (3-20%). ABCB1, ABCG2 and VEGFA mRNA expression levels were assessed by RT-PCR and protein was localized by IHC. ABCB1 is expressed most highly in the first trimester placenta (p < 0.05), whereas ABCG2 expression does not change significantly over pregnancy. P-gp and BCRP staining is present in the syncytiotrophoblast and in cytotrophoblasts. ABCG2 mRNA is increased in hyperoxic (20%) conditions after 48 h (p < 0.05). In contrast, hypoxia (3%) did not change ABCB1 mRNA expression but significantly increased VEGFA mRNA (p < 0.05). Hypoxia resulted in increased BCRP staining in cytotrophoblasts and in the microvillous membrane of the syncytium. Whereas, hypoxia resulted in increased P-gp staining in proliferating cytotrophoblasts. We conclude that placental multidrug resistance expression, specifically ABCG2, is regulated by oxygen tension in the first trimester. It is possible that changes in placental oxygen supply are capable of altering fetal drug exposure especially during early pregnancy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Down-regulation of ABCG2 and ABCB4 transporters in the placenta of rats exposed to cadmium

    PubMed Central

    Liu, Lili; Zhou, Liang; Hu, Shuiwang; Zhou, Shanyu; Deng, Yingyu; Dong, Ming; Huang, Jianxun; Zeng, Yuli; Chen, Xiaoyan; Zhao, Na; Li, Hongling; Ding, Zhenhua

    2016-01-01

    As a maternal and developmental toxicant, cadmium (Cd) possesses weak penetrability through the placental barrier. However, the underlying mechanism remains unclear. To gain insight into the protein molecules associated with Cd toxicity in placenta and explore their roles in Cd transportation, a reproductive animal experiment was carried out using Sprague-Dawley rats. We performed proteomic analysis of the placenta by Difference Gel Electrophoresis (DIGE) combined with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Tandem Mass Spectroscopy (MALDI-TOF/TOF MS). The DIGE assay identified 15 protein spots that were differentially expressed with a greater than 1.5-fold change in placenta of Cd-treated rats compared to the control rats. Based on the expression patterns and biological functions of the proteins, we selected the ABCG2 and ABCB4 transporter proteins for further analysis. Western blot analysis showed that Cd exposure could down-regulate the expression of ABCG2 and ABCB4 in the placenta. There was a negative dose-response relationship between Cd exposure and the expression of ABCG2 or ABCB4 protein. These results indicated that down-regulation of ABCG2 and ABCB4 transporters may regulate Cd across through placenta and thus affect the in vivo toxic effect of Cd to fetus. PMID:27203216

  3. PDK2 and ABCG2 genes polymorphisms are correlated with blood glucose levels and uric acid in Tibetan gout patients.

    PubMed

    Ren, Y C; Jin, T B; Sun, X D; Geng, T T; Zhang, M X; Wang, L; Feng, T; Kang, L L; Chen, C

    2016-02-11

    Previous studies have shown that the PDK2 and ABCG2 genes play important roles in many aspects of gout development in European populations. However, a detailed genotype-phenotype analysis was not performed. The aim of the present study was to investigate the potential association between variants in these two genes and metabolism-related quantitative phenotypes relevant to gout in a Chinese Tibetan population. In total, 316 Chinese Tibetan gout patients were recruited from rheumatology outpatient clinics and 6 single nucleotide polymorphisms in PDK2 and ABCG2 were genotyped, which were possible etiologic variants as identified in the HapMap Chinese Han Beijing population. A significant difference in blood glucose levels was detected between different genotypes of rs2728109 (P = 0.005) in the PDK2 gene. We also detected a significant difference in the mean serum uric levels between different genotypes of rs3114018 (P = 0.004) in the ABCG2 gene. All P values remained significant after Bonferroni's correction for multiple testing. Our data demonstrate potential roles for PDK2 and ABCG2 polymorphisms in the metabolic phenotypes of Tibetan gout patients, which may provide new insights into the etiology of gout. Further studies are required to confirm these findings.

  4. Interaction of mammary bovine ABCG2 with AFB1 and its metabolites and regulation by PCB 126 in a MDCKII in vitro model.

    PubMed

    Manzini, L; Halwachs, S; Girolami, F; Badino, P; Honscha, W; Nebbia, C

    2017-02-14

    The ATP-binding cassette efflux transporter ABCG2 plays a key role in the mammary excretion of drugs and toxins in humans and animals. Aflatoxins (AF) are worldwide contaminants of food and feed commodities, while PCB 126 is a dioxin-like PCB which may contaminate milk and dairy products. Both compounds are known human carcinogens. The interactions between AF and bovine ABCG2 (bABCG2) as well as the effects of PCB 126 on its efflux activity have been investigated by means of the Hoechst H33342 transport assay in MDCKII cells stably expressing mammary bABCG2. Both AFB1 and its main milk metabolite AFM1 showed interaction with bABCG2 even at concentrations approaching the legal limits in feed and food commodities. Moreover, PCB 126 significantly enhanced bABCG2 functional activity. Specific inhibitors of either AhR (CH233191) or ABCG2 (Ko143) were able to reverse the PCB 126-induced increase in bABCG2 transport activity, showing the specific upregulation of the efflux protein by the AhR pathway. The incubation of PCB 126-pretreated cells with AFM1 was able to substantially reverse such effect, with still unknown mechanism(s). Overall, results from this study point to AFB1 and AFM1 as likely bABCG2 substrates. The PCB 126-dependent increased activity of the transporter could enhance the ABCG2-mediated excretion into dairy milk of chemicals (i.e., drugs and toxins) potentially harmful to neonates and consumers.

  5. Deregulation of the miR-222-ABCG2 regulatory module in tongue squamous cell carcinoma contributes to chemoresistance and enhanced migratory/invasive potential.

    PubMed

    Zhao, Luodan; Ren, Yuexin; Tang, Haikuo; Wang, Wei; He, Qianting; Sun, Jingjing; Zhou, Xiaofeng; Wang, Anxun

    2015-12-29

    Chemoresistance is often associated with other clinical characteristics such as enhanced migratory/invasive potential. However, the correlation and underlying molecular mechanisms remain unclear. The aim of this study was to elucidate the function of the miR-222-ABCG2 pathway in the correlation between cisplatin (DDP) resistance and enhanced cell migration/invasion in tongue squamous cell carcinoma (TSCC). Using TSCC cell lines and primary cultures from TSCC cases, we first confirmed the correlation among DDP resistance (measured by IC50 values and ABCG2/ERCC1 expression), migratory/invasive potential (assessed by migration/invasion assays) and miR-222 expression. In TSCC cells, siRNA-mediated ABCG2 knockdown led to enhanced DDP responsiveness and reduced migratory/invasive potential, whereas ABCG2 overexpression induced DDP resistance and enhanced cell migration/invasion. Luciferase assays revealed that ABCG2 is a direct target of miR-222. In addition to reducing cell migration/invasion, functional analyses in TSCC cells indicated that miR-222 can reduce expression of the ABCG2 gene and enhance DDP responsiveness. However, co-transfection with ABCG2 cDNA restored both DDP resistance and migration/invasion. Moreover, miR-222 mimics and ABCG2 siRNA inhibited tumor growth and lung metastasis in vivo. Thus, our results verified that DDP resistance is correlated with enhanced migratory/invasive potential in TSCC. ABCG2 is a direct target of miR-222,and deregulation of the miR-222-ABCG2 regulatory module in TSCC contributes to both DDP resistance and enhanced migratory/invasive potential.

  6. Effect of bovine ABCG2 Y581S polymorphism on concentrations in milk of enrofloxacin and its active metabolite ciprofloxacin.

    PubMed

    Otero, J A; García-Mateos, D; de la Fuente, A; Prieto, J G; Álvarez, A I; Merino, G

    2016-07-01

    The ATP-binding cassette transporter G2 (ABCG2) is involved in the secretion of several drugs into milk. The bovine Y581S ABCG2 polymorphism increases the secretion into milk of the fluoroquinolone danofloxacin in Holstein cows. Danofloxacin and enrofloxacin are the fluoroquinolones most widely used in veterinary medicine. Both enrofloxacin (ENRO) and its active metabolite ciprofloxacin (CIPRO) reach milk at relatively high concentrations. The aim of this work was to study the effect of the bovine Y581S ABCG2 polymorphism on in vitro transport as well as on concentrations in plasma and in milk of ENRO and CIPRO. Experiments using cells overexpressing bovine ABCG2 showed the effects of ABCG2 on the transport of CIPRO, demonstrating more efficient in vitro transport of this antimicrobial by the S581 variant as compared with the Y581 variant. Animal studies administering 2.5mg/kg of ENRO subcutaneously to Y/Y 581 and Y/S 581 cows revealed that concentrations in plasma of ENRO and CIPRO were significantly lower in Y/S animals. Regardless of the genotype, the antimicrobial profile in milk after the administration of ENRO was predominantly of CIPRO. With respect to the genotype effects on the amounts of drugs present in milk, AUC0-24 values were more than 1.2 times higher in Y/S cows for ENRO and 2.2 times for CIPRO, indicating a greater capacity of Y581S to transfer these drugs into milk. These results emphasize the clinical relevance of this polymorphism as a factor affecting the concentrations in plasma and in milk of drugs of importance in veterinary medicine.

  7. ABCG2 and ABCB1 Limit the Efficacy of Dasatinib in a PDGF-B-Driven Brainstem Glioma Model.

    PubMed

    Mittapalli, Rajendar K; Chung, Alexander H; Parrish, Karen E; Crabtree, Donna; Halvorson, Kyle G; Hu, Guo; Elmquist, William F; Becher, Oren J

    2016-05-01

    Dasatinib is a multikinase inhibitor in clinical trials for glioma, and thus far has failed to demonstrate significant efficacy. We investigated whether the ABC efflux transporters ABCG2 and ABCB1 expressed in the blood-brain barrier (BBB), are limiting the efficacy of dasatinib in the treatment of glioma using genetic and pharmacologic approaches. We utilized a genetic brainstem glioma mouse model driven by platelet-derived growth factor-B and p53 loss using abcg2/abcb1 wild-type (ABC WT) or abcg2/abcb1 knockout mice (ABC KO). First, we observed that brainstem glioma tumor latency is significantly prolonged in ABC KO versus ABC WT mice (median survival of 47 vs. 34 days). Dasatinib treatment nearly doubles the survival of brainstem glioma-bearing ABC KO mice (44 vs. 80 days). Elacridar, an ABCG2 and ABCB1 inhibitor, significantly increases the efficacy of dasatinib in brainstem glioma-bearing ABC WT mice (42 vs. 59 days). Pharmacokinetic analysis demonstrates that dasatinib delivery into the normal brain, but not into the tumor core, is significantly increased in ABC KO mice compared with ABC WT mice. Surprisingly, elacridar did not significantly increase dasatinib delivery into the normal brain or the tumor core of ABC WT mice. Next, we demonstrate that the tight junctions of the BBB of this model are compromised as assessed by tissue permeability to Texas Red dextran. Finally, elacridar increases the cytotoxicity of dasatinib independent of ABCG2 and ABCB1 expression in vitro In conclusion, elacridar improves the efficacy of dasatinib in a brainstem glioma model without significantly increasing its delivery to the tumor core. Mol Cancer Ther; 15(5); 819-29. ©2016 AACR. ©2016 American Association for Cancer Research.

  8. ABCG2 and ABCB1 limit the efficacy of dasatinib in a PDGF-B driven brainstem glioma model

    PubMed Central

    Mittapalli, Rajendar K; Chung, Alexander H; Parrish, Karen E; Crabtree, Donna; Halvorson, Kyle G.; Hu, Guo; Elmquist, William F; Becher, Oren J

    2016-01-01

    Dasatinib is a multi-kinase inhibitor in clinical trials for glioma, and thus far failed to demonstrate significant efficacy. We investigated whether the ABC efflux transporters, ABCG2 and ABCB1, expressed in the blood-brain barrier (BBB), are limiting the efficacy of dasatinib in the treatment of glioma using genetic and pharmacological approaches. We utilized a genetic brainstem glioma mouse model driven by platelet-derived growth factor-B and p53 loss using abcg2/abcb1 wild type (ABC WT) or abcg2/abcb1 knockout mice (ABC KO). First, we observed that brainstem glioma tumor latency is significantly prolonged in ABC KO versus ABC WT mice (median survival of 47 vs. 34 days). Dasatinib treatment nearly doubles the survival of brainstem glioma-bearing ABC KO mice (44 vs. 80 days). Elacridar, an ABCG2 and ABCB1 inhibitor, significantly increases the efficacy of dasatinib in brainstem glioma-bearing ABC WT mice (42 vs. 59 days). Pharmacokinetic analysis demonstrates that dasatinib delivery into the normal brain, but not into the tumor core, is significantly increased in ABC KO mice compared to ABC WT mice. Surprisingly, elacridar did not significantly increase dasatinib delivery into the normal brain or the tumor core of ABC WT mice. Next, we demonstrate that the tight junctions of the BBB of this model are compromised as assessed by tissue permeability to Texas Red dextran. Lastly, elacridar increases the cytotoxicity of dasatinib independent of ABCG2 and ABCB1 expression in vitro. In conclusion, elacridar improves the efficacy of dasatinib in a brainstem glioma model without significantly increasing its delivery to the tumor core. PMID:26883271

  9. Mithramycin represses basal and cigarette smoke-induced expression of ABCG2 and inhibits stem cell signaling in lung and esophageal cancer cells.

    PubMed

    Zhang, Mary; Mathur, Aarti; Zhang, Yuwei; Xi, Sichuan; Atay, Scott; Hong, Julie A; Datrice, Nicole; Upham, Trevor; Kemp, Clinton D; Ripley, R Taylor; Wiegand, Gordon; Avital, Itzak; Fetsch, Patricia; Mani, Haresh; Zlott, Daniel; Robey, Robert; Bates, Susan E; Li, Xinmin; Rao, Mahadev; Schrump, David S

    2012-08-15

    Cigarette smoking at diagnosis or during therapy correlates with poor outcome in patients with lung and esophageal cancers, yet the underlying mechanisms remain unknown. In this study, we observed that exposure of esophageal cancer cells to cigarette smoke condensate (CSC) led to upregulation of the xenobiotic pump ABCG2, which is expressed in cancer stem cells and confers treatment resistance in lung and esophageal carcinomas. Furthermore, CSC increased the side population of lung cancer cells containing cancer stem cells. Upregulation of ABCG2 coincided with increased occupancy of aryl hydrocarbon receptor, Sp1, and Nrf2 within the ABCG2 promoter, and deletion of xenobiotic response elements and/or Sp1 sites markedly attenuated ABCG2 induction. Under conditions potentially achievable in clinical settings, mithramycin diminished basal as well as CSC-mediated increases in AhR, Sp1, and Nrf2 levels within the ABCG2 promoter, markedly downregulated ABCG2, and inhibited proliferation and tumorigenicity of lung and esophageal cancer cells. Microarray analyses revealed that mithramycin targeted multiple stem cell-related pathways in vitro and in vivo. Collectively, our findings provide a potential mechanistic link between smoking status and outcome of patients with lung and esophageal cancers, and support clinical use of mithramycin for repressing ABCG2 and inhibiting stem cell signaling in thoracic malignancies.

  10. The combination of quinazoline and chalcone moieties leads to novel potent heterodimeric modulators of breast cancer resistance protein (BCRP/ABCG2).

    PubMed

    Kraege, Stefanie; Stefan, Katja; Juvale, Kapil; Ross, Thomas; Willmes, Thomas; Wiese, Michael

    2016-07-19

    During the last decade it has been found that chalcones and quinazolines are promising inhibitors of ABCG2. The combination of these two scaffolds offers a new class of heterocyclic compounds with potentially high inhibitory activity against ABCG2. For this purpose we investigated 22 different heterodimeric derivatives. In this series only methoxy groups were used as substituents as these had been proven superior for inhibitory activity of chalcones. All compounds were tested for their inhibitory activity, specificity and cytotoxicity. The most potent ABCG2 inhibitor in this series showed an IC50 value of 0.19 μM. It possesses low cytotoxicity (GI50 = 93 μM), the ability to reverse MDR and is nearly selective toward ABCG2. Most compounds containing dimethoxy groups showed slight activity against ABCB1 too. Among these three compounds (17, 19 and 24) showed even higher activity toward ABCB1 than ABCG2. All inhibitors were further screened for their effect on basal ATPase activity. Although the basal ATPase activity was partially stimulated, the compounds were not transported by ABCG2. Thus, quinazoline-chalcones are a new class of effective ABCG2 inhibitors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  11. Correlation between clinical response to sorafenib in hepatocellular carcinoma treatment and polymorphisms of P-glycoprotein (ABCB1) and of breast cancer resistance protein (ABCG2): monocentric study.

    PubMed

    Tandia, Mahamadou; Mhiri, Asma; Paule, Bernard; Saffroy, Raphaël; Cailliez, Valérie; Noé, Gaëlle; Farinotti, Robert; Bonhomme-Faivre, Laurence

    2017-04-01

    We studied the relation between the polymorphism of P-glycoprotein (P-gp) and of breast cancer resistance protein (BCRP), encoded by ABCB1 and ABCG2 genes, respectively, and the pharmacokinetic variability and clinical response during the treatment with sorafenib of hepatocellular carcinoma. At the Paul Brousse Hospital in Villejuif, France, 47 consecutive patients with advanced HCC treated with a single agent sorafenib, were enrolled. Sorafenib exposure was measured by its plasma concentration 3 h after oral administration of 400 mg (bid) by liquid chromatography. All enrolled patients were genotyped for ABCB1 (rs2032582; rs1045642) and ABCG2 (rs2231137; rs2231142; rs2622604) by blood genomic DNA extraction and Mass ARRAY genotyping. The clinical response was evaluated after 3months of treatment according to the RECIST criteria. Significant associations between sorafenib exposure and the studied polymorphisms were observed for ABCB1 3435C>T, ABCG2 34G>A, ABCG2 1143C>T and ABCG2 421C>A, but not for ABCB1 2677G>TA SNP. In heterozygous patients for ABCB1 3435 C>T, ABCG2 34 G>A and ABCG2 1143 C>T polymorphisms were significantly associated with the lowest sorafenib plasma levels. Those patients presented a tendency to have the best clinical evolution. Heterozygous forms of the studied polymorphisms could be associated with a better therapeutic response.

  12. Structural determinants of peripheral O-arylcarbamate FAAH inhibitors render them dual substrates for Abcb1 and Abcg2 and restrict their access to the brain.

    PubMed

    Moreno-Sanz, Guillermo; Barrera, Borja; Armirotti, Andrea; Bertozzi, Sine M; Scarpelli, Rita; Bandiera, Tiziano; Prieto, Julio G; Duranti, Andrea; Tarzia, Giorgio; Merino, Gracia; Piomelli, Daniele

    2014-09-01

    The blood-brain barrier (BBB) is the main entry route for chemicals into the mammalian central nervous system (CNS). Two transmembrane transporters of the ATP-binding cassette (ABC) family - breast cancer resistance protein (ABCG2 in humans, Abcg2 in rodents) and P-glycoprotein (ABCB1 in humans, Abcb1 in rodents) - play a key role in mediating this process. Pharmacological and genetic evidence suggests that Abcg2 prevents CNS access to a group of highly potent and selective O-arylcarbamate fatty-acid amidohydrolase (FAAH) inhibitors, which include the compound URB937 (cyclohexylcarbamic acid 3'-carbamoyl-6-hydroxybiphenyl-3-yl ester). To define structure-activity relationships of the interaction of these molecules with Abcg2, in the present study we tested various peripherally restricted and non-restricted O-arylcarbamate FAAH inhibitors for their ability to serve as transport substrates in monolayer cultures of Madin-Darby Canine Kidney-II (MDCKII) cells over-expressing Abcg2. Surprisingly, we found that the majority of compounds tested - even those able to enter the CNS in vivo - were substrates for Abcg2 in vitro. Additional experiments in MDCKII cells overexpressing ABCB1 revealed that only those compounds that were dual substrates for ABCB1 and Abcg2 in vitro were also peripherally restricted in vivo. The extent of such restriction seems to depend upon other physicochemical features of the compounds, in particular the polar surface area. Consistent with these in vitro results, we found that URB937 readily enters the brain in dual knockout mice lacking both Abcg2 and Abcb1, whereas it is either partially or completely excluded from the brain of mice lacking either transporter alone. The results suggest that Abcg2 and Abcb1 act together to restrict the access of URB937 to the CNS.

  13. Structural determinants of peripheral O-arylcarbamate FAAH inhibitors render them dual substrates for Abcb1 and Abcg2 and restrict their access to the brain

    PubMed Central

    Moreno-Sanz, Guillermo; Barrera, Borja; Armirotti, Andrea; Bertozzi, Sine M.; Scarpelli, Rita; Bandiera, Tiziano; Prieto, Julio G.; Duranti, Andrea; Tarzia, Giorgio; Merino, Gracia

    2014-01-01

    The blood-brain barrier (BBB) is the main entry route for chemicals into the mammalian central nervous system (CNS). Two transmembrane transporters of the ATP-binding cassette (ABC) family – Breast Cancer Resistance Protein (ABCG2 in humans, Abcg2 in rodents) and P-glycoprotein (ABCB1 in humans, Abcb1 in rodents) – play a key role in mediating this process. Pharmacological and genetic evidence suggests that Abcg2 prevents CNS access to a group of highly potent and selective O-arylcarbamate fatty-acid amidohydrolase (FAAH) inhibitors, which include the compound URB937 (cyclohexylcarbamic acid 3′-carbamoyl-6-hydroxybiphenyl-3-yl ester). To define structure-activity relationships of the interaction of these molecules with Abcg2, in the present study we tested various peripherally restricted and non-restricted O-arylcarbamate FAAH inhibitors for their ability to serve as transport substrates in monolayer cultures of Madin-Darby Canine Kidney-II (MDCKII) cells over-expressing Abcg2. Surprisingly, we found that the majority of compounds tested – even those able to enter the CNS in vivo – were substrates for Abcg2 in vitro. Additional experiments in MDCKII cells overexpressing ABCB1 revealed that only those compounds that were dual substrates for ABCB1 and Abcg2 in vitro were also peripherally restricted in vivo. The extent of such restriction seems to depend upon other physicochemical features of the compounds, in particular the polar surface area. Consistent with these in vitro results, we found that URB937 readily enters the brain in dual knockout mice lacking both Abcg2 and Abcb1, whereas it is either partially or completely excluded from the brain of mice lacking either transporter alone. The results suggest that Abcg2 and Abcb1 act together to restrict the access of URB937 to the CNS. PMID:24993496

  14. The ABCG2 Efflux Transporter in the Mammary Gland Mediates Veterinary Drug Secretion across the Blood-Milk Barrier into Milk of Dairy Cows.

    PubMed

    Mahnke, Hanna; Ballent, Mariana; Baumann, Sven; Imperiale, Fernanda; von Bergen, Martin; Lanusse, Carlos; Lifschitz, Adrian L; Honscha, Walther; Halwachs, Sandra

    2016-05-01

    In human and mice ATP-binding cassette efflux transporter ABCG2 represents the main route for active drug transport into milk. However, there is no detailed information on the role of ABCG2 in drug secretion and accumulation in milk of dairy animals. We therefore examined ABCG2-mediated drug transport in the bovine mammary gland by parallel pharmacokinetic studies in lactating Jersey cows and in vitro flux studies using the anthelmintic drug monepantel (MNP) as representative bovine ABCG2 (bABCG2) drug substrate. Animals received MNP (Zolvix, Novartis Animal Health Inc.) once (2.5 mg/kg per os) and the concentrations of MNP and the active MNP metabolite MNPSO2 were assessed by high-performance liquid chromatography. Compared with the parent drug MNP, we detected higher MNPSO2 plasma concentrations (expressed as area under the concentration-versus-time curve). Moreover, we observed MNPSO2 excretion into milk of dairy cows with a high milk-to-plasma ratio of 6.75. In mechanistic flux assays, we determined a preferential time-dependent basolateral-to-apical (B > A) MNPSO2 transport across polarized Madin-Darby canine kidney II cells-bABCG2 monolayers using liquid chromatography coupled with tandem mass spectrometry analysis. The B > A MNPSO2 transport was significantly inhibited by the ABCG2 inhibitor fumitremorgin C in bABCG2- but not in mock-transduced MDCKII cells. Additionally, the antibiotic drug enrofloxacin, the benzimidazole anthelmintic oxfendazole and the macrocyclic lactone anthelmintic moxidectin caused a reduction in the MNPSO2(B > A) net efflux. Altogether, this study indicated that therapeutically relevant drugs like the anthelmintic MNP represent substrates of the bovine mammary ABCG2 transporter and may thereby be actively concentrated in dairy milk. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  15. Oleic acid increases intestinal absorption of the BCRP/ABCG2 substrate, mitoxantrone, in mice.

    PubMed

    Aspenström-Fagerlund, Bitte; Tallkvist, Jonas; Ilbäck, Nils-Gunnar; Glynn, Anders W

    2015-09-02

    The efflux transporter breast cancer resistance protein (BCRP/ABCG2) decrease intestinal absorption of many food toxicants. Oleic acid increases absorption of the specific BCRP substrate mitoxantrone (MXR), and also BCRP gene expression in human intestinal Caco-2 cells, suggesting that oleic acid affect the BCRP function. Here, we investigated the effect of oleic acid on intestinal absorption of MXR in mice. Mice were orally dosed with 2.4g oleic acid/kg b.w. and 1mg MXR/kg b.w., and sacrificed 30, 60, 90 or 120min after exposure, or were exposed to 0.6, 2.4 or 4.8g oleic acid/kg b.w. and 1mg MXR/kg b.w., and sacrificed 90min after exposure. Mice were also treated with Ko143 together with MXR and sacrificed after 60min, as a positive control of BCRP-mediated effects on MXR absorption. Absorption of MXR increased after exposure to oleic acid at all doses, and also after exposure to Ko143. Intestinal BCRP gene expression tended to increase 120min after oleic acid exposure. Our results in mice demonstrate that oleic acid decreases BCRP-mediated efflux, causing increased intestinal MXR absorption in mice. These findings may have implications in humans, concomitantly exposed to oleic acid and food contaminants that, similarly as MXR, are substrates of BCRP.

  16. Interactions of ABCG2 (BCRP) with epidermal growth factor receptor kinase inhibitors developed for molecular imaging.

    PubMed

    Qawasmi, Israa; Shmuel, Miriam; Eyal, Sara

    2014-01-01

    The objective of this study was to investigate in vitro the interactions between novel epidermal growth factor receptor kinase inhibitors (EGFRIs) developed for positron emission tomography (PET) imaging and the major efflux transporter breast cancer resistance protein (BCRP/ABCG2). Seven compounds were evaluated, using the ATPase activity assays and Madin-Darbey canine kidney (MDCK) cells overexpressing BCRP. Five of the tested compounds activated BCRP ATPase to various extent. Overexpression of BCRP conferred resistance to ML04, ML06, methoxy-Br-ML03, and PEG6-ML05 (IC50 values for inhibition of control cell proliferation 2.1 ± 0.6, 2.2 ± 0.7, 1.8 ± 1.2, and 2.8 ± 3.1 μM, respectively, compared to >50 μM in MDCK-BCRP cells). At submicromolar concentrations, none of the EGFRIs significantly inhibited BCRP. Immunoblotting studies indicated that BCRP expression is evident in cell lines utilized for in vivo tumor grafting in small animal PET imaging studies. Thus, the intensity of EGFRIs radioactivity signals previously observed in tumor xenografts reflects an interplay between transporter-mediated distribution of the probe into tumor cells and target binding. Concomitant use of efflux transporter inhibitors may help distinguish between the contribution of efflux transport and EGFR binding to the tissue signal.

  17. Interactions of ABCG2 (BCRP) with epidermal growth factor receptor kinase inhibitors developed for molecular imaging

    PubMed Central

    Qawasmi, Israa; Shmuel, Miriam; Eyal, Sara

    2014-01-01

    The objective of this study was to investigate in vitro the interactions between novel epidermal growth factor receptor kinase inhibitors (EGFRIs) developed for positron emission tomography (PET) imaging and the major efflux transporter breast cancer resistance protein (BCRP/ABCG2). Seven compounds were evaluated, using the ATPase activity assays and Madin-Darbey canine kidney (MDCK) cells overexpressing BCRP. Five of the tested compounds activated BCRP ATPase to various extent. Overexpression of BCRP conferred resistance to ML04, ML06, methoxy-Br-ML03, and PEG6-ML05 (IC50 values for inhibition of control cell proliferation 2.1 ± 0.6, 2.2 ± 0.7, 1.8 ± 1.2, and 2.8 ± 3.1 μM, respectively, compared to >50 μM in MDCK-BCRP cells). At submicromolar concentrations, none of the EGFRIs significantly inhibited BCRP. Immunoblotting studies indicated that BCRP expression is evident in cell lines utilized for in vivo tumor grafting in small animal PET imaging studies. Thus, the intensity of EGFRIs radioactivity signals previously observed in tumor xenografts reflects an interplay between transporter-mediated distribution of the probe into tumor cells and target binding. Concomitant use of efflux transporter inhibitors may help distinguish between the contribution of efflux transport and EGFR binding to the tissue signal. PMID:25484865

  18. The Human Breast Cancer Resistance Protein (BCRP/ABCG2) Shows Conformational Changes with Mitoxantrone

    PubMed Central

    Rosenberg, Mark F.; Bikadi, Zsolt; Chan, Janice; Liu, Xiaoping; Ni, Zhanglin; Cai, Xiaokun; Ford, Robert C.; Mao, Qingcheng

    2010-01-01

    Summary BCRP/ABCG2 mediates efflux of drugs and xenobiotics. BCRP was expressed in Pichia pastoris, purified to > 90% homogeneity, and subjected to two-dimensional (2-D) crystallization. The 2-D crystals showed a p121 symmetry and projection maps were determined at 5-Å resolution by electron cryomicroscopy. Two crystal forms with and without mitoxantrone were observed with unit cell dimensions of a = 55.4 Å, b = 81.4 Å, γ = 89.8°, and a = 57.3 Å, b =88.0 Å, γ = 89.7°, respectively. The projection map without mitoxantrone revealed an asymmetric structure with ring-shaped density features probably corresponding to a bundle of transmembrane α-helices, and appeared more open and less symmetric than the map with mitroxantrone. The open and closed inward-facing forms of BCRP were generated by homology modeling, representing the substrate-free and substrate-bound conformations in the absence of nucleotide, respectively. These models are consistent with the experimentally observed conformational change upon substrate binding. PMID:20399185

  19. Sorafenib overcomes irinotecan resistance in colorectal cancer by inhibiting the ABCG2 drug-efflux pump.

    PubMed

    Mazard, Thibault; Causse, Annick; Simony, Joelle; Leconet, Wilhem; Vezzio-Vie, Nadia; Torro, Adeline; Jarlier, Marta; Evrard, Alexandre; Del Rio, Maguy; Assenat, Eric; Martineau, Pierre; Ychou, Marc; Robert, Bruno; Gongora, Celine

    2013-10-01

    Despite recent advances in the treatment of colorectal cancer (CRC), tumor resistance is a frequent cause of chemotherapy failure. Therefore, new treatment options are needed to improve survival of patients with irinotecan-refractory CRCs, particularly those bearing KRAS mutations that preclude the use of anti-EGFR therapies. In this study, we investigated whether sorafenib could reverse irinotecan resistance, thereby enhancing the therapeutic efficacy of routinely used irinotecan-based chemotherapy. We used both in vitro (the HCT116, SW48, SW620, and HT29 colon adenocarcinoma cell lines and four SN-38-resistant HCT-116 and SW48 clones) and in vivo models (nude mice xenografted with SN-38-resistant HCT116 cells) to test the efficacy of sorafenib alone or in combination with irinotecan or its active metabolite, SN-38. We have shown that sorafenib improved the antitumoral activity of irinotecan in vitro, in both parental and SN-38-resistant colon adenocarcinoma cell lines independently of their KRAS status, as well as in vivo, in xenografted mice. By inhibiting the drug-efflux pump ABCG2, sorafenib favors irinotecan intracellular accumulation and enhances its toxicity. Moreover, we found that sorafenib improved the efficacy of irinotecan by inhibiting the irinotecan-mediated p38 and ERK activation. In conclusion, our results show that sorafenib can suppress resistance to irinotecan and suggest that sorafenib could be used to overcome resistance to irinotecan-based chemotherapies in CRC, particularly in KRAS-mutated tumors.

  20. Influence of the ABCG2 gout risk 141 K allele on urate metabolism during a fructose challenge.

    PubMed

    Dalbeth, Nicola; House, Meaghan E; Gamble, Gregory D; Pool, Bregina; Horne, Anne; Purvis, Lauren; Stewart, Angela; Merriman, Marilyn; Cadzow, Murray; Phipps-Green, Amanda; Merriman, Tony R

    2014-01-30

    Both genetic variation in ATP-binding cassette sub-family G member 2 (ABCG2) and intake of fructose-containing beverages are major risk factors for hyperuricemia and gout. This study aimed to test the hypothesis that the ABCG2 gout risk allele 141 K promotes the hyperuricaemic response to fructose loading. Healthy volunteers (n = 74) provided serum and urine samples immediately before and 30, 60, 120 and 180 minutes after ingesting a 64 g fructose solution. Data were analyzed based on the presence or absence of the ABCG2 141 K gout risk allele. The 141 K risk allele was present in 23 participants (31%). Overall, serum urate (SU) concentrations during the fructose load were similar in those with and without the 141 K allele (PSNP = 0.15). However, the 141 K allele was associated with a smaller increase in SU following fructose intake (PSNP <0.0001). Those with the 141 K allele also had a smaller increase in serum glucose following the fructose load (PSNP = 0.002). Higher fractional excretion of uric acid (FEUA) at baseline and throughout the fructose load was observed in those with the 141 K risk allele (PSNP <0.0001). However, the change in FEUA in response to fructose was not different in those with and without the 141 K risk allele (PSNP = 0.39). The 141 K allele effects on serum urate and glucose were more pronounced in Polynesian participants and in those with a body mass index ≥25 kg/m². In contrast to the predicted responses for a hyperuricemia/gout risk allele, the 141 K allele is associated with smaller increases in SU and higher FEUA following a fructose load. The results suggest that ABCG2 interacts with extra-renal metabolic pathways in a complex manner to regulate SU and gout risk. The study was registered by the Australian Clinical Trials Registry (ACTRN12610001036000).

  1. An automated method measures variability in P-glycoprotein and ABCG2 densities across brain regions and brain matter.

    PubMed

    Kannan, Pavitra; Schain, Martin; Kretzschmar, Warren W; Weidner, Lora; Mitsios, Nicholas; Gulyás, Balázs; Blom, Hans; Gottesman, Michael M; Innis, Robert B; Hall, Matthew D; Mulder, Jan

    2017-06-01

    Changes in P-glycoprotein and ABCG2 densities may play a role in amyloid-beta accumulation in Alzheimer's disease. However, previous studies report conflicting results from different brain regions, without correcting for changes in vessel density. We developed an automated method to measure transporter density exclusively within the vascular space, thereby correcting for vessel density. We then examined variability in transporter density across brain regions, matter, and disease using two cohorts of post-mortem brains from Alzheimer's disease patients and age-matched controls. Changes in transporter density were also investigated in capillaries near plaques and on the mRNA level. P-glycoprotein density varied with brain region and matter, whereas ABCG2 density varied with brain matter. In temporal cortex, P-glycoprotein density was 53% lower in Alzheimer's disease samples than in controls, and was reduced by 35% in capillaries near plaque deposits within Alzheimer's disease samples. ABCG2 density was unaffected in Alzheimer's disease. No differences were detected at the transcript level. Our study indicates that region-specific changes in transporter densities can occur globally and locally near amyloid-beta deposits in Alzheimer's disease, providing an explanation for conflicting results in the literature. When differences in region and matter are accounted for, changes in density can be reproducibly measured using our automated method.

  2. Two novel SNPs of the ABCG2 gene and its associations with milk traits in Chinese Holsteins.

    PubMed

    Yue, Wangping; Fang, Xingtang; Zhang, Chunlei; Pang, Yonghong; Xu, Haixia; Gu, Chuanwen; Shao, Ruying; Lei, Chuzhao; Chen, Hong

    2011-06-01

    The ATP-binding cassette transporter ABCG2 (also known as breast cancer resistance protein, BCRP) belongs to the ATP-binding cassette (ABC) family of transmembrane drug transporters, playing a crucial role in the protection of various cells and tissues against xenotoxins and/or endotoxins. Recently, several studies have proposed it as the potential gene underlying the QTL on bovine chromosome 6. Hence, in this study, the PCR-SSCP method was applied to detect two polymorphisms (A → C and A → G) in the target sequence coding nucleotide-binding domain (NBD) region of ABCG2 and evaluate its associations with milk production traits and mastitis-related traits among Chinese Holsteins. In the analyzed population, the allelic frequencies for the A and B alleles were 0.5990 and 0.4010, respectively and the genotypic frequencies were in Hardy-Weinberg disequilibrium (P < 0.01). Moreover, significant statistical relationships between the polymorphisms of ABCG2 gene and following traits, including milk yields, milk protein percentage and somatic cell scores (SCS), were found (P < 0.05). When compared with AA genotype, BB genotype was associated with higher milk yields during 1st and 2nd lactations, as well as lower milk protein percentage and SCS. Thus, BB genotype is suggested to be a molecular marker for superior milk performance.

  3. Target therapy of multiple myeloma by PTX-NPs and ABCG2 antibody in a mouse xenograft model

    PubMed Central

    Xue, Jun; Zhan, Xi; Shi, Fangfang; Li, Miao; Wu, Songyan; Luo, Shouhua; Zhang, Tianzhu; Zhang, Yu; Ming, Ji; Gu, Ning

    2015-01-01

    Multiple myeloma (MM) remains to be an incurable disease. The purpose of this study was to evaluate the effect of ABCG2 monoclonal antibody (McAb) combined with paclitaxel (PTX) conjugated with Fe3O4 nanoparticles (NPs) on MM progressed from cancer stem cells (CSCs)in non-obese-diabetic/severe-combined-immunodeficiency (NOD/SCID) mouse model. Mice were injected with MM CSCs as marked by CD138−CD34− phenotypes through tail veins. The developed MM mice were examined by micro-computer tomography scanning, ultrasonography and enzyme-linked immunosorbent analysis. These mice were then intravenously treated with different combinations of NPs, PTX, McAb, PTX-NPs and melphalan/prednisone once a week for four weeks. The injected mice developed characteristic MM-associated syndromes, including lytic bone lesions, renal damages and proteinuria. All the treated mice showed decrease in bone lesions, renal damages and anemia but increase in apoptosis compared with the mice treated with NPs only. In particular, the treatment with ABCG2 McAb plus PTX-NPs induced the strongest therapeutic response and had an efficacy even better than that of melphalan/prednisone, a conventional regimen for MM patients. These data suggest that PTX-NPs with ABCG2 McAb can be developed into potential treatment regimens for patients with relapsed/refractory MM. PMID:26314844

  4. SOX4 contributes to the progression of cervical cancer and the resistance to the chemotherapeutic drug through ABCG2

    PubMed Central

    Sun, R; Jiang, B; Qi, H; Zhang, X; Yang, J; Duan, J; Li, Y; Li, G

    2015-01-01

    SOX4, a member of the SOX (sex-determining region Y-related HMG box) transcription factor family, has been reported to be abnormally expressed in a wide variety of cancers, and to exert a pleiotropic function. However, its function in progression of cervical cancer (CC) remains unknown. In this study, we found that SOX4 was highly expressed in CC cells and tissues, and overexpression of SOX4 in CC CaSki cells enhanced tumor clone formation and cell proliferation, and accelerated cell cycle progress. Meanwhile, downregulation of SOX4 by shRNA in CaSki cells inhibited cell proliferation, and slowed cell cycle progress, indicating that SOX4 contributes to the development of CC. In addition, SOX4 overexpression by gene transfer reduced the sensitivity of CaSki cells in response to the chemotherapeutic drug cisplatin, and SOX4 downregulation by RNA interference increased the sensitivity of CaSki cells in response to cisplatin. Moreover, SOX4 overexpression upregulated multiple drug resistant gene ABCG2, and SOX4 downregulation inhibited ABCG2 expression. Taken together, these results suggested that SOX4 functions to modulate cancer proliferation by regulation of cell cycle, and inhibit cancer cell sensitivity to therapeutic drug via upregulation of ABCG2. Thus, SOX4 may be a target for CC chemotherapy. PMID:26583330

  5. Target therapy of multiple myeloma by PTX-NPs and ABCG2 antibody in a mouse xenograft model.

    PubMed

    Yang, Cuiping; Xiong, Fei; Dou, Jun; Xue, Jun; Zhan, Xi; Shi, Fangfang; Li, Miao; Wu, Songyan; Luo, Shouhua; Zhang, Tianzhu; Zhang, Yu; Ming, Ji; Gu, Ning

    2015-09-29

    Multiple myeloma (MM) remains to be an incurable disease. The purpose of this study was to evaluate the effect of ABCG2 monoclonal antibody (McAb) combined with paclitaxel (PTX) conjugated with Fe3O4 nanoparticles (NPs) on MM progressed from cancer stem cells (CSCs) in non-obese-diabetic/severe-combined-immunodeficiency (NOD/SCID) mouse model. Mice were injected with MM CSCs as marked by CD138-CD34- phenotypes through tail veins. The developed MM mice were examined by micro-computer tomography scanning, ultrasonography and enzyme-linked immunosorbent analysis. These mice were then intravenously treated with different combinations of NPs, PTX, McAb, PTX-NPs and melphalan/prednisone once a week for four weeks. The injected mice developed characteristic MM-associated syndromes, including lytic bone lesions, renal damages and proteinuria. All the treated mice showed decrease in bone lesions, renal damages and anemia but increase in apoptosis compared with the mice treated with NPs only. In particular, the treatment with ABCG2 McAb plus PTX-NPs induced the strongest therapeutic response and had an efficacy even better than that of melphalan/prednisone, a conventional regimen for MM patients. These data suggest that PTX-NPs with ABCG2 McAb can be developed into potential treatment regimens for patients with relapsed/refractory MM.

  6. Metabolic Interactions of Purine Derivatives with Human ABC Transporter ABCG2: Genetic Testing to Assess Gout Risk.

    PubMed

    Ishikawa, Toshihisa; Aw, Wanping; Kaneko, Kiyoko

    2013-11-04

    In mammals, excess purine nucleosides are removed from the body by breakdown in the liver and excretion from the kidneys. Uric acid is the end product of purine metabolism in humans. Two-thirds of uric acid in the human body is normally excreted through the kidney, whereas one-third undergoes uricolysis (decomposition of uric acid) in the gut. Elevated serum uric acid levels result in gout and could be a risk factor for cardiovascular disease and diabetes. Recent studies have shown that human ATP-binding cassette transporter ABCG2 plays a role of renal excretion of uric acid. Two non-synonymous single nucleotide polymorphisms (SNPs), i.e., 421C>A (major) and 376C>T (minor), in the ABCG2 gene result in impaired transport activity, owing to ubiquitination-mediated proteosomal degradation and truncation of ABCG2, respectively. These genetic polymorphisms are associated with hyperuricemia and gout. Allele frequencies of those SNPs are significantly higher in Asian populations than they are in African and Caucasian populations. A rapid and isothermal genotyping method has been developed to detect the SNP 421C>A, where one drop of peripheral blood is sufficient for the detection. Development of simple genotyping methods would serve to improve prevention and early therapeutic intervention for high-risk individuals in personalized healthcare.

  7. Influence of the dual ABCB1 and ABCG2 inhibitor tariquidar on the disposition of oral imatinib in mice

    PubMed Central

    Gardner, Erin R; Smith, Nicola F; Figg, William D; Sparreboom, Alex

    2009-01-01

    Background Imatinib, a tyrosine kinase inhibitor currently approved for treatment of several malignancies, has been shown to be a substrate for multiple efflux-transporter proteins, including ABCB1 (P-glycoprotein) and ABCG2 (BCRP). The effect of inhibiting these transporters on tissue exposure to imatinib remains unclear. Objective To assess the role of these transporters on drug disposition, 50 mg/kg imatinib was administered to Balb/C mice, 30 minutes after receiving tariquidar (10 mg/kg), an inhibitor of both ABCB1 and ABCG2, or vehicle, via oral gavage. Methods Quantitative determination of imatinib in mouse plasma, liver and brain was performed using a newly-developed and validated liquid-chromatography-mass spectrometric method. Results: Exposure to imatinib was 2.2-fold higher in plasma, liver and brain in mice that received tariquidar, as compared to those that received the vehicle (P = 0.001). The peak plasma concentration did not increase substantially, suggesting that tariquidar is affecting the distribution, metabolism and/or excretion of imatinib, rather than absorption. Though tariquidar increased the absolute exposure of imatinib, the brain-to-plasma ratio of imatinib was unaffected. Conclusion This study suggests that intentional inhibition of ABCB1 and ABCG2 function at the blood-brain barrier is unlikely to significantly improve clinical outcome of imatinib with currently used dosing regimens. PMID:19591692

  8. Three-dimensional structure of the human breast cancer resistance protein (BCRP/ABCG2) in an inward-facing conformation

    PubMed Central

    Rosenberg, Mark F.; Bikadi, Zsolt; Hazai, Eszter; Starborg, Tobias; Kelley, Lawrence; Chayen, Naomi E.; Ford, Robert C.; Mao, Qingcheng

    2015-01-01

    ABCG2 is an efflux drug transporter that plays an important role in drug resistance and drug disposition. In this study, the first three-dimensional structure of human full-length ABCG2 analysed by electron crystallography from two-dimensional crystals in the absence of nucleotides and transported substrates is reported at 2 nm resolution. In this state, ABCG2 forms a symmetric homodimer with a noncrystallographic twofold axis perpendicular to the two-dimensional crystal plane, as confirmed by subtomogram averaging. This configuration suggests an inward-facing configuration similar to murine ABCB1, with the nucleotide-binding domains (NBDs) widely separated from each other. In the three-dimensional map, densities representing the long cytoplasmic extensions from the transmembrane domains that connect the NBDs are clearly visible. The structural data have allowed the atomic model of ABCG2 to be refined, in which the two arms of the V-shaped ABCG2 homodimeric complex are in a more closed and narrower conformation. The structural data and the refined model of ABCG2 are compatible with the biochemical analysis of the previously published mutagenesis studies, providing novel insight into the structure and function of the transporter. PMID:26249353

  9. Degree of kinase inhibition achieved in vitro by imatinib and nilotinib is decreased by high levels of ABCB1 but not ABCG2.

    PubMed

    Eadie, Laura N; Saunders, Verity A; Hughes, Timothy P; White, Deborah L

    2013-03-01

    Imatinib and nilotinib interact with ABCB1 and ABCG2. However, whether they are substrates or inhibitors is a source of conjecture. Here, in vitro, Bcr-Abl kinase inhibition was used to elucidate the impact of ABCB1/ABCG2 overexpression on imatinib and nilotinib transport. High levels of ABCB1 protein in K562-Dox cells resulted in a significantly increased 50% inhibitory concentration (IC(50)) compared with parental K562 cells for imatinib (IC(50)(IM); 9 µM to 19 µM, p = 0.002) and nilotinib (IC(50)(NIL); 345 nM to 620 nM, p = 0.013). This difference was abrogated by ABCB1 inhibitors. However, overexpression of ABCG2 did not significantly increase IC(50)(IM) or IC(50)(NIL) or significantly decrease IC(50) upon ABCG2 inhibition. Inhibition of ABCB1 but not ABCG2 resulted in a substantial increase in intracellular nilotinib when used at 150 nM but no increase when used at 2 µM. Imatinib and nilotinib appear to be transported by ABCB1 but do not interact strongly with ABCG2. Furthermore, ABCB1 efflux of nilotinib may be concentration-dependent with transport occurring at clinically relevant concentrations.

  10. Three-dimensional structure of the human breast cancer resistance protein (BCRP/ABCG2) in an inward-facing conformation.

    PubMed

    Rosenberg, Mark F; Bikadi, Zsolt; Hazai, Eszter; Starborg, Tobias; Kelley, Lawrence; Chayen, Naomi E; Ford, Robert C; Mao, Qingcheng

    2015-08-01

    ABCG2 is an efflux drug transporter that plays an important role in drug resistance and drug disposition. In this study, the first three-dimensional structure of human full-length ABCG2 analysed by electron crystallography from two-dimensional crystals in the absence of nucleotides and transported substrates is reported at 2 nm resolution. In this state, ABCG2 forms a symmetric homodimer with a noncrystallographic twofold axis perpendicular to the two-dimensional crystal plane, as confirmed by subtomogram averaging. This configuration suggests an inward-facing configuration similar to murine ABCB1, with the nucleotide-binding domains (NBDs) widely separated from each other. In the three-dimensional map, densities representing the long cytoplasmic extensions from the transmembrane domains that connect the NBDs are clearly visible. The structural data have allowed the atomic model of ABCG2 to be refined, in which the two arms of the V-shaped ABCG2 homodimeric complex are in a more closed and narrower conformation. The structural data and the refined model of ABCG2 are compatible with the biochemical analysis of the previously published mutagenesis studies, providing novel insight into the structure and function of the transporter.

  11. Extra-Renal Elimination of Uric Acid via Intestinal Efflux Transporter BCRP/ABCG2

    PubMed Central

    Hosomi, Atsushi; Nakanishi, Takeo; Fujita, Takuya; Tamai, Ikumi

    2012-01-01

    Urinary excretion accounts for two-thirds of total elimination of uric acid and the remainder is excreted in feces. However, the mechanism of extra-renal elimination is poorly understood. In the present study, we aimed to clarify the mechanism and the extent of elimination of uric acid through liver and intestine using oxonate-treated rats and Caco-2 cells as a model of human intestinal epithelium. In oxonate-treated rats, significant amounts of externally administered and endogenous uric acid were recovered in the intestinal lumen, while biliary excretion was minimal. Accordingly, direct intestinal secretion was thought to be a substantial contributor to extra-renal elimination of uric acid. Since human efflux transporter BCRP/ABCG2 accepts uric acid as a substrate and genetic polymorphism causing a decrease of BCRP activity is known to be associated with hyperuricemia and gout, the contribution of rBcrp to intestinal secretion was examined. rBcrp was confirmed to transport uric acid in a membrane vesicle study, and intestinal regional differences of expression of rBcrp mRNA were well correlated with uric acid secretory activity into the intestinal lumen. Bcrp1 knockout mice exhibited significantly decreased intestinal secretion and an increased plasma concentration of uric acid. Furthermore, a Bcrp inhibitor, elacridar, caused a decrease of intestinal secretion of uric acid. In Caco-2 cells, uric acid showed a polarized flux from the basolateral to apical side, and this flux was almost abolished in the presence of elacridar. These results demonstrate that BCRP contributes at least in part to the intestinal excretion of uric acid as extra-renal elimination pathway in humans and rats. PMID:22348008

  12. A novel xenobiotic responsive element regulated by aryl hydrocarbon receptor is involved in the induction of BCRP/ABCG2 in LS174T cells.

    PubMed

    Tompkins, Leslie M; Li, Haishan; Li, Linhao; Lynch, Caitlin; Xie, Yi; Nakanishi, Takeo; Ross, Douglas D; Wang, Hongbing

    2010-12-01

    Induction of the breast cancer resistance protein (BCRP/ABCG2) expression has been found in various tissues and cell-types after exposure to chemicals including 17β-estradiol, rosiglitazone, imatinib, as well as aryl hydrocarbon receptor (AhR) activators such as 2,3,7,8-tetrachlorodibenzodioxin, 3-methylcholanthrene (3MC), and omeprazole. However, the mechanism(s) underlying AhR-related induction of ABCG2 is largely unknown. Here, we demonstrate the AhR-dependent induction of ABCG2 expression in human colon adenocarcinoma LS174T cells. Importantly, a novel distal AhR-responsive element (AhRE5) located -2357/-2333bp upstream of the ABCG2 transcriptional start site has been identified and characterized as a functional unit pivotal to 3MC-mediated induction of ABCG2. Cell-based reporter assays revealed that deletion of AhRE5 and 4 dramatically attenuated 3MC-induced activation of ABCG2 reporter activity, while further deletion of the proximal AhRE3 and 2 only moderately changed the luciferase activities. Notably, site-directed mutation of the AhRE5 in the BCRP-3.8kb reporter construct alone resulted in approximately 80% decrease in 3MC activation of the ABCG2 promoter; additional mutation of the AhRE4 site had negligible effect on the ABCG2 promoter activity. Moreover, chromatin immunoprecipitation assays demonstrated that treatment with 3MC significantly enhanced the recruitment of AhR to the AhRE5 occupied region, and mutation of the AhRE5 site clearly dissociated AhR protein from this promoter region. Together, these data show that the novel distal AhRE5 is critical for AhR-mediated transcriptional activation of ABCG2 gene expression in LS174T cells, and it may offer new strategies for early identification of ABCG2 inducers, which would be of benefit for preventing transporter-associated drug-drug interactions.

  13. A Novel Xenobiotic Responsive Element Regulated by Aryl Hydrocarbon Receptor is Involved in the Induction of BCRP/ABCG2 in LS174T cells

    PubMed Central

    Tompkins, Leslie M; Li, Haishan; Li, Linhao; Lynch, Caitlin; Xie, Yi; Nakanishi, Takeo; Ross, Douglas D; Wang, Hongbing

    2010-01-01

    Induction of the breast cancer resistance protein (BCRP/ABCG2) expression has been found in various tissues and cell-types after exposure to chemicals including 17β-estradiol, rosiglitazone, imatinib, as well as aryl hydrocarbon receptor (AhR) activators such as 2,3,7,8-tetrachlorodibenzodioxin, 3-methylcholanthrene (3MC), and omeprazole. However, the mechanism(s) underlying AhR-related induction of ABCG2 is largely unknown. Here, we demonstrate the AhR-dependent induction of ABCG2 expression in human colon adenocarcinoma LS174T cells. Importantly, a novel distal AhR responsive element (AhRE5) located −2357/−2333 bp upstream of the ABCG2 transcriptional start site has been identified and characterized as a functional unit pivotal to 3MC-mediated induction of ABCG2. Cell-based reporter assays revealed that deletion of AhRE5 and 4 dramatically attenuated 3MC-induced activation of ABCG2 reporter activity, while further deletion of the proximal AhRE3 and 2 only moderately changed the luciferase activities. Notably, site-directed mutation of the AhRE5 in the BCRP-3.8kb reporter construct alone resulted in approximately 80% decrease in 3MC activation of the ABCG2 promoter; additional mutation of the AhRE4 site had negligible effect on the ABCG2 promoter activity. Moreover, chromatin immunoprecipitation assays demonstrated that treatment with 3MC significantly enhanced the recruitment of AhR to the AhRE5 occupied region, and mutation of the AhRE5 site clearly dissociated AhR protein from this promoter region. Together, these data show that the novel distal AhRE5 is critical for AhR-mediated transcriptional activation of ABCG2 gene expression in LS174T cells, and it may offer new strategies for early identification of ABCG2 inducers, which would be of benefit for preventing transporter-associated drug-drug interactions. PMID:20804740

  14. Double-transduced MDCKII cells to study human P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) interplay in drug transport across the blood-brain barrier.

    PubMed

    Poller, Birk; Wagenaar, Els; Tang, Seng Chuan; Schinkel, Alfred H

    2011-04-04

    P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) combination knockout mice display disproportionately increased brain penetration of shared substrates, including topotecan and several tyrosine kinase inhibitors, compared to mice deficient for only one transporter. To better study the interplay of both transporters also in vitro, we generated a transduced polarized MDCKII cell line stably coexpressing substantial levels of human ABCB1 and ABCG2 (MDCKII-ABCB1/ABCG2). Next, we measured concentration-dependent transepithelial transport of topotecan, sorafenib and sunitinib. By blocking either one or both of the transporters simultaneously, using specific inhibitors, we aimed to mimic the ABCB1-ABCG2 interplay at the blood-brain barrier in wild-type, single or combination knockout mice. ABCB1 and ABCG2 contributed to similar extents to topotecan transport, which was only partly saturable. For sorafenib transport, ABCG2 was the major determinant at low concentrations. However, saturation of ABCG2-mediated transport occurred at higher sorafenib concentrations, where ABCB1 was still fully active. Furthermore, sunitinib was transported equally by ABCB1 and ABCG2 at low concentrations, but ABCG2-mediated transport became saturated at lower concentrations than ABCB1-mediated transport. The relative impact of these transporters can thus be affected by the applied drug concentrations. A comparison of the in vitro observed (inverse) transport ratios and cellular accumulation of the drugs at low concentrations with in vivo brain penetration data from corresponding Abcb1a/1b⁻/⁻, Abcg2⁻/⁻ and Abcb1a/1b;Abcg2⁻/⁻ mouse strains revealed very similar qualitative patterns for each of the tested drugs. MDCKII-ABCB1/ABCG2 cells thus present a useful in vitro model to study the interplay of ABCB1 and ABCG2.

  15. Effects of triclabendazole on secretion of danofloxacin and moxidectin into the milk of sheep: role of triclabendazole metabolites as inhibitors of the ruminant ABCG2 transporter.

    PubMed

    Barrera, Borja; González-Lobato, Lucía; Otero, Jon A; Real, Rebeca; Prieto, Julio G; Álvarez, Ana I; Merino, Gracia

    2013-11-01

    ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP) mediates drug-drug interactions that affect the secretion of drugs into milk. The aims of this study were: (1) to determine whether the major plasma metabolites of the flukicide triclabendazole (TCBZ), triclabendazole sulfoxide (TCBZSO) and triclabendazole sulfone (TCBZSO2), inhibit ovine and bovine ABCG2 and its Y581S variant in vitro, and (2) to examine whether coadministration of TCBZ with the ABCG2 substrates danofloxacin (a fluoroquinolone) and moxidectin (a milbemycin) affects the secretion of these drugs into the milk of sheep. TCBZSO and TCBZSO2 inhibited ruminant ABCG2 in vitro by reversing the reduced mitoxantrone accumulation and reducing basal to apical transport of nitrofurantoin in cells transduced with bovine variants (S581 and Y581) and the ovine variant of ABCG2. Coadministration of TCBZ with moxidectin or danofloxacin to sheep resulted in significantly reduced levels of moxidectin, but not danofloxacin, in the milk of TCBZ-treated sheep compared to sheep administered moxidectin or danofloxacin alone. The milk area under concentration time curve (AUC 0-48 h) was 2.99±1.41 μg h/mL in the group treated with TCBZ and moxidectin, and 7.75±3.58 μg h/mL in the group treated with moxidectin alone. The AUC (0-48 h) milk/plasma ratio was 37% lower in the group treated with TCBZ and moxidectin (7.34±1.51) than in the group treated with moxidectin alone (11.68±3.61). TCBZ metabolites appear to inhibit ruminant ABCG2 and affect the secretion of ABCG2 substrates into milk of sheep.

  16. The rs2231142 variant of the ABCG2 gene is associated with uric acid levels and gout among Japanese people.

    PubMed

    Yamagishi, Kazumasa; Tanigawa, Takeshi; Kitamura, Akihiko; Köttgen, Anna; Folsom, Aaron R; Iso, Hiroyasu

    2010-08-01

    Recent genome-wide association and functional studies have shown that the ABCG2 gene encodes for a urate transporter, and a common causal ABCG2 variant, rs2231142, leads to elevated uric acid levels and prevalent gout among Whites and Blacks. We examined whether this finding is observed in a Japanese population, since Asians have a high reported prevalence of the T-risk allele. A total of 3923 Japanese people from the Circulatory Risk in Communities Study aged 40-90 years were genotyped for rs2231142. Associations of the rs2231142 variant with serum uric acid levels and prevalence of gout and hyperuricaemia were examined. The frequency of the T-risk allele was 31% in this Japanese sample. Multivariable adjusted mean uric acid levels were 7-9 micromol/l higher for TG and TT than GG carriers (P-additive = 0.0006). The multivariable-adjusted odds ratio (OR) of prevalent gout was 1.37 (95% CI 0.68, 2.76) for TG and 4.37 (95% CI 1.98, 9.62) for TT compared with the GG carriers (P-additive = 0.001). When evaluating the combined outcome of hyperuricaemia and gout, the respective ORs were 1.40 (95% CI 1.04, 1.87) for TG and 1.88 (95% CI 1.23, 2.89) for TT carriers. The population attributable risk was 29% for gout and 19% for gout and/or hyperuricaemia. The association of the causal ABCG2 rs2231142 variant with uric acid levels and gout was confirmed in a sample of Japanese ancestry. Our study emphasizes the importance of this common causal variant in a population with a high risk allele frequency, especially as more Japanese adopt a Western lifestyle with a concomitant increase in mean serum uric acid levels.

  17. Trough concentration and ABCG2 polymorphism are better to predict imatinib response in chronic myeloid leukemia: a meta-analysis.

    PubMed

    Jiang, Zhi-Ping; Zhao, Xie-Lan; Takahashi, Naoto; Angelini, Sabrina; Dubashi, Biswajit; Sun, Li; Xu, Ping

    2017-01-01

    The present study aimed to conduct a series of meta-analyses to investigate the influence of imatinib trough concentration (C0), as well as ABCB1 and ABCG2 polymorphisms, on the clinical response in patients with chronic myeloid leukemia (CML). A literature search was conducted using the PubMed and Cochrane electronic databases to locate relevant papers from 2003 onward. Then, an initial meta-analysis of 14 studies involving 2184 patients was conducted to understand the effect of imatinib mesylate (IM) C0 on clinical outcome in CML patients. Subsequently, a series of meta-analyses were performed, including up to 23 studies with 2577 patients, on the effect of genetic polymorphisms of ABCB1 and ABCG2 on the clinical response to IM. Meta-analysis revealed that patients who achieved a major molecular response (MMR) have a significantly higher IM C0 than those who failed to achieve an MMR. We also found that the patients who achieved a complete cytogenic response (CCyR) have a significantly higher IM C0 than those who did not achieve a CCyR. However, no significant difference in IM C0 was found between the complete molecular response and non-complete molecular response groups. Additional analysis showed that ABCG2 421 variant A allele was significantly associated with a higher rate of MMR and overall response, especially in Asian patients. Meta-analysis did not reveal a correlation between ABCB1 C3435T and C1236T polymorphisms with any clinical response to IM. However, the G2677T/A polymorphism could play a role in IM response in the recessive model. This meta-analysis demonstrates that there was a significant correlation between the IM trough concentration and clinical responses, especially MMR and CCyR, in CML patients. Furthermore, we found that the probability of successful treatment was correlated with the ABCG2 C421A polymorphism, at least within the Asian population. We failed to determine an association between ABCB1 polymorphisms and IM response, although the

  18. Abcb1a but not Abcg2 played a predominant role in limiting the brain distribution of Huperzine A in mice.

    PubMed

    Li, Jiajun; Yue, Mei; Zhou, Dandan; Wang, Meiyu; Zhang, Hongjian

    2017-09-01

    Huperzine A has been used for improving symptoms of Alzheimer's disease. Its cholinergic side effect is thought to be an exaggerated pharmacological outcome linked to its high brain or CNS concentrations. Although Huperzine A is brain penetrable, its interaction with efflux transporters (ABCB1 and ABCG2) has not been fully investigated. The aim of the present study was to characterize roles of ABCB1 and ABCG2 in the transmembrane transport of Huperzine A and identify a rate limiting step in its brain distribution. Data obtained from stably transfected MDCK II cells showed that Huperzine A is a substrate of ABCB1 but not ABCG2. ABCB1 inhibitors significantly inhibited ABCB1 mediated efflux of Huperzine A. In Abcb1a(-/-) mice, the brain to plasma concentration ratio of Huperzine A was significantly increased as compared to the wild type mice, while there were no obvious differences between the wild type and Abcg2(-/-) mice. Taken together, the present study demonstrated that ABCB1 but not ABCG2 played a predominant role in the efflux of Huperzine A across BBB. The current finding is clinically relevant as changes in ABCB1 activity in the presence of ABCB1 inhibitors or genetic polymorphism may affect efficacy and safety of Huperzine A. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Milk secretion of nitrofurantoin, as a specific BCRP/ABCG2 substrate, in assaf sheep: modulation by isoflavones.

    PubMed

    Pérez, M; Real, R; Mendoza, G; Merino, G; Prieto, J G; Alvarez, A I

    2009-10-01

    Studies on residues in milk used for human consumption have increased due to health concerns and priority interest in the control of potentially risky drugs. The protein BCRP/ABCG2, present in the mammary epithelia, actively extrudes drugs into milk and can be modulated by isoflavones. Nitrofurantoin is a specific BCRP substrate which is actively excreted into milk by this transporter. In this research, we studied nitrofurantoin transport into milk in four experimental groups: G1-calves fed forage with isoflavones; G2-calves fed forage with isoflavones and administered exogenous genistein and daidzein; G3-calves fed forage without isoflavones; G4-calves fed forage without isoflavones and administered exogenous genistein and daidzein. Results show increased levels of nitrofurantoin in milk from calves without isoflavones (G3) and decreased nitrofurantoin residues in milk when isoflavones were present, either by forage (G1 and G2) or by exogenous administration (G4). The values of C(max) in milk were significantly lower in those groups with isoflavones in forage (G1, G2). Plasma levels were low and unmodified among the groups. Inter-individual variation was high. All these results seem to point to a feasible control of drug secretion into milk through isoflavones in the diet when the drug is a good BCRP/ABCG2 substrate.

  20. Pelitinib (EKB-569) targets the up-regulation of ABCB1 and ABCG2 induced by hyperthermia to eradicate lung cancer

    PubMed Central

    To, Kenneth K W; Poon, Daniel C; Wei, Yuming; Wang, Fang; Lin, Ge; Fu, Liwu

    2015-01-01

    Background and Purpose Pelitinib is a potent irreversible EGFR TK inhibitor currently in clinical trials for the treatment of lung cancer. Hyperthermia has been applied concomitantly with chemotherapy and radiotherapy to enhance treatment outcome. In this study, we investigated the ability of the combination of pelitinib with other conventional anticancer drugs to specifically target cancer cells with up-regulated efflux transporters ABCB1/ABCG2 after hyperthermia as a novel way to eradicate the cancer stem-like cells responsible for cancer recurrence. Experimental Approach Alterations in intracellular topotecan accumulation, the efflux of fluorescent probe substrates, expression and ATPase activity of ABCB1/ABCG2 and tumoursphere formation capacity of side population (SP) cells sorted after hyperthermia were examined to elucidate the mechanism of pelitinib-induced chemosensitization. Key Results While pelitinib did not modulate ABCB1/ABCG2 expressions, the combination of pelitinib with transporter substrate anticancer drugs induced more marked apoptosis, specifically in cells exposed to hyperthermia. The flow cytometric assay showed that both ABCB1- and ABCG2-mediated drug effluxes were significantly inhibited by pelitinib in a concentration-dependent manner. The inhibition kinetics suggested that pelitinib is a competitive inhibitor of ABCB1/ABCG2, which is consistent with its ability to stimulate their ATPase activity. SP cells sorted after hyperthermia were found to be more resistant to anticancer drugs, presumably due to the up-regulation of ABCB1 and ABCG2. Importantly, pelitinib specifically enhanced the chemosensitivity but reduced the tumoursphere formation capacity of these SP cells. Conclusions and Implications This study demonstrated a novel approach, exploiting drug resistance, to selectively kill cancer stem-like cells after hyperthermia. PMID:25988710

  1. Targeted therapeutic effect of anti-ABCG2 antibody combined with nano silver and vincristine on mouse myeloma cancer stem cells

    NASA Astrophysics Data System (ADS)

    Dou, Jun; He, Xiangfeng; Liu, Yunjing; Huang, Zhihai; Yang, Cuiping; Shi, Fangfang; Chen, Dengyu; Gu, Ning

    2013-12-01

    Studies from hematopoietic origin malignancies have demonstrated that multiple myeloma contain a rare population of cancer stem cells (CSCs) that are responsible for tumor multiresistance and recurrence. The goal of this study was to investigate targeted therapeutic effect of anti-ABCG2 monoclonal antibody (McAb) combined with silver nanoparticles (AgNPs) and vincristine (VCR) on myeloma CSCs. The characteristics of CD44+ CD24- cells that were isolated from the SP2/0 cells using magnetic activated cell sorting system were first identified. The results showed that the CD44+ CD24- cells exhibited higher proliferation, more colony formation, more side population fraction, and stronger tumorigenicity in BALB/c mice than the control cells. Moreover, CD44+ CD24- cells markedly up-regulated the ABCG2 expression, however, anti-ABCG2 McAb combined with AgNPs and VCR effectively inhibited the CD44+ CD24- cell growth and prolonged the survival of myeloma-bearing mice. We concluded that the CD44+ CD24- cells in mouse myeloma SP2/0 cell line posses CSC properties. Anti-ABCG2 McAb combined with AgNPs and VCR provide an efficient targeted therapeutic method for inhibiting myeloma CD44+ CD24- CSC growth in mice.

  2. Significantly increased expression of OCT4 and ABCG2 in spheroid body-forming cells of the human gastric cancer MKN-45 cell line.

    PubMed

    Liu, Jianming; Wang, Lei; Ma, Lilin; Xu, Junfei; Liu, Chun; Zhang, Jianguo; Liu, Jie; Chen, Ruixin

    2013-10-01

    The cancer stem cell (CSC) theory hypothesizes that CSCs are the cause of tumor formation, recurrence and metastasis. Key to the study of CSCs is their isolation and identification. The present study investigated whether spheroid body-forming cells in the human gastric cancer (GC) MKN-45 cell line are enriched for CSC properties, and also assessed the expression of the candidate CSC markers, octamer-binding transcription factor-4 (OCT4) and adenosine triphosphate-binding cassette transporter G2 (ABCG2) in the MKN-45 spheroid body cells. The MKN-45 cells were plated in a stem cell-conditioned culture system to allow for spheroid body formation. The expression levels of OCT4 and ABCG2 in the spheroid body cells were assessed by qPCR, western blot analysis and immunofluorescence staining, while the tumorigenicity of the spheroid body-forming cells was assessed by in vivo xenograft studies in nude mice. The MKN-45 cells were able to form spheroid bodies when cultured in stem cell-conditioned medium. The spheroid body-forming cells showed a significantly higher (P<0.01) expression of OCT4 and ABCG2 compared with the parental cells. These data suggest that the spheroid body cells from the MKN-45 GC cell line cultured in stem cell-conditioned medium possessed gastric CSC properties. The co-expression of OCT4 and ABCG2 by these cells may represent the presence of a subpopulation of gastric CSCs.

  3. Co-expression of CD44 and ABCG2 in spheroid body-forming cells of gastric cancer cell line MKN45.

    PubMed

    Liu, Jianming; Ma, Lilin; Xu, Junfei; Liu, Chun; Zhang, Jianguo; Liu, Jie; Chen, Ruixin; Zhou, Youlang

    2013-01-01

    The cancer stem cell (CSC) theory hypothesizes that CSCs are regarded as the cause of tumor formation, recurrence and metastasis. This study aimed to investigate whether spheroid body-forming cells in human gastric cancer cell were enriched for CSC properties, and to assess the expression of candidate CSC markers, cluster of differentiation 44 (CD44) and adenosine triphosphate binding cassette transporter G 2 (ABCG2) in the MKN45 spheroid body cells. Human gastric cancer cell line MKN45 were plated in stem cell conditioned culture system allowed for spheroid body forming. The expression levels of CD44 and ABCG2 in the spheroid body cells were assessed by quantitative real-time PCR, western blot analysis and immunofluorescence staining, and the tumorigenicity of the spheroid body-forming cells were assessed by in vivo xenograft studies in nude mice. The MKN45 cells could form spheroid bodies cultured in stem cell conditioned medium. The spheroid body-forming cells showed a significantly greater (p <0.05) expression of CD44 and ABCG2 than the parental cells. Spheroid body cells from gastric cancer cell line MKN45 cultured in stem cell conditioned medium possessed gastric CSC properties. The cells co-expressed of CD44 and ABCG2 might represent a subpopulation of gastric CSCs.

  4. Telmisartan increases systemic exposure to rosuvastatin after single and multiple doses, and in vitro studies show telmisartan inhibits ABCG2-mediated transport of rosuvastatin.

    PubMed

    Hu, Miao; Lee, Hon-Kit; To, Kenneth K W; Fok, Benny S P; Wo, Siu-Kwan; Ho, Chung-Shun; Wong, Chun-Kwok; Zuo, Zhong; Chan, Thomas Y K; Chan, Juliana C N; Tomlinson, Brian

    2016-12-01

    The ATP-binding cassette transporter G2 (ABCG2) plays an important role in the disposition of rosuvastatin. Telmisartan, a selective angiotension-II type 1 (AT1) receptor blocker, inhibits the transport capacity of ABCG2, which may result in drug interactions. This study investigated the pharmacokinetic interaction between rosuvastatin and telmisartan and the potential mechanism. In this two-phase fixed-order design study, healthy subjects received single doses of 10 mg rosuvastatin at baseline and after telmisartan 40 mg daily for 14 days. Patients with hyperlipidaemia who had been taking rosuvastatin 10 mg daily for at least 4 weeks were given telmisartan 40 mg daily for 14 days together with rosuvastatin. Plasma concentrations of rosuvastatin were measured over 24 h before and after telmisartan administration. In vitro experiments using a bidirectional transport assay were performed to investigate the involvement of ABCG2 in the interaction. Co-administration of telmisartan significantly increased the maximum plasma concentration (C max) and the area under the plasma concentration-time curve (AUC) of rosuvastatin by 71 and 26 %, respectively. The T max values were reduced after administration of telmisartan. There was no significant difference in the interaction of rosuvastatin with telmisartan between healthy volunteers and patients receiving long-term rosuvastatin therapy or among subjects with the different ABCG2 421 C>A genotypes. The in vitro experiment demonstrated that telmisartan inhibited ABCG2-mediated efflux of rosuvastatin. This study demonstrated that telmisartan significantly increased the systemic exposure to rosuvastatin after single and multiple doses.

  5. Piperazinobenzopyranones and phenalkylaminobenzopyranones: potent inhibitors of breast cancer resistance protein (ABCG2).

    PubMed

    Boumendjel, Ahcène; Nicolle, Edwige; Moraux, Thomas; Gerby, Bastien; Blanc, Madeleine; Ronot, Xavier; Boutonnat, Jean

    2005-11-17

    In continuing research that led us to identify chromanone derivatives (J. Med. Chem. 2003, 46, 2125) as P-glycoprotein inhibitors, we obtained analogues able to modulate multidrug resistance (MDR) mediated by the breast cancer resistance protein (BCRP). The linkage of 5-hydroxybenzopyran-4-one to piperazines or phenalkylamines affords highly potent inhibitors of BCRP. By using sensitive (HCT116) and resistant colon cancer cells expressing BCRP, we evaluated the effect of 14 benzopyranone (chromone) derivatives on the accumulation and the cytotoxic effect of the anticancer drug, mitoxantrone. At 10 microM, three compounds increased both intracellular accumulation and cytotoxicity of mitoxantrone in HCT116/R cells with a comparable rate as fumitremorgin C and Gleevec used as reference inhibitors. The most potent molecules 5b and 5c are still active at 1 microM, whereas FTC shows weak inhibition. These molecules do not induce cell death as shown by the cell cycle distribution study, which makes them potential candidates for in vivo studies.

  6. Interaction of Isoflavones with the BCRP/ABCG2 Drug Transporter

    PubMed Central

    Bircsak, Kristin M; Aleksunes, Lauren M

    2015-01-01

    This review will provide a comprehensive overview of the interactions between dietary isoflavones and the ATP-binding cassette (ABC) G2 efflux transporter, which is also named the breast cancer resistance protein (BCRP). Expressed in a variety of organs including the liver, kidneys, intestine, and placenta, BCRP mediates the disposition and excretion of numerous endogenous chemicals and xenobiotics. Isoflavones are a class of naturally-occurring compounds that are found at high concentrations in commonly consumed foods and dietary supplements. A number of isoflavones, including genistein and daidzein and their metabolites, interact with BCRP as substrates, inhibitors, and/or modulators of gene expression. To date, a variety of model systems have been employed to study the ability of isoflavones to serve as substrates and inhibitors of BCRP; these include whole cells, inverted plasma membrane vesicles, in situ organ perfusion, as well as in vivo rodent and sheep models. Evidence suggests that BCRP plays a role in mediating the disposition of isoflavones and in particular, their conjugated forms. Furthermore, as inhibitors, these compounds may aid in reversing multidrug resistance and sensitizing cancer cells to chemotherapeutic drugs. This review will also highlight the consequences of altered BCRP expression and/or function on the pharmacokinetics and toxicity of chemicals following isoflavone exposure. PMID:26179608

  7. Adenovirus adenine nucleotide translocator-2 shRNA effectively induces apoptosis and enhances chemosensitivity by the down-regulation of ABCG2 in breast cancer stem-like cells.

    PubMed

    Jang, Ji Young; Kim, Min Kyoung; Jeon, Yoon Kyung; Joung, Yoon Ki; Park, Ki Dong; Kim, Chul Woo

    2012-04-30

    Cancer stem cells (CSCs) are resistant to chemo- and radio-therapy, and can survive to regenerate new tumors. This is an important reason why various anti- cancer therapies often fail to completely control tumors, although they kill and eliminate the bulk of cancer cells. In this study, we determined whether or not adenine nucleotide translocator-2 (ANT2) suppression could also be effective in inducing cell death of breast cancer stem-like cells. A sub-population (SP; CD44+/ CD24-) of breast cancer cells has been reported to have stem/progenitor cell properties. We utilized the adeno- ANT2 shRNA virus to inhibit ANT2 expression and then observed the treatment effect in a SP of breast cancer cell line. In this study, MCF7, MDA-MB-231 cells, and breast epithelial cells (MCF10A) mesenchymally-transdifferentiated through E-cadherin knockdown were used. ANT2 expression was high in both stem-like cells and non-stem-like cells of MCF7 and MDA-MB-231 cells, and was induced and up-regulated by mesenchymal transdifferentiation in MCF10A cells (MCF10A(EMT)). Knockdown of ANT2 by adeno-shRNA virus efficiently induced apoptotic cell death in the stem-like cells of MCF7 and MDA-MB-231 cells, and MCF10A(EMT). Stem-like cells of MCF7 and MDA-MB-231, and MCF10A(EMT) cells exhibited increased drug (doxorubicin) resistance, and expressed a multi-drug resistant related molecule, ABCG2, at a high level. Adeno-ANT2 shRNA virus markedly sensitized the stem-like cells of MCF7 and MDA-MB-231, and the MCF10A(EMT) cells to doxorubicin, which was accompanied by down-regulation of ABCG2. Our results suggest that ANT2 suppression by adeno-shRNA virus is an effective strategy to induce cell death and increase the chemosensitivity of stem-like cells in breast cancer.

  8. Intestinal Ciprofloxacin Efflux: The Role of Breast Cancer Resistance Protein (ABCG2)

    PubMed Central

    Wright, J. A.; O'Reilly, D. A.; Sherlock, D. J.; Coleman, T.; Simmons, N. L.

    2011-01-01

    Intestinal secretory movement of the fluoroquinolone antibiotic, ciprofloxacin, may limit its oral bioavailability. Active ATP-binding cassette (ABC) transporters such as breast cancer resistance protein (BCRP) have been implicated in ciprofloxacin transport. The aim of this study was to test the hypothesis that BCRP alone mediates intestinal ciprofloxacin secretion. The involvement of ABC transport proteins in ciprofloxacin secretory flux was investigated with the combined use of transfected cell lines [bcrp1/BCRP-Madin-Darby canine kidney II (MDCKII) and multidrug resistance-related protein 4 (MRP4)-human embryonic kidney (HEK) 293] and human intestinal Caco-2 cells, combined with pharmacological inhibition using 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6, 7,12,12a-octahydropyrazino[1′,2′:1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester (Ko143), cyclosporine, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), and verapamil as ABC-selective inhibitors. In addition, the regional variation in secretory capacity was investigated using male Han Wistar rat intestine mounted in Ussing chambers, and the first indicative measurements of ciprofloxacin transport by ex vivo human jejunum were made. Active, Ko143-sensitive ciprofloxacin secretion was observed in bcrp1-MDCKII cell layers, but in low-passage (BCRP-expressing) Caco-2 cell layers only a 54% fraction was Ko143-sensitive. Ciprofloxacin accumulation was lower in MRP4-HEK293 cells than in the parent line, indicating that ciprofloxacin is also a substrate for this transporter. Ciprofloxacin secretion by Caco-2 cell layers was not inhibited by MK571. Secretory flux showed marked regional variability in the rat intestine, increasing from the duodenum to peak in the ileum. Ciprofloxacin secretion was present in human jejunum and was reduced by Ko143 but showed marked interindividual variability. Ciprofloxacin is a substrate for human

  9. Tyrosine kinase inhibitor resistance in cancer: role of ABC multidrug transporters.

    PubMed

    Ozvegy-Laczka, Csilla; Cserepes, Judit; Elkind, N Barry; Sarkadi, Balázs

    2005-01-01

    Recent antitumor drug research has seen the development of a large variety of tyrosine kinase inhibitors (TKIs) with increasing specificity and selectivity. These are highly promising agents for specific inhibition of malignant cell growth and metastasis. However, their therapeutic potential also depends on access to their intracellular targets, which may be significantly affected by certain ABC membrane transporters. It has been recently shown that several human multidrug transporter ABC proteins interact with specific TKIs, and the ABCG2 transporter has an especially high affinity for some of these kinase inhibitors. These results indicate that multidrug resistance protein modulation by TKIs may be an important factor in the treatment of cancer patients; moreover, the extrusion of TKIs by multidrug transporters may result in tumor cell TKI resistance. Interaction with multidrug resistance ABC transporters may also significantly modify the pharmacokinetics and toxicity of TKIs in patients.

  10. Involvement of breast cancer resistance protein (BCRP/ABCG2) in the secretion of danofloxacin into milk: interaction with ivermectin.

    PubMed

    Real, R; Egido, E; Pérez, M; González-Lobato, L; Barrera, B; Prieto, J G; Alvarez, A I; Merino, G

    2011-08-01

    Danofloxacin, a veterinary fluoroquinolone antimicrobial drug, is actively secreted into milk by an as yet unknown mechanism. One of the main determinants of active drug secretion into milk is the transporter (BCRP/ABCG2). The main purpose was to determine whether danofloxacin is an in vitro substrate for Bcrp1/BCRP and to assess its involvement in danofloxacin secretion into milk. In addition, the role of potential drug-drug interactions in this process was assessed using ivermectin. Danofloxacin was transported in vitro by Bcrp1/BCRP, and ivermectin efficiently blocked this transport. Experiments with Bcrp1(-/-) mice showed no evidence of the involvement of Bcrp1 in plasma pharmacokinetics of danofloxacin. However, the milk concentration and milk-to-plasma ratio of danofloxacin were almost twofold higher in wild-type compared with Bcrp1(-/-) mice. The in vivo interaction with ivermectin was studied in sheep after co-administration of danofloxacin (1.25 mg/kg, i.m.) and ivermectin (0.2 mg/kg, s.c.). Ivermectin had no significant effect on the plasma levels of danofloxacin but significantly decreased danofloxacin concentrations in milk by almost 40%. Concomitant administration of multiple drugs, often used in veterinary therapy, may not only affect their pharmacological activity but also their secretion into milk, because of potential drug-drug interactions mediated by BCRP. © 2010 Blackwell Publishing Ltd.

  11. A New Strategy to Rapidly Evaluate Kinetics of Glucuronide Efflux by Breast Cancer Resistance Protein (BCRP/ABCG2)

    PubMed Central

    Wu, Baojian; Jiang, Wen; Yin, Taijun; Gao, Song

    2013-01-01

    Purpose The efflux transporter breast cancer resistance protein (BCRP/ABCG2) plays an important role in excretion of anionic drugs and metabolites including glucuronides in humans. Methods In this article, our recently published cell model (i.e., HeLa cells over-expressing UGT1A9 (HeLa1A9)) is used to determine the kinetic parameters of BCRP-mediated transport of glucuronides. Results After incubation of the aglycone with the cells, a steady-state (i.e., zero-order or near zero-order) excretion of its glucuronide is rapidly achieved and then maintained. Kinetic profiling with different (intracellular) glucuronide concentrations and their corresponding excretion rates is enabled by varying the concentration of the aglycone, which allows for the determination of kinetic parameters responsible for BCRP-mediated efflux of glucuronides. This approach was validated theoretically using a cellular pharmacokinetic model incorporating various enzymatic and transporter-mediated kinetic processes. It was also validated experimentally in that kinetic parameters of efflux of glucuronides of 6-hydroxyflavone and 4-methylumberiferone in the HeLa1A9 cell model were shown to be consistent with those derived with BCRP-overexpressing membrane vesicles. Conclusion This study provides a new strategy for rapidly evaluating the kinetics of glucuronide efflux by BCRP. PMID:22752253

  12. Development of a model for functional studies of ABCG2 (breast cancer resistance protein) efflux employing a standard BeWo clone (B24).

    PubMed

    Crowe, Andrew; Keelan, Jeffrey A

    2012-10-01

    Human choriocarcinoma-derived BeWo cells express high levels of breast cancer resistance protein (BCRP/ABCG2) with no functional P-glycoprotein (P-gp) (ABCB1) activity, making them a potential model to study bidirectional ABCG2-mediated drug transport. However, the original BeWo clone (B24) available to researchers does not form confluent monolayers with tight junctions required by the model. Our aim was to adapt culture conditions to attempt to generate confluent BeWo monolayers for drug transport studies using the standard B24 clone. BeWo cells (B24; American Type Culture collection [ATCC]) were cultured in six-well plates or polycarbonate millicell inserts in a number of media formulations, growth supplements, and basement membrane substitutes. Cells were examined for confluence by microscopy, and transepithelial electrical resistance (TEER) was measured daily; monolayer permeability was assessed when TEER had stabilized. Optimal growth rates were achieved in culture conditions consisting of Medium 199 (M199) supplemented with epidermal growth factor (EGF; 20 ng/mL), vitamin supplements, and 10% fetal calf serum (FCS) with collagen coating. A TEER of 170 Ω in 0.6 cm(2) inserts was achieved 2 weeks after seeding under optimal conditions. The cell-impermeable diffusion marker 5(6) carboxy-2,7dichlorodihydrofluorescein (C-DCDHF) had a permeability coefficient of 3.5×10(-6) cm/s, indicative of minimal paracellular permeability. ABCG2 expression, as determined by immunoblotting, remained unaffected by confluency. In conclusion, we describe culture conditions for the B24 BeWo clone that facilitate the formation of monolayers with tighter junctions and reduced paracellular transport compared to previously published models. These growth conditions provide a good model of ABCG2-mediated drug transport in a human placental cell line.

  13. The gut microbiota ellagic acid-derived metabolite urolithin A and its sulfate conjugate are substrates for the drug efflux transporter breast cancer resistance protein (ABCG2/BCRP).

    PubMed

    González-Sarrías, Antonio; Miguel, Verónica; Merino, Gracia; Lucas, Ricardo; Morales, Juan C; Tomás-Barberán, Francisco; Alvarez, Ana I; Espín, Juan C

    2013-05-08

    The breast cancer resistance protein (BCRP/ABCG2) is a drug efflux transporter that can affect the pharmacological and toxicological properties of many molecules. Urolithins, metabolites produced by the gut microbiota from ellagic acid (EA) and ellagitannins, have been acknowledged with in vivo anti-inflammatory and cancer chemopreventive properties. This study evaluated whether urolithins (Uro-A, -B, -C, and -D) and their main phase II metabolites Uro-A sulfate, Uro-A glucuronide, and Uro-B glucuronide as well as their precursor EA were substrates for ABCG2/BCRP. Parental and Bcrp1-transduced MDCKII cells were used for active transport assays. Uro-A and, to a lesser extent, Uro-A sulfate showed a significant increase in apically directed translocation in Bcrp1-transduced cells. Bcrp1 did not show affinity for the rest of the tested compounds. Data were confirmed for murine, human, bovine, and ovine BCRP-transduced subclones as well as with the use of the selective BCRP inhibitor Ko143. The transport inhibition by Uro-A was analyzed by flow cytometry compared to Ko143 using the antineoplastic agent mitoxantrone as a model substrate. Results showed that Uro-A was able to inhibit mitoxantrone transport in a dose-dependent manner. This study reports for the first time that Uro-A and its sulfate conjugate are ABCG2/BCRP substrates. The results suggest that physiologically relevant concentrations of these gut microbiota-derived metabolites could modulate ABCG2/BCRP-mediated transport processes and mechanisms of cancer drug resistance. Further in vivo investigations are warranted.

  14. The effects of ABCG2 on the viability, proliferation and paracrine actions of kidney side population cells under oxygen-glucose deprivation.

    PubMed

    Liu, Hong-Bao; Meng, Qiu-Hong; Du, De-Wei; Sun, Ji-Feng; Wang, Jian-Bo; Han, Hua

    2014-01-01

    Bcrp1/ABCG2 is exclusively expressed in side population (SP) cells, however, it has not been fully elucidated whether it has an impact on the viability, proliferation and paracrine actions in kidney SP cells under oxygen-glucose deprivation (OGD) followed by reoxygenation. In this study, we found that 2-h OGD did not injure SP cells (sub-lethal OGD) but induced SP cells proliferation 48 and 72 h after reoxygenation; whereas 4-h OGD markedly injured the cells (lethal OGD) and led to apoptosis 24-72 h after reoxygenation. Fumitremorgin C, an inhibitor of ABCG2, attenuated both the proliferation and viability of SP cells. Sub-lethal and lethal OGD induced the increase in the secretion of vascular endothelial growth factor, insulin-like growth factor 1, hepatocyte growth factor, and stromal cell-derived factor-1α in kidney SP cells, which was inhibited by Fumitremorgin C. Collectively, these findings provide evidence for a crucial role for the ABCG2 expression in the viability, proliferation and paracrine actions of kidney SP cells after OGD.

  15. Structure-activity relationships of chromone derivatives toward the mechanism of interaction with and inhibition of breast cancer resistance protein ABCG2.

    PubMed

    Winter, Evelyn; Lecerf-Schmidt, Florine; Gozzi, Gustavo; Peres, Basile; Lightbody, Mark; Gauthier, Charlotte; Ozvegy-Laczka, Csilla; Szakacs, Gergely; Sarkadi, Balazs; Creczynski-Pasa, Tânia B; Boumendjel, Ahcène; Di Pietro, Attilio

    2013-12-27

    We recently identified a chromone derivative, 5-(4-bromobenzyloxy)-2-(2-(5-methoxyindolyl)ethyl-1-carbonyl)-4H-chromen-4-one, named here as chromone 1, as a potent, selective, nontoxic, and nontransported inhibitor of ABCG2-mediated drug efflux (Valdameri et al. J. Med. Chem. 2012, 55, 966). We have now synthesized a series of 14 derivatives to study the structure-activity relationships controlling both drug efflux and ATPase activity of ABCG2 and to elucidate their molecular mechanism of interaction and inhibition. It was found that the 4-bromobenzyloxy substituent at position 5 and the methoxyindole are important for both inhibition of mitoxantrone efflux and inhibition of basal ATPase activity. Quite interestingly, methylation of the central amide nitrogen strongly altered the high affinity for ABCG2 and the complete inhibition of mitoxantrone efflux and coupled ATPase activity. These results allowed the identification of a critical central inhibitory moiety of chromones that has never been investigated previously in any series of inhibitors.

  16. The breast cancer resistance protein (BCRP/ABCG2) affects pharmacokinetics, hepatobiliary excretion, and milk secretion of the antibiotic nitrofurantoin.

    PubMed

    Merino, Gracia; Jonker, Johan W; Wagenaar, Els; van Herwaarden, Antonius E; Schinkel, Alfred H

    2005-05-01

    Nitrofurantoin is a commonly used urinary tract antibiotic prescribed to lactating woman. It is actively transported into human and rat milk by an unknown mechanism. Our group has demonstrated an important role of the breast cancer resistance protein (BCRP/ABCG2) in the secretion of xenotoxins into the milk. This ATP-binding cassette drug efflux transporter extrudes xenotoxins from cells in intestine, liver, mammary gland, and other organs, affecting the pharmacological and toxicological behavior of many compounds. We investigated whether Bcrp1 is involved in the pharmacokinetic profile of nitrofurantoin and its active secretion into the milk. Using polarized cell lines, we found that nitrofurantoin is efficiently transported by murine Bcrp1 and human BCRP. After oral administration of 10 mg/kg nitrofurantoin, the area under the plasma concentrationtime curve in Bcrp1 knockout mice was almost 4-fold higher than in wild-type mice (148.8 +/- 30.4 versus 37.5 +/- 6.8 min x microg/ml); and after i.v. administration (5 mg/kg), 2-fold higher (139.2 +/- 22.0 versus 73.9 +/- 9.0 min x microg/ml). Hepatobiliary excretion of nitrofurantoin was almost abolished in Bcrp1 knockout mice (9.6 +/- 3.2 versus 0.2 +/- 0.1% in wild-type and Bcrp1 knockout mice, respectively). The milk-to-plasma ratio of nitrofurantoin was almost 80 times higher in wild-type compared with Bcrp1 knockout lactating female mice (45.7 +/- 16.2 versus 0.6 +/- 0.1). Nitrofurantoin elimination via milk was quantitatively even higher than hepatobiliary elimination. We conclude that Bcrp1 is an important determinant for the bioavailability of nitrofurantoin and the main mechanism involved in its hepatobiliary excretion and secretion into the milk.

  17. Breast cancer resistance protein (BCRP/ABCG2) transports fluoroquinolone antibiotics and affects their oral availability, pharmacokinetics, and milk secretion.

    PubMed

    Merino, Gracia; Alvarez, Ana I; Pulido, Mivis M; Molina, Antonio J; Schinkel, Alfred H; Prieto, Julio G

    2006-04-01

    The breast cancer resistance protein (BCRP/ABCG2) is an ATP-binding cassette drug efflux transporter that extrudes xenotoxins from cells in intestine, liver, mammary gland, and other organs, affecting the pharmacological and toxicological behavior of many compounds, including their secretion into the milk. The purpose of this study was to determine whether three widely used fluoroquinolone antibiotics (ciprofloxacin, ofloxacin, and norfloxacin) are substrates of Bcrp1/BCRP and to investigate the possible role of this transporter in the in vivo pharmacokinetic profile of these compounds and their secretion into the milk. Using polarized cell lines, we found that ciprofloxacin, ofloxacin, and norfloxacin are transported by mouse Bcrp1 and human BCRP. In vivo pharmacokinetic studies showed that the ciprofloxacin plasma concentration was more than 2-fold increased in Bcrp1(-/-) compared with wild-type mice (1.77 +/- 0.73 versus 0.85 +/- 0.39 microg/ml, p < 0.01) after oral administration of ciprofloxacin (10 mg/kg). The area under the plasma concentration-time curve in Bcrp1(-/-) mice was 1.5-fold higher than that in wild-type mice (48.63 +/- 5.66 versus 33.10 +/- 4.68 min x microg/ml, p < 0.05) after i.v. administration (10 mg/kg). The milk concentration and milk/plasma ratio of ciprofloxacin were 2-fold higher in wild-type than in Bcrp1(-/-) lactating mice. We conclude that Bcrp1 is one of the determinants for the bioavailability of fluoroquinolones and their secretion into the milk.

  18. The antiepileptic drug lamotrigine is a substrate of mouse and human breast cancer resistance protein (ABCG2).

    PubMed

    Römermann, Kerstin; Helmer, Renate; Löscher, Wolfgang

    2015-06-01

    Resistance to antiepileptic drugs (AEDs) is the major problem in the treatment of epilepsy. One hypothesis to explain AED resistance suggests that seizure-induced overexpression of efflux transporters at the blood-brain barrier (BBB) restricts AEDs to reach their brain targets. Various studies examined whether AEDs are substrates of P-glycoprotein (Pgp; MDR1; ABCB1), whereas information about the potential role of breast cancer resistance protein (BCRP; ABCG2) is scanty. We used a highly sensitive in vitro assay (concentration equilibrium transport assay; CETA) with MDCKII cells transduced with murine Bcrp1 or human BCRP to evaluate whether AEDs are substrates of this major efflux transporter. Six of 7 AEDs examined, namely phenytoin, phenobarbital, carbamazepine, levetiracetam, topiramate, and valproate, were not transported by Bcrp at therapeutic concentrations, whereas lamotrigine exhibited a marked asymmetric, Bcrp-mediated transport in the CETA, which could be almost completely inhibited with the Bcrp inhibitor Ko143. Significant but less marked transport of lamotrigine was determined in MDCK cells transfected with human BCRP. Lamotrigine is also a substrate of human Pgp, so that this drug is the first AED that has been identified as a dual substrate of the two major human efflux transporters at the BBB. Previous in vivo studies have demonstrated a synergistic or cooperative role of Pgp and Bcrp in the efflux of dual substrates at the BBB, so that transport of lamotrigine by Pgp and BCRP may be an important mechanism of pharmacoresistance in epilepsy patients in whom both transporters are overexpressed.

  19. Kinetic Modeling of ABCG2 Transporter Heterogeneity: A Quantitative, Single-Cell Analysis of the Side Population Assay

    PubMed Central

    Prasanphanich, Adam F.; White, Douglas E.; Gran, Margaret A.

    2016-01-01

    The side population (SP) assay, a technique used in cancer and stem cell research, assesses the activity of ABC transporters on Hoechst staining in the presence and absence of transporter inhibition, identifying SP and non-SP cell (NSP) subpopulations by differential staining intensity. The interpretation of the assay is complicated because the transporter-mediated mechanisms fail to account for cell-to-cell variability within a population or adequately control the direct role of transporter activity on staining intensity. We hypothesized that differences in dye kinetics at the single-cell level, such as ABCG2 transporter-mediated efflux and DNA binding, are responsible for the differential cell staining that demarcates SP/NSP identity. We report changes in A549 phenotype during time in culture and with TGFβ treatment that correlate with SP size. Clonal expansion of individually sorted cells re-established both SP and NSPs, indicating that SP membership is dynamic. To assess the validity of a purely kinetics-based interpretation of SP/NSP identity, we developed a computational approach that simulated cell staining within a heterogeneous cell population; this exercise allowed for the direct inference of the role of transporter activity and inhibition on cell staining. Our simulated SP assay yielded appropriate SP responses for kinetic scenarios in which high transporter activity existed in a portion of the cells and little differential staining occurred in the majority of the population. With our approach for single-cell analysis, we observed SP and NSP cells at both ends of a transporter activity continuum, demonstrating that features of transporter activity as well as DNA content are determinants of SP/NSP identity. PMID:27851764

  20. Kinetic Modeling of ABCG2 Transporter Heterogeneity: A Quantitative, Single-Cell Analysis of the Side Population Assay.

    PubMed

    Prasanphanich, Adam F; White, Douglas E; Gran, Margaret A; Kemp, Melissa L

    2016-11-01

    The side population (SP) assay, a technique used in cancer and stem cell research, assesses the activity of ABC transporters on Hoechst staining in the presence and absence of transporter inhibition, identifying SP and non-SP cell (NSP) subpopulations by differential staining intensity. The interpretation of the assay is complicated because the transporter-mediated mechanisms fail to account for cell-to-cell variability within a population or adequately control the direct role of transporter activity on staining intensity. We hypothesized that differences in dye kinetics at the single-cell level, such as ABCG2 transporter-mediated efflux and DNA binding, are responsible for the differential cell staining that demarcates SP/NSP identity. We report changes in A549 phenotype during time in culture and with TGFβ treatment that correlate with SP size. Clonal expansion of individually sorted cells re-established both SP and NSPs, indicating that SP membership is dynamic. To assess the validity of a purely kinetics-based interpretation of SP/NSP identity, we developed a computational approach that simulated cell staining within a heterogeneous cell population; this exercise allowed for the direct inference of the role of transporter activity and inhibition on cell staining. Our simulated SP assay yielded appropriate SP responses for kinetic scenarios in which high transporter activity existed in a portion of the cells and little differential staining occurred in the majority of the population. With our approach for single-cell analysis, we observed SP and NSP cells at both ends of a transporter activity continuum, demonstrating that features of transporter activity as well as DNA content are determinants of SP/NSP identity.

  1. Dual mTORC1 and mTORC2 inhibitor Palomid 529 penetrates the blood-brain barrier without restriction by ABCB1 and ABCG2.

    PubMed

    Lin, Fan; Buil, Levi; Sherris, David; Beijnen, Jos H; van Tellingen, Olaf

    2013-09-01

    Palomid 529, a novel dual mTORC1/2 inhibitor has displayed interesting activities in experimental models and is a candidate for clinical evaluation. We have assessed the interaction of Palomid 529 with ATP-binding cassette (ABC) drug efflux transporters ABCB1 (P-gp/P-glycoprotein) and ABCG2 (BCRP/Breast Cancer Resistant Protein) by in vitro transwell assays, and their effects on the brain penetration using drug disposition analysis of i.v. and oral Palomid 529 in wild-type (WT) and Abcb1 and/or Abcg2 knockout (KO) mice. Palomid 529 lacked affinity for these transporters in vitro, in contrast to GDC-0941, a small molecule PI3K inhibitor, which we used as control substance for in vitro transport. The plasma AUCi.v. of micronized and DMSO formulated Palomid 529 was similar in WT and KO mice. Importantly, the brain and brain tumor concentration of Palomid 529 at a high dose (54 mg/kg) was also similar in both strains, whereas a less than 1.4-fold difference (p < 0.05) was found at the low (5.4 mg/kg) dose. Because of poor solubility, the oral bioavailability of micronized Palomid 529 was only 5%. Olive oil or spray-dried formulation greatly improved the bioavailability up to 50%. Finally, Palomid 529 effectively inhibits the orthotopic U87 glioblastoma growth. In summary, Palomid 529 is the first mTOR targeting drug lacking affinity for ABCB1/ABCG2 and having good brain penetration. This warrants further evaluation of Palomid 529 for treatment of high-grade gliomas and other intracranial malignancies.

  2. Population Pharmacokinetics of Oral Topotecan in Infants and Very Young Children with Brain Tumors Demonstrates a Role of ABCG2 rs4148157 on the Absorption Rate Constant

    PubMed Central

    Roberts, Jessica K.; Birg, Anna V.; Lin, Tong; Daryani, Vinay M.; Panetta, John C.; Broniscer, Alberto; Robinson, Giles W.; Gajjar, Amar J.

    2016-01-01

    For infants and very young children with brain tumors, chemotherapy after surgical resection is the main treatment due to neurologic and neuroendocrine adverse effects from whole brain irradiation. Topotecan, an anticancer drug with antitumor activity against pediatric brain tumors, can be given intravenous or orally. However, high interpatient variability in oral drug bioavailability is common in children less than 3 years old. Therefore, this study aimed to determine the population pharmacokinetics of oral topotecan in infants and very young children, specifically evaluating the effects of age and ABCG2 and ABCB1 on the absorption rate constant (Ka), as well as other covariate effects on all pharmacokinetic parameters. A nonlinear mixed effects model was implemented in Monolix 4.3.2 (Lixoft, Orsay, France). A one-compartment model with first-order input and first-order elimination was found to adequately characterize topotecan lactone concentrations with population estimates as [mean (S.E.)]; Ka = 0.61 (0.11) h−1, apparent volume of distribution (V/F) = 40.2 (7.0) l, and apparent clearance (CL/F) = 40.0 (2.9) l/h. After including the body surface area in the V/F and CL/F as a power model centered on the population median, the ABCG2 rs4148157 allele was found to play a significant role in the value of Ka. Patients homozygous or heterozygous for G>A demonstrated a Ka value 2-fold higher than their GG counterparts, complemented with a 2-fold higher maximal concentration as well. These results demonstrate a possible role for the ABCG2 rs4148157 allele in the pharmacokinetics of oral topotecan in infants and very young children, and warrants further investigation. PMID:27052877

  3. Acyclovir is a substrate for the human breast cancer resistance protein (BCRP/ABCG2): implications for renal tubular transport and acyclovir-induced nephrotoxicity.

    PubMed

    Gunness, Patrina; Aleksa, Katarina; Koren, Gideon

    2011-09-01

    The human breast cancer resistance protein (BCRP/ABCG2) is widely expressed in human tissues, including the kidney. In mice, Bcrp1 (murine BCRP ortholog) mediates the transport of acyclovir into breast milk. It is plausible that acyclovir is also a substrate for the human BCRP. The objective of the study was to determine whether acyclovir is a substrate for human BCRP. Transfected human embryonic kidney (HEK293) cells (containing the wild-type ABCG2 gene) were exposed to [8-(14)C]acyclovir (1 µmol/L) in the presence or absence of the BCRP inhibitor fumitremorgin C (FTC). Intracellular acyclovir accumulation was assessed using a liquid scintillation counter. Coexposure to FTC resulted in a significant (5-fold) increase in the intracellular accumulation of acyclovir. The results suggest that acyclovir is a substrate for human BCRP. The study is the first to provide direct evidence for the role of human BCRP in acyclovir transport and its potential significance with respect to renal tubular transport of acyclovir and the direct renal tubular insult induced by the drug.

  4. Cobalt Chloride Induces Expression and Function of Breast Cancer Resistance Protein (BCRP/ABCG2) in Human Renal Proximal Tubular Epithelial Cell Line HK-2.

    PubMed

    Nishihashi, Katsuki; Kawashima, Kei; Nomura, Takami; Urakami-Takebayashi, Yumiko; Miyazaki, Makoto; Takano, Mikihisa; Nagai, Junya

    2017-01-01

    The human breast cancer resistance protein (BCRP/ABCG2), a member of the ATP-binding cassette transporter family, is a drug transporter restricting absorption and enhancing excretion of many compounds including anticancer drugs. The cis-regulatory elements in the BCRP promoter include a hypoxia response element, i.e., the DNA binding site for hypoxia-inducible factor-1 (HIF-1). In this study, we investigated the effect of cobalt chloride, a chemical inducer of HIF-1α, on the expression and function of BCRP in human renal proximal tubular cell line HK-2. Cobalt chloride treatment significantly increased the mRNA expression of not only glucose transporter 1 (GLUT1), a typical HIF-1 target gene mRNA, but also ABCG2 mRNA in HK-2 cells. The BCRP inhibitor Ko143-sensitive accumulation of BCRP substrates such as Hoechst33342 and mitoxantrone was significantly enhanced by cobalt chloride treatment. In addition, treatment with cobalt chloride significantly increased the Ko143-sensitive accumulation of fluorescein isothiocyanate-labeled methotrexate in HK-2 cells. Furthermore, cobalt chloride treatment attenuated the cytotoxicity induced by mitoxantrone and methotrexate, which might be, at least in part, due to the increase in BCRP-mediated transport activity via HIF-1 activation. These findings indicate that HIF-1 activation protects renal proximal tubular cells against BCRP substrate-induced cytotoxicity by enhancing the expression and function of BCRP in renal proximal tubular cells.

  5. Polycyclic aromatic hydrocarbons in food--efflux of the conjugated biomarker 1-hydroxypyrene is mediated by Breast Cancer Resistance Protein (ABCG2) in human intestinal Caco-2 cells.

    PubMed

    Hessel, Stefanie; Lampen, Alfonso; Seidel, Albrecht

    2013-12-01

    Polycyclic aromatic hydrocarbons (PAHs) are well-known food contaminants comprising compounds with carcinogenic properties. Pyrene (PYR) is an important non-carcinogenic PAH because its metabolites are frequently used as biomarkers to assess human PAH exposure. This study investigated for the first time the formation and transport of PYR metabolites in the human small intestinal Caco-2 cell model using HPLC technique. The intermediate phase I metabolite 1-hydroxypyrene formed by cytochrome P450 monooxygenases is subsequently conjugated by phase II enzymes to the water-soluble PYR 1-glucuronide as minor and PYR 1-sulfate as major metabolites. The formation of the latter is mediated by human sulfotransferases 1A1*Arg, 1A2*1, 1A3, and 1B1. Caco-2 monolayer experiments revealed a predominantly apical transport of both conjugates mediated by the breast cancer resistance protein (BCRP/ABCG2). Additional treatment with several aryl hydrocarbon receptor (AhR) agonists indicate an AhR-driven induction of PYR-metabolizing enzymes and/or ABCG2. Overall, this study provides advanced mechanistic insights into the bioavailability of PYR and underlines a key role of the human small intestinal epithelium for the first pass metabolism of contaminants in food. Copyright © 2013 Elsevier Ltd. All rights reserved.

  6. The Full-Size ABCG Transporters Nb-ABCG1 and Nb-ABCG2 Function in Pre- and Postinvasion Defense against Phytophthora infestans in Nicotiana benthamiana.

    PubMed

    Shibata, Yusuke; Ojika, Makoto; Sugiyama, Akifumi; Yazaki, Kazufumi; Jones, David A; Kawakita, Kazuhito; Takemoto, Daigo

    2016-05-01

    The sesquiterpenoid capsidiol is the major phytoalexin produced by Nicotiana and Capsicum species. Capsidiol is produced in plant tissues attacked by pathogens and plays a major role in postinvasion defense by inhibiting pathogen growth. Using virus-induced gene silencing-based screening, we identified two Nicotiana benthamiana (wild tobacco) genes encoding functionally redundant full-size ABCG (PDR-type) transporters, Nb-ABCG1/PDR1 and Nb-ABCG2/PDR2, which are essential for resistance to the potato late blight pathogen Phytophthora infestans Silencing of Nb-ABCG1/2 compromised secretion of capsidiol, revealing Nb-ABCG1/2 as probable exporters of capsidiol. Accumulation of plasma membrane-localized Nb-ABCG1 and Nb-ABCG2 was observed at the site of pathogen penetration. Silencing of EAS (encoding 5-epi-aristolochene synthase), a gene for capsidiol biosynthesis, reduced resistance to P. infestans, but penetration by P. infestans was not affected. By contrast, Nb-ABCG1/2-silenced plants showed reduced penetration defense, indicating that Nb-ABCG1/2 are involved in preinvasion defense against P. infestans Plastidic GGPPS1 (geranylgeranyl diphosphate synthase) was also found to be required for preinvasion defense, thereby suggesting that plastid-produced diterpene(s) are the antimicrobial compounds active in preinvasion defense. These findings suggest that N. benthamiana ABCG1/2 are involved in the export of both antimicrobial diterpene(s) for preinvasion defense and capsidiol for postinvasion defense against P. infestans.

  7. Additive composite ABCG2, SLC2A9 and SLC22A12 scores of high-risk alleles with alcohol use modulate gout risk.

    PubMed

    Tu, Hung-Pin; Chung, Chia-Min; Min-Shan Ko, Albert; Lee, Su-Shin; Lai, Han-Ming; Lee, Chien-Hung; Huang, Chung-Ming; Liu, Chiu-Shong; Ko, Ying-Chin

    2016-09-01

    The aim of the present study was to evaluate the contribution of urate transporter genes and alcohol use to the risk of gout/tophi. Eight variants of ABCG2, SLC2A9, SLC22A12, SLC22A11 and SLC17A3 were genotyped in male individuals in a case-control study with 157 gout (33% tophi), 106 asymptomatic hyperuricaemia and 295 control subjects from Taiwan. The multilocus profiles of the genetic risk scores for urate gene variants were used to evaluate the risk of asymptomatic hyperuricaemia, gout and tophi. ABCG2 Q141K (T), SLC2A9 rs1014290 (A) and SLC22A12 rs475688 (C) under an additive model and alcohol use independently predicted the risk of gout (respective odds ratio for each factor=2.48, 2.03, 1.95 and 2.48). The additive composite Q141K, rs1014290 and rs475688 scores of high-risk alleles were associated with gout risk (P<0.0001). We observed the supramultiplicative interaction effect of genetic urate scores and alcohol use on gout and tophi risk (P for interaction=0.0452, 0.0033). The synergistic effect of genetic urate score 5-6 and alcohol use indicates that these combined factors correlate with gout and tophi occurrence.

  8. The Full-Size ABCG Transporters Nb-ABCG1 and Nb-ABCG2 Function in Pre- and Postinvasion Defense against Phytophthora infestans in Nicotiana benthamiana

    PubMed Central

    Shibata, Yusuke; Ojika, Makoto; Sugiyama, Akifumi; Yazaki, Kazufumi; Jones, David A.; Kawakita, Kazuhito

    2016-01-01

    The sesquiterpenoid capsidiol is the major phytoalexin produced by Nicotiana and Capsicum species. Capsidiol is produced in plant tissues attacked by pathogens and plays a major role in postinvasion defense by inhibiting pathogen growth. Using virus-induced gene silencing-based screening, we identified two Nicotiana benthamiana (wild tobacco) genes encoding functionally redundant full-size ABCG (PDR-type) transporters, Nb-ABCG1/PDR1 and Nb-ABCG2/PDR2, which are essential for resistance to the potato late blight pathogen Phytophthora infestans. Silencing of Nb-ABCG1/2 compromised secretion of capsidiol, revealing Nb-ABCG1/2 as probable exporters of capsidiol. Accumulation of plasma membrane-localized Nb-ABCG1 and Nb-ABCG2 was observed at the site of pathogen penetration. Silencing of EAS (encoding 5-epi-aristolochene synthase), a gene for capsidiol biosynthesis, reduced resistance to P. infestans, but penetration by P. infestans was not affected. By contrast, Nb-ABCG1/2-silenced plants showed reduced penetration defense, indicating that Nb-ABCG1/2 are involved in preinvasion defense against P. infestans. Plastidic GGPPS1 (geranylgeranyl diphosphate synthase) was also found to be required for preinvasion defense, thereby suggesting that plastid-produced diterpene(s) are the antimicrobial compounds active in preinvasion defense. These findings suggest that N. benthamiana ABCG1/2 are involved in the export of both antimicrobial diterpene(s) for preinvasion defense and capsidiol for postinvasion defense against P. infestans. PMID:27102667

  9. The bovine ATP-binding cassette transporter ABCG2 Tyr581Ser single-nucleotide polymorphism increases milk secretion of the fluoroquinolone danofloxacin.

    PubMed

    Otero, Jon A; Real, Rebeca; de la Fuente, Álvaro; Prieto, Julio G; Marqués, Margarita; Álvarez, Ana I; Merino, Gracia

    2013-03-01

    The bovine adenosine triphosphate-binding cassette transporter G2 (ABCG2/breast cancer resistance protein) polymorphism Tyr581Ser (Y581S) has recently been shown to increase in vitro transepithelial transport of antibiotics. Since this transporter has been extensively related to the active secretion of drugs into milk, the potential in vivo effect of this polymorphism on secretion of xenobiotics in livestock could have striking consequences for milk production, the dairy industry, and public health. Our purpose was to study the in vivo effect of this polymorphism on the secretion of danofloxacin, a widely used veterinary antibiotic, into milk. Danofloxacin (1.25 mg/kg) was administered to six Y/Y 581 homozygous and six Y/S 581 heterozygous lactating cows, and plasma and milk samples were collected and analyzed by high-performance liquid chromatography. No differences were found in the pharmacokinetic parameters of danofloxacin in plasma between the two groups of animals. In contrast, Y/S heterozygous cows showed a 2-fold increase in danofloxacin levels in milk. In addition, the pharmacokinetic elimination parameters, mean residence time and elimination half-life, were significantly lower in the milk of the animals carrying the Y/S polymorphism. These in vivo results are in agreement with our previously published in vitro data, which showed a greater capacity of the S581 variant in accumulation assays, and demonstrate, for the first time, an important effect of the Y581S single-nucleotide polymorphism on antibiotic secretion into cow milk. These findings could be extended to other ABCG2 substrates, and may be relevant for the treatment of mastitis and for the design of accurate and novel strategies to handle milk residues.

  10. Association of Functional Polymorphism rs2231142 (Q141K) in the ABCG2 Gene With Serum Uric Acid and Gout in 4 US Populations

    PubMed Central

    Zhang, Lili; Spencer, Kylee L.; Voruganti, V. Saroja; Jorgensen, Neal W.; Fornage, Myriam; Best, Lyle G.; Brown-Gentry, Kristin D.; Cole, Shelley A.; Crawford, Dana C.; Deelman, Ewa; Franceschini, Nora; Gaffo, Angelo L.; Glenn, Kimberly R.; Heiss, Gerardo; Jenny, Nancy S.; Kottgen, Anna; Li, Qiong; Liu, Kiang; Matise, Tara C.; North, Kari E.; Umans, Jason G.; Kao, W. H. Linda

    2013-01-01

    A loss-of-function mutation (Q141K, rs2231142) in the ATP-binding cassette, subfamily G, member 2 gene (ABCG2) has been shown to be associated with serum uric acid levels and gout in Asians, Europeans, and European and African Americans; however, less is known about these associations in other populations. Rs2231142 was genotyped in 22,734 European Americans, 9,720 African Americans, 3,849 Mexican Americans, and 3,550 American Indians in the Population Architecture using Genomics and Epidemiology (PAGE) Study (2008–2012). Rs2231142 was significantly associated with serum uric acid levels (P = 2.37 × 10−67, P = 3.98 × 10−5, P = 6.97 × 10−9, and P = 5.33 × 10−4 in European Americans, African Americans, Mexican Americans, and American Indians, respectively) and gout (P = 2.83 × 10−10, P = 0.01, and P = 0.01 in European Americans, African Americans, and Mexican Americans, respectively). Overall, the T allele was associated with a 0.24-mg/dL increase in serum uric acid level (P = 1.37 × 10−80) and a 1.75-fold increase in the odds of gout (P = 1.09 × 10−12). The association between rs2231142 and serum uric acid was significantly stronger in men, postmenopausal women, and hormone therapy users compared with their counterparts. The association with gout was also significantly stronger in men than in women. These results highlight a possible role of sex hormones in the regulation of ABCG2 urate transporter and its potential implications for the prevention, diagnosis, and treatment of hyperuricemia and gout. PMID:23552988

  11. Upregulated miR-132 in Lgr5(+) gastric cancer stem cell-like cells contributes to cisplatin-resistance via SIRT1/CREB/ABCG2 signaling pathway.

    PubMed

    Zhang, Lanfang; Guo, Xiaohe; Zhang, Dezhong; Fan, Yingying; Qin, Lei; Dong, Shuping; Zhang, Lanfang

    2017-09-01

    Cisplatin resistance has long been a major problem that restricts its use. A novel paradigm in tumor biology suggests that gastric tumor chemo-resistance is driven by gastric cancer stem cell-like (GCSCs). Growing evidence has indicated that microRNAs (miRNAs) contributes to chemo-resistance in gastric cancer (GC). Here, Lgr5(+) cells derived from gastric cancer cell lines displayed stem cell-like features. Flow cytometry demonstrated the presence of a variable fraction of Lgr5 in 19 out of 20 GC specimens. By comparing the miRNA expression profiles of Lgr5(+) GCSCs and Lrg5(-) cells, we established the upregulation of miR-132 in Lgr5(+) GCSCs. The enhanced miR-132 expression correlated chemo-resistance in GC patients. Kaplan-Meier survival curve showed that patients with low miR-132 expression survived obviously longer. Functional assays results indicated that miR-132 promoted cisplatin resistance in Lgr5(+) GCSCs in vitro and in vivo. Further dual-luciferase reporter gene assays revealed that SIRT1 was the direct target of miR-132. The expression of miR-132 was inversely correlated with SIRT1 in gastric cancer specimens. Furthermore, through PCR array we discovered ABCG2 was one of the downstream targets of SIRT1. Overexpression of SIRT1 down-regulated ABCG2 expression by promoting the de-acetylation of the transcription factor CREB. CREB was further activated ABCG2 via binding to the promoter of ABCG2 to induce transcription. Thus, we concluded that miR-132 regulated SIRT1/CREB/ABCG2 signaling pathway contributing to the cisplatin resistance and might serve as a novel therapeutic target against gastric cancer. © 2017 Wiley Periodicals, Inc.

  12. Elevated expression of Nrf2 mediates multidrug resistance in CD133+ head and neck squamous cell carcinoma stem cells

    PubMed Central

    Lu, Bao-Cai; Li, Jing; Yu, Wen-Fa; Zhang, Guo-Zheng; Wang, Hui-Min; Ma, Hui-Min

    2016-01-01

    Enhanced expression of the ATP-binding cassette (ABC) transporter protein ABC sub-family G member 2 (ABCG2) in cancer stem cells (CSCs) plays a major role in chemotherapeutic drug efflux, which results in therapy failure and tumor relapse. In addition to downregulating apoptosis in CSCs, it has been reported that the transcriptional upregulation of the redox sensing factor Nrf2 is involved in the upregulation of ABCG2 expression and consequent chemoresistance. The current study investigated the presence of cancer stem-like side population (SP) cells from head and neck squamous cell carcinoma (HNSCC) samples, and evaluated the Nrf2 expression profile and multidrug resistance properties of HNSCC stem cells. Fluorescence-activated cell sorting was used for SP cells detection, while reverse transcription-polymerase chain reaction was used for the analysis of Nrf2 expression. The present study identified ~2.1% SP cells present in HNSCC specimens, which were positive for cluster of differentiation (CD)133 expression and displayed significantly elevated messenger RNA expression of Nrf2, compared with non-SP cells. These data suggest that the ABC transporter ABCG2 is highly upregulated in SP cells, and this results in multidrug resistance. In addition, these CD133+ cells underwent rapid proliferation and exhibited high self-renewal and tumorigenic properties. Taken together, the present findings suggest that elevated expression of Nrf2 mediated drug resistance in HNSCC CSCs, which may be one of the causative factors for cancer treatment failure. Therefore, novel anti-cancer drugs that downregulate the Nrf2 signaling pathway could effectively improve the treatment and survival rate of patients with HNSCC. PMID:28101198

  13. Changing the expression vector of multidrug resistance genes is related to neoadjuvant chemotherapy response.

    PubMed

    Litviakov, Nicolay V; Cherdyntseva, Nadezhda V; Tsyganov, Matvey M; Denisov, Evgeny V; Garbukov, Evgeny Y; Merzliakova, Marina K; Volkomorov, Victor V; Vtorushin, Sergey V; Zavyalova, Marina V; Slonimskaya, Elena M; Perelmuter, Vladimir M

    2013-01-01

    We aimed to examine the association between alterations in multidrug resistance (MDR) gene expression, measured before and after neoadjuvant chemotherapy (NAC), and short-term response in a cohort of stage IIA-IIIC breast cancer patients (n = 84). All patients were treated with two to four preoperative cycles of FAC (5-fluorouracil-adriamycin-cyclophosphamide), CAX (cyclophosphamide-adriamycin-xeloda) or taxane regimes. The expression levels of key MDR genes (ABCB1, ABCC1, ABCC2, ABCC3, ABCC5, ABCG1, ABCG2, GSTP1, and MVP) were evaluated in both tumor tissues obtained pre-therapy and in specimens removed by final surgery, using TaqMan-based quantitative reverse transcriptase PCR. No significant difference in the average level of MDR gene expression in paired breast tumors before and after NAC was found when analyzed in both responsive and non-responsive patients. There was no correlation between the expression levels of MDR genes in pre-NAC tumors and immediate NAC response. In the group with tumor responses, we found a statistically significant downregulation of expression of ABCB1, ABCC1, ABCC2, ABCC5, ABCG1, ABCG2, GSTP1, and MVP genes following NAC in FAC and CAX-treated patients (67-93% of cases). In contrast, we found that expression of these genes was upregulated after NAC, mostly in non-responsive patients (55-96% of cases). Responsiveness to taxotere was related to reduced levels of ABCB1, ABCC2, ABCG1, ABCG2, and MVP mRNA in tumor samples collected after chemotherapy. Our results suggest that reductions in MDR gene expression in post-NAC samples in comparison with pre-NAC are associated with tumor response to FAC and CAX as well as taxotere-based NAC, while patients displaying MDR gene upregulation had resistance to therapy.

  14. Interaction of enrofloxacin with breast cancer resistance protein (BCRP/ABCG2): influence of flavonoids and role in milk secretion in sheep.

    PubMed

    Pulido, Mivis M; Molina, Antonio J; Merino, Gracia; Mendoza, Gracia; Prieto, Julio G; Alvarez, Ana I

    2006-08-01

    The ATP-binding cassette (ABC) transporter breast cancer resistance protein (BCRP)/ABCG2 is a high-capacity efflux transporter with wide substrate specificity located in apical membranes of epithelia, which is involved in drug availability. BCRP is responsible for the active secretion of clinically and toxicologically important substrates to milk. The present study shows BCRP expression in sheep and cow by immunoblotting with MAb (BXP-53). Vanadate-sensitive ATPase activity with specific BCRP substrates and inhibitors was measured in bovine mammary gland homogenates. To assess the role of BCRP in ruminant mammary gland we tested the fluoroquinolone enrofloxacin (ENRO). In polarized cell lines, ENRO was transported by Bcrp1/BCRP with secretory/absorptive ratios of 6.5 and 2 respectively. The efflux was blocked by the BCRP inhibitor Ko143. ENRO pharmacokinetics in plasma and milk was studied in sheep after co-administration of drug (2.5 mg/kg, i.v.) and genistein (0.8 mg/kg, i.m.) or albendazole sulfoxide (2 mg/kg, i.v) as BCRP inhibitors. Concomitant administration of BCRP inhibitors with ENRO had no significant effect on the plasma disposition kinetics of ENRO but decreased ENRO concentrations in milk.

  15. IL-17A exacerbates cisplatin-based resistance of OVCA via upregulating the expression of ABCG2 and MDR1 through Gli1-mediated Hh signaling.

    PubMed

    Niu, Xiulong; Liu, Wenxing; Wang, Yue; Liu, Xiaomei; Zhang, Hongjian; Li, Zhijun; Li, Hongzhao; Iwakura, Yoichiro; Deng, Weimin

    2016-07-18

    The major obstacle of the tumor chemotherapy, including ovarian cancer (OVCA), is drug resistance. However, the relevance of IL-17A with drug-resistance of OVCA has been poorly elaborated. In this study, we used 2 human OVCA cell lines to investigate the effects of IL-17A on cisplatin (CDDP or DDP)-based resistance in OVCA cells and the underlying mechanisms. Meanwhile, IL-17A-deficient mice and ID8 were used to verify the IL-17A's effects on OVCA chemo-resistance in vivo. Moreover, the relationship between IL-17A level and relevant indices were primarily assessed in ovarian specimens from 55 patients with OVCA. We found that rhIL-17A exacerbated DDP-based resistance of OVCA cells via up-regulating the expression of ABCG2 and MDR1 through Gli1-mediated Hh signal pathway. Animal experiment demonstrated that IL-17A significantly recede DDP-based treatment for ID8 tumor. Similar results were observed in preliminary clinical investigation. Our findings suggest that inhibiting IL-17A/IL-17RA-Gli1 signal may improve the resistance of OVCA to DDP.

  16. Phenyltetrazolyl-phenylamides: Substituent impact on modulation capability and selectivity toward the efflux protein ABCG2 and investigation of interaction with the transporter.

    PubMed

    Köhler, Sebastian C; Silbermann, Katja; Wiese, Michael

    2016-11-29

    We recently presented a novel class of ABCG2 modulators based on the third-generation ABCB1 inhibitor tariquidar bearing a 2,5-linked tetrazole instead of an amid linker. We investigated the modulating potential of the compound class by enlarging the substitution pattern on the outer phenyl rings of the scaffold. To identify the structural conditions for achieving a high response, we decided to determine the individual influence of substituents on the scaffold using monosubstituted derivatives. While electron withdrawing groups (with a few exceptions) and bulky moieties decreased the modulating potency, small electron donating groups ensured a high activity level. Interestingly, the unsubstituted derivative 32 reached a similar inhibitory potential as the best derivatives in the previous study. Enzyme kinetic assays indicated that our derivatives have the same binding site as reference inhibitor Ko143. They were found to interact competitively and non-competitively with the substrates Hoechst 33342 and pheophorbide A, respectively. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  17. Mutations in the bovine ABCG2 and the ovine MSTN gene added to the few quantitative trait nucleotides identified in farm animals: a mini-review.

    PubMed

    Braunschweig, M H

    2010-01-01

    The progress in molecular genetics in animal breeding is moderately effective as compared to traditional animal breeding using quantitative genetic approaches. There is an extensive disparity between the number of reported quantitative trait loci (QTLs) and their linked genetic variations in cattle, pig, and chicken. The identification of causative mutations affecting quantitative traits is still very challenging and hampered by the cloudy relationship between genotype and phenotype. There are relatively few reports in which a successful identification of a causative mutation for an animal production trait was demonstrated. The examples that have attracted considerable attention from the animal breeding community are briefly summarized and presented in a table. In this mini-review, the recent progress in mapping quantitative trait nucleotides (QTNs) are reviewed, including the ABCG2 gene mutation that underlies a QTL for fat and protein content and the ovine MSTN gene mutation that causes muscular hypertrophy in Texel sheep. It is concluded that the progress in molecular genetics might facilitate the elucidation of the genetic architecture of QTLs, so that also the high-hanging fruits can be harvested in order to contribute to efficient and sustainable animal production.

  18. Enhancement of the Effect of Methyl Pyropheophorbide-a-Mediated Photodynamic Therapy was Achieved by Increasing ROS via Inhibition of Nrf2-HO-1 or Nrf2-ABCG2 Signaling.

    PubMed

    Tian, Si; Yong, Min; Zhu, Jiang; Zhang, Li; Pan, Li; Chen, Qing; Li, Kai-Ting; Kong, Yu-Han; Jiang, Yuan; Yu, Ting-He; Yu, Le-Hua; Bai, Ding-Qun

    2017-03-27

    Emerging evidence indicates that the transcription factor nuclear factor-E2-related factor 2 (-NRF2) plays an essential role in cellular defense against oxidative stress; its activation has been related to cytoprotection. Here, we investigated the role of Nrf2 in improving the efficacy of methyl pyropheophorbide-a-mediated photodynamic therapy (Mppa-PDT) via the downregulation of Nrf2 in human ovarian cancer A2780 cells and SKOV3 cells. We found that Nrf2 translocated from the cytoplasm to the nucleus in vitro and in vivo, and the expression of Nrf2 and P-Nrf2 increased through a possible mechanism regulated by mitogen-activated protein kinase (MAPK) after Mppa-PDT treatment. Furthermore, cytotoxicity and apoptosis induced by Mppa-PDT increased after Nrf2down-regulation. Nrf2 down -regulation increased reactive oxygen species (ROS) levels by attenuating antioxidants or pumping Mppa out of cells, which resulted from the inhibition of Nrf2-HO-1 or Nrf2-ABCG2 signaling. In addition, SKOV3 cells exhibited increased resistance to Mppa-PDT, and the expression levels of P-Nrf2 and ABCG2 were higher in SKOV3 cells than in A2780 cells, suggesting that Nrf2-ABCG2 signaling might be involved in the intrinsic resistanceto Mppa-PDT. Taken together, these results provided evidence that Nrf2 down-regulation can enhance the effect of Mppa-PDT.

  19. Up-regulation of hepatic ABCC2, ABCG2, CYP1A1 and GST in multixenobiotic-resistant killifish (Fundulus heteroclitus) from the Sydney Tar Ponds, Nova Scotia, Canada.

    PubMed

    Paetzold, S Christine; Ross, Neil W; Richards, Robert C; Jones, Martha; Hellou, Jocelyne; Bard, Shannon M

    2009-07-01

    Cellular defence against accumulation of toxic xenobiotics includes metabolism by phase I and II enzymes and export of toxicants and their metabolites via ATP-binding cassette (ABC) transporters. Liver gene expression of representatives of these three protein groups was examined in a population of multixenobiotic-resistant killifish (Fundulus heteroclitus) from the Sydney Tar Ponds, Nova Scotia, Canada. The Tar Ponds are heavily polluted with polycyclic aromatic hydrocarbons, polychlorinated biphenyls and heavy metals. The relationship among ABC transporters ABCB1, ABCB11, ABCC2, ABCG2, phase I enzyme cytochrome P4501A1 (CYP1A1) and phase II enzyme glutathione-S-transferase (GST-mu) was investigated by quantifying hepatic transcript abundance. In Tar Pond killifish, hepatic mRNA expression levels of ABCC2, ABCG2, CYP1A1 and GST-mu were elevated compared to reference sites, suggesting that hydrophobic contaminants undergo phase I and II metabolism and are then excreted into the bile of these fish. Hepatic ABCB1 and ABCB11 mRNA were not up-regulated in Tar Pond fish compared to two reference sites, indicating that these two proteins are not involved in conferring multixenobiotic resistance to Tar Pond killifish. The results suggest instead that liver up-regulation of phase I and II enzymes and complementary ABC transporters ABCC2 and ABCG2 may confer contaminant resistance to Tar Pond fish.

  20. Breast cancer resistance protein (ABCG2) in clinical pharmacokinetics and drug interactions: practical recommendations for clinical victim and perpetrator drug-drug interaction study design.

    PubMed

    Lee, Caroline A; O'Connor, Meeghan A; Ritchie, Tasha K; Galetin, Aleksandra; Cook, Jack A; Ragueneau-Majlessi, Isabelle; Ellens, Harma; Feng, Bo; Taub, Mitchell E; Paine, Mary F; Polli, Joseph W; Ware, Joseph A; Zamek-Gliszczynski, Maciej J

    2015-04-01

    Breast cancer resistance protein (BCRP; ABCG2) limits intestinal absorption of low-permeability substrate drugs and mediates biliary excretion of drugs and metabolites. Based on clinical evidence of BCRP-mediated drug-drug interactions (DDIs) and the c.421C>A functional polymorphism affecting drug efficacy and safety, both the US Food and Drug Administration and European Medicines Agency recommend preclinical evaluation and, when appropriate, clinical assessment of BCRP-mediated DDIs. Although many BCRP substrates and inhibitors have been identified in vitro, clinical translation has been confounded by overlap with other transporters and metabolic enzymes. Regulatory recommendations for BCRP-mediated clinical DDI studies are challenging, as consensus is lacking on the choice of the most robust and specific human BCRP substrates and inhibitors and optimal study design. This review proposes a path forward based on a comprehensive analysis of available data. Oral sulfasalazine (1000 mg, immediate-release tablet) is the best available clinical substrate for intestinal BCRP, oral rosuvastatin (20 mg) for both intestinal and hepatic BCRP, and intravenous rosuvastatin (4 mg) for hepatic BCRP. Oral curcumin (2000 mg) and lapatinib (250 mg) are the best available clinical BCRP inhibitors. To interrogate the worst-case clinical BCRP DDI scenario, study subjects harboring the BCRP c.421C/C reference genotype are recommended. In addition, if sulfasalazine is selected as the substrate, subjects having the rapid acetylator phenotype are recommended. In the case of rosuvastatin, subjects with the organic anion-transporting polypeptide 1B1 c.521T/T genotype are recommended, together with monitoring of rosuvastatin's cholesterol-lowering effect at baseline and DDI phase. A proof-of-concept clinical study is being planned by a collaborative consortium to evaluate the proposed BCRP DDI study design.

  1. Inhibitory effect of epirubicin-loaded lipid microbubbles with conjugated anti-ABCG2 antibody combined with therapeutic ultrasound on multiple myeloma cancer stem cells.

    PubMed

    Shi, Fangfang; Yang, Fang; He, Xiangfeng; Zhang, Ying; Wu, Songyan; Li, Miao; Zhang, Yunxia; Di, Wu; Dou, Jun; Gu, Ning

    2016-01-01

    Ultrasound-targeted microbubble destruction (UTMD) technique is thought to improve the chemotherapeutic agent delivery from microbubbles (MBs) in tumor tissues and reduce the side effects in non-tumor tissues. Multiple myeloma (MM) is a bone marrow cancer and remains to be an incurable disease. In this study, we used the UTMD technique to investigate the inhibitory effect of our developed novel reagent on MM cancer stem cells (CD138(-)CD34(-)MM CSCs) that are MM cells with CD138(-)CD34(-) phenotypes, responsible for MM-initiating potential, drug resistance and eventual relapse. The preparatory steps of novel reagent was first epirubicin (EPI)-loaded in the lipid MBs that was consisted of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]-biotin, dipalmitoyl-phosphatidylglycerol and 25-NBD-cholesterol, then anti-ABCG2 monoclonal antibody (mAb) was conjugated onto the MB surface to form EPI-MBs+mAb. CD138(-)CD34(-)MM CSCs were isolated from human MM RPMI 8226 cell line by the magnetic associated cell sorting method. The results showed that the attenuated proliferation, migration and invasion ability, and increased apoptosis were observed when MM CSCs were incubated with a various agents. EPI-MBs+mAb combined with therapeutic ultrasound significantly promoted the MM CSC apoptosis compared with EPI, EPI-MBs alone or EPI-MBs+mAb without ultrasound exposure. These results suggest that the developed EPI-MBs+mAb combined with therapeutic ultrasound remarkably induced MM CSC apoptosis in vitro.

  2. Mouse breast cancer resistance protein (Bcrp1/Abcg2) mediates etoposide resistance and transport, but etoposide oral availability is limited primarily by P-glycoprotein.

    PubMed

    Allen, John D; Van Dort, Sonja C; Buitelaar, Marije; van Tellingen, Olaf; Schinkel, Alfred H

    2003-03-15

    The breast cancer resistance protein [BCRP (BCRP/ABCG2)] has not previously been directly identified as a source of resistance to epipodophyllotoxins.However, when P-glycoprotein (P-gp)- and Mrp1-deficient mouse fibroblast and kidney cell lines were selected for resistance to etoposide, amplification and overexpression of Bcrp1 emerged as the dominant resistance mechanism in five of five cases. Resistance was accompanied by reduced intracellular etoposide accumulation. Bcrp1 sequence in all of the resistant lines was wild-type in the region spanning the R482 mutation hot spot known to alter the substrate specificity of mouse Bcrp1 (mouse cognate of BCRP) and human BCRP. Transduced wild-type Bcrp1 cDNA mediated resistance to etoposide and teniposide in fibroblast lines and trans-epithelial etoposide transport in polarized Madin-Darby canine kidney II cells. Bcrp1-mediated etoposide resistance was reversed by two structurally different BCRP/Bcrp1 inhibitors, GF120918 and Ko143. BCRP/Bcrp1 (inhibition) might thus impact on the antitumor activity and pharmacokinetics of epipodophyllotoxins. However, treatment of P-gp-deficient mice with GF120918 did not improve etoposide oral uptake, suggesting that Bcrp1 activity is not a major limiting factor in this process. In contrast, use of GF120918 to inhibit P-gp in wild-type mice increased the plasma levels of etoposide after oral administration 4-5-fold. It may thus be worthwhile to test inhibition of P-gp in humans to improve the oral availability of etoposide.

  3. Mouse ATP-Binding Cassette (ABC) Transporters Conferring Multi-Drug Resistance.

    PubMed

    Li, Shuaizhang; Zhang, Wen; Yin, Xuejiao; Xing, Shilai; Xie, Heidi Qunhui; Cao, Zhengyu; Zhao, Bin

    2015-01-01

    The ABC (ATP-binding cassette) transporter is one of the largest and most ancient protein families with members functioning from protozoa to human. The resistance of cancer and tumor cells to anticancer drugs is due to the over-expression of some ABC transporters, which may finally lead to chemotherapy failure. The mouse ABC transporters are classified into seven subfamilies by phylogenetic analysis. The mouse ABC transporter gene, alias, chromosomal location and function have been determined. Within the ABC super-family, the MDR transporters (Abcb1, Abcc1, Abcg2) in mouse models have been proved to be valuable to investigate the biochemistry and physiological functions. This review concentrates on the multidrug resistance of mouse ABC transporters in cancer and tumor cells.

  4. Mouse ATP-Binding Cassette (ABC) Transporters Conferring Multi-Drug Resistance

    PubMed

    Shuaizhang, L I; Zhang, Wen; Yin, Xuejiao; Xing, Shilai; Xie, Qunhui; Cao, Zhengyu; Zhao, Bin

    2015-04-28

    The ABC (ATP-binding cassette) transporter is one of the largest and most ancient protein families with members functioning from protozoa to human. The resistance of cancer and tumor cells to anticancer drugs is due to the over-expression of some ABC transporters, which may finally lead to chemotherapy failure. The mouse ABC transporters are classified into seven subfamilies by phylogenetic analysis. The mouse ABC transporter gene, alias, chromosomal location and function have been determined. Within the ABC super-family, the MDR transporters (Abcb1, Abcc1, Abcg2) in mouse models have been proved to be valuable to investigate the biochemistry and physiological functions. This review concentrates on the multidrug resistance of mouse ABC transporters in cancer and tumor cells.

  5. N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) as a chemical ATP-binding cassette transporter family G member 2 (Abcg2) knockout model to study nitrofurantoin transfer into milk.

    PubMed

    Wang, Lipeng; Leggas, Markos; Goswami, Mamta; Empey, Philip E; McNamara, Patrick J

    2008-12-01

    Genetic knockout mice studies suggested ATP-binding cassette transporter family G member 2 (ABCG2)/Abcg2 translocates nitrofurantoin at the mammary-blood barrier, resulting in drug accumulation in milk. The purpose of this study was to establish the role of Abcg2 in nitrofurantoin accumulation in rat milk using N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918) as a "chemical knockout" equivalent. The inhibitory effect of GF120918 was verified in Madin-Darby canine kidney II cells stably expressing rat Abcg2 with Hoechst 33342 and nitrofurantoin flux in Transwells. Nitrofurantoin was infused (0.5 mg/h) in the absence and presence of GF120918 (10 mg/kg in dimethyl sulfoxide) to Sprague-Dawley lactating female rats using a balanced crossover design. Administration of GF120918 increased nitrofurantoin concentration in serum (from 443 +/- 51 to 650 +/- 120 ng/ml) and decreased concentration in milk (from 18.1 +/- 0.9 to 1.9 +/- 1.2 microg/ml), resulting in corresponding mean values for milk to serum concentration ratio (M/S) of 41.4 +/- 19.1 versus 3.04 +/- 2.27 in the absence and presence of GF120918 (p < 0.05), respectively. There was a decrease in systemic clearance with GF120918 (2.8 +/- 0.5 l/h/kg) compared with vehicle controls (4.1 +/- 0.5 l/h/kg; p < 0.05). Western blot analysis revealed good expression of Abcg2 and no P-glycoprotein (P-gp) expression in mammary gland, whereas immunohistochemistry confirmed the apical expression of Abcg2 in lactating mammary gland epithelia. Nitrofurantoin active transport into rat milk can be inhibited by GF120918 resulting in a 10-fold lower M/S. Although GF120918 inhibits both Abcg2 and P-gp, the high expression of Abcg2 and the absence of detectable P-gp expression in lactating mammary gland validate an important role for Abcg2 in nitrofurantoin accumulation in rat milk. GF120918 is particularly useful as a rat chemical knockout model to

  6. Factors predisposing to coma and delirium: fentanyl and midazolam exposure; CYP3A5, ABCB1, and ABCG2 genetic polymorphisms; and inflammatory factors.

    PubMed

    Skrobik, Yoanna; Leger, Caroline; Cossette, Mariève; Michaud, Veronique; Turgeon, Jacques

    2013-04-01

    Delirium and sedative-induced coma are described as incremental manifestations of cerebral dysfunction. Both may be associated with sedative or opiate doses and pharmacokinetic or pharmacogenetic variables, such as drug plasma levels (exposure), drug metabolism, and/or their transport across the blood-brain barrier. To compare biological and drug treatment characteristics in patients with coma and/or delirium while in the ICU. In 99 patients receiving IV fentanyl, midazolam, or both, we evaluated drug doses, covariates likely to influence drug effects (age, body mass index, and renal and hepatic dysfunction); delirium risk factors; concomitant administration of CYP3A and P-glycoprotein substrates/inhibitors; ABCB1, ABCG2, and CYP3A5 genetic polymorphisms; and fentanyl and midazolam plasma levels. Delirium and coma were evaluated daily. In patients with only coma (n=15), only delirium (n=7), and neither ever (n=14), we measured plasma levels of tumor necrosis factor-α, interleukin (IL)-1β, IL-1RA, IL-6, IL-8, IL-10, IL-17,macrophage inflammatory protein-1β, and monocyte chemotactic protein-1. Time to first coma was associated with fentanyl and midazolam doses (p=0.03 and p=0.01, respectively). The number of days in coma was associated with the number of days of coadministration of CYP3A inhibitors (r=0.30; p=0.006). Plasma levels of fentanyl were higher in patients with clinical coma (3.7±4.7 vs. 2.0±1.8 ng/mL, p=0.0001) as were midazolam plasma levels (1050±2232 vs. 168±249 ng/mL, p=0.0001). Delirium occurrence was unrelated to midazolam administration, cumulative doses, or serum levels. Days with delirium were associated with days of coadministration of P-glycoprotein inhibitor (r=0.35; p=0.0004). Delirious patients had higher levels of the inflammatory mediator IL-6 than comatose patients (129.3 vs. 35.0 pg/mL, p=0.05). Coma is associated with fentanyl and midazolam exposure; delirium is unrelated to midazolam and may be linked to inflammatory status

  7. ABCG2/BCRP decreases the transfer of a food-born chemical carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in perfused term human placenta

    SciTech Connect

    Myllynen, Paeivi Kummu, Maria; Kangas, Tiina; Ilves, Mika; Immonen, Elina; Rysae, Jaana; Pirilae, Rauna; Lastumaeki, Anni; Vaehaekangas, Kirsi H.

    2008-10-15

    We have studied the role of ATP binding cassette (ABC) transporters in fetal exposure to carcinogens using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) a known substrate for ABC transporters as a model compound. In perfusion of human term placenta, transfer of {sup 14}C-PhIP (2 {mu}M) through the placenta resulted in fetal-to-maternal concentration ratio (FM ratio) of 0.72 {+-} 0.09 at 6 h. The specific ABCG2 inhibitor KO143 increased the transfer of {sup 14}C-PhIP from maternal to fetal circulation (FM ratio 0.90 {+-} 0.08 at 6 h, p < 0.05) while the ABCC1/ABCC2 inhibitor probenecid had no effect (FM ratio at 6 h 0.75 {+-} 0.10, p = 0.84). There was a negative correlation between the expression of ABCG2 protein in perfused tissue and the FM ratio of {sup 14}C-PhIP (R = - 0.81, p < 0.01) at the end of the perfusion. The expression of ABCC2 protein did not correlate with FM ratio of PhIP (R: - 0.11, p = 0.76). In addition, PhIP induced the expression of ABC transporters in BeWo cells at mRNA level. In conclusion, our data indicates that ABCG2 decreases placental transfer of {sup 14}C-PhIP in perfused human placenta. Also, PhIP may modify ABC transporter expression in choriocarinoma cells.

  8. Upregulation of miR-199a/b contributes to cisplatin resistance via Wnt/β-catenin-ABCG2 signaling pathway in ALDHA1(+) colorectal cancer stem cells.

    PubMed

    Chen, Binghe; Zhang, Dezhong; Kuai, Jun; Cheng, Mingkun; Fang, Xiangjie; Li, Guangyan

    2017-06-01

    Cisplatin resistance in colorectal cancer largely results from the colorectal cancer stem cells which could be targeted to improve the efficacy of chemotherapy. MicroRNAs are possible modulators of cancer stem cell characteristics and maybe involved in the retention of cancer stem cell chemoresistance. The aim of this study was to investigate the biological function of miR-199a/b on cisplatin resistance in colorectal cancer stem cells and its related mechanisms. Here, ALDHA1(+) cells from primary colorectal cancer tissues behaved similar to cancer stem cells and were chemoresistant to cisplatin. The presence of a variable fraction of ALDHA1 was detected in 9 out of 10 colorectal cancer specimens. Significantly, increased miR-199a/b expression was detected in ALDHA1(+) colorectal cancer stem cells, accompanied by a downregulation of Gsk3β and an overexpression of β-catenin and ABCG2. In patient cohort, enhanced miR-199a/b expression in colorectal cancer tissues was associated with cisplatin response and poor patient survival. In addition, 80% of colorectal cancer samples showed lower level of Gsk3β than their adjacent normal counterparts. Furthermore, Gsk3β was the direct target of miR-199a/b. MiR-199a/b regulated Wnt/β-catenin pathway by targeting Gsk3β in ALDHA1(+) colorectal cancer stem cells. By blocking Wnt/β-catenin pathway, we implied that ABCG2 lies downstream of Wnt/β-catenin pathway. ABCG2 was further demonstrated to contribute cisplatin resistance in ALDHA1(+) colorectal cancer stem cells and can be regulated by miR-199a/b. Thus, our data suggested that upregulation of miR-199a/b in ALDHA1(+) colorectal cancer stem cells contributed to cisplatin resistance via Wnt/β-catenin-ABCG2 signaling, which sheds new light on understanding the mechanism of cisplatin resistance in colorectal cancer stem cells and facilitates the development of potential therapeutics against colorectal cancer.

  9. Development and Validation of a Computational Model Ensemble for the Early Detection of BCRP/ABCG2 Substrates during the Drug Design Stage.

    PubMed

    Gantner, Melisa E; Peroni, Roxana N; Morales, Juan F; Villalba, María L; Ruiz, María E; Talevi, Alan

    2017-08-28

    Breast Cancer Resistance Protein (BCRP) is an ATP-dependent efflux transporter linked to the multidrug resistance phenomenon in many diseases such as epilepsy and cancer and a potential source of drug interactions. For these reasons, the early identification of substrates and nonsubstrates of this transporter during the drug discovery stage is of great interest. We have developed a computational nonlinear model ensemble based on conformational independent molecular descriptors using a combined strategy of genetic algorithms, J48 decision tree classifiers, and data fusion. The best model ensemble consists in averaging the ranking of the 12 decision trees that showed the best performance on the training set, which also demonstrated a good performance for the test set. It was experimentally validated using the ex vivo everted rat intestinal sac model. Five anticonvulsant drugs classified as nonsubstrates for BRCP by the model ensemble were experimentally evaluated, and none of them proved to be a BCRP substrate under the experimental conditions used, thus confirming the predictive ability of the model ensemble. The model ensemble reported here is a potentially valuable tool to be used as an in silico ADME filter in computer-aided drug discovery campaigns intended to overcome BCRP-mediated multidrug resistance issues and to prevent drug-drug interactions.

  10. Sulfonation of raloxifene in HEK293 cells overexpressing SULT1A3: Involvement of breast cancer resistance protein (BCRP/ABCG2) and multidrug resistance-associated protein 4 (MRP4/ABCC4) in excretion of sulfate metabolites.

    PubMed

    Zhou, Xiaotong; Wang, Shaoxiang; Sun, Hua; Wu, Baojian

    2015-12-01

    Excretion of sulfate metabolites is an essential process in disposition of raloxifene via the sulfonation pathway. However, the transporters responsible for excretion of raloxifene sulfates remain undefined. Here, sulfonation of raloxifene and excretion of its sulfate metabolites were investigated using SULT1A3-overexpressing HEK293 cells (or SULT293 cells) with significant expression of BCRP and MRP4. SULT293 cell lysate catalyzed the sulfonation of raloxifene at both 6-OH and 4'-OH groups, generating raloxifene-6-sulfate (R-6-S) and raloxifene-4'-sulfate (R-4'-S), respectively. Sulfate formation followed the Michaelis-Menten kinetics (Km = 0.49 μM and Vmax = 5.79 pmol/min/mg for R-6-S; Km = 0.33 μM and Vmax = 1.25 pmol/min/mg for R-4'-S). As expected, the recombinant SULT1A3 enzyme showed a high similarity in raloxifene sulfonation profiles with the lysate preparation. Ko143, a selective inhibitor of BCRP, significantly decreased the excretion rates of raloxifene sulfates (maximal 66.1%) while increasing the intracellular sulfates (maximal 282%). As a result, the apparent efflux clearance (CLef,app, representing the efflux efficiency of raloxifene sulfates) was substantially reduced (maximal 85.6%). Likewise, the pan-MRP inhibitor MK-571 significantly deceased the excretion rates (maximal 69.6%) and CLef,app values (maximal 96.0%) of raloxifene sulfates while increasing the intracellular sulfates (maximal 667%). Further, the short-hairpin RNA (shRNA) targeting BCRP significantly reduced (maximal 35.0%) sulfate excretion. Use of BCRP shRNA also caused significant decreases (maximal 52.5%) in the CLef,app values. Silencing of MRP4 by shRNA led to a substantial alteration in sulfate disposition (i.e., 28.6-37.8% reductions in sulfate excretion, 30.5-59.3% elevations in intracellular sulfates, and 44.8-47.7% deceases in CLef,app values). In conclusion, two sulfate metabolites R-6-S and R-4'-S were generated from raloxifene in SULT293 cells. Cellular excretion of the raloxifene sulfates was mainly mediated by BCRP and MRP4.

  11. Neurogenic and neuro-protective potential of a novel subpopulation of peripheral blood-derived CD133+ ABCG2+CXCR4+ mesenchymal stem cells: development of autologous cell-based therapeutics for traumatic brain injury.

    PubMed

    Nichols, Joan E; Niles, Jean A; DeWitt, Douglas; Prough, Donald; Parsley, Margaret; Vega, Stephanie; Cantu, Andrea; Lee, Eric; Cortiella, Joaquin

    2013-01-06

    Nervous system injuries comprise a diverse group of disorders that include traumatic brain injury (TBI). The potential of mesenchymal stem cells (MSCs) to differentiate into neural cell types has aroused hope for the possible development of autologous therapies for central nervous system injury. In this study we isolated and characterized a human peripheral blood derived (HPBD) MSC population which we examined for neural lineage potential and ability to migrate in vitro and in vivo. HPBD CD133+, ATP-binding cassette sub-family G member 2 (ABCG2)+, C-X-C chemokine receptor type 4 (CXCR4)+ MSCs were differentiated after priming with β-mercaptoethanol (β-ME) combined with trans-retinoic acid (RA) and culture in neural basal media containing basic fibroblast growth factor (FGF2) and epidermal growth factor (EGF) or co-culture with neuronal cell lines. Differentiation efficiencies in vitro were determined using flow cytometry or fluorescent microscopy of cytospins made of FACS sorted positive cells after staining for markers of immature or mature neuronal lineages. RA-primed CD133+ABCG2+CXCR4+ human MSCs were transplanted into the lateral ventricle of male Sprague-Dawley rats, 24 hours after sham or traumatic brain injury (TBI). All animals were evaluated for spatial memory performance using the Morris Water Maze (MWM) Test. Histological examination of sham or TBI brains was done to evaluate MSC survival, migration and differentiation into neural lineages. We also examined induction of apoptosis at the injury site and production of MSC neuroprotective factors. CD133+ABCG2+CXCR4+ MSCs consistently expressed markers of neural lineage induction and were positive for nestin, microtubule associated protein-1β (MAP-1β), tyrosine hydroxylase (TH), neuron specific nuclear protein (NEUN) or type III beta-tubulin (Tuj1). Animals in the primed MSC treatment group exhibited MWM latency results similar to the uninjured (sham) group with both groups showing improvements in

  12. Effect of polymorphisms in CYP3A4, PPARA, NR1I2, NFKB1, ABCG2 and SLCO1B1 on the pharmacokinetics of lovastatin in healthy Chinese volunteers.

    PubMed

    Zhao, Guilian; Liu, Mei; Wu, Xiujun; Li, Guofei; Qiu, Feng; Gu, Jingkai; Zhao, Limei

    2017-01-01

    This study examined whether gene polymorphisms (CYP3A4, ABCG2, SLCO1B1, NR1I2, PPARA and NFKB1) influenced the pharmacokinetics of lovastatin in Chinese healthy subjects. Plasma concentrations of lovastatin and lovastatin acid were quantified using LC/MS/MS. PPARA c.208+3819 G allele carriers had approximately twofold higher AUC0-∞ and Cmax of lovastatin than wild-type (PPARA c.208+3819 AA) subjects. After adjustment for the PPARA variants, subjects with the SLCO1B1 521TT genotype had approximately 50% lower AUC0-∞ of lovastatin acid than those with 521TC/CC genotypes, while the AUC0-∞ of lovastatin lactone in NFKB1-94 DD wild-type carriers was twofold higher than in mutant homozygotes carriers. Gene polymorphisms of PPARA, SLCO1B1 and NFKB1 affected the pharmacokinetics of lovastatin.

  13. Human multidrug resistance ABCB and ABCG transporters: participation in a chemoimmunity defense system.

    PubMed

    Sarkadi, Balázs; Homolya, László; Szakács, Gergely; Váradi, András

    2006-10-01

    In this review we give an overview of the physiological functions of a group of ATP binding cassette (ABC) transporter proteins, which were discovered, and still referred to, as multidrug resistance (MDR) transporters. Although they indeed play an important role in cancer drug resistance, their major physiological function is to provide general protection against hydrophobic xenobiotics. With a highly conserved structure, membrane topology, and mechanism of action, these essential transporters are preserved throughout all living systems, from bacteria to human. We describe the general structural and mechanistic features of the human MDR-ABC transporters and introduce some of the basic methods that can be applied for the analysis of their expression, function, regulation, and modulation. We treat in detail the biochemistry, cell biology, and physiology of the ABCB1 (MDR1/P-glycoprotein) and the ABCG2 (MXR/BCRP) proteins and describe emerging information related to additional ABCB- and ABCG-type transporters with a potential role in drug and xenobiotic resistance. Throughout this review we demonstrate and emphasize the general network characteristics of the MDR-ABC transporters, functioning at the cellular and physiological tissue barriers. In addition, we suggest that multidrug transporters are essential parts of an innate defense system, the "chemoimmunity" network, which has a number of features reminiscent of classical immunology.

  14. Pyrrolopyrimidine Derivatives as Novel Inhibitors of Multidrug Resistance-Associated Protein 1 (MRP1, ABCC1).

    PubMed

    Schmitt, Sven Marcel; Stefan, Katja; Wiese, Michael

    2016-04-14

    Five series of pyrrolo[3,2-d]pyrimidines were synthesized and evaluated with respect to potency and selectivity toward multidrug resistance-associated protein 1 (MRP1, ABCC1). This transport protein is a major target to overcome multidrug resistance in cancer patients. We investigated differently substituted pyrrolopyrimidines using the doxorubicin selected and MRP1 overexpressing small cell lung cancer cell line H69 AR in a calcein AM and daunorubicin cell accumulation assay. New compounds with high potency and selectivity were identified. Piperazine residues at position 4 bearing large phenylalkyl side chains proved to be beneficial for MRP1 inhibition. Its replacement by an amino group led to decreased activity. Aliphatic and aliphatic-aromatic variations at position 5 and 6 revealed compounds with IC50 values in high nanomolar range. All investigated compounds had low affinity toward P-glycoprotein (P-gp, ABCB1). Pyrrolopyrimidines with small substituents showed moderate inhibition against breast cancer resistance protein (BCRP, ABCG2).

  15. Role of UGT1A1*6, UGT1A1*28 and ABCG2 c.421C>A polymorphisms in irinotecan-induced neutropenia in Asian cancer patients.

    PubMed

    Jada, Srinivasa Rao; Lim, Robert; Wong, Chiung Ing; Shu, Xiaochen; Lee, Soo Chin; Zhou, Qingyu; Goh, Boon Cher; Chowbay, Balram

    2007-09-01

    The objectives of the present study were (i) to study the pharmacogenetics of UGT1A1*6, UGT1A1*28 and ABCG2 c.421C>A in three distinct healthy Asian populations (Chinese, Malays and Indians), and (ii) to investigate the polygenic influence of these polymorphic variants in irinotecan-induced neutropenia in Asian cancer patients. Pharmacokinetic and pharmacogenetic analyses were done after administration of irinotecan as a 90-min intravenous infusion of 375 mg/m(2) once every 3 weeks (n = 45). Genotypic-phenotypic correlates showed a non-significant influence of UGT1A1*28 and ABCG2 c.421C>A polymorphisms on the pharmacokinetics of SN-38 (P > 0.05), as well as severity of neutropenia (P > 0.05). Significantly higher exposure levels to SN-38 (P = 0.018), lower relative extent of glucuronidation (REG; P = 0.006) and higher biliary index (BI; P = 0.003) were found in cancer patients homozygous for the UGT1A1*6 allele compared with patients harboring the reference genotype. The mean absolute neutrophil count (ANC) was 85% lower and the prevalence of grade 4 neutropenia (ANC < or = 500/microL) was 27% in patients homozygous for UGT1A1*6 compared with the reference group. Furthermore, the presence of the UGT1A1*6 allele was associated with an approximately 3-fold increased risk of developing severe grade 4 neutropenia compared with patients harboring the reference genotype. These exploratory findings suggest that homozygosity for UGT1A1*6 allele may be associated with altered SN-38 disposition and may increase the risk of severe neutropenia in Asian cancer patients, particularly in the Chinese cancer patients who comprised 80% (n = 36) of the patient population in the present study.

  16. Activity of the dietary flavonoid, apigenin, against multidrug-resistant tumor cells as determined by pharmacogenomics and molecular docking.

    PubMed

    Saeed, Mohamed; Kadioglu, Onat; Khalid, Hassan; Sugimoto, Yoshikazu; Efferth, Thomas

    2015-01-01

    Apigenin is a common dietary flavonoid with considerable cytotoxic activity in vitro and in vivo. Despite many mechanistic studies, less is known about resistance factors hampering apigenin's activity. We investigated the ATP-binding cassette (ABC) transporters BCRP/ABCG2, P-glycoprotein/ABCB1 and its close relative ABCB5. Multidrug-resistant cells overexpressing these ABC transporters were not cross-resistant toward apigenin. Moreover, apigenin inhibited not only P-glycoprotein but also BCRP by increasing cellular uptake of doxorubicin and synergistic inhibition of cell viability in combination with doxorubicin or docetaxel in multidrug-resistant cells. To perform in silico molecular docking studies, we first generated homology models for human P-glycoprotein and ABCB5 based on the crystal structure of murine P-glycoprotein. Their nucleotide binding domains (NDBs) revealed the highest degrees of sequence homologies (89%-100%), indicating that ATP binding and cleavage is of crucial importance for ABC transporters. Molecular docking of apigenin bound to the NDBs of P-glycoprotein and ABCB5 in molecular docking studies. Hence, apigenin may compete with ATP for NDB-binding leading to energy depletion to fuel the transport of ABC transporter substrates. Furthermore, we performed COMPARE and hierarchical cluster analyses of transcriptome-wide mRNA expression profiles of the National Cancer Institute tumor cell line panel. Microarray-based mRNA expressions of genes of diverse biological functions (signal transduction, transcriptional regulation, ubiquitination, autophagy, metabolic activity, xenobiotic detoxification and microtubule formation) significantly predicted responsiveness of tumor cells to apigenin. In conclusion, apigenin's activity is not hampered by classical mechanisms of multidrug resistance and the inhibition of ABC transporters by apigenin indicates that apigenin may overcome multidrug resistance in otherwise refractory tumors.

  17. Imidazoacridinone-dependent lysosomal photodestruction: a pharmacological Trojan horse approach to eradicate multidrug-resistant cancers

    PubMed Central

    Adar, Y; Stark, M; Bram, E E; Nowak-Sliwinska, P; van den Bergh, H; Szewczyk, G; Sarna, T; Skladanowski, A; Griffioen, A W; Assaraf, Y G

    2012-01-01

    Multidrug resistance (MDR) remains a primary hindrance to curative cancer therapy. Thus, introduction of novel strategies to overcome MDR is of paramount therapeutic significance. Sequestration of chemotherapeutics in lysosomes is an established mechanism of drug resistance. Here, we show that MDR cells display a marked increase in lysosome number. We further demonstrate that imidazoacridinones (IAs), which are cytotoxic fluorochromes, undergo a dramatic compartmentalization in lysosomes because of their hydrophobic weak base nature. We hence developed a novel photoactivation-based pharmacological Trojan horse approach to target and eradicate MDR cancer cells based on photo-rupture of IA-loaded lysosomes and tumor cell lysis via formation of reactive oxygen species. Illumination of IA-loaded cells resulted in lysosomal photodestruction and restoration of parental cell drug sensitivity. Lysosomal photodestruction of MDR cells overexpressing the key MDR efflux transporters ABCG2, ABCB1 or ABCC1 resulted in 10- to 52-fold lower IC50 values of various IAs, thereby restoring parental cell sensitivity. Finally, in vivo application of this photodynamic therapy strategy after i.v. injection of IAs in human ovarian tumor xenografts in the chorioallantoic membrane model revealed selective destruction of tumors and their associated vasculature. These findings identify lysosomal sequestration of IAs as an Achilles heel of MDR cells that can be harnessed to eradicate MDR tumor cells via lysosomal photodestruction. PMID:22476101

  18. Breast cancer resistance protein (Bcrp1/Abcg2) reduces systemic exposure of the dietary carcinogens aflatoxin B1, IQ and Trp-P-1 but also mediates their secretion into breast milk.

    PubMed

    van Herwaarden, Antonius E; Wagenaar, Els; Karnekamp, Barbara; Merino, Gracia; Jonker, Johan W; Schinkel, Alfred H

    2006-01-01

    The breast cancer resistance protein (BCRP/ABCG2) usually protects the body from a wide variety of environmental and dietary xenotoxins by reducing their net uptake from intestine and by increasing their hepatobiliary, intestinal and renal elimination. BCRP is also highly expressed in lactating mammary glands in mice, and this expression is conserved in cows and humans. As a result, BCRP substrates can be secreted into milk. We investigated whether different classes of dietary carcinogens are substrates of Bcrp1/BCRP and the implications for systemic exposure and breast milk contamination. Using polarized cell lines, we found that Bcrp1 transports the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and the potent human hepatocarcinogen aflatoxin B1, and decreases their cellular accumulation up to 10-fold. In vivo pharmacokinetic studies showed that [14C]IQ, [14C]Trp-P-1 and [3H]aflatoxin B1 plasma levels were substantially lower in wild-type compared with Bcrp1-/- mice, after both oral and intravenous administration, demonstrating that Bcrp1 restricts systemic exposure to these carcinogens. Moreover, Bcrp1 mediates transfer of [14C]IQ, [14C]Trp-P-1 and [3H]aflatoxin into milk, with 3.4+/-0.6, 2.6+/-0.3 and 3.8+/-0.5-fold higher milk to plasma ratios, respectively, in lactating wild-type versus Bcrp1-/- mice. We have thus identified Bcrp1/BCRP as one of the molecular mechanisms by which heterocyclic amines and aflatoxin are transferred into milk, thereby posing a health risk to breast-fed infants and dairy consumers. Paradoxically, Bcrp1/BCRP appears to have both protective and adverse roles with respect to exposure to dietary carcinogens.

  19. The effect of low pH on breast cancer resistance protein (ABCG2)-mediated transport of methotrexate, 7-hydroxymethotrexate, methotrexate diglutamate, folic acid, mitoxantrone, topotecan, and resveratrol in in vitro drug transport models.

    PubMed

    Breedveld, Pauline; Pluim, Dick; Cipriani, Greta; Dahlhaus, Femke; van Eijndhoven, Maria A J; de Wolf, Cornelia J F; Kuil, Annemieke; Beijnen, Jos H; Scheffer, George L; Jansen, Gerrit; Borst, Piet; Schellens, Jan H M

    2007-01-01

    Some cellular uptake systems for (anti)folates function optimally at acidic pH. We have tested whether this also applies to efflux from cells by breast cancer resistance protein (BCRP; ABCG2), which has been reported to transport folic acid, methotrexate, and methotrexate di- and triglutamate at physiological pH. Using Spodoptera frugiperda-BCRP membrane vesicles, we showed that the ATP-dependent vesicular transport of 1 muM methotrexate by BCRP is 5-fold higher at pH 5.5 than at physiological pH. The transport of methotrexate was saturable at pH 5.5, with apparent Km and Vmax values of 1.3 +/- 0.2 mM and 44 +/- 2.5 nmol/mg of protein/min, respectively, but was linear with drug concentration at pH 7.3 up to 6 mM methotrexate. In contrast to recent reports, we did not detect transport of methotrexate diglutamate at physiological pH, but we did find transport at pH 5.5. We also found that 7-hydroxy-methotrexate, the major metabolite of methotrexate, is transported by BCRP both at physiological pH and (more efficiently) at low pH. The pH effect was also observed in intact BCRP-overexpressing cells: we found a 3-fold higher level of resistance to both methotrexate and the prototypical BCRP substrate mitoxantrone at pH 6.5 as at physiological pH. Furthermore, with MDCKII-BCRP monolayers, we found that resveratrol, which is a neutral compound at pH < or = 7.4, is efficiently transported by BCRP at pH 6.0, whereas we did not detect active transport at pH 7.4. We conclude that BCRP transports substrate drugs more efficiently at low pH, independent of the dissociation status of the substrate.

  20. Effect of variations in the amounts of P-glycoprotein (ABCB1), BCRP (ABCG2) and CYP3A4 along the human small intestine on PBPK models for predicting intestinal first pass.

    PubMed

    Bruyère, Arnaud; Declèves, Xavier; Bouzom, Francois; Ball, Kathryn; Marques, Catie; Treton, Xavier; Pocard, Marc; Valleur, Patrice; Bouhnik, Yoram; Panis, Yves; Scherrmann, Jean-Michel; Mouly, Stephane

    2010-10-04

    It is difficult to predict the first-pass effect in the human intestine due to a lack of scaling factors for correlating in vitro and in vivo data. We have quantified cytochrome P450/3A4 (CYP3A4) and two ABC transporters, P-glycoprotein (P-gp, ABCB1) and the breast cancer resistant protein BCRP (ABCG2), throughout the human small intestine to determine the scaling factors for predicting clearance from intestinal microsomes and develop a physiologically based pharmacokinetic (PBPK) model. CYP3A4, P-gp and BCRP proteins were quantified by Western blotting and/or enzyme activities in small intestine samples from 19 donors, and mathematical trends of these expressions with intestinal localization were established. Microsome fractions were prepared and used to calculate the amount of microsomal protein per gram of intestine (MPPGI). Our results showed a trend in CYP3A4 expression decrease from the upper to the lower small intestine while P-gp expression is increasing. In contrast, BCRP expression did not vary significantly with position, but varied greatly between individuals. The MPPGI (mg microsomal protein per centimeter intestine) remained constant along the length of the small intestine, at about 1.55 mg/cm. Moreover, intrinsic clearance measured with specific CYP3A4 substrates (midazolam and an in-house Servier drug) and intestinal microsomes was well correlated with the amount of CYP3A4 (R(2) > 0.91, p < 0.01). In vivo data were more accurately predicted using PBPK models of blood concentrations of these two substrates based on the segmental distributions of these enzymes and MPPGI determined in this study. Thus, these mathematical trends can be used to predict drug absorption at different intestinal sites and their metabolism can be predicted with the MPPGI.

  1. mRNA expression profile of multidrug-resistant genes in acute lymphoblastic leukemia of children, a prognostic value for ABCA3 and ABCA2.

    PubMed

    Rahgozar, Soheila; Moafi, Alireza; Abedi, Marjan; Entezar-E-Ghaem, Mansureh; Moshtaghian, Jamal; Ghaedi, Kamran; Esmaeili, Abolghasem; Montazeri, Fatemeh

    2014-01-01

    Multidrug resistance (MDR) is an important cause of treatment failure in acute lymphoblastic leukemia (ALL). The ABC family of membrane transporters is proposed, albeit with controversy, to be involved in this process. The present study aims to investigate the mRNA expression profile of several genes of this family, including ABCA2, ABCA3, ABCB1/MDR1, MRP1/ABCC1, MRP3/ABCC3, ABCG2/BCRP, and the intracellular transporter MVP/LRP, in childhood ALL, and to evaluate their association with response to therapy. Some genes in the present research are being studied for the first time in Iran. Using quantitative real-time PCR, we evaluated 27 children with ALL at diagnosis and 15 children with normal bone marrow. The status of response to therapy was assessed one year after the onset of therapy through investigating the IgH/TCRγ gene rearrangements. Our findings indicate a considerable and direct relationship between mRNA expression levels of ABCA2, ABCA3, MDR1, and MRP1 genes and positive minimal residual disease (MRD) measured after one year of treatment. Statistical analysis revealed that expression of these genes higher than the cutoff point will raise the risk of MRD by 15-, 6.25-, 12-, and 9-fold, respectively. No relationship was found between of MVP/LRP, MRP3 and ABCG2 genes expression and ALL prognoses. Considering the direct and significant relationship between the increased expression of ABCA2, ABCA3, MDR1, and MRP1 genes and positive risk of MRD in children with ALL, evaluating the expression profile of these genes on diagnosis may identify high risk individuals and help plan a more efficient treatment strategy.

  2. mRNA expression profile of multidrug-resistant genes in acute lymphoblastic leukemia of children, a prognostic value for ABCA3 and ABCA2

    PubMed Central

    Rahgozar, Soheila; Moafi, Alireza; Abedi, Marjan; Entezar-e-ghaem, Mansureh; Moshtaghian, Jamal; Ghaedi, Kamran; Esmaeili, Abolghasem; Montazeri, Fatemeh

    2014-01-01

    Multidrug resistance (MDR) is an important cause of treatment failure in acute lymphoblastic leukemia (ALL). The ABC family of membrane transporters is proposed, albeit with controversy, to be involved in this process. The present study aims to investigate the mRNA expression profile of several genes of this family, including ABCA2, ABCA3, ABCB1/MDR1, MRP1/ABCC1, MRP3/ABCC3, ABCG2/BCRP, and the intracellular transporter MVP/LRP, in childhood ALL, and to evaluate their association with response to therapy. Some genes in the present research are being studied for the first time in Iran. Using quantitative real-time PCR, we evaluated 27 children with ALL at diagnosis and 15 children with normal bone marrow. The status of response to therapy was assessed one year after the onset of therapy through investigating the IgH/TCRγ gene rearrangements. Our findings indicate a considerable and direct relationship between mRNA expression levels of ABCA2, ABCA3, MDR1, and MRP1 genes and positive minimal residual disease (MRD) measured after one year of treatment. Statistical analysis revealed that expression of these genes higher than the cutoff point will raise the risk of MRD by 15-, 6.25-, 12-, and 9-fold, respectively. No relationship was found between of MVP/LRP, MRP3 and ABCG2 genes expression and ALL prognoses. Considering the direct and significant relationship between the increased expression of ABCA2, ABCA3, MDR1, and MRP1 genes and positive risk of MRD in children with ALL, evaluating the expression profile of these genes on diagnosis may identify high risk individuals and help plan a more efficient treatment strategy. PMID:24145140

  3. Effects of genetic variants in UGT1A1, SLCO1B3, ABCB1, ABCC2, ABCG2, ORM1 on PK/PD of telmisartan in Chinese patients with mild to moderate essential hypertension
.

    PubMed

    Pei, Qi; Yang, Liu; Tan, Hong-Yi; Liu, Shi-Kun; Liu, Yang; Huang, Lu; Li, Rong-Hui; Wan, Qian; Huang, Jie; Guo, Cheng-Xian; Zuo, Xiao-Cong; Li, Jingle; Yang, Guo-Ping

    2017-08-01

    This study aimed to understand the effects of single nucleotide polymorphisms (SNPs) in UGT1A1, SLCO1B3, ABCB1, ABCC2, ABCG2, and ORM1 on the pharmacokinetics (PK) (plasma concentration) and pharmacodynamics (PD) (blood pressure) of telmisartan in Chinese patients. 58 Han Chinese patients (aged 45 - 72 years) with mild to moderate essential hypertension were included and received 80 mg/day telmisartan for 4 weeks. The plasma concentration and genetic variants were determined by LC/MS/MS and MALDI-TOF mass spectrometry, respectively. Multivariable linear analysis was used to examine the relationships between PK/PD and genetic variants. Females showed a significantly higher AUClast than males (n = 22, 4,879.48 ± 3,449.33 h×ng/mL vs. n = 36, 2,715.59 ± 2,223.77 h×ng/mL, p = 0.047). Amongst all genetic variants investigated, the patients with UGT1A1 rs4124874 AA (n = 11, 1,730.51 ± 1,325.79 h×ng/mL) had a significantly lower AUClast compared with patients with UGT1A1 rs4124874 CC+AC (n = 19 + 28, 4,177.44 ± 3,222.11 h×ng/mL and 3,810.82 ± 2,960.43 h×ng/mL, p = 0.027). None of the SNPs investigated was associated with the PD responses to telmisartan. Variation of UGT1A1 (rs4124874) affects PK of telmisartan in Chinese patients, highlighting the value of genetic testing in precision medicine as the telmisartan dose could be adjusted based on UGT1A1 genetic variations.
.

  4. Cytotoxicity of South-African medicinal plants towards sensitive and multidrug-resistant cancer cells.

    PubMed

    Saeed, Mohamed E M; Meyer, Marion; Hussein, Ahmed; Efferth, Thomas

    2016-06-20

    Traditional medicine plays a major role for primary health care worldwide. Cancer belongs to the leading disease burden in industrialized and developing countries. Successful cancer therapy is hampered by the development of resistance towards established anticancer drugs. In the present study, we investigated the cytotoxicity of 29 extracts from 26 medicinal plants of South-Africa against leukemia cell lines, most of which are used traditionally to treat cancer and related symptoms. We have investigated the plant extracts for their cytotoxic activity towards drug-sensitive parental CCRF-CEM leukemia cells and their multidrug-resistant P-glycoprotein-overexpressing subline, CEM/ADR5000 by means of the resazurin assay. A panel of 60 NCI tumor cell lines have been investigated for correlations between selected phytochemicals from medicinal plants and the expression of resistance-conferring genes (ABC-transporters, oncogenes, tumor suppressor genes). Seven extracts inhibited both cell lines (Acokanthera oppositifolia, Hypoestes aristata, Laurus nobilis, Leonotis leonurus, Plectranthus barbatus, Plectranthus ciliates, Salvia apiana). CEM/ADR5000 cells exhibited a low degree of cross-resistance (3.35-fold) towards the L. leonurus extract, while no cross-resistance was observed to other plant extracts, although CEM/ADR5000 cells were highly resistant to clinically established drugs. The log10IC50 values for two out of 14 selected phytochemicals from these plants (acovenoside A and ouabain) of 60 tumor cell lines were correlated to the expression of ABC-transporters (ABCB1, ABCB5, ABCC1, ABCG2), oncogenes (EGFR, RAS) and tumor suppressors (TP53). Sensitivity or resistance of the cell lines were not statistically associated with the expression of these genes, indicating that multidrug-resistant, refractory tumors expressing these genes may still respond to acovenoside A and ouabain. The bioactivity of South African medicinal plants may represent a basis for the development

  5. Multidrug Resistant Acinetobacter

    PubMed Central

    Manchanda, Vikas; Sanchaita, Sinha; Singh, NP

    2010-01-01

    Emergence and spread of Acinetobacter species, resistant to most of the available antimicrobial agents, is an area of great concern. It is now being frequently associated with healthcare associated infections. Literature was searched at PUBMED, Google Scholar, and Cochrane Library, using the terms ‘Acinetobacter Resistance, multidrug resistant (MDR), Antimicrobial Therapy, Outbreak, Colistin, Tigecycline, AmpC enzymes, and carbapenemases in various combinations. The terms such as MDR, Extensively Drug Resistant (XDR), and Pan Drug Resistant (PDR) have been used in published literature with varied definitions, leading to confusion in the correlation of data from various studies. In this review various mechanisms of resistance in the Acinetobacter species have been discussed. The review also probes upon the current therapeutic options, including combination therapies available to treat infections due to resistant Acinetobacter species in adults as well as children. There is an urgent need to enforce infection control measures and antimicrobial stewardship programs to prevent the further spread of these resistant Acinetobacter species and to delay the emergence of increased resistance in the bacteria. PMID:20927292

  6. Multidrug-Resistant TB

    PubMed Central

    Cox, Helen; Coomans, Fons

    2016-01-01

    Abstract The right to enjoy the benefits of scientific progress (REBSP) is a little-known but potentially valuable right that can contribute to rights-based approaches to addressing multidrug-resistant TB (MDR-TB). We argue that better understanding of the REBSP may help to advance legal and civil society action for health rights. While the REBSP does not provide an individual entitlement to have a new drug developed for MDR-TB, it sets up entitlements to expect a state to establish a legislative and policy framework aimed at developing scientific capacity to address the most important health issues and at disseminating the outcomes of scientific research. By making scientific findings available and accessible, people can be enabled to claim the use of science for social benefits. Inasmuch as the market fails to address neglected diseases such as MDR-TB, the REBSP provides a potential counterbalance to frame a positive obligation on states to both marshal their own resources and to coordinate the actions of multiple other actors towards this goal, including non-state actors. While the latter do not hold the same level of accountability as states, the REBSP can still enable the recognition of obligations at a level of “soft law” responsibilities. PMID:27780997

  7. A molecular understanding of ATP-dependent solute transport by multidrug resistance-associated protein MRP1.

    PubMed

    Chang, Xiu-bao

    2007-03-01

    Over a million new cases of cancers are diagnosed each year in the United States and over half of these patients die from these devastating diseases. Thus, cancers cause a major public health problem in the United States and worldwide. Chemotherapy remains the principal mode to treat many metastatic cancers. However, occurrence of cellular multidrug resistance (MDR) prevents efficient killing of cancer cells, leading to chemotherapeutic treatment failure. Numerous mechanisms of MDR exist in cancer cells, such as intrinsic or acquired MDR. Overexpression of ATP-binding cassette (ABC) drug transporters, such as P-glycoprotein (P-gp or ABCB1), breast cancer resistance protein (BCRP or ABCG2) and/or multidrug resistance-associated protein (MRP1 or ABCC1), confers an acquired MDR due to their capabilities of transporting a broad range of chemically diverse anticancer drugs. In addition to their roles in MDR, there is substantial evidence suggesting that these drug transporters have functions in tissue defense. Basically, these drug transporters are expressed in tissues important for absorption, such as in lung and gut, and for metabolism and elimination, such as in liver and kidney. In addition, these drug transporters play an important role in maintaining the barrier function of many tissues including blood-brain barrier, blood-cerebral spinal fluid barrier, blood-testis barrier and the maternal-fetal barrier. Thus, these ATP-dependent drug transporters play an important role in the absorption, disposition and elimination of the structurally diverse array of the endobiotics and xenobiotics. In this review, the molecular mechanism of ATP-dependent solute transport by MRP1 will be addressed.

  8. Pyrrolopyrimidine derivatives and purine analogs as novel activators of Multidrug Resistance-associated Protein 1 (MRP1, ABCC1).

    PubMed

    Schmitt, Sven Marcel; Stefan, Katja; Wiese, Michael

    2017-01-01

    Multidrug resistance (MDR) is the main cause of diminished success in cancer chemotherapy. ABC transport proteins are considered to be one important factor of MDR. Besides P-glycoprotein (P-gp, ABCB1) and Breast Cancer Resistance Protein (BCRP, ABCG2), Multidrug Resistance-associated Protein 1 (MRP1, ABCC1) is associated with non-response to chemotherapy in different cancers. While considerable effort was spent in overcoming MDR during the last two decades, almost nothing is known with respect to activators of transport proteins. In this work we present certain pyrrolo[3,2-d]pyrimidine derivatives with variations at positions 4 and 5 and purine analogs with variations at position 6 as novel activators of MRP1-mediated transport of the MRP1 substrate calcein AM and the anticancer drug daunorubicin in low nanomolar concentration range. Two different MRP1 overexpressing cell lines were used, the doxorubicin-selected human lung cancer cell line H69 AR and the transfected Madin-Darby Canine Kidney cell line MDCK II MRP1. No effect was observed in the sensitive counterparts H69 and MDCK II wild type (wt). Derivatives with higher molecular weight possessed also inhibitory properties at low micromolar concentrations, although most compounds were rather poor MRP1 inhibitors. Purine analogs derived from potent MRP1 inhibitors of the pyrrolopyrimidine class showed equal activating, but no inhibiting effects at all. All tested compounds were non-toxic and had only minor impact on P-gp or BCRP, showing no inhibition or activation. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Effect of Prostaglandin E2 on Multidrug Resistance Transporters In Human Placental Cells

    PubMed Central

    Lee, Gene T.; Dong, Yafeng; Zhou, Helen; He, Lily; Weiner, Carl P.

    2014-01-01

    Prostaglandin (PG) E2, a major product of cyclooxygenase (COX)-2, acts as an immunomodulator at the maternal-fetal interface during pregnancy. It exerts biologic function through interaction with E-prostanoid (EP) receptors localized to the placenta. The activation of the COX-2/PGE2/EP signal pathway can alter the expression of the ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 [P-glycoprotein (Pgp); gene: ABCB1], and breast cancer resistance protein (BCRP; gene: ABCG2), which function to extrude drugs and xenobiotics from cells. In the placenta, PGE2-mediated changes in ABC transporter expression could impact fetal drug exposure. Furthermore, understanding the signaling cascades involved could lead to strategies for the control of Pgp and BCRP expression levels. We sought to determine the impact of PGE2 signaling mechanisms on Pgp and BCRP in human placental cells. The treatment of placental cells with PGE2 up-regulated BCRP expression and resulted in decreased cellular accumulation of the fluorescent substrate Hoechst 33342. Inhibiting the EP1 and EP3 receptors with specific antagonists attenuated the increase in BCRP. EP receptor signaling results in activation of transcription factors, which can affect BCRP expression. Although PGE2 decreased nuclear factor κ-light chain-enhancer of activated B activation and increased activator protein 1, chemical inhibition of these inflammatory transcription factors did not blunt BCRP up-regulation by PGE2. Though PGE2 decreased Pgp mRNA, Pgp expression and function were not significantly altered. Overall, these findings suggest a possible role for PGE2 in the up-regulation of placental BCRP expression via EP1 and EP3 receptor signaling cascades. PMID:25261564

  10. Effect of prostaglandin E2 on multidrug resistance transporters in human placental cells.

    PubMed

    Mason, Clifford W; Lee, Gene T; Dong, Yafeng; Zhou, Helen; He, Lily; Weiner, Carl P

    2014-12-01

    Prostaglandin (PG) E2, a major product of cyclooxygenase (COX)-2, acts as an immunomodulator at the maternal-fetal interface during pregnancy. It exerts biologic function through interaction with E-prostanoid (EP) receptors localized to the placenta. The activation of the COX-2/PGE2/EP signal pathway can alter the expression of the ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 [P-glycoprotein (Pgp); gene: ABCB1], and breast cancer resistance protein (BCRP; gene: ABCG2), which function to extrude drugs and xenobiotics from cells. In the placenta, PGE2-mediated changes in ABC transporter expression could impact fetal drug exposure. Furthermore, understanding the signaling cascades involved could lead to strategies for the control of Pgp and BCRP expression levels. We sought to determine the impact of PGE2 signaling mechanisms on Pgp and BCRP in human placental cells. The treatment of placental cells with PGE2 up-regulated BCRP expression and resulted in decreased cellular accumulation of the fluorescent substrate Hoechst 33342. Inhibiting the EP1 and EP3 receptors with specific antagonists attenuated the increase in BCRP. EP receptor signaling results in activation of transcription factors, which can affect BCRP expression. Although PGE2 decreased nuclear factor κ-light chain-enhancer of activated B activation and increased activator protein 1, chemical inhibition of these inflammatory transcription factors did not blunt BCRP up-regulation by PGE2. Though PGE2 decreased Pgp mRNA, Pgp expression and function were not significantly altered. Overall, these findings suggest a possible role for PGE2 in the up-regulation of placental BCRP expression via EP1 and EP3 receptor signaling cascades. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  11. Association of Multi-Drug Resistance Gene Polymorphisms with Pancreatic Cancer Outcome

    PubMed Central

    Tanaka, Motofumi; Okazaki, Taro; Suzuki, Hideo; Abbruzzese, James L.; Li, Donghui

    2010-01-01

    BACKGROUND The purpose of this study was to identify single nucleotide polymorphisms (SNPs) of multi-drug resistance genes that are associated with clinical outcome in patients with potentially resectable pancreatic adenocarcinoma who were treated with preoperative gemcitabine-based chemoradiotherapy at M.D. Anderson Cancer Center. METHODS We selected 8 SNPs of 7 drug resistance genes; MDR1 (ABCB1), MRP1-5 (ABCC1-5), and BCRP (ABCG2), which have been reported to be important in mediating drug resistance. Genotype was determined by the Taqman method. The associations of genotype with tumor response to therapy and overall survival (OS) were evaluated using log-rank test, Cox regression, and logistic regression models. RESULTS MRP5 A-2G AA genotype showed significant association with OS (log-rank P =.010). The hazard ratio (95% confidence interval) was 1.65 (1.11-2.45) after adjusting for clinical predictors. The MRP2 G40A GG genotype had a weak association with reduced OS (log-rank P =.097). A combined effect of the two genotypes on OS was observed. Patients with none of the adverse genotypes had a median survival time (MST) of 34.0 months, and those with 1-2 deleterious alleles had a significantly lower MST of 20.7 months, respectively (log-rank P =.006). MRP2 G40A GG genotype was also significantly associated with poor histological response to chemoradiotherapy (P =.028). CONCLUSIONS These observations suggest a potential role of polymorphic variants of drug resistance genes to predict a therapeutic efficacy and survival of patients with potentially resectable pancreatic cancer. PMID:20922799

  12. Multidrug resistance proteins restrain the intestinal absorption of trans-resveratrol in rats.

    PubMed

    Juan, M Emília; González-Pons, Eulalia; Planas, Joana M

    2010-03-01

    trans-Resveratrol, a natural antioxidant, has been described as a nutraceutic compound with important beneficial effects on health, but its low oral bioavailability hinders its therapeutic activity. Here, we studied the mechanisms of apical transport of trans-resveratrol in enterocytes and the role of ATP-binding cassette (ABC) transporters in the secretion of resveratrol glucuronide and sulfate resulting from the rapid intracellular metabolism. An intestinal perfusion method with recirculation in vivo was used in rats. Jejunal loops were perfused with increasing concentrations of trans-resveratrol and results showed that its uptake occurs by simple diffusion without the participation of a mediated transport. The apparent diffusion constant was 8.1 +/- 0.3 microL/(5 min.mg dry weight). The glycoprotein-P (Pgp, ABCB1), multidrug resistance-associated protein 2 (MRP2, ABCC2), and breast cancer resistance protein (BCRP, ABCG2) located in the apical membrane of enterocytes were investigated using specific inhibitors. The Pgp inhibitors verapamil (5 micromol/L) and cyclosporin A (5 micromol/L) did not affect the efflux of trans-resveratrol and its conjugates. The MRP2 inhibitors probenecid (2 mmol/L) and MK571 (10 micromol/L) reduced the efflux of glucuronide by 61 and 55%, respectively, and of sulfate by 43 and 28%, respectively. The BCRP inhibitor Ko143 (0.5 micromol/L) decreased the secretion of glucuronide by 64% and of sulfate by 46%. Our experiments identify MRP2 and BCRP as the 2 apical transporters involved in the efflux of resveratrol conjugates.

  13. Multidrug toxicity involving sumatriptan.

    PubMed

    Knittel, Jessica L; Vorce, Shawn P; Levine, Barry; Hughes, Rhome L; Bosy, Thomas Z

    2015-01-01

    A multidrug fatality involving sumatriptan is reported. Sumatriptan is a tryptamine derivative that acts at 5-HT(1B/1D) receptors and is used for the treatment of migraines. The decedent was a 21-year-old white female found dead in bed by her spouse. No signs of physical trauma were observed and a large number of prescription medications were discovered at the scene. Toxicological analysis of the central blood revealed sumatriptan at a concentration of 1.03 mg/L. Following therapeutic dosing guidelines, sumatriptan concentrations do not exceed 0.095 mg/L. Sumatriptan was isolated by solid-phase extraction and analyzed using liquid chromatography-tandem mass spectrometry in multiple reaction monitoring mode. A tissue distribution study was completed with the following concentrations measured: 0.61 mg/L in femoral blood, 0.56 mg/L in iliac blood, 5.01 mg/L in urine, 0.51 mg/kg in liver, 3.66 mg/kg in kidney, 0.09 mg/kg in heart, 0.32 mg/kg in spleen, 0.01 mg/kg in brain, 15.99 mg/kg in lung and 78.54 mg/45 mL in the stomach contents. Carisoprodol, meprobamate, fluoxetine, doxylamine, orphenadrine, dextromethorphan and hydroxyzine were also present in the blood at the following concentrations: 3.35, 2.36, 0.63, 0.19, 0.06, 0.55 and 0.16 mg/L. The medical examiner ruled the cause of death as acute mixed drug toxicity and the manner of death as accident.

  14. The choreography of multidrug export.

    PubMed

    Doshi, Rupak; Gutmann, Daniel A P; Khoo, Yvonne S K; Fagg, Lisa A; van Veen, Hendrik W

    2011-06-01

    Multidrug transporters have a crucial role in causing the drug resistance that can arise in infectious micro-organisms and tumours. These integral membrane proteins mediate the export of a broad range of unrelated compounds from cells, including antibiotics and anticancer agents, thus reducing the concentration of these compounds to subtoxic levels in target cells. In spite of intensive research, it is not clear exactly how multidrug transporters work. The present review focuses on recent advancements in the biochemistry and structural biology of bacterial and human multidrug ABC (ATP-binding cassette) transporters. These advancements point to a common mechanism in which polyspecific drug-binding surfaces in the membrane domains are alternately exposed to the inside and outside surface of the membrane in response to the ATP-driven dimerization of nucleotide-binding domains and their dissociation following ATP hydrolysis.

  15. Biochemistry of Bacterial Multidrug Efflux Pumps

    PubMed Central

    Kumar, Sanath; Varela, Manuel F.

    2012-01-01

    Bacterial pathogens that are multi-drug resistant compromise the effectiveness of treatment when they are the causative agents of infectious disease. These multi-drug resistance mechanisms allow bacteria to survive in the presence of clinically useful antimicrobial agents, thus reducing the efficacy of chemotherapy towards infectious disease. Importantly, active multi-drug efflux is a major mechanism for bacterial pathogen drug resistance. Therefore, because of their overwhelming presence in bacterial pathogens, these active multi-drug efflux mechanisms remain a major area of intense study, so that ultimately measures may be discovered to inhibit these active multi-drug efflux pumps. PMID:22605991

  16. Hepatocyte SLAMF3 reduced specifically the multidrugs resistance protein MRP-1 and increases HCC cells sensitization to anti-cancer drugs

    PubMed Central

    Eugenio, Mélanie Simoes; Demey, Baptiste; Singh, Amrathlal Rabbind; Ossart, Christèle; Bagami, Mohammed Al; Regimbeau, Jean-Marc; Nguyen-Khac, Eric; Naassila, Mickael

    2016-01-01

    Multidrug resistance MDR proteins (MRPs) are members of the C family of a group of proteins named ATP binding cassette (ABC) transporters. MRPs can transport drugs including anticancer drugs, nucleoside analogs, antimetabolites and tyrosine kinase inhibitors. Drugs used in HCC therapy, such as tyrosine kinase inhibitor sorafenib, are substrates of uptake and/or efflux transporters. Variable expression of MRPs at the plasma membrane of tumor cells may contribute to drug resistance and subsequent clinical response. Recently, we reported that the hepatocyte SLAMF3 expression (Signaling Lymphocytic Activation Molecule Family member 3) was reduced in tumor cells from hepatocellular carcinoma (HCC) compared to its high expression in adjacent tissues. In the present study, we make a strong correlation between induced SLAMF3 overexpression and the specific loss of MRP-1 expression and its functionalities as a drugs resistance transporter. No changes were observed on expression of ABCG2 and MDR. More importantly, we highlight a strong inverse correlation between MRP-1 and SLAMF3 expression in patients with HCC. We propose that the SLAMF3 overexpression in cancerous cells could represent a potential therapeutic strategy to improve the drugs sensibility of resistant cells and thus control the therapeutic failure in HCC patients. PMID:27081035

  17. Hepatocyte SLAMF3 reduced specifically the multidrugs resistance protein MRP-1 and increases HCC cells sensitization to anti-cancer drugs.

    PubMed

    Fouquet, Grégory; Debuysscher, Véronique; Ouled-Haddou, Hakim; Eugenio, Mélanie Simoes; Demey, Baptiste; Singh, Amrathlal Rabbind; Ossart, Christèle; Al Bagami, Mohammed; Regimbeau, Jean-Marc; Nguyen-Khac, Eric; Naassila, Mickael; Marcq, Ingrid; Bouhlal, Hicham

    2016-05-31

    Multidrug resistance MDR proteins (MRPs) are members of the C family of a group of proteins named ATP binding cassette (ABC) transporters. MRPs can transport drugs including anticancer drugs, nucleoside analogs, antimetabolites and tyrosine kinase inhibitors. Drugs used in HCC therapy, such as tyrosine kinase inhibitor sorafenib, are substrates of uptake and/or efflux transporters. Variable expression of MRPs at the plasma membrane of tumor cells may contribute to drug resistance and subsequent clinical response. Recently, we reported that the hepatocyte SLAMF3 expression (Signaling Lymphocytic Activation Molecule Family member 3) was reduced in tumor cells from hepatocellular carcinoma (HCC) compared to its high expression in adjacent tissues. In the present study, we make a strong correlation between induced SLAMF3 overexpression and the specific loss of MRP-1 expression and its functionalities as a drugs resistance transporter. No changes were observed on expression of ABCG2 and MDR. More importantly, we highlight a strong inverse correlation between MRP-1 and SLAMF3 expression in patients with HCC. We propose that the SLAMF3 overexpression in cancerous cells could represent a potential therapeutic strategy to improve the drugs sensibility of resistant cells and thus control the therapeutic failure in HCC patients.

  18. Pien Tze Huang Overcomes Multidrug Resistance and Epithelial-Mesenchymal Transition in Human Colorectal Carcinoma Cells via Suppression of TGF-β Pathway

    PubMed Central

    Shen, Aling; Chen, Hongwei; Chen, Youqin; Lin, Wei; Liu, Liya; Sferra, Thomas J.; Peng, Jun

    2014-01-01

    The traditional Chinese medicine formula Pien Tze Huang (PZH) has long been used as a folk remedy for cancer. To elucidate the mode of action of PZH against cancer, in the present study we used a 5-FU resistant human colorectal carcinoma cell line (HCT-8/5-FU) to evaluate the effects of PZH on multidrug resistance (MDR) and epithelial-mesenchymal transition (EMT) as well as the activation of TGF-β pathway. We found that PZH dose-dependently inhibited the viability of HCT-8/5-FU cells which were insensitive to treatment of 5-FU and ADM, demonstrating the ability of PZH to overcome chemoresistance. Furthermore, PZH increased the intercellular accumulation of Rhodamine-123 and downregulated the expression of ABCG2 in HCT-8/5-FU cells. In addition, drug resistance induced the process of EMT in HCT-8 cells as evidenced by EMT-related morphological changes and alteration in the expression of EMT-regulatory factors, which however was neutralized by PZH treatment. Moreover, PZH inhibited MDR/EMT-enhanced migration and invasion capabilities of HCT-8 cells in a dose-dependent manner and suppressed MDR-induced activation of TGF-β signaling in HCT-8/5-FU cells. Taken together, our study suggests that PZH can effectively overcome MDR and inhibit EMT in human colorectal carcinoma cells via suppression of the TGF-β pathway. PMID:25505925

  19. Pien Tze Huang Overcomes Multidrug Resistance and Epithelial-Mesenchymal Transition in Human Colorectal Carcinoma Cells via Suppression of TGF-β Pathway.

    PubMed

    Shen, Aling; Chen, Hongwei; Chen, Youqin; Lin, Jiumao; Lin, Wei; Liu, Liya; Sferra, Thomas J; Peng, Jun

    2014-01-01

    The traditional Chinese medicine formula Pien Tze Huang (PZH) has long been used as a folk remedy for cancer. To elucidate the mode of action of PZH against cancer, in the present study we used a 5-FU resistant human colorectal carcinoma cell line (HCT-8/5-FU) to evaluate the effects of PZH on multidrug resistance (MDR) and epithelial-mesenchymal transition (EMT) as well as the activation of TGF-β pathway. We found that PZH dose-dependently inhibited the viability of HCT-8/5-FU cells which were insensitive to treatment of 5-FU and ADM, demonstrating the ability of PZH to overcome chemoresistance. Furthermore, PZH increased the intercellular accumulation of Rhodamine-123 and downregulated the expression of ABCG2 in HCT-8/5-FU cells. In addition, drug resistance induced the process of EMT in HCT-8 cells as evidenced by EMT-related morphological changes and alteration in the expression of EMT-regulatory factors, which however was neutralized by PZH treatment. Moreover, PZH inhibited MDR/EMT-enhanced migration and invasion capabilities of HCT-8 cells in a dose-dependent manner and suppressed MDR-induced activation of TGF-β signaling in HCT-8/5-FU cells. Taken together, our study suggests that PZH can effectively overcome MDR and inhibit EMT in human colorectal carcinoma cells via suppression of the TGF-β pathway.

  20. Proton-dependent multidrug efflux systems.

    PubMed Central

    Paulsen, I T; Brown, M H; Skurray, R A

    1996-01-01

    Multidrug efflux systems display the ability to transport a variety of structurally unrelated drugs from a cell and consequently are capable of conferring resistance to a diverse range of chemotherapeutic agents. This review examines multidrug efflux systems which use the proton motive force to drive drug transport. These proteins are likely to operate as multidrug/proton antiporters and have been identified in both prokaryotes and eukaryotes. Such proton-dependent multidrug efflux proteins belong to three distinct families or superfamilies of transport proteins: the major facilitator superfamily (MFS), the small multidrug resistance (SMR) family, and the resistance/ nodulation/cell division (RND) family. The MFS consists of symporters, antiporters, and uniporters with either 12 or 14 transmembrane-spanning segments (TMS), and we show that within the MFS, three separate families include various multidrug/proton antiport proteins. The SMR family consists of proteins with four TMS, and the multidrug efflux proteins within this family are the smallest known secondary transporters. The RND family consists of 12-TMS transport proteins and includes a number of multidrug efflux proteins with particularly broad substrate specificity. In gram-negative bacteria, some multidrug efflux systems require two auxiliary constituents, which might enable drug transport to occur across both membranes of the cell envelope. These auxiliary constituents belong to the membrane fusion protein and the outer membrane factor families, respectively. This review examines in detail each of the characterized proton-linked multidrug efflux systems. The molecular basis of the broad substrate specificity of these transporters is discussed. The surprisingly wide distribution of multidrug efflux systems and their multiplicity in single organisms, with Escherichia coli, for instance, possessing at least nine proton-dependent multidrug efflux systems with overlapping specificities, is examined. We also

  1. Binding and inhibition of drug transport proteins by heparin: a potential drug transporter modulator capable of reducing multidrug resistance in human cancer cells.

    PubMed

    Chen, Yunliang; Scully, Michael; Petralia, Gloria; Kakkar, Ajay

    2014-01-01

    A major problem in cancer treatment is the development of resistance to chemotherapeutic agents, multidrug resistance (MDR), associated with increased activity of transmembrane drug transporter proteins which impair cytotoxic treatment by rapidly removing the drugs from the targeted cells. Previously, it has been shown that heparin treatment of cancer patients undergoing chemotherapy increases survival. In order to determine whether heparin is capable reducing MDR and increasing the potency of chemotherapeutic drugs, the cytoxicity of a number of agents toward four cancer cell lines (a human enriched breast cancer stem cell line, two human breast cancer cell lines, MCF-7 and MDA-MB-231, and a human lung cancer cell line A549) was tested in the presence or absence of heparin. Results demonstrated that heparin increased the cytotoxicity of a range of chemotherapeutic agents. This effect was associated with the ability of heparin to bind to several of the drug transport proteins of the ABC and non ABC transporter systems. Among the ABC system, heparin treatment caused significant inhibition of the ATPase activity of ABCG2 and ABCC1, and of the efflux function observed as enhanced intracellular accumulation of specific substrates. Doxorubicin cytoxicity, which was enhanced by heparin treatment of MCF-7 cells, was found to be under the control of one of the major non-ABC transporter proteins, lung resistance protein (LRP). LRP was also shown to be a heparin-binding protein. These findings indicate that heparin has a potential role in the clinic as a drug transporter modulator to reduce multidrug resistance in cancer patients.

  2. Role of multidrug resistance in photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Diddens, Heyke C.

    1992-06-01

    Multidrug resistance in cancer chemotherapy is a well established phenomenon. One of the most common phenotypical changes in acquired or intrinsic multidrug resistance in human tumor cells is the overexpression of the mdrl gene product P-glycoprotein, which acts as an active efflux pump. Increased levels of P-glycoprotein are associated with resistance to a variety of anticancer drugs commonly used in tumor chemotherapy like anthracyclins, vinca- alcaloids, epipodophyllotoxins or actinomycin D. We investigated the efficacy or photodynamic therapy in the treatment of tumor cells expressing the multidrug resistance phenotype. Our data show that multidrug resistant cells are highly cross resistant to the phototoxic stain rhodamine 123 but exhibit only low degrees of cross resistance (2 - 3 -folds) to the photosensitizers Photosan-3, Clorin-2, methylene blue and meso-tetra (4- sulfonatophenyl) porphine (TPPS4). Resistance is associated with a decrease in intracellular accumulation of the photosensitizer. Verapamil, a membrane active compound known to enhance drug sensitivity in multidrug resistant cells by inhibition of P-glycoprotein, also increases phototoxicity in multidrug resistant cells. Our results imply that tumors expressing the multidrug resistance phenotype might fail to respond to photochemotherapy with rhodamine 123. On the other hand, multidrug resistance may not play an important role in photodynamic therapy with Photosan-3, Chlorin-2, methylene blue or TPPS4.

  3. Identification of the hepatic efflux transporters of organic anions using double-transfected Madin-Darby canine kidney II cells expressing human organic anion-transporting polypeptide 1B1 (OATP1B1)/multidrug resistance-associated protein 2, OATP1B1/multidrug resistance 1, and OATP1B1/breast cancer resistance protein.

    PubMed

    Matsushima, Soichiro; Maeda, Kazuya; Kondo, Chihiro; Hirano, Masaru; Sasaki, Makoto; Suzuki, Hiroshi; Sugiyama, Yuichi

    2005-09-01

    Until recently, it was generally believed that the transport of various organic anions across the bile canalicular membrane was mainly mediated by multidrug resistance-associated protein 2 (MRP2/ABCC2). However, a number of new reports have shown that some organic anions are also substrates of multidrug resistance 1 (MDR1/ABCB1) and/or breast cancer resistance protein (BCRP/ABCG2), implying MDR1 and BCRP could also be involved in the biliary excretion of organic anions in humans. In the present study, we constructed new double-transfected Madin-Darby canine kidney II (MDCKII) cells expressing organic anion-transporting polypeptide 1B1 (OATP1B1)/MDR1 and OATP1B1/BCRP, and we investigated the transcellular transport of four kinds of organic anions, estradiol-17beta-d-glucuronide (EG), estrone-3-sulfate (ES), pravastatin (PRA), and cerivastatin (CER), to identify which efflux transporters mediate the biliary excretion of compounds using double-transfected cells. We observed the vectorial transport of EG and ES in all the double transfectants. MRP2 showed the highest efflux clearance of EG among these efflux transporters, whereas BCRP-mediated clearance of ES was the highest in these double transfectants. In addition, two kinds of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, CER and PRA, were also substrates of all these efflux transporters. The rank order of the efflux clearance of PRA mediated by each transporter was the same as that of EG, whereas the contribution of MDR1 to the efflux of CER was relatively greater than for PRA. This experimental system is very useful for identifying which transporters are involved in the biliary excretion of organic anions that cannot easily penetrate the plasma membrane.

  4. Structural and mechanistic diversity of multidrug transporters.

    PubMed

    Mousa, Jarrod J; Bruner, Steven D

    2016-10-26

    Covering: 2009 to mid 2016Multidrug transporters are common and prevalent in all orders of life, having diverse functions from the removal of toxins, resistance to cytotoxins, and the transport of specific eluents. In addition, multidrug transporters pose a significant threat to modern medicine. Able to transport structurally diverse small molecule drugs, these transporters are implicated in antibiotic resistant strains of bacteria, as well as chemotherapeutic-resistance cancer cells. Although important in such resistance, a relatively small number of multidrug transporters have been structurally characterized, primarily due to the difficulty in purifying and crystallizing active membrane proteins and protein complexes. This review will cover recent structural breakthroughs in the past six years that have led to increased knowledge of the mechanisms of multidrug transporter chemistry, and the role of these transporters in exporting secondary metabolites.

  5. Molecular Properties of Bacterial Multidrug Transporters

    PubMed Central

    Putman, Monique; van Veen, Hendrik W.; Konings, Wil N.

    2000-01-01

    One of the mechanisms that bacteria utilize to evade the toxic effects of antibiotics is the active extrusion of structurally unrelated drugs from the cell. Both intrinsic and acquired multidrug transporters play an important role in antibiotic resistance of several pathogens, including Neisseria gonorrhoeae, Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae. Detailed knowledge of the molecular basis of drug recognition and transport by multidrug transport systems is required for the development of new antibiotics that are not extruded or of inhibitors which block the multidrug transporter and allow traditional antibiotics to be effective. This review gives an extensive overview of the currently known multidrug transporters in bacteria. Based on energetics and structural characteristics, the bacterial multidrug transporters can be classified into five distinct families. Functional reconstitution in liposomes of purified multidrug transport proteins from four families revealed that these proteins are capable of mediating the export of structurally unrelated drugs independent of accessory proteins or cytoplasmic components. On the basis of (i) mutations that affect the activity or the substrate specificity of multidrug transporters and (ii) the three-dimensional structure of the drug-binding domain of the regulatory protein BmrR, the substrate-binding site for cationic drugs is predicted to consist of a hydrophobic pocket with a buried negatively charged residue that interacts electrostatically with the positively charged substrate. The aromatic and hydrophobic amino acid residues which form the drug-binding pocket impose restrictions on the shape and size of the substrates. Kinetic analysis of drug transport by multidrug transporters provided evidence that these proteins may contain multiple substrate-binding sites. PMID:11104814

  6. Multidrug resistance: an emerging crisis.

    PubMed

    Tanwar, Jyoti; Das, Shrayanee; Fatima, Zeeshan; Hameed, Saif

    2014-01-01

    The resistance among various microbial species (infectious agents) to different antimicrobial drugs has emerged as a cause of public health threat all over the world at a terrifying rate. Due to the pacing advent of new resistance mechanisms and decrease in efficiency of treating common infectious diseases, it results in failure of microbial response to standard treatment, leading to prolonged illness, higher expenditures for health care, and an immense risk of death. Almost all the capable infecting agents (e.g., bacteria, fungi, virus, and parasite) have employed high levels of multidrug resistance (MDR) with enhanced morbidity and mortality; thus, they are referred to as "super bugs." Although the development of MDR is a natural phenomenon, the inappropriate use of antimicrobial drugs, inadequate sanitary conditions, inappropriate food-handling, and poor infection prevention and control practices contribute to emergence of and encourage the further spread of MDR. Considering the significance of MDR, this paper, emphasizes the problems associated with MDR and the need to understand its significance and mechanisms to combat microbial infections.

  7. Multifunctional micelle delivery system for overcoming multidrug resistance of doxorubicin.

    PubMed

    Qin, Li; Wu, Lei; Jiang, Shanshan; Yang, Dandan; He, Huiyang; Zhang, Fang; Zhang, Peng

    2017-09-13

    Doxorubicin, as an anthracycline, plays an important role in chemotherapy. But multidrug resistance tremendously retards the anticancer effect of doxorubicin and results in the failure of chemotherapy. Multifunctional micelles emerge as a valid strategy to load doxorubicin by physical encapsulation or chemical binding to be delivered to cancer cells against multidrug resistance. In this review, mechanism of multidrug resistance of doxorubicin is simply described. Multifunctional co-delivery micelles of doxorubicin and main multidrug resistance modulators have been summarized in detail. Doxorubicin-loaded multifunctional polymeric micelles are also introduced to alleviate multidrug resistance of doxorubicin, in which polymers act as multidrug resistance modulators.

  8. Deciphering the molecular basis of multidrug recognition: crystal structures of the Staphylococcus aureus multidrug binding transcription regulator QacR.

    PubMed

    Schumacher, Maria A; Brennan, Richard G

    2003-03-01

    Multidrug transporters and their transcriptional regulators are key components of bacterial multidrug resistance (MDR). How these multidrug binding proteins can recognize such chemically disparate compounds represents a fascinating question from a structural standpoint and an important question in future drug development efforts. The Staphylococcus aureus multidrug binding regulator, QacR, is soluble and recognizes an especially wide range of structurally dissimilar compounds, properties making it an ideal model system for deciphering the molecular basis of multidrug recognition. Recent structures of QacR have afforded the first view of any MDR protein bound to multiple drugs, revealing key structural features of multidrug recognition, including a multisite binding pocket.

  9. Multidrug transporters as drug targets.

    PubMed

    Liang, X-J; Aszalos, A

    2006-08-01

    Transport molecules can significantly affect the pharmacodynamics and pharmacokinetics of drugs. An important transport molecule, the 170 kDa P-glycoprotein (Pgp), is constitutively expressed at several organ sites in the human body. Pgp is expressed at the blood-brain barrier, in the kidneys, liver, intestines and in certain T cells. Other transporters such as the multidrug resistance protein 1 (MRP1) and MRP2 also contribute to drug distribution in the human body, although to a lesser extent than Pgp. These three transporters, and especially Pgp, are often targets of drugs. Pgp can be an intentional or unintentional target. It is directly targeted when one wants to block its function by a modifier drug so that another drug, also a substrate of Pgp, can penetrate the cell membrane, which would otherwise be impermeable. Unintentional targeting occurs when several drugs are administered to a patient and as a consequence, the physiological function of Pgp is blocked at different organ sites. Like Pgp, MRP1 also has the capacity to mediate transport of many drugs and other compounds. MRP1 has a protective role in preventing accumulation of toxic compounds and drugs in epithelial tissue covering the choroid plexus/cerebrospinal fluid compartment, oral epithelium, sertoli cells, intesticular tubules and urinary collecting duct cells. MRP2 primarily transports weakly basic drugs and bilirubin from the liver to bile. Most compounds that efficiently block Pgp have only low affinity for MRP1 and MRP2. There are only a few effective and specific MRP inhibitors available. Drug targeting of these transporters may play a role in cancer chemotherapy and in the pharmacokinetics of substrate drugs.

  10. Interactions between riluzole and ABCG2/BCRP transporter.

    PubMed

    Milane, Aline; Vautier, Sarah; Chacun, Hélène; Meininger, Vincent; Bensimon, Gilbert; Farinotti, Robert; Fernandez, Christine

    2009-03-06

    Amyotrophic lateral sclerosis (ALS) is a neurodegenerative fatal disease. Drugs used in this disease need to cross the blood-brain barrier (BBB). Only riluzole is approved for ALS treatment. We have investigated riluzole as a breast cancer resistance protein (BCRP) substrate by studying its brain transport in CF1 mdr1a (-/-) mice and its intracellular uptake on BeWo cells (human placental choriocarcinoma cell line). We have also investigated the effect of riluzole on BCRP expression level and on its activity using the prazocin as a test probe for brain transport and intracellular uptake. Assays on mdr1a (-/-) mice and BeWo cells showed a higher uptake of riluzole when pretreated with a BCRP inhibitor. After repeated doses of riluzole, BCRP activity was increased in CF1 mdr1a (-/-) mice, riluzole uptake was decrease and both BCRP expression and activity were increased in BeWo cells. In conclusion, we report in this study that riluzole is transported by BCRP at the BBB level and can enhance its function. These results taken with our previous studies on riluzole and P-glycoprotein show that drug-drug interactions between riluzole and efflux transporters substrates may occur at the BBB level and should be taken into account in future clinical trial design in ALS.

  11. Inhibition of Multidrug Resistance-Linked P-Glycoprotein (ABCB1) Function by 5′-Fluorosulfonylbenzoyl 5′-Adenosine: Evidence for an ATP Analog That Interacts With Both Drug-Substrate- and Nucleotide-Binding Sites†

    PubMed Central

    Ohnuma, Shinobu; Chufan, Eduardo; Nandigama, Krishnamachary; Miller Jenkins, Lisa M.; Durell, Stewart R.; Appella, Ettore; Sauna, Zuben E.; Ambudkar, Suresh V.

    2011-01-01

    5′-fluorosulfonylbenzonyl 5′-adenosine (FSBA) is an ATP analog that covalently modifies several residues in the nucleotide-binding domains (NBDs) of several ATPases, kinases and other proteins. P-glycoprotein (P-gp, ABCB1) is a member of the ATP-binding cassette (ABC) transporter superfamily that utilizes energy from ATP hydrolysis for the efflux of amphipathic anticancer agents from cancer cells. We investigated the interactions of FSBA with P-gp to study the catalytic cycle of ATP hydrolysis. Incubation of P-gp with FSBA inhibited ATP hydrolysis (IC50= 0.21 mM) and the binding of 8-azido[α–32P]ATP (IC50= 0.68 mM). In addition, 14C-FSBA crosslinks to P-gp, suggesting that FSBA-mediated inhibition of ATP hydrolysis is irreversible due to covalent modification of P-gp. However, when the NBDs were occupied with a saturating concentration of ATP prior to treatment, FSBA stimulated ATP hydrolysis by P-gp. Furthermore, FSBA inhibited the photocrosslinking of P-gp with [125I]-Iodoaryl-azidoprazosin (IAAP; IC50 = 0.17 mM). As IAAP is a transport substrate for P-gp, this suggests that FSBA affects not only the NBDs, but also the transport-substrate site in the transmembrane domains. Consistent with these results, FSBA blocked efflux of rhodamine 123 from P-gp-expressing cells. Additionally, mass spectrometric analysis identified FSBA crosslinks to residues within or nearby the NBDs but not in the transmembrane domains and docking of FSBA in a homology model of human P-gp NBDs supports the biochemical studies. Thus, FSBA is an ATP analog that interacts with both the drug-binding and ATP-binding sites of P-gp, but fluorosulfonyl-mediated crosslinking is observed only at the NBDs. PMID:21452853

  12. AKT as locus of cancer multidrug resistance and fragility.

    PubMed

    Radisavljevic, Ziv

    2013-04-01

    Complexity and robustness of cancer hypoxic microenvironment are supported by the robust signaling networks of autocrine and paracrine elements creating powerful interactome for multidrug resistance. These elements generate a positive feedback loops responsible for the extreme robustness and multidrug resistance in solid cancer, leukemia, myeloma, and lymphoma. Phosphorylated AKT is a cancer multidrug resistance locus. Targeting that locus by oxidant/antioxidant balance modulation, positive feedback loops are converted into negative feedback loops, leading to disappearance of multidrug resistance. This is a new principle for targeting cancer multidrug resistance by the locus chemotherapy inducing a phenomenon of loops conversion. Copyright © 2012 Wiley Periodicals, Inc.

  13. Ceramide glycosylation potentiates cellular multidrug resistance.

    PubMed

    Liu, Y Y; Han, T Y; Giuliano, A E; Cabot, M C

    2001-03-01

    Ceramide glycosylation, through glucosylceramide synthase (GCS), allows cellular escape from ceramide-induced programmed cell death. This glycosylation event confers cancer cell resistance to cytotoxic anticancer agents [Liu, Y. Y., Han, T. Y., Giuliano, A. E., and M. C. Cabot. (1999) J. Biol. Chem. 274, 1140-1146]. We previously found that glucosylceramide, the glycosylated form of ceramide, accumulates in adriamycin-resistant breast carcinoma cells, in vinblastine-resistant epithelioid carcinoma cells, and in tumor specimens from patients showing poor response to chemotherapy. Here we show that multidrug resistance can be increased over baseline and then totally reversed in human breast cancer cells by GCS gene targeting. In adriamycin-resistant MCF-7-AdrR cells, transfection of GCS upgraded multidrug resistance, whereas transfection of GCS antisense markedly restored cellular sensitivity to anthracyclines, Vinca alkaloids, taxanes, and other anticancer drugs. Sensitivity to the various drugs by GCS antisense transfection increased 7- to 240-fold and was consistent with the resumption of ceramide-caspase-apoptotic signaling. GCS targeting had little influence on cellular sensitivity to either 5-FU or cisplatin, nor did it modify P-glycoprotein expression or rhodamine-123 efflux. GCS antisense transfection did enhance rhodamine-123 uptake compared with parent MCF-7-AdrR cells. This study reveals that GCS is a novel mechanism of multidrug resistance and positions GCS antisense as an innovative force to overcome multidrug resistance in cancer chemotherapy.

  14. Multidrug-Resistant Mycobacterium tuberculosis, Southwestern Colombia

    PubMed Central

    Nieto, Luisa Maria; Rozo, Juan C.; Forero, Liliana; van Soolingen, Dick

    2011-01-01

    Using spoligotyping, we identified 13 genotypes and 17 orphan types among 160 Mycobacterium tuberculosis isolates from patients in Valle del Cauca, Colombia. The Beijing genotype represented 15.6% of the isolates and was correlated with multidrug-resistant tuberculosis, female sex of the patients, and residence in Buenaventura and may represent a new public health threat. PMID:21762581

  15. Multidrug Resistance: Physiological Principles and Nanomedical Solutions

    PubMed Central

    Storm, Gert; Kiessling, Fabian; Lammers, Twan

    2014-01-01

    Multidrug (MDR) resistance is a pathophysiological phenomenon employed by cancer cells which limits the prolonged and effective use of chemotherapeutic agents. MDR is primarily based on the over-expression of drug efflux pumps in the cellular membrane. Prominent examples of such efflux pumps, which belong to the ATP-binding cassette (ABC) superfamily of proteins, are Pgp (P-glycoprotein) and MRP (multidrug resistance-associated protein), nowadays officially known as ABCB1 and ABCC1. Over the years, several strategies have been evaluated to overcome MDR, based not only on the use of low-molecular-weight MDR modulators, but also on the implementation of 1-100(0) nm-sized drug delivery systems. In the present manuscript, after introducing the most important physiological principles of MDR, we summarize prototypic nanomedical strategies to overcome multidrug resistance, including the use of carrier materials with intrinsic anti-MDR properties, the use of nanomedicines to modify the mode of cellular uptake, and the co-formulation of chemotherapeutic drugs together with low- and high-molecular-weight MDR inhibitors within a single drug delivery system. While certain challenges still need to be overcome before such constructs and concepts can be widely applied in the clinic, the insights obtained and the progress made strongly suggest that nanomedicine formulations hold significant potential for improving the treatment of multidrug-resistant malignancies. PMID:24120954

  16. [Travellers and multi-drug resistance bacteria].

    PubMed

    Takeshita, Nozomi

    2012-02-01

    The number of international travellers has increased. There is enormous diversity in medical backgrounds, purposes of travel, and travelling styles among travellers. Travellers are hospitalized abroad because of exotic and common diseases via medical tourism. This is one way of transporting and importing human bacteria between countries, including multi-drug resistant organisms. In developing countries, the antimicrobial resistance in Shigella sp. and Salmonella sp. have been a problem, because of this trend, the first choice of antibiotics has changed in some countries. Community acquired infections as well as hospital acquired infections with MRSA, multi-drug resistance (MDR) Pseudomonas aeruginosa, and ESBL have been a problem. This review will discuss the risk of MDR bacterial infectious diseases for travellers.

  17. Multidrug-resistant Acinetobacter meningitis in children

    PubMed Central

    Shah, Ira; Kapdi, Muznah

    2016-01-01

    Acinetobacter species have emerged as one of the most troublesome pathogens for healthcare institutions globally. In more recent times, nosocomial infections involving the central nervous system, skin and soft tissue, and bone have emerged as highly problematic. Acinetobacter species infection is common in intensive care units; however, Acinetobacter baumannii meningitis is rarely reported. Here, we report two cases of Acinetobacter baumannii meningitis which was multidrug resistance and ultimately required the carbapenem group of drugs for the treatment.

  18. Dissection of mechanistic principles of a secondary multidrug efflux protein.

    PubMed

    Fluman, Nir; Ryan, Christopher M; Whitelegge, Julian P; Bibi, Eitan

    2012-09-14

    Multidrug transporters are ubiquitous efflux pumps that provide cells with defense against various toxic compounds. In bacteria, which typically harbor numerous multidrug transporter genes, the majority function as secondary multidrug/proton antiporters. Proton-coupled secondary transport is a fundamental process that is not fully understood, largely owing to the obscure nature of proton-transporter interactions. Here we analyzed the substrate/proton coupling mechanism in MdfA, a model multidrug/proton antiporter. By measuring the effect of protons on substrate binding and by directly measuring proton binding and release, we show that substrates and protons compete for binding to MdfA. Our studies strongly suggest that competition is an integral feature of secondary multidrug transport. We identified the proton-binding acidic residue and show that, surprisingly, the substrate binds at a different site. Together, the results suggest an interesting mode of indirect competition as a mechanism of multidrug/proton antiport.

  19. Multidrug efflux pumps in Staphylococcus aureus and their clinical implications.

    PubMed

    Jang, Soojin

    2016-01-01

    Antibiotic resistance is rapidly spreading among bacteria such as Staphylococcus aureus, an opportunistic bacterial pathogen that causes a variety of diseases in humans. For the last two decades, bacterial multidrug efflux pumps have drawn attention due to their potential association with clinical multidrug resistance. Numerous researchers have demonstrated efflux-mediated resistance in vitro and in vivo and found novel multidrug transporters using advanced genomic information about bacteria. This article aims to provide a concise summary of multidrug efflux pumps and their important clinical implications, focusing on recent findings concerning S. aureus efflux pumps.

  20. Multidrug resistance in pediatric urinary tract infections.

    PubMed

    Gaspari, Romolo J; Dickson, Eric; Karlowsky, James; Doern, Gary

    2006-01-01

    Urinary tract infections (UTIs) represent a common infection in the pediatric population. Escherichia coli is the most common uropathogen in children, and antimicrobial resistance in this species complicates the treatment of pediatric UTIs. Despite the impact of resistance on empiric antibiotic choice, there is little data on multidrug resistance in pediatric patients. In this paper, we describe characteristics of multidrug-resistant E. coli in pediatric patients using a large national database of uropathogens antimicrobial sensitivities. Antimicrobial susceptibility patterns to commonly prescribed antibiotics were performed on uropathogens isolated from children presenting to participating hospitals between 1999 and 2001. Data were analyzed separately for four pediatric age groups. Single and multidrug resistance to ampicillin, amoxicillin-clavulanate, cefazolin, ciprofloxacin, nitrofurantoin, and trimethoprim-sulfamethoxazole (TMP-SMX) were performed on all specimens. There were a total of 11,341 E. coli urine cultures from 343 infants (0-4 weeks), 1,801 toddlers (5 weeks-24 months), 6,742 preteens (2-12 years), and 2,455 teens (13-17 years). E. coli resistance to ampicillin peaked in toddlers (52.8%) but was high in preteens (52.1%), infants (50.4%), and teens (40.6%). Resistance to two or more antibiotics varied across age groups, with toddlers (27%) leading preteens (23.1%), infants (21%), and teens (15.9%). Resistance to three or more antibiotics was low in all age groups (range 3.1-5.2%). The most common co-resistance in all age groups was ampicillin/TMP-SMZ. In conclusion, less than half of all pediatric UTIs are susceptible to all commonly used antibiotics. In some age groups, there is a significant percentage of co-resistance between the two most commonly used antibiotics (ampicillin and TMP-SMZ).

  1. Multidrug-resistant tuberculosis in central Asia.

    PubMed

    Cox, Helen Suzanne; Orozco, Juan Daniel; Male, Roy; Ruesch-Gerdes, Sabine; Falzon, Dennis; Small, Ian; Doshetov, Darebay; Kebede, Yared; Aziz, Mohammed

    2004-05-01

    Multidrug-resistant tuberculosis (MDR-TB) has emerged as a major threat to TB control, particularly in the former Soviet Union. To determine levels of drug resistance within a directly observed treatment strategy (DOTS) program supported by Médecins Sans Frontières in two regions in Uzbekistan and Turkmenistan, Central Asia, we conducted a cross-sectional survey of smear-positive TB patients in selected districts of Karakalpakstan (Uzbekistan) and Dashoguz (Turkmenistan). High levels of MDR-TB were found in both regions. In Karakalpakstan, 14 (13%) of 106 new patients were infected with MDR-TB; 43 (40%) of 107 previously treated patients were similarly infected. The proportions for Dashoguz were 4% (4/105 patients) and 18% (18/98 patients), respectively. Overall, 27% of patients with positive smear results whose infections were treated through the DOTS program in Karakalpakstan and 11% of similar patients in Dashoguz were infected with multidrug-resistant strains of TB on admission. These results show the need for concerted action by the international community to contain transmission and reduce the effects of MDR-TB.

  2. Reversal of multidrug resistance by surfactants.

    PubMed Central

    Woodcock, D. M.; Linsenmeyer, M. E.; Chojnowski, G.; Kriegler, A. B.; Nink, V.; Webster, L. K.; Sawyer, W. H.

    1992-01-01

    Cremophor EL, a pharmacologically inactive solubilising agent, has been shown to reverse multidrug resistance (MDR). Using flow cytometric evaluation of equilibrium intracellular levels of daunorubicin (DNR), we found that eight other surface active agents will also reverse MDR. All the active detergents contain polyethoxylated moieties but have no similarities in their hydrophobic components. The properties of three polyethoxylated surfactants that showed the lowest toxicities, Cremophor, Tween 80 and Solutol HS15, were examined in more detail. The concentrations of Tween 80 and Solutol required to reverse DNR exclusion were 10-fold lower than for Cremophor. However while concentrations greater than or equal to 1:10(2) of the former two surfactants resulted in breakdown of cells, even 1:10 of Cremophor did not lyse cells. Studies of the effects of Cremophor on the uptake and efflux of DNR in normal and MDR cell types showed that Cremophor increases intracellular DNR primarily by locking the rapid efflux from the cells. This blockage of drug efflux may be mediated by a substantial alteration in the fluidity of cell membranes induced by Cremophor, as shown by decreased fluorescence anisotropy of a membrane probe. Consistent with these data, coinjection of adriamycin plus Cremophor into mice carrying a multidrug resistant P388 transplantable tumour significantly increased the survival time of the mice compared with adriamycin treatment alone. PMID:1637678

  3. Role of multidrug transporters in neurotherapeutics

    PubMed Central

    Jose, Manna; Thomas, Sanjeev V.

    2009-01-01

    Acquired resistance to antibiotics and other chemotherapeutic agents is a major problem in the practice of neurology and other branches of medicine. There are several mechanisms by which drug resistance is acquired. Multidrug transporters are important glycoproteins located in the cell membrane that actively transport small lipophilic molecules from one side of the cell membrane to the other, most often from the inside to the outside of a cell. They have important protective role yet may prove inconvenient in chemotherapy. In epilepsy and other disorders this mechanism augments the elimination of drugs from their target cells and leads to drug resistance. In this review, we have discussed the biochemical characteristics of multidrug transporters and the mechanisms by which these membrane bound proteins transport their target molecules from one side to the other side of the cell membrane. We have also briefly discussed the application of this knowledge in the understanding of drug resistance in various clinical situations with particular reference to neurological disorders. These proteins located in the placenta have important role in preventing the transplacental movement of drugs in to the fetus which may result in congenital malformations or other defects. The molecular genetic mechanisms that govern the expression of these important proteins are discussed briefly. The potential scope to develop targeted chemotherapeutic agents is also discussed. PMID:20142853

  4. NANOPREPARATIONS TO OVERCOME MULTIDRUG RESISTANCE IN CANCER

    PubMed Central

    Patel, Niravkumar R.; Pattni, Bhushan S.; Abouzeid, Abraham H.; Torchilin, Vladimir P.

    2013-01-01

    Multidrug resistance is the most widely exploited phenomenon by which cancer eludes chemotherapy. Broad variety of factors, ranging from the cellular ones, such as over-expression of efflux transporters, defective apoptotic machineries, and altered molecular targets, to the physiological factors such as higher interstitial fluid pressure, low extracellular pH, and formation of irregular tumor vasculature are responsible for multidrug resistance. A combination of various undesirable factors associated with biological surroundings together with poor solubility and instability of many potential therapeutic small & large molecules within the biological systems and systemic toxicity of chemotherapeutic agents has necessitated the need for nano-preparations to optimize drug delivery. The physiology of solid tumors presents numerous challenges for successful therapy. However, it also offers unique opportunities for the use of nanotechnology. Nanoparticles, up to 400 nm in size, have shown great promise for carrying, protecting and delivering potential therapeutic molecules with diverse physiological properties. In this review, various factors responsible for the MDR and the use of nanotechnology to overcome the MDR, the use of spheroid culture as well as the current technique of producing micro tumor tissues in vitro are discussed in detail. PMID:23973912

  5. Multidrug-resistant Tuberculosis in Central Asia

    PubMed Central

    Orozco, Juan Daniel; Male, Roy; Ruesch-Gerdes, Sabine; Falzon, Dennis; Small, Ian; Doshetov, Darebay; Kebede, Yared; Aziz, Mohammed

    2004-01-01

    Multidrug-resistant tuberculosis (MDR-TB) has emerged as a major threat to TB control, particularly in the former Soviet Union. To determine levels of drug resistance within a directly observed treatment strategy (DOTS) program supported by Médecins Sans Frontières in two regions in Uzbekistan and Turkmenistan, Central Asia, we conducted a cross-sectional survey of smear-positive TB patients in selected districts of Karakalpakstan (Uzbekistan) and Dashoguz (Turkmenistan). High levels of MDR-TB were found in both regions. In Karakalpakstan, 14 (13%) of 106 new patients were infected with MDR-TB; 43 (40%) of 107 previously treated patients were similarly infected. The proportions for Dashoguz were 4% (4/105 patients) and 18% (18/98 patients), respectively. Overall, 27% of patients with positive smear results whose infections were treated through the DOTS program in Karakalpakstan and 11% of similar patients in Dashoguz were infected with multidrug-resistant strains of TB on admission. These results show the need for concerted action by the international community to contain transmission and reduce the effects of MDR-TB. PMID:15200821

  6. Effect of oxygen on multidrug resistance in term human placenta.

    PubMed

    Javam, M; Audette, M C; Iqbal, M; Bloise, E; Gibb, W; Matthews, S G

    2014-05-01

    The placenta contains efflux transporters, including P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), that limit the passage of xenobiotics, certain hormones and nutrients from the maternal to the fetal circulation. The expression of these transporters changes with gestational age, yet the mechanisms involved remain unknown. However, the changes in P-gp and BCRP transporter expression coincide with those of oxygen tension in the placenta, and oxygen tension has been shown to modulate P-gp and BCRP expression in other tissues. The objective of this study was to investigate the effects of oxygen tension on P-gp and BCRP expression in the term human placenta. Following equilibration in culture (96 h), term placental explants (n = 7) were cultured in 3% or 20% oxygen for 24 and 48 h. Culture medium was collected every 24 h to measure lactate dehydrogenase (LDH; explant viability) and human chorionic gonadotropin (hCG; syncytiotrophoblast function). P-gp (encoded by ABCB1) and BCRP (encoded by ABCG2) protein and mRNA, as well as VEGFA mRNA were measured using western blot and qRT-PCR. P-gp localization was determined using immunofluorescence. Oxygen tension had a significant effect on P-gp expression, with ABCB1/P-gp mRNA and protein levels increased in the hypoxic condition (3% O2) after 48 h (p < 0.05). VEGFA mRNA was elevated by hypoxia at both 24 and 48 h (p < 0.05). In contrast, placental ABCG2/BCRP mRNA and protein expression were stable with changes in oxygen tension. We identified profound differences in the glycosylation of P-gp between cultured and non-cultured placental tissue, with cultured explants expressing deglycosylated P-gp. These findings demonstrate that, at term, the expression of placental P-gp, is regulated by oxygen tension. This suggests that changes in oxygenation of the placenta in the third trimester may alter levels of placental P-gp, and in doing so alter fetal exposure to P-gp substrates, including xenobiotics and certain

  7. Studies on pyrrolopyrimidines as selective inhibitors of multidrug-resistance-associated protein in multidrug resistance.

    PubMed

    Wang, Shouming; Folkes, Adrian; Chuckowree, Irina; Cockcroft, Xiaoling; Sohal, Sukhjit; Miller, Warren; Milton, John; Wren, Stephen P; Vicker, Nigel; Depledge, Paul; Scott, John; Smith, Lyndsay; Jones, Hazel; Mistry, Prakash; Faint, Richard; Thompson, Deanne; Cocks, Simon

    2004-03-11

    Multidrug resistance mediated by P-glycoprotein (Pgp) or multidrug-resistance-associated protein (MRP) remains a major obstacle for successful treatment of cancer. Inhibition of Pgp and MRP transport is important for high efficacy of anticancer drugs. While several Pgp inhibitors have entered clinical trials, the development of specific MRP1 inhibitors is still in its infancy. In our screening program, we have identified a pyrrolopyrimidine (4) as a novel and selective MRP1 inhibitor. Subsequent SAR work on the 4-position of the template revealed the phenethylpiperazine side chain as a potent replacement of the benzylthio group of the lead molecule. Introduction of groups at the 2-position seems to have no detrimental effect on activity. Modifications to the nitrile group at the 7-position resulted in the identification of analogues with groups, such as amides, with superior pharmacokinetic profiles. In vivo efficacy has been demonstrated by xenograft studies on selected compounds.

  8. BRCA2-deficient sarcomatoid mammary tumors exhibit multidrug resistance.

    PubMed

    Jaspers, Janneke E; Sol, Wendy; Kersbergen, Ariena; Schlicker, Andreas; Guyader, Charlotte; Xu, Guotai; Wessels, Lodewyk; Borst, Piet; Jonkers, Jos; Rottenberg, Sven

    2015-02-15

    Pan- or multidrug resistance is a central problem in clinical oncology. Here, we use a genetically engineered mouse model of BRCA2-associated hereditary breast cancer to study drug resistance to several types of chemotherapy and PARP inhibition. We found that multidrug resistance was strongly associated with an EMT-like sarcomatoid phenotype and high expression of the Abcb1b gene, which encodes the drug efflux transporter P-glycoprotein. Inhibition of P-glycoprotein could partly resensitize sarcomatoid tumors to the PARP inhibitor olaparib, docetaxel, and doxorubicin. We propose that multidrug resistance is a multifactorial process and that mouse models are useful to unravel this.

  9. Structural basis of RND-type multidrug exporters.

    PubMed

    Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

    2015-01-01

    Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient

  10. Structural basis of RND-type multidrug exporters

    PubMed Central

    Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

    2015-01-01

    Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient

  11. Multidrug Efflux Systems in Microaerobic and Anaerobic Bacteria

    PubMed Central

    Xu, Zeling; Yan, Aixin

    2015-01-01

    Active drug efflux constitutes an important mechanism of antibiotic and multidrug resistance in bacteria. Understanding the distribution, expression, and physiological functions of multidrug efflux pumps, especially under physiologically and clinically relevant conditions of the pathogens, is the key to combat drug resistance. In animal hosts, most wounded, infected and inflamed tissues display low oxygen tensions. In this article, we summarize research development on multidrug efflux pumps in the medicinally relevant microaerobic and anaerobic pathogens and their implications in the effort to combat drug-resistant infections. PMID:27025630

  12. Breaking the Spell: Combating Multidrug Resistant 'Superbugs'.

    PubMed

    Khan, Shahper N; Khan, Asad U

    2016-01-01

    Multidrug-resistant (MDR) bacteria have become a severe threat to community wellbeing. Conventional antibiotics are getting progressively more ineffective as a consequence of resistance, making it imperative to realize improved antimicrobial options. In this review we emphasized the microorganisms primarily reported of being resistance, referred as ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacteriaceae) accentuating their capacity to "escape" from routine antimicrobial regimes. The upcoming antimicrobial agents showing great potential and can serve as alternative therapeutic options are discussed. We also provided succinct overview of two evolving technologies; specifically network pharmacology and functional genomics profiling. Furthermore, In vivo imaging techniques can provide novel targets and a real time tool for potential lead molecule assessment. The employment of such approaches at prelude of a drug development process, will enables more informed decisions on candidate drug selection and will maximize or predict therapeutic potential before clinical testing.

  13. Epidemiology and Treatment of Multidrug Resistant Tuberculosis

    PubMed Central

    Mitnick, Carole D.; Appleton, Sasha C.; Shin, Sonya S.

    2010-01-01

    Multidrug resistant tuberculosis is now thought to afflict between 1 and 2 million patients annually. Although significant regional variability in the distribution of disease has been recorded, surveillance data are limited by several factors. The true burden of disease is likely underestimated. Nevertheless, the estimated burden is substantial enough to warrant concerted action. A range of approaches is possible, but all appropriate interventions require scale-up of laboratories and early treatment with regimens containing a sufficient number of second-line drugs. Ambulatory treatment for most patients, and improved infection control, can facilitate scale-up with decreased risk of nosocomial transmission. Several obstacles have been considered to preclude worldwide scale-up of treatment, mostly attributable to inadequate human, drug, and financial resources. Further delays in scale-up, however, risk continued generation and transmission of resistant tuberculosis, as well as associated morbidity and mortality. PMID:18810684

  14. Yeast ABC proteins involved in multidrug resistance.

    PubMed

    Piecuch, Agata; Obłąk, Ewa

    2014-03-01

    Pleiotropic drug resistance is a complex phenomenon that involves many proteins that together create a network. One of the common mechanisms of multidrug resistance in eukaryotic cells is the active efflux of a broad range of xenobiotics through ATP-binding cassette (ABC) transporters. Saccharomyces cerevisiae is often used as a model to study such activity because of the functional and structural similarities of its ABC transporters to mammalian ones. Numerous ABC transporters are found in humans and some are associated with the resistance of tumors to chemotherapeutics. Efflux pump modulators that change the activity of ABC proteins are the most promising candidate drugs to overcome such resistance. These modulators can be chemically synthesized or isolated from natural sources (e.g., plant alkaloids) and might also be used in the treatment of fungal infections. There are several generations of synthetic modulators that differ in specificity, toxicity and effectiveness, and are often used for other clinical effects.

  15. Radioiodinated agents for imaging multidrug resistant tumors.

    PubMed

    Kortylewicz, Zbigniew P; Augustine, Ann M; Nearman, Jessica; McGarry, Jonathon; Baranowska-Kortylewicz, Janina

    2009-03-01

    Diagnostic agents enabling characterization of multidrug resistance (MDR) in tumors can aid in the selection of chemotherapy regimens. We report here synthesis and evaluation of radiopharmaceuticals based on the second-generation MDR-reversing drug MS-209. 5-[3-{4-(2-Phenyl-2-(4'-[(125)I]iodo-phenyl)acetyl)piperazin-1-yl}-2-hydroxypropoxy]quino-line (17) was prepared from the 4'-tributylstannyl precursor (16) in >95% radiochemical yield. (16) was synthesized in a six-step process with the overall yield of 25%. In vitro studies were conducted in MES-SA (drug-sensitive) and MES-SA/Dx5 (MDR) human uterine sarcoma cell lines. In vivo studies were performed in athymic mice bearing MES-SA and MES-SA/Dx5 xenografts. The uptake of (17) is higher in MES-SA than MES-SA/Dx5 cells. The uptake and efflux of (17) depend on temperature and concentration, and indicate active transport mechanism(s). Incubation of drug sensitive MES-SA cells with verapamil or (15), a nonradioactive analog of (17), alters the cellular retention of radioactivity only marginally. However, MES-SA/Dx5 cells retain approximately 12% more of (17) when incubated with 10 muM verapamil. The addition of (15) or high concentrations of (17) also increase the uptake of (17) in MES-SA/Dx5 up to 200%, depending on the concentration and temperature. The dependence of (17) uptake on the MDR status is also evident in the ex vivo binding studies. In vivo tests in mice xenografted simultaneously with both tumor cell lines indicate distinct pharmacokinetics for each tumor. The absorption half-life in MES-SA/Dx5 xenograft is approximately 10x shorter and the mean residence time approximately 50% shorter compared to MES-SA xenograft in the same mouse. Radioiodinated derivatives of MS-209 appear to be good indicators of multidrug resistance.

  16. Multidrug-resistant typhoid fever: a review.

    PubMed

    Zaki, Syed Ahmed; Karande, Sunil

    2011-05-28

    Multidrug-resistant typhoid fever (MDRTF) is defined as typhoid fever caused by Salmonella enterica serovar Typhi strains (S. Typhi), which are resistant to the first-line recommended drugs for treatment such as chloramphenicol, ampicillin and trimethoprim-sulfamethoxazole. Since the mid-1980s, MDRTF has caused outbreaks in several countries in the developing world, resulting in increased morbidity and mortality, especially in affected children below five years of age and those who are malnourished. Two methods were used to gather the information presented in this article. First PubMed was searched for English language references to published relevant articles. Secondly, chapters on typhoid fever in standard textbooks of paediatric infectious diseases and preventive and social medicine were reviewed. Although there are no pathognomonic clinical features of MDRTF at the onset of the illness, high fever ( > 104°F), toxaemia, abdominal distension, abdominal tenderness, hepatomegaly and splenomegaly are often reported. The gold standard for the diagnosis of MDRTF is bacterial isolation of the organism in blood cultures. Ciprofloxacin and ceftriaxone are the drugs most commonly used for treatment of MDRTF and produce good clinical results. MDRTF remains a major public health problem, particularly in developing countries. Mass immunization in endemic areas with either the oral live attenuated Typhi 21a or the injectable unconjugated Vi typhoid vaccine, rational use of antibiotics, improvement in public sanitation facilities, availability of clean drinking water, promotion of safe food handling practices and public health education are vital in the prevention of MDRTF.

  17. Management of multidrug resistant bacterial endemic.

    PubMed

    Zahar, J-R; Lesprit, P

    2014-09-01

    The fight against multi-drug resistant Gram-negative bacilli (MDRGNB), especially extended-spectrum β-lactamase producing Enterobacteriaceae, is about to be lost in our country. The emergence of new resistance mechanisms to carbapenems in these Enterobacteriaceae exposes patients to a risk of treatment failure without any other therapeutic options. This dramatic situation is paradoxical because we are well aware of the 2 major factors responsible for this situation: 1) MDRO cross-transmission, associated with a low compliance to standard precautions, especially hand hygiene, and 2) overexposure of patients to antibiotics. The implementation of a "search and isolate" policy, which was justified to control the spread of some MDRO that remained rare in the country, was not associated with a better adherence to standard precautions. The antibiotic policy and the measures implemented to control antibiotic consumptions have rarely been enforced and have shown inconsistent results. Notably, no significant decrease of antibiotic consumption has been observed. There is no excuse for these poor results, because some authors evaluating the effectiveness of programs for the control of MDRO have reported their positive effects on antimicrobial resistance without any detrimental effects. It is now urgent to deal with the 2 major factors by establishing an educational and persuasive program with quantified and opposable objectives. Firstly, we have to improve the observance of hand hygiene above 70%. Secondly, we have to define and reach a target for the reduction of antibiotic consumption both in community and in hospital settings.

  18. Diversity among multidrug-resistant enterococci.

    PubMed Central

    Murray, B. E.

    1998-01-01

    Enterococci are associated with both community- and hospital-acquired infections. Even though they do not cause severe systemic inflammatory responses, such as septic shock, enterococci present a therapeutic challenge because of their resistance to a vast array of antimicrobial drugs, including cell-wall active agents, all commercially available aminoglycosides, penicillin and ampicillin, and vancomycin. The combination of the latter two occurs disproportionately in strains resistant to many other antimicrobial drugs. The propensity of enterococci to acquire resistance may relate to their ability to participate in various forms of conjugation, which can result in the spread of genes as part of conjugative transposons, pheromone-responsive plasmids, or broad host-range plasmids. Enterococcal hardiness likely adds to resistance by facilitating survival in the environment (and thus enhancing potential spread from person to person) of a multidrug-resistant clone. The combination of these attributes within the genus Enterococcus suggests that these bacteria and their resistance to antimicrobial drugs will continue to pose a challenge. PMID:9452397

  19. Mechanisms of multidrug resistance in cancer.

    PubMed

    Gillet, Jean-Pierre; Gottesman, Michael M

    2010-01-01

    The development of multidrug resistance (MDR) to chemotherapy remains a major challenge in the treatment of cancer. Resistance exists against every effective anticancer drug and can develop by numerous mechanisms including decreased drug uptake, increased drug efflux, activation of detoxifying systems, activation of DNA repair mechanisms, evasion of drug-induced apoptosis, etc. In the first part of this chapter, we briefly summarize the current knowledge on individual cellular mechanisms responsible for MDR, with a special emphasis on ATP-binding cassette transporters, perhaps the main theme of this textbook. Although extensive work has been done to characterize MDR mechanisms in vitro, the translation of this knowledge to the clinic has not been crowned with success. Therefore, identifying genes and mechanisms critical to the development of MDR in vivo and establishing a reliable method for analyzing clinical samples could help to predict the development of resistance and lead to treatments designed to circumvent it. Our thoughts about translational research needed to achieve significant progress in the understanding of this complex phenomenon are therefore discussed in a third section. The pleotropic response of cancer cells to chemotherapy is summarized in a concluding diagram.

  20. Tripartite assembly of RND multidrug efflux pumps

    NASA Astrophysics Data System (ADS)

    Daury, Laetitia; Orange, François; Taveau, Jean-Christophe; Verchère, Alice; Monlezun, Laura; Gounou, Céline; Marreddy, Ravi K. R.; Picard, Martin; Broutin, Isabelle; Pos, Klaas M.; Lambert, Olivier

    2016-02-01

    Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB-OprM and Escherichia coli AcrAB-TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA-MexB-TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components.

  1. Protein abundance of clinically relevant multidrug transporters along the entire length of the human intestine.

    PubMed

    Drozdzik, Marek; Gröer, Christian; Penski, Jette; Lapczuk, Joanna; Ostrowski, Marek; Lai, Yurong; Prasad, Bhagwat; Unadkat, Jashvant D; Siegmund, Werner; Oswald, Stefan

    2014-10-06

    Intestinal transporters are crucial determinants in the oral absorption of many drugs. We therefore studied the mRNA expression (N = 33) and absolute protein content (N = 10) of clinically relevant transporters in healthy epithelium of the duodenum, the proximal and distal jejunum and ileum, and the ascending, transversal, descending, and sigmoidal colon of six organ donors (24-54 years). In the small intestine, the abundance of nearly all studied proteins ranged between 0.2 and 1.6 pmol/mg with the exception of those of OCT3 (<0.1 pmol/mg) and PEPT1 (2.6-4.9 pmol/mg) that accounted for ∼50% of all measured transporters. OATP1A2 was not detected in any intestinal segment. ABCB1, ABCG2, PEPT1, and ASBT were significantly more abundant in jejunum and ileum than in colon. In contrast to this, the level of expression of ABCC2, ABCC3, and OCT3 was found to be highest in colon. Site-dependent differences in the levels of gene and protein expression were observed for ABCB1 and ASBT. Significant correlations between mRNA and protein levels have been found for ABCG2, ASBT, OCT3, and PEPT1 in the small intestine. Our data provide further physiological pieces of the puzzle required to predict intestinal drug absorption in humans.

  2. Structural basis and dynamics of multidrug recognition in a minimal bacterial multidrug resistance system

    PubMed Central

    Habazettl, Judith; Allan, Martin; Jensen, Pernille Rose; Sass, Hans-Jürgen; Thompson, Charles J.; Grzesiek, Stephan

    2014-01-01

    TipA is a transcriptional regulator found in diverse bacteria. It constitutes a minimal autoregulated multidrug resistance system against numerous thiopeptide antibiotics. Here we report the structures of its drug-binding domain TipAS in complexes with promothiocin A and nosiheptide, and a model of the thiostrepton complex. Drug binding induces a large transition from a partially unfolded to a globin-like structure. The structures rationalize the mechanism of promiscuous, yet specific, drug recognition: (i) a four-ring motif present in all known TipA-inducing antibiotics is recognized specifically by conserved TipAS amino acids; and (ii) the variable part of the antibiotic is accommodated within a flexible cleft that rigidifies upon drug binding. Remarkably, the identified four-ring motif is also the major interacting part of the antibiotic with the ribosome. Hence the TipA multidrug resistance mechanism is directed against the same chemical motif that inhibits protein synthesis. The observed identity of chemical motifs responsible for antibiotic function and resistance may be a general principle and could help to better define new leads for antibiotics. PMID:25489067

  3. The prevalence of antiretroviral multidrug resistance in highly active antiretroviral therapy-treated patients with HIV/AIDS between 2004 and 2009 in South Korea.

    PubMed

    Choi, Ju-yeon; Kwon, Oh-Kyung; Choi, Byeong-Sun; Kee, Mee-Kyung; Park, Mina; Kim, Sung Soon

    2014-06-01

    Highly active antiretroviral therapy (HAART) including protease inhibitors (PIs) has been used in South Korea since 1997. Currently, more than 20 types of antiretroviral drugs are used in the treatment of human immunodeficiency virus-infected/acquired immune deficiency syndrome patients in South Korea. Despite the rapid development of various antiretroviral drugs, many drug-resistant variants have been reported after initiating HAART, and the efficiency of HAART is limited by these variants. To investigate and estimate the annual antiretroviral drug resistance and prevalence of antiretroviral multi-class drug resistance in Korean patients with experience of treatment. The amplified HIV-1 pol gene in 535 patients requested for genotypic drug resistance testing from 2004 to 2009 by the Korea Centers for Disease Control and Prevention was sequenced and analyzed annually and totally. The prevalence of antiretroviral drug resistance was estimated based on "SIR" interpretation of the Stanford sequence database. Of viruses derived from 787 specimens, 380 samples (48.3%) showed at least one drug class-related resistance. Predicted NRTI drug resistance was highest at 41.9%. NNRTI showed 27.2% resistance with 23.3% for PI. The percent of annual drug resistance showed similar pattern and slightly declined except 2004 and 2005. The prevalence of multi-class drug resistance against each drug class was: NRTI/NNRTI/PI, 9.8%; NRTI/PI, 21.9%; NNRTI/PI, 10.4%; and NRTI/NNRTI, 21.5%. About 50% and less than 10% of patients infected with HIV-1 have multidrug and multiclass resistance linked to 16 antiretroviral drugs, respectively. The significance of this study lies in its larger-scale examination of the prevalence of drug-resistant variants and multidrug resistance in HAART-experienced patients in South Korea. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Bacterial Multidrug Efflux Pumps: Much More Than Antibiotic Resistance Determinants.

    PubMed

    Blanco, Paula; Hernando-Amado, Sara; Reales-Calderon, Jose Antonio; Corona, Fernando; Lira, Felipe; Alcalde-Rico, Manuel; Bernardini, Alejandra; Sanchez, Maria Blanca; Martinez, Jose Luis

    2016-02-16

    Bacterial multidrug efflux pumps are antibiotic resistance determinants present in all microorganisms. With few exceptions, they are chromosomally encoded and present a conserved organization both at the genetic and at the protein levels. In addition, most, if not all, strains of a given bacterial species present the same chromosomally-encoded efflux pumps. Altogether this indicates that multidrug efflux pumps are ancient elements encoded in bacterial genomes long before the recent use of antibiotics for human and animal therapy. In this regard, it is worth mentioning that efflux pumps can extrude a wide range of substrates that include, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals or bacterial metabolites, among others. In the current review, we present information on the different functions that multidrug efflux pumps may have for the bacterial behaviour in different habitats as well as on their regulation by specific signals. Since, in addition to their function in non-clinical ecosystems, multidrug efflux pumps contribute to intrinsic, acquired, and phenotypic resistance of bacterial pathogens, the review also presents information on the search for inhibitors of multidrug efflux pumps, which are currently under development, in the aim of increasing the susceptibility of bacterial pathogens to antibiotics.

  5. Structural basis for the blockade of MATE multidrug efflux pumps

    DOE PAGES

    Radchenko, Martha; Symersky, Jindrich; Nie, Rongxin; ...

    2015-08-06

    Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H+ or Na+ electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.0 Å resolution X-ray structures of a protonation-mimetic mutant of an H+-coupled DinF transporter, as well as of an H+-coupled DinF and a Na+-coupled NorM transporters in complexes with verapamil, a small-molecule pharmaceutical that inhibits MATE-mediated multidrug extrusion. Combining structure-inspired mutational and functional studies, we confirm the biological relevance of our crystalmore » structures, reveal the mechanistic differences among MATE transporters, and suggest how verapamil inhibits MATE-mediated multidrug efflux. Our findings offer insights into how MATE transporters extrude chemically and structurally dissimilar drugs and could inform the design of new strategies for tackling multidrug resistance.« less

  6. Structural basis for the blockade of MATE multidrug efflux pumps

    SciTech Connect

    Radchenko, Martha; Symersky, Jindrich; Nie, Rongxin; Lu, Min

    2015-08-06

    Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H+ or Na+ electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.0 Å resolution X-ray structures of a protonation-mimetic mutant of an H+-coupled DinF transporter, as well as of an H+-coupled DinF and a Na+-coupled NorM transporters in complexes with verapamil, a small-molecule pharmaceutical that inhibits MATE-mediated multidrug extrusion. Combining structure-inspired mutational and functional studies, we confirm the biological relevance of our crystal structures, reveal the mechanistic differences among MATE transporters, and suggest how verapamil inhibits MATE-mediated multidrug efflux. Our findings offer insights into how MATE transporters extrude chemically and structurally dissimilar drugs and could inform the design of new strategies for tackling multidrug resistance.

  7. Structural basis for the blockade of MATE multidrug efflux pumps

    NASA Astrophysics Data System (ADS)

    Radchenko, Martha; Symersky, Jindrich; Nie, Rongxin; Lu, Min

    2015-08-01

    Multidrug and toxic compound extrusion (MATE) transporters underpin multidrug resistance by using the H+ or Na+ electrochemical gradient to extrude different drugs across cell membranes. MATE transporters can be further parsed into the DinF, NorM and eukaryotic subfamilies based on their amino-acid sequence similarity. Here we report the 3.0 Å resolution X-ray structures of a protonation-mimetic mutant of an H+-coupled DinF transporter, as well as of an H+-coupled DinF and a Na+-coupled NorM transporters in complexes with verapamil, a small-molecule pharmaceutical that inhibits MATE-mediated multidrug extrusion. Combining structure-inspired mutational and functional studies, we confirm the biological relevance of our crystal structures, reveal the mechanistic differences among MATE transporters, and suggest how verapamil inhibits MATE-mediated multidrug efflux. Our findings offer insights into how MATE transporters extrude chemically and structurally dissimilar drugs and could inform the design of new strategies for tackling multidrug resistance.

  8. Bacterial Multidrug Efflux Pumps: Much More Than Antibiotic Resistance Determinants

    PubMed Central

    Blanco, Paula; Hernando-Amado, Sara; Reales-Calderon, Jose Antonio; Corona, Fernando; Lira, Felipe; Alcalde-Rico, Manuel; Bernardini, Alejandra; Sanchez, Maria Blanca; Martinez, Jose Luis

    2016-01-01

    Bacterial multidrug efflux pumps are antibiotic resistance determinants present in all microorganisms. With few exceptions, they are chromosomally encoded and present a conserved organization both at the genetic and at the protein levels. In addition, most, if not all, strains of a given bacterial species present the same chromosomally-encoded efflux pumps. Altogether this indicates that multidrug efflux pumps are ancient elements encoded in bacterial genomes long before the recent use of antibiotics for human and animal therapy. In this regard, it is worth mentioning that efflux pumps can extrude a wide range of substrates that include, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals or bacterial metabolites, among others. In the current review, we present information on the different functions that multidrug efflux pumps may have for the bacterial behaviour in different habitats as well as on their regulation by specific signals. Since, in addition to their function in non-clinical ecosystems, multidrug efflux pumps contribute to intrinsic, acquired, and phenotypic resistance of bacterial pathogens, the review also presents information on the search for inhibitors of multidrug efflux pumps, which are currently under development, in the aim of increasing the susceptibility of bacterial pathogens to antibiotics. PMID:27681908

  9. Phorbol esters induce multidrug resistance in human breast cancer cells

    SciTech Connect

    Fine, R.L.; Patel, J.; Chabner, B.A.

    1988-01-01

    Mechanisms responsible for broad-based resistance to antitumor drugs derived from natural products (multidrug resistance) are incompletely understood. Agents known to reverse the multidrug-resistant phenotype (verapamil and trifluoperazine) can also inhibit the activity of protein kinase C. When the authors assayed human breast cancer cell lines for protein kinase C activity, they found that enzyme activity was 7-fold higher in the multidrug-resistance cancer cells compared with the control, sensitive parent cells. Exposure of drug-sensitive cells to the phorbol ester phorbol 12,13-dibutyate (P(BtO)/sub 2/) led to an increase in protein kinase C activity and induced a drug-resistance phenotype, whereas exposure of drug-resistant cells to P(BtO)/sub 2/ further increased drug resistance. In sensitive cells, this increased resistance was accomplished by a 3.5-fold increased phosphorylation of a 20-kDa particulate protein and a 35-40% decreased intracellular accumulation of doxorubicin and vincristine. P(BtO)/sub 2/ induced resistance to agents involved in the multidrug-resistant phenotype (doxorubicin and vincristine) but did not affect sensitivity to an unrelated alkylating agent (melphalan). The increased resistance was partially or fully reversible by the calcium channel blocker verapamil and by the calmodulin-antagonist trifluoperazine. These data suggest that stimulation of protein kinase C playus a role in the drug-transport changes in multidrug-resistant cells. This may occur through modulation of an efflux pump by protein phosphorylation.

  10. Salvage therapy for multidrug-resistant tuberculosis.

    PubMed

    Seung, K J; Becerra, M C; Atwood, S S; Alcántara, F; Bonilla, C A; Mitnick, C D

    2014-05-01

    Treatment of multidrug-resistant tuberculosis (MDR-TB), defined as Mycobacterium tuberculosis resistant to both isoniazid and rifampicin, is challenging under the best of circumstances, and particularly in resource-limited settings. For patients who remain persistently sputum-culture-positive despite therapy with second-line TB drugs, treatment options are limited, especially if disease is too advanced for resective surgery. Salvage therapy refers to the design of a regimen combining new and previously used drugs in a final effort to attain sputum conversion before declaring treatment to have failed. We retrospectively evaluated the outcomes of salvage therapy in 213 Peruvian patients. Salvage regimens included a median of two new drugs (range 1-6) and nine (range 5-13) total (new plus previously used) drugs. The most frequently used new drug was moxifloxacin, followed by capreomycin, amoxicillin-clavulanate, kanamycin and clarithromycin. Culture conversion occurred in 65 (30.5%) patients. Salvage regimens that included moxifloxacin were significantly more likely to be followed by culture conversion (OR 2.2; p 0.02). Later-generation fluoroquinolones such as moxifloxacin should be used in salvage therapy but also in the initial treatment of MDR-TB, if the best clinical strategy is to use the most effective drugs when the patient has the best chance for cure. New TB drugs are most likely to be initially used in salvage patients, in conditions similar to those described here. Close bacteriological monitoring of these patients will be essential, as useful information about the best way to use these new drugs can be gained from analysis of salvage therapy cohorts.

  11. A light in multidrug resistance: photodynamic treatment of multidrug-resistant tumors.

    PubMed

    Capella, Márcia Alves Marques; Capella, Luiz Sabbatini

    2003-01-01

    The major drawback of cancer chemotherapy is the development of multidrug-resistant (MDR) tumor cells, which are cross-resistant to a broad range of structurally and functionally unrelated agents, making it difficult to treat these tumors. In the last decade, a number of authors have studied the effects of photodynamic therapy (PDT), a combination of visible light with photosensitizing agents, on MDR cells. The results, although still inconclusive, have raised the possibility of treating MDR tumors by PDT. This review examines the growing literature concerning the responses of MDR cells to PDT, while stressing the need for the development of new photosensitizers that possess the necessary characteristics for the photodynamic treatment of this class of tumor. Copyright 2003 National Science Council, ROC and S. Karger AG, Basel

  12. Multidrug-Resistant Gram-Negative Bacilli: Infection Control Implications.

    PubMed

    Adler, Amos; Friedman, N Deborah; Marchaim, Dror

    2016-12-01

    Antimicrobial resistance is a common iatrogenic complication of both modern life and medical care. Certain multidrug resistant and extensively drug resistant Gram-negative organisms pose the biggest challenges to health care today, predominantly owing to a lack of therapeutic options. Containing the spread of these organisms is challenging, and in reality, the application of multiple control measures during an evolving outbreak makes it difficult to measure the relative impact of each measure. This article reviews the usefulness of various infection control measures in containing the spread of multidrug-resistant Gram-negative bacilli. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Multidrug resistant tuberculosis diagnosed by synovial fluid analysis.

    PubMed

    van Zeller, M; Monteiro, R; Ramalho, J; Almeida, I; Duarte, R

    2012-01-01

    Tuberculosis remains a major public health problem worldwide. HIV co-infection is contributing to an increased incidence of the disease, particularly that caused by multidrug resistant strains of Mycobacterium tuberculosis (MT). We describe an HIV-infected patient with pleural and lymph node tuberculosis diagnosed by pleural effusion characteristics and biopsy specimens, without MT identification, that further presented with knee-joint involvement. Arthrocentesis allowed MT isolation and drug susceptibility testing, resulting in a diagnosis of multidrug-resistant tuberculosis and an appropriate treatment regimen. MT identification and drug susceptibility tests are very important, especially for HIV co-infected patients.

  14. Bedaquiline: a novel antitubercular drug for multidrug-resistant tuberculosis.

    PubMed

    Nagabushan, H; Roopadevi, H S

    2014-01-01

    Multidrug-resistant and extensively drug-resistant tuberculosis (TB) are emerging global health threats. Bedaquiline is a new antituberculous drug belonging to the diarylquinoline class that efficiently inhibits the adenosine triphosphate synthase enzyme of Mycobacterium tuberculosis. It is a bactericidal and long-acting drug. It inhibits both dormant as well as replicating bacterial sub-populations and thus shortens the duration of TB treatment. This drug has been approved by the Food and Drug Administration in December 2012 for the management of multidrug resistant-TB. The drug marks the introduction of a new addition to the TB armamentarium after four decades.

  15. Modulation of Bacterial Multidrug Resistance Efflux Pumps of the Major Facilitator Superfamily

    PubMed Central

    Kumar, Sanath; Mukherjee, Mun Mun; Varela, Manuel F.

    2013-01-01

    Bacterial infections pose a serious public health concern, especially when an infectious disease has a multidrug resistant causative agent. Such multidrug resistant bacteria can compromise the clinical utility of major chemotherapeutic antimicrobial agents. Drug and multidrug resistant bacteria harbor several distinct molecular mechanisms for resistance. Bacterial antimicrobial agent efflux pumps represent a major mechanism of clinical resistance. The major facilitator superfamily (MFS) is one of the largest groups of solute transporters to date and includes a significant number of bacterial drug and multidrug efflux pumps. We review recent work on the modulation of multidrug efflux pumps, paying special attention to those transporters belonging primarily to the MFS. PMID:25750934

  16. Multidrug-Resistant Acinetobacter baumannii Harboring OXA-24 Carbapenemase, Spain

    PubMed Central

    Acosta, Joshi; Merino, María; Viedma, Esther; Poza, Margarita; Sanz, Francisca; Otero, Joaquín R.; Chaves, Fernando

    2011-01-01

    In February 2006, a patient colonized with a multidrug-resistant sequence type 56 Acinetobacter baumannii strain was admitted to a hospital in Madrid, Spain. This strain spread rapidly and caused a large outbreak in the hospital. Clinicians should be alert for this strain because its spread would have serious health consequences. PMID:21749771

  17. Characterization of a multidrug-resistant, novel Bacteroides genomospecies.

    PubMed

    Salipante, Stephen J; Kalapila, Aley; Pottinger, Paul S; Hoogestraat, Daniel R; Cummings, Lisa; Duchin, Jeffrey S; Sengupta, Dhruba J; Pergam, Steven A; Cookson, Brad T; Butler-Wu, Susan M

    2015-01-01

    Metronidazole- and carbapenem-resistant Bacteroides fragilis are rare in the United States. We isolated a multidrug-resistant anaerobe from the bloodstream and intraabdominal abscesses of a patient who had traveled to India. Whole-genome sequencing identified the organism as a novel Bacteroides genomospecies. Physicians should be aware of the possibility for concomitant carbapenem- and metronidazole-resistant Bacteroides infections.

  18. [Multi-drug resistant bacteria, a complex mechanism].

    PubMed

    Hilaire, Jean-Christophe

    2013-01-01

    Bacteria are said to be multidrug resistant when they are only sensitive to a small number of antibiotics used as treatments. This problem of resistance appeared in hospitals soon after antibiotics were first used. In the 1960s, strains of staphylococcus became resistant to penicillin.

  19. Multidrug-Resistant Staphylococcus aureus in US Meat and Poultry

    PubMed Central

    Waters, Andrew E.; Contente-Cuomo, Tania; Buchhagen, Jordan; Liu, Cindy M.; Watson, Lindsey; Pearce, Kimberly; Foster, Jeffrey T.; Bowers, Jolene; Driebe, Elizabeth M.; Engelthaler, David M.; Keim, Paul S.

    2011-01-01

    We characterized the prevalence, antibiotic susceptibility profiles, and genotypes of Staphylococcus aureus among US meat and poultry samples (n = 136). S. aureus contaminated 47% of samples, and multidrug resistance was common among isolates (52%). S. aureus genotypes and resistance profiles differed significantly among sample types, suggesting food animal–specific contamination. PMID:21498385

  20. Epidemiology of Primary Multidrug-Resistant Tuberculosis, Vladimir Region, Russia.

    PubMed

    Ershova, Julia V; Volchenkov, Grigory V; Kaminski, Dorothy A; Somova, Tatiana R; Kuznetsova, Tatiana A; Kaunetis, Natalia V; Cegielski, J Peter; Kurbatova, Ekaterina V

    2015-11-01

    We studied the epidemiology of drug-resistant tuberculosis (TB) in Vladimir Region, Russia, in 2012. Most cases of multidrug-resistant TB (MDR TB) were caused by transmission of drug-resistant strains, and >33% were in patients referred for testing after mass radiographic screening. Early diagnosis of drug resistance is essential for preventing transmission of MDR TB.

  1. Multidrug Resistant Mycobacterium leprae from Patients with Leprosy

    PubMed Central

    Maeda, Shinji; Matsuoka, Masanori; Nakata, Noboru; Kai, Masanori; Maeda, Yumi; Hashimoto, Ken; Kimura, Hiroaki; Kobayashi, Kazuo; Kashiwabara, Yoshiko

    2001-01-01

    Sequences of the folP1, rpoB, and gyrA genes were analyzed for 88 isolates of Mycobacterium leprae from leprosy patients in Japan, Haiti, Indonesia, Pakistan, and the Philippines. Thirteen isolates (14.8%) showed representative mutations in more than two genes, suggesting the emergence of multidrug-resistant M. leprae. PMID:11709358

  2. Multidrug-Resistant Acinetobacter baumannii in Veterinary Clinics, Germany

    PubMed Central

    Prenger-Berninghoff, Ellen; Weiss, Reinhard; van der Reijden, Tanny; van den Broek, Peterhans; Baljer, Georg; Dijkshoorn, Lenie

    2011-01-01

    An increase in prevalence of multidrug-resistant Acinetobacter spp. in hospitalized animals was observed at the Justus-Liebig-University (Germany). Genotypic analysis of 56 isolates during 2000–2008 showed 3 clusters that corresponded to European clones I–III. Results indicate spread of genotypically related strains within and among veterinary clinics in Germany. PMID:21888812

  3. Invasive Infections with Multidrug-Resistant Yeast Candida auris, Colombia

    PubMed Central

    Morales-López, Soraya E.; Parra-Giraldo, Claudia M.; Ceballos-Garzón, Andrés; Martínez, Heidys P.; Rodríguez, Gerson J.; Álvarez-Moreno, Carlos A.

    2017-01-01

    Candida auris is an emerging multidrug-resistant fungus that causes a wide range of symptoms. We report finding 17 cases of C. auris infection that were originally misclassified but correctly identified 27.5 days later on average. Patients with a delayed diagnosis of C. auris had a 30-day mortality rate of 35.2%. PMID:27983941

  4. bmr3, a third multidrug transporter gene of Bacillus subtilis.

    PubMed Central

    Ohki, R; Murata, M

    1997-01-01

    A third multidrug transporter gene named bmr3 was cloned from Bacillus subtilis. Although Bmr3 shows relatively low homology to Bmr and Blt, the substrate specificities of these three transporters overlap. Northern hybridization analysis showed that expression of the bmr3 gene was dependent on the growth phase. PMID:9023234

  5. Multidrug-Resistant Pathogens in Hospitalized Syrian Children

    PubMed Central

    Kassem, Diana Faour; Hoffmann, Yoav; Shahar, Naama; Ocampo, Smadar; Salomon, Liora; Zonis, Zeev

    2017-01-01

    Since 2013, wounded and ill children from Syria have received treatment in Israel. Screening cultures indicated that multidrug-resistant (MDR) pathogens colonized 89 (83%) of 107 children. For 58% of MDR infections, the pathogen was similar to that identified during screening. MDR screening of these children is valuable for purposes of isolation and treatment. PMID:27618479

  6. Multidrug-Resistant Tuberculosis, Somalia, 2010–2011

    PubMed Central

    Sindani, Ireneaus; Fitzpatrick, Christopher; Falzon, Dennis; Suleiman, Bashir; Arube, Peter; Adam, Ismail; Baghdadi, Samiha; Bassili, Amal

    2013-01-01

    In a nationwide survey in 2011, multidrug-resistant tuberculosis (MDR TB) was found in 5.2% and 40.8% of patients with new and previously treated TB, respectively. These levels of drug resistance are among the highest ever documented in Africa and the Middle East. This finding presents a serious challenge for TB control in Somalia. PMID:23621911

  7. Molecular Surveillance for Multidrug-Resistant Plasmodium falciparum, Cambodia

    PubMed Central

    Shah, Naman K.; Alker, Alisa P.; Sem, Rithy; Susanti, Agustina Ika; Muth, Sinuon; Maguire, Jason D.; Duong, Socheat; Ariey, Frederic; Meshnick, Steven R.

    2008-01-01

    We conducted surveillance for multidrug-resistant Plasmodium falciparum in Cambodia during 2004–2006 by assessing molecular changes in pfmdr1. The high prevalence of isolates with multiple pfmdr1 copies found in western Cambodia near the Thai border, where artesunate–mefloquine therapy failures occur, contrasts with isolates from eastern Cambodia, where this combination therapy remains highly effective. PMID:18826834

  8. Multidrug-resistant tuberculosis, Somalia, 2010-2011.

    PubMed

    Sindani, Ireneaus; Fitzpatrick, Christopher; Falzon, Dennis; Suleiman, Bashir; Arube, Peter; Adam, Ismail; Baghdadi, Samiha; Bassili, Amal; Zignol, Matteo

    2013-03-01

    In a nationwide survey in 2011, multidrug-resistant tuberculosis (MDR TB) was found in 5.2% and 40.8% of patients with new and previously treated TB, respectively. These levels of drug resistance are among the highest ever documented in Africa and the Middle East. This finding presents a serious challenge for TB control in Somalia.

  9. ABC multidrug transporters in schistosomes and other parasitic flatworms.

    PubMed

    Greenberg, Robert M

    2013-12-01

    Schistosomiasis, a neglected tropical disease affecting hundreds of millions, is caused by parasitic flatworms of the genus Schistosoma. Treatment and control of schistosomiasis relies almost exclusively on a single drug, praziquantel (PZQ), a dangerous situation for a disease of this magnitude. Though PZQ is highly effective overall, it has drawbacks, and reports of worms showing PZQ resistance, either induced in the laboratory or isolated from the field, are disconcerting. Multidrug transporters underlie multidrug resistance (MDR), a phenomenon in which resistance to a single drug is accompanied by unexpected cross-resistance to several structurally unrelated compounds. Some of the best studied multidrug transporters are members of the ancient and very large ATP-binding cassette (ABC) superfamily of efflux transporters. ABC multidrug transporters such as P-glycoprotein (Pgp; ABCB1) are also associated with drug resistance in parasites, including helminths such as schistosomes. In addition to their association with drug resistance, however, ABC transporters also function in a wide variety of physiological processes in metazoans. In this review, we examine recent studies that help define the role of schistosome ABC transporters in regulating drug susceptibility, and in normal schistosome physiology, including reproduction and excretory activity. We postulate that schistosome ABC transporters could be useful targets for compounds that enhance the effectiveness of current therapeutics as well as for agents that act as antischistosomals on their own. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  10. ABC multidrug transporters in schistosomes and other parasitic flatworms

    PubMed Central

    Greenberg, Robert M.

    2013-01-01

    Schistosomiasis, a neglected tropical disease affecting hundreds of millions, is caused by parasitic flatworms of the genus Schistosoma. Treatment and control of schistosomiasis relies almost exclusively on a single drug, praziquantel (PZQ), a dangerous situation for a disease of this magnitude. Though PZQ is highly effective overall, it has drawbacks, and reports of worms showing PZQ resistance, either induced in the laboratory or isolated from the field, are disconcerting. Multidrug transporters underlie multidrug resistance (MDR), a phenomenon in which resistance to a single drug is accompanied by unexpected cross-resistance to several structurally unrelated compounds. Some of the best studied multidrug transporters are members of the ancient and very large ATP-binding cassette (ABC) superfamily of efflux transporters. ABC multidrug transporters such as P-glycoprotein (Pgp; ABCB1) are also associated with drug resistance in parasites, including helminths such as schistosomes. In addition to their association with drug resistance, however, ABC transporters also function in a wide variety of physiological processes in metazoans. In this review, we examine recent studies that help define the role of schistosome ABC transporters in regulating drug susceptibility, and in normal schistosome physiology, including reproduction and excretory activity. We postulate that schistosome ABC transporters could be useful targets for compounds that enhance the effectiveness of current therapeutics as well as for agents that act as antischistosomals on their own. PMID:23474413

  11. Invasive Infections with Multidrug-Resistant Yeast Candida auris, Colombia.

    PubMed

    Morales-López, Soraya E; Parra-Giraldo, Claudia M; Ceballos-Garzón, Andrés; Martínez, Heidys P; Rodríguez, Gerson J; Álvarez-Moreno, Carlos A; Rodríguez, José Y

    2017-01-01

    Candida auris is an emerging multidrug-resistant fungus that causes a wide range of symptoms. We report finding 17 cases of C. auris infection that were originally misclassified but correctly identified 27.5 days later on average. Patients with a delayed diagnosis of C. auris had a 30-day mortality rate of 35.2%.

  12. Breast Cancer Resistance Protein (ABCG2) Determines Distribution of Genistein Phase II Metabolites: Reevaluation of the Roles of ABCG2 in the Disposition of Genistein

    PubMed Central

    Yang, Zhen; Zhu, Wei; Gao, Song; Yin, Taijun; Jiang, Wen

    2012-01-01

    It was recently proposed that the improved oral bioavailability of genistein aglycone and conjugates in Bcrp1(−/−) mice is mainly due to increased intestinal absorption of aglycone and subsequent elevated exposure to conjugation enzymes. Here we tested this proposed mechanism and found that intestinal absorption of genistein aglycone did not increase in Bcrp1(−/−) mice compared with wild-type mice using an in situ mouse intestinal perfusion model and that inhibition of breast cancer resistance protein (BCRP) in Caco-2 cells also did not significantly increase permeability or intracellular concentration of aglycone. Separately, we showed that 5- to 10-fold increases in exposures of conjugates and somewhat lower fold increases (<2-fold) in exposures of aglycone were apparent after both oral and intraperitoneal administration in Bcrp1(−/−) mice. In contrast, the intestinal and biliary excretion of genistein conjugates significantly decreased in Bcrp1(−/−) mice without corresponding changes in aglycone excretion. Likewise, inhibition of BCRP functions in Caco-2 cells altered polarized excretion of genistein conjugates by increasing their basolateral excretion. We further found that genistein glucuronides could be hydrolyzed back to genistein, whereas sulfates were stable in blood. Because genistein glucuronidation rates were 110% (liver) and 50% (colon) higher and genistein sulfation rates were 40% (liver) and 42% (colon) lower in Bcrp1(−/−) mice, the changes in genistein exposures are not mainly due to changes in enzyme activities. In conclusion, improved bioavailability of genistein and increased plasma area under the curve of its conjugates in Bcrp1(−/−) mice is due to altered distribution of genistein conjugates to the systemic circulation. PMID:22736306

  13. Molecular fingerprinting of multidrug-resistant Salmonella enterica serotype Typhi.

    PubMed Central

    Hampton, M. D.; Ward, L. R.; Rowe, B.; Threlfall, E. J.

    1998-01-01

    For epidemiologic investigations, the primary subdivision of Salmonella Typhi is vi-phage typing; 106 Vi-phage types are defined. For multidrug-resistant strains the most common types have been M1 (Pakistan) and E1 (India, Pakistan, Bangladesh, and the Arabian Gulf); a strain untypable with the Vi phages has been responsible for a major epidemic in Tajikistan. Most often, isolates from the Indian subcontinent have been resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracyclines, and trimethoprim; but in the 1997 Tajikistan outbreak, the epidemic strain was also resistant to ciprofloxacin. For multidrug-resistant strains, subdivision within phage type can be achieved by plasmid profile typing and pulsed-field gel electrophoresis. PMID:9621206

  14. Lipids modulate the conformational dynamics of a secondary multidrug transporter

    PubMed Central

    Martens, Chloé; Stein, Richard A; Masureel, Matthieu; Roth, Aurélie; Mishra, Smriti; Dawaliby, Rosie; Konijnenberg, Albert; Sobott, Frank; Govaerts, Cédric; Mchaourab, Hassane S

    2017-01-01

    Direct interactions with lipids have emerged as key determinants of the folding, structure and function of membrane proteins, but an understanding of how lipids modulate protein dynamics is still lacking. Here, we systematically explored the effects of lipids on the conformational dynamics of the proton-powered, multidrug transporter LmrP from Lactococcus lactis utilizing the pattern of distances between spin label pairs previously shown to fingerprint alternating access of the protein. We uncover at the molecular level how the lipid headgroups shape the conformational energy landscape of the transporter. The model emerging from our data hypothesizes a direct interaction between lipid headgroups and a conserved motif of charged residues that control the conformational equilibrium through an interplay of electrostatic interactions within the protein. Together, our data lay the foundation for a comprehensive model of secondary multidrug transport in lipid bilayers. PMID:27399258

  15. Multidrug-resistant Salmonella Typhimurium in Four Animal Facilities

    PubMed Central

    Wright, Jennifer G.; Tengelsen, Leslie A.; Smith, Kirk E.; Bender, Jeff B.; Frank, Rodney K.; Grendon, John H.; Rice, Daniel H.; Thiessen, Ann Marie B.; Gilbertson, Catherine Jo; Sivapalasingam, Sumathi; Barrett, Timothy J.; Besser, Thomas E.; Hancock, Dale D.

    2005-01-01

    In 1999 and 2000, 3 state health departments reported 4 outbreaks of gastrointestinal illness due to Salmonella enterica serotype Typhimurium in employees, clients, and client animals from 3 companion animal veterinary clinics and 1 animal shelter. More than 45 persons and companion animals became ill. Four independent investigations resulted in the testing of 19 human samples and >200 animal samples; 18 persons and 36 animals were culture-positive for S. Typhimurium. One outbreak was due to multidrug-resistant S. Typhimurium R-type ACKSSuT, while the other 3 were due to multidrug-resistant S. Typhimurium R-type ACSSuT DT104. This report documents nosocomial transmission of S. Typhimurium and demonstrates that companion animal facilities may serve as foci of transmission for salmonellae between animals and humans if adequate precautions are not followed. PMID:16102313

  16. RND multidrug efflux pumps: what are they good for?

    PubMed

    Alvarez-Ortega, Carolina; Olivares, Jorge; Martínez, José L

    2013-01-01

    Multidrug efflux pumps are chromosomally encoded genetic elements capable of mediating resistance to toxic compounds in several life forms. In bacteria, these elements are involved in intrinsic and acquired resistance to antibiotics. Unlike other well-known horizontally acquired antibiotic resistance determinants, genes encoding for multidrug efflux pumps belong to the core of bacterial genomes and thus have evolved over millions of years. The selective pressure stemming from the use of antibiotics to treat bacterial infections is relatively recent in evolutionary terms. Therefore, it is unlikely that these elements have evolved in response to antibiotics. In the last years, several studies have identified numerous functions for efflux pumps that go beyond antibiotic extrusion. In this review we present some examples of these functions that range from bacterial interactions with plant or animal hosts, to the detoxification of metabolic intermediates or the maintenance of cellular homeostasis.

  17. Successful Treatment of Multiple Multidrug Resistant Intracranial Tuberculomata

    PubMed Central

    Goldberg, Hazel F.; Mellick, Ross S.; Post, Jeffrey J.

    2016-01-01

    A 21-year-old Bangladesh-born man presented with a month history of evolving neurological symptoms in the context of a six-month history of fever, night sweats, and axillary lymphadenopathy. He was subsequently diagnosed with multiple multidrug resistant intracranial tuberculomata and was successfully treated over two years. Intracranial multidrug resistant tuberculosis has a high mortality and successful treatment is rarely reported. Management is complex and requires consideration of the penetration and likely effect of antituberculous agents within the central nervous system. We discuss the role of various antituberculous agents, the duration of therapy, the utility of corticosteroids, the value of intrathecal and systemic therapy, and the need for rapid diagnosis. PMID:28127479

  18. RND multidrug efflux pumps: what are they good for?

    PubMed Central

    Alvarez-Ortega, Carolina; Olivares, Jorge; Martínez, José L.

    2013-01-01

    Multidrug efflux pumps are chromosomally encoded genetic elements capable of mediating resistance to toxic compounds in several life forms. In bacteria, these elements are involved in intrinsic and acquired resistance to antibiotics. Unlike other well-known horizontally acquired antibiotic resistance determinants, genes encoding for multidrug efflux pumps belong to the core of bacterial genomes and thus have evolved over millions of years. The selective pressure stemming from the use of antibiotics to treat bacterial infections is relatively recent in evolutionary terms. Therefore, it is unlikely that these elements have evolved in response to antibiotics. In the last years, several studies have identified numerous functions for efflux pumps that go beyond antibiotic extrusion. In this review we present some examples of these functions that range from bacterial interactions with plant or animal hosts, to the detoxification of metabolic intermediates or the maintenance of cellular homeostasis. PMID:23386844

  19. Combination Approaches to Combat Multi-Drug Resistant Bacteria

    PubMed Central

    Worthington, Roberta J.; Melander, Christian

    2013-01-01

    The increasing prevalence of infections caused by multi-drug resistant bacteria is a global health problem that is exacerbated by the dearth of novel classes of antibiotics entering the clinic over the past 40 years. Herein we describe recent developments toward combination therapies for the treatment of multi-drug resistant bacterial infections. These efforts include antibiotic-antibiotic combinations, and the development of adjuvants that either directly target resistance mechanisms such as the inhibition of β-lactamase enzymes, or indirectly target resistance by interfering with bacterial signaling pathways such as two-component systems. We also discuss screening of libraries of previously approved drugs to identify non-obvious antimicrobial adjuvants. PMID:23333434

  20. Community-Acquired Pneumonia Due to Multidrug- and Non-Multidrug-Resistant Pseudomonas aeruginosa.

    PubMed

    Cillóniz, Catia; Gabarrús, Albert; Ferrer, Miquel; Puig de la Bellacasa, Jorge; Rinaudo, Mariano; Mensa, Josep; Niederman, Michael S; Torres, Antoni

    2016-08-01

    Pseudomonas aeruginosa is not a frequent pathogen in community-acquired pneumonia (CAP). However, in patients with severe CAP, P aeruginosa can be the etiology in 1.8% to 8.3% of patients, with a case-fatality rate of 50% to 100%. We describe the prevalence, clinical characteristics, outcomes, and risk factors associated with CAP resulting from multidrug-resistant (MDR) and non-MDR P aeruginosa. Prospective observational study of 2,023 consecutive adult patients with CAP with definitive etiology. P aeruginosa was found in 77 (4%) of the 2,023 cases with microbial etiology. In 22 (32%) of the 68 cases of P aeruginosa with antibiogram data, the isolates were MDR. Inappropriate therapy was present in 49 (64%) cases of P aeruginosa CAP, including 17/22 (77%) cases of MDR P aeruginosa CAP. Male sex, chronic respiratory disease, C-reactive protein <12.35 mg/dL, and pneumonia severity index risk class IV to V were independently associated with P aeruginosa CAP. Prior antibiotic treatment was more frequent in MDR P aeruginosa CAP compared with non-MDR P aeruginosa (58% vs 29%, P = .029), and was the only risk factor associated with CAP resulting from MDR P aeruginosa. In the multivariate analysis, age ≥65 years, CAP resulting from P aeruginosa, chronic liver disease, neurologic disease, nursing home, criteria of ARDS, acute renal failure, ICU admission, and inappropriate empiric treatment were the factors associated with 30-day mortality. P aeruginosa is an individual risk factor associated with mortality in CAP. The risk factors described can help clinicians to suspect P aeruginosa and MDR P aeruginosa. Copyright © 2016 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

  1. Isolation of multidrug-resistant Salmonella in Singapore

    PubMed Central

    Phoon, Yee Wei; Chan, Yuen Yue Candice; Koh, Tze Hsien

    2015-01-01

    Multidrug-resistant Salmonella is a well-recognised problem worldwide, especially in developing countries such as India, where non-typhoidal Salmonella infections and enteric fever are endemic. Antimicrobial resistance, particularly to fluoroquinolones, is common and leads to the frequent use of alternative agents, such as azithromycin. We herein describe the first reported case of azithromycin-resistant Salmonella gastroenteritis in a Singaporean patient. PMID:26311915

  2. Tackling Threats and Future Problems of Multidrug-Resistant Bacteria.

    PubMed

    Medina, Eva; Pieper, Dietmar Helmut

    2016-01-01

    With the advent of the antibiotic era, the overuse and inappropriate consumption and application of antibiotics have driven the rapid emergence of multidrug-resistant pathogens. Antimicrobial resistance increases the morbidity, mortality, length of hospitalization and healthcare costs. Among Gram-positive bacteria, Staphylococcus aureus (MRSA) and multidrug-resistant (MDR) Mycobacterium tuberculosis, and among the Gram-negative bacteria, extended-spectrum beta-lactamase (ESBLs)-producing bacteria have become a major global healthcare problem in the 21st century. The pressure to use antibiotics guarantees that the spread and prevalence of these as well as of future emerging multidrug-resistant pathogens will be a persistent phenomenon. The unfeasibility of reversing antimicrobial resistance back towards susceptibility and the critical need to treat bacterial infection in modern medicine have burdened researchers and pharmaceutical companies to develop new antimicrobials effective against these difficult-to-treat multidrug-resistant pathogens. However, it can be anticipated that antibiotic resistance will continue to develop more rapidly than new agents to treat these infections become available and a better understanding of the molecular, evolutionary and ecological mechanisms governing the spread of antibiotic resistance is needed. The only way to curb the current crisis of antimicrobial resistance will be to develop entirely novel strategies to fight these pathogens such as combining antimicrobial drugs with other agents that counteract and obstruct the antibiotic resistant mechanisms expressed by the pathogen. Furthermore, as many antibiotics are often inappropriately prescribed, a more personalized approach based on precise diagnosis tools will ensure that proper treatments can be promptly applied leading to more targeted and effective therapies. However, in more general terms, also the overall use and release of antibiotics in the environment needs to be

  3. Multidrug-Resistant Escherichia fergusonii: a Case of Acute Cystitis▿

    PubMed Central

    Savini, Vincenzo; Catavitello, Chiara; Talia, Marzia; Manna, Assunta; Pompetti, Franca; Favaro, Marco; Fontana, Carla; Febbo, Fabio; Balbinot, Andrea; Di Berardino, Fabio; Di Bonaventura, Giovanni; Di Zacomo, Silvia; Esattore, Francesca; D'Antonio, Domenico

    2008-01-01

    We report a case in which Escherichia fergusonii, an emerging pathogen in various types of infections, was associated with cystitis in a 52-year-old woman. The offending strain was found to be multidrug resistant. Despite in vitro activity, beta-lactam treatment failed because of a lack of patient compliance with therapy. The work confirms the pathogenic potential of E. fergusonii. PMID:18256229

  4. Multidrug Resistant Shigella flexneri Infection Simulating Intestinal Intussusception

    PubMed Central

    Sreenivasan, Srirangaraj; Kali, Arunava; Pradeep, Jothimani

    2016-01-01

    Shigella enteritis remains an important cause of mortality and morbidity in all age groups, in developing as well as developed countries. Owing to the emerging resistance to multiple antibiotics among Shigella spp., it has been recognized as a major global public health concern and warrants constant monitoring of its resistance pattern. We report a case of segmental ileitis caused by non.-ESBL producing multidrug resistant Shigella flexneri in an infant clinically mimicking intussusception, which was effectively treated by ceftriaxone. PMID:27013815

  5. Multidrug Resistant Shigella flexneri Infection Simulating Intestinal Intussusception.

    PubMed

    Sreenivasan, Srirangaraj; Kali, Arunava; Pradeep, Jothimani

    2016-01-01

    Shigella enteritis remains an important cause of mortality and morbidity in all age groups, in developing as well as developed countries. Owing to the emerging resistance to multiple antibiotics among Shigella spp., it has been recognized as a major global public health concern and warrants constant monitoring of its resistance pattern. We report a case of segmental ileitis caused by non.-ESBL producing multidrug resistant Shigella flexneri in an infant clinically mimicking intussusception, which was effectively treated by ceftriaxone.

  6. Imipenem: a potent inducer of multidrug resistance in Acinetobacter baumannii.

    PubMed

    Kuo, Han-Yueh; Chang, Kai-Chih; Kuo, Jai-Wei; Yueh, Hui-Wen; Liou, Ming-Li

    2012-01-01

    This study investigated the progression of multidrug resistance upon exposure to imipenem in Acinetobacter baumannii. Eighteen A. baumannii strains, including two reference strains (ATCC 19606 and ATCC 17978), four clinical strains (AB56, AB242, AB273 and AB279) and 12 antibiotic-selected mutant strains, were used in this study. Imipenem-selected mutants were generated from imipenem-susceptible strains (ATCC 19606, ATCC 17978 and AB242) by multistep selection resistance. Amikacin-, ciprofloxacin-, colistin-, meropenem- and ceftazidime-selected mutants were also generated from the two reference strains and were used for comparison. Antibiotic susceptibilities in the absence and presence of the efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone (CCCP) and 1-(1-naphthylmethyl)-piperazine (NMP) were examined in the three imipenem-selected mutants and the three clinical multidrug-resistant (MDR) isolates. Expression profiles of the antimicrobial resistance genes in the imipenem-selected mutants and their parental strains were also determined. The results showed that imipenem was more likely, compared with the other antibiotics, to induce a MDR phenotype in the two reference strains. Differences in OXA-51-like carbapenemase, efflux pumps or/and AmpC β-lactamase expression were observed in the three imipenem-selected mutants. Moreover, a reduction in imipenem or amikacin resistance was observed when the imipenem-selected mutants and clinical isolates were exposed to NMP and CCCP. This study concluded that imipenem might be a potent inducer of multidrug resistance in A. baumannii strains. OXA-51-like carbapenemase combined with other resistance mechanisms may contribute to the development of multidrug resistance in A. baumannii. Monitoring the use of carbapenems is required to reduce the spread of MDR A. baumannii in hospitals.

  7. Multidrug-Resistant Candida: Epidemiology, Molecular Mechanisms, and Treatment.

    PubMed

    Arendrup, Maiken Cavling; Patterson, Thomas F

    2017-08-15

    Invasive Candida infections remain an important cause of morbidity and mortality, especially in hospitalized and immunocompromised or critically ill patients. A limited number of antifungal agents from only a few drug classes are available to treat patients with these serious infections. Resistance can be either intrinsic or acquired. Resistance mechanisms are not exchanged between Candida; thus, acquired resistance either emerges in response to an antifungal selection pressure in the individual patient or, more rarely, occur due to horizontal transmission of resistant strains between patients. Although multidrug resistance is uncommon, increasing reports of multidrug resistance to the azoles, echinocandins, and polyenes have occurred in several Candida species, most notably Candida glabrata and more recently Candida auris. Drivers are overall antifungal use, subtherapeutic drug levels at sites of infection/colonization, drug sequestration in the biofilm matrix, and, in the setting of outbreaks, suboptimal infection control. Moreover, recent research suggests that DNA mismatch repair gene mutations may facilitate acquisition of resistance mutations in C. glabrata specifically. Diagnosis of antifungal-resistant Candida infections is critical to the successful management of patients with these infections. Reduction of unnecessary use of antifungals via antifungal stewardship is critical to limit multidrug resistance emergence. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  8. Bacterial multidrug efflux pumps: mechanisms, physiology and pharmacological exploitations.

    PubMed

    Sun, Jingjing; Deng, Ziqing; Yan, Aixin

    2014-10-17

    Multidrug resistance (MDR) refers to the capability of bacterial pathogens to withstand lethal doses of structurally diverse drugs which are capable of eradicating non-resistant strains. MDR has been identified as a major threat to the public health of human being by the World Health Organization (WHO). Among the four general mechanisms that cause antibiotic resistance including target alteration, drug inactivation, decreased permeability and increased efflux, drug extrusion by the multidrug efflux pumps serves as an important mechanism of MDR. Efflux pumps not only can expel a broad range of antibiotics owing to their poly-substrate specificity, but also drive the acquisition of additional resistance mechanisms by lowering intracellular antibiotic concentration and promoting mutation accumulation. Over-expression of multidrug efflux pumps have been increasingly found to be associated with clinically relevant drug resistance. On the other hand, accumulating evidence has suggested that efflux pumps also have physiological functions in bacteria and their expression is subject tight regulation in response to various of environmental and physiological signals. A comprehensive understanding of the mechanisms of drug extrusion, and regulation and physiological functions of efflux pumps is essential for the development of anti-resistance interventions. In this review, we summarize the development of these research areas in the recent decades and present the pharmacological exploitation of efflux pump inhibitors as a promising anti-drug resistance intervention.

  9. A Salmonella nanoparticle mimic overcomes multidrug resistance in tumours

    PubMed Central

    Mercado-Lubo, Regino; Zhang, Yuanwei; Zhao, Liang; Rossi, Kyle; Wu, Xiang; Zou, Yekui; Castillo, Antonio; Leonard, Jack; Bortell, Rita; Greiner, Dale L.; Shultz, Leonard D.; Han, Gang; McCormick, Beth A.

    2016-01-01

    Salmonella enterica serotype Typhimurium is a food-borne pathogen that also selectively grows in tumours and functionally decreases P-glycoprotein (P-gp), a multidrug resistance transporter. Here we report that the Salmonella type III secretion effector, SipA, is responsible for P-gp modulation through a pathway involving caspase-3. Mimicking the ability of Salmonella to reverse multidrug resistance, we constructed a gold nanoparticle system packaged with a SipA corona, and found this bacterial mimic not only accumulates in tumours but also reduces P-gp at a SipA dose significantly lower than free SipA. Moreover, the Salmonella nanoparticle mimic suppresses tumour growth with a concomitant reduction in P-gp when used with an existing chemotherapeutic drug (that is, doxorubicin). On the basis of our finding that the SipA Salmonella effector is fundamental for functionally decreasing P-gp, we engineered a nanoparticle mimic that both overcomes multidrug resistance in cancer cells and increases tumour sensitivity to conventional chemotherapeutics. PMID:27452236

  10. Bacterial Multidrug Efflux Pumps of the Major Facilitator Superfamily as Targets for Modulation.

    PubMed

    Kumar, Sanath; He, Guixin; Kakarla, Prathusha; Shrestha, Ugina; Ranjana, K C; Ranaweera, Indrika; Willmon, T Mark; Barr, Sharla R; Hernandez, Alberto J; Varela, Manuel F

    2016-01-01

    Causative agents of infectious disease that are multidrug resistant bacterial pathogens represent a serious public health concern due to the increasingly difficult nature of achieving efficacious clinical treatments. Of the various acquired and intrinsic antimicrobial agent resistance determinants, integral-membrane multidrug efflux pumps of the major facilitator superfamily constitute a major mechanism of bacterial resistance. The major facilitator superfamily (MFS) encompasses thousands of known related secondary active and passive solute transporters, including multidrug efflux pumps, from bacteria to humans. This review article addresses recent developments involving the targeting by various modulators of bacterial multidrug efflux pumps from the major facilitator superfamily. It is currently of tremendous interest to modulate bacterial multidrug efflux pumps in order to eventually restore the clinical efficacy of therapeutic agents against recalcitrant bacterial infections. Such MFS multidrug efflux pumps are good targets for modulation.

  11. Linsitinib (OSI-906) antagonizes ATP-binding cassette subfamily G member 2 and subfamily C member 10-mediated drug resistance.

    PubMed

    Zhang, Hui; Kathawala, Rishil J; Wang, Yi-Jun; Zhang, Yun-Kai; Patel, Atish; Shukla, Suneet; Robey, Robert W; Talele, Tanaji T; Ashby, Charles R; Ambudkar, Suresh V; Bates, Susan E; Fu, Li-Wu; Chen, Zhe-Sheng

    2014-06-01

    In this study we investigated the effect of linsitinib on the reversal of multidrug resistance (MDR) mediated by the overexpression of the ATP-binding cassette (ABC) subfamily members ABCB1, ABCG2, ABCC1 and ABCC10. Our results indicate for the first time that linsitinib significantly potentiate the effect of anti-neoplastic drugs mitoxantrone (MX) and SN-38 in ABCG2-overexpressing cells; paclitaxel, docetaxel and vinblastine in ABCC10-overexpressing cells. Linsitinib moderately enhanced the cytotoxicity of vincristine in cell lines overexpressing ABCB1, whereas it did not alter the cytotoxicity of substrates of ABCC1. Furthermore, linsitinib significantly increased the intracellular accumulation and decreased the efflux of [(3)H]-MX in ABCG2-overexpressing cells and [(3)H]-paclitaxel in ABCC10-overexpressing cells. However, linsitinib, at a concentration that reversed MDR, did not significantly alter the expression levels of either the ABCG2 or ABCC10 transporter proteins. Furthermore, linsitinib did not significantly alter the intracellular localization of ABCG2 or ABCC10. Moreover, linsitinib stimulated the ATPase activity of ABCG2 in a concentration-dependent manner. Overall, our study suggests that linsitinib attenuates ABCG2- and ABCC10-mediated MDR by directly inhibiting their function as opposed to altering ABCG2 or ABCC10 protein expression.

  12. Substituted chromones as highly potent nontoxic inhibitors, specific for the breast cancer resistance protein.

    PubMed

    Valdameri, Glaucio; Genoux-Bastide, Estelle; Peres, Basile; Gauthier, Charlotte; Guitton, Jérôme; Terreux, Raphaël; Winnischofer, Sheila M B; Rocha, Maria E M; Boumendjel, Ahcène; Di Pietro, Attilio

    2012-01-26

    A series of 13 disubstituted chromones was synthesized. Two types of substituents, on each side of the scaffold, contributed to both the potency of ABCG2 inhibition and the cytotoxicity. The best compound, 5-(4-bromobenzyloxy)-2-(2-(5-methoxyindolyl)ethyl-1-carbonyl)-4H-chromen-4-one (6g), displayed high-affinity inhibition and low cytotoxicity, giving a markedly high therapeutic index. The chromone derivative specifically inhibited ABCG2 versus other multidrug ABC transporters and was not transported. It constitutes a highly promising candidate for in vivo chemosensitization of ABCG2-expressing tumors.

  13. Epidemiologic analysis: Prophylaxis and multidrug-resistance in surgery.

    PubMed

    Solís-Téllez, H; Mondragón-Pinzón, E E; Ramírez-Marino, M; Espinoza-López, F R; Domínguez-Sosa, F; Rubio-Suarez, J F; Romero-Morelos, R D

    Surgical site infection is defined as an infection related to the surgical procedure in the area of manipulation occurring within the first 30 postoperative days. The diagnostic criteria include: purulent drainage, isolation of microorganisms, and signs of infection. To describe the epidemiologic characteristics and differences among the types of prophylactic regimens associated with hospital-acquired infections at the general surgery service of a tertiary care hospital. The electronic case records of patients that underwent general surgery at a tertiary care hospital within the time frame of January 1, 2013 and December 31, 2014 were reviewed. A convenience sample of 728 patients was established and divided into the following groups: Group 1: n=728 for the epidemiologic study; Group 2: n=638 for the evaluation of antimicrobial prophylaxis; and Group 3: n=50 for the evaluation of multidrug-resistant bacterial strains in the intensive care unit. The statistical analysis was carried out with the SPSS 19 program, using the Mann-Whitney U test and the chi-square test. A total of 728 procedures were performed (65.9% were elective surgeries). Three hundred twelve of the patients were males and 416 were females. Only 3.98% of the patients complied with the recommended antimicrobial prophylaxis, and multidrug-resistant bacterial strains were found in the intensive care unit. A single prophylactic dose is effective, but adherence to this recommendation was not adequate. The prophylactic guidelines are not strictly adhered to in our environment. There was a significant association between the development of nosocomial infections from multidrug-resistant germs and admission to the intensive care unit. Copyright © 2016 Asociación Mexicana de Gastroenterología. Publicado por Masson Doyma México S.A. All rights reserved.

  14. Presence of Multidrug Resistant Enteric Bacteria in Dairy Farm Topsoil

    PubMed Central

    Burgos, J. M.; Ellington, B. A.; Varela, M. F.

    2008-01-01

    In addition to human and veterinary medicine, antibiotics are extensively used in agricultural settings, such as for treatment of infections, growth enhancement and prophylaxis in food animals, leading to selection of drug and multidrug resistant bacteria. In order to help circumvent the problem of bacterial antibiotic resistance, it is first necessary to understand the scope of the problem. However, is it not fully understood how widespread antibiotic resistant bacteria are in agricultural settings. The lack of such surveillance data is especially evident in dairy farm environments, such as soil. It is also unknown to what extent various physiological modulators, such as salycilate, a component of aspirin and known model modulator of multiple antibiotic resistance (mar) genes, influence bacterial multidrug resistance. We isolated and identified enteric soil bacteria from local dairy farms within Roosevelt County, NM, determined the resistance profiles to antibiotics associated with mar, such as chloramphenicol, nalidixic acid, penicillin G and tetracycline. We then purified and characterized plasmid DNA and detected mar phenotypic activity. The minimal inhibitory concentrations (MICs) of antibiotics for the isolates ranged between 6 - >50 μg/mL for chloramphenicol, 2–8 μg/mL for nalidixic acid, 25- >300 μg/mL for penicillin G and 1- > 80 μg/mL for tetracycline. On the other hand, the many of the isolates had significantly enhanced MICs for the same antibiotics in the presence of 5 mM salycilate. Plasmid DNA extracted from 12 randomly chosen isolates ranged in size between 6 and 12.5kb and in several cases conferred resistances to chloramphenicol and penicillin G. It is concluded that enteric bacteria from dairy farm topsoil are multi-drug resistant and harbor antibiotic resistance plasmids. A role for dairy topsoil in zoonosis is suggested, thus implicating this environment as a reservoir for bacterial resistance development against clinically relevant

  15. A Multidrug ABC Transporter with a Taste for Salt

    PubMed Central

    Gutmann, Daniel A. P.; Venter, Henrietta; Barrera, Nelson P.; Seeger, Markus A.; Woebking, Barbara; Matak-Vinkovic, Dijana; Balakrishnan, Lekshmy; Yao, Yao; U, Edmond C. Y.; Shilling, Richard A.; Robinson, Carol V.; Thorn, Peter; van Veen, Hendrik W.

    2009-01-01

    Background LmrA is a multidrug ATP-binding cassette (ABC) transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1) in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H+-dependent, secondary-active transport reaction. The mechanism and physiological context of this reaction are not known. Methodology/Principal Findings We examined ion transport by LmrA in electrophysiological experiments and in transport studies using radioactive ions and fluorescent ion-selective probes. Here we show that LmrA itself can transport NaCl by a similar secondary-active mechanism as observed for ethidium bromide, by mediating apparent H+-Na+-Cl− symport. Remarkably, LmrA activity significantly enhances survival of high-salt adapted lactococcal cells during ionic downshift. Conclusions/Significance The observations on H+-Na+-Cl− co-transport substantiate earlier suggestions of H+-coupled transport by LmrA, and indicate a novel link between the activity of LmrA and salt stress. Our findings demonstrate the relevance of investigations into the bioenergetics of substrate translocation by ABC transporters for our understanding of fundamental mechanisms in this superfamily. This study represents the first use of electrophysiological techniques to analyze substrate transport by a purified multidrug transporter. PMID:19593434

  16. Reversers of the multidrug resistance transporter P-glycoprotein.

    PubMed

    Stein, Wilfred D

    2002-05-01

    Multidrug resistance can arise from the presence of the membrane-bound pump, P-glycoprotein, in a tumor. Major efforts have been made to develop inhibitors of this pump, and a number of promising blockers have reached late stages of clinical trials. The kinetics of the inhibition of P-glycoprotein is complex, with binding sites that can interact synergistically. Reversers of increased affinity and specificity could, in principle, be developed on the basis of these synergies, and offer some promise in cancer therapeutics.

  17. Molecular Dynamics Computer Simulations of Multidrug RND Efflux Pumps.

    PubMed

    Ruggerone, Paolo; Vargiu, Attilio V; Collu, Francesca; Fischer, Nadine; Kandt, Christian

    2013-01-01

    Over-expression of multidrug efflux pumps of the Resistance Nodulation Division (RND) protein super family counts among the main causes for microbial resistance against pharmaceuticals. Understanding the molecular basis of this process is one of the major challenges of modern biomedical research, involving a broad range of experimental and computational techniques. Here we review the current state of RND transporter investigation employing molecular dynamics simulations providing conformational samples of transporter components to obtain insights into the functional mechanism underlying efflux pump-mediated antibiotics resistance in Escherichia coli and Pseudomonas aeruginosa.

  18. Recurrence after treatment for pulmonary multidrug-resistant tuberculosis.

    PubMed

    Becerra, Mercedes C; Appleton, Sasha C; Franke, Molly F; Chalco, Katiuska; Bayona, Jaime; Murray, Megan B; Mitnick, Carole D

    2010-09-15

    We estimated the proportion of recurrence within 2 years among adults cured by individualized multidrug-resistant tuberculosis regimens in Peru. Among 310 individuals with at least 24 months of follow-up, 16 experienced an episode of recurrent tuberculosis. If we assume the worst for treatment effectiveness-that all 16 episodes were caused by the original tuberculosis strain-then 5.2% (95% confidence interval, 3.0%-8.2%) experienced true relapse. This is an upper-bound estimate of relapse on which new regimens must improve.

  19. Clonal distribution of multidrug-resistant Enterobacter cloacae.

    PubMed

    Girlich, Delphine; Poirel, Laurent; Nordmann, Patrice

    2015-04-01

    A multilocus sequence typing (MLST) scheme including 7 housekeeping genes was used to evaluate whether the current spread of multidrug-resistant Enterobacter cloacae isolates worldwide might be associated to specific successful clones. Fifty E. cloacae clinical isolates of worldwide origin, with various β-lactamase content, and recovered at different periods of time were studied. Forty-four sequence types were identified, highlighting a high clonal diversity with 3 main lineages. This study revealed that a precise identification of the isolates by sequencing of the chromosomal ampC gene of E. cloacae would provide a significant added value to improve the reliability of the MLST scheme.

  20. Clinical implications of the global multidrug-resistant tuberculosis epidemic.

    PubMed

    Kumar, Kartik; Abubakar, Ibrahim

    2015-12-01

    Multidrug-resistant tuberculosis (MDR TB) is a significant threat to global health estimated to account for nearly half a million new cases and over 200,000 deaths in 2013. The number of MDR TB cases in the UK has risen over the last 15 years, with ever more complex clinical cases and associated challenging public health and societal implications. In this review, we provide an overview of the epidemiology of MDR TB globally and in the UK, outline the clinical management of MDR TB and summarise recent advances in diagnostics and prospects for new treatment.

  1. [Multidrug-resistant tuberculosis: current epidemiology, therapeutic regimens, new drugs].

    PubMed

    Gómez-Ayerbe, C; Vivancos, M J; Moreno, S

    2016-09-01

    Multidrug and extensively resistant tuberculosis are especially severe forms of the disease for which no efficacious therapy exists in many cases. All the countries in the world have registered cases, although most of them are diagnosed in resource-limited countries from Asia, Africa and South America. For adequate treatment, first- and second-line antituberculosis drugs have to be judiciously used, but the development of new drugs with full activity, good tolerability and little toxicity is urgently needed. There are some drugs in development, some of which are already available through expanded-access programs.

  2. Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes.

    PubMed

    Ghisalberti, Didier; Masi, Muriel; Pagès, Jean-Marie; Chevalier, Jacqueline

    2005-03-25

    Chloramphenicol has been reported to act as an inducer of the multidrug resistance in Escherichia coli. A resistant variant able to grow on plates containing 64 microg/ml chloramphenicol was obtained from the Enterobacter aerogenes ATCC 13048-type strain. Chloramphenicol resistance was due to an active efflux of this antibiotic and it was associated with resistance to fluoroquinolones and tetracycline, but not to aminoglycoside or beta-lactam antibiotics. MDR in the chloramphenicol-resistant variant is linked to the overexpression of the major AcrAB-TolC efflux system. This overexpression seems unrelated to the global Mar and the local AcrR regulatory pathways.

  3. Molecular Dynamics Computer Simulations of Multidrug RND Efflux Pumps

    PubMed Central

    Ruggerone, Paolo; Vargiu, Attilio V.; Collu, Francesca; Fischer, Nadine; Kandt, Christian

    2013-01-01

    Over-expression of multidrug efflux pumps of the Resistance Nodulation Division (RND) protein super family counts among the main causes for microbial resistance against pharmaceuticals. Understanding the molecular basis of this process is one of the major challenges of modern biomedical research, involving a broad range of experimental and computational techniques. Here we review the current state of RND transporter investigation employing molecular dynamics simulations providing conformational samples of transporter components to obtain insights into the functional mechanism underlying efflux pump-mediated antibiotics resistance in Escherichia coli and Pseudomonas aeruginosa. PMID:24688701

  4. On the physics of multidrug efflux through a biomolecular complex

    NASA Astrophysics Data System (ADS)

    Mishima, Hirokazu; Oshima, Hiraku; Yasuda, Satoshi; Amano, Ken-ichi; Kinoshita, Masahiro

    2013-11-01

    Insertion and release of a solute into and from a vessel comprising biopolymers is a fundamental function in a biological system. A typical example is found in a multidrug efflux transporter. "Multidrug efflux" signifies that solutes such as drug molecules with diverse properties can be handled. In our view, the mechanism of the multidrug efflux is not chemically specific but rather has to be based on a physical factor. In earlier works, we showed that the spatial distribution of the solute-vessel potential of mean force (PMF) induced by the solvent plays imperative roles in the insertion/release process. The PMF can be decomposed into the energetic and entropic components. The entropic component, which originates from the translational displacement of solvent molecules, is rather insensitive to the solute-solvent and vessel inner surface-solvent affinities. This feature is not shared with the energetic component. When the vessel inner surface is neither solvophobic nor solvophilic, the solvents within the vessel cavity and in the bulk offer almost the same environment to any solute with solvophobicity or solvophilicity, and the energetic component becomes much smaller than the entropic component (i.e., the latter predominates over the former). Our idea is that the multidrug efflux can be realized if the insertion/release process is accomplished by the entropic component exhibiting the insensitivity to the solute properties. However, we have recently argued that the entropic release of the solute is not feasible as long as the vessel geometry is fixed. Here we consider a model of TolC, a cylindrical vessel possessing an entrance at one end and an exit at the other end for the solute. The spatial distribution of the PMF is calculated by employing the three-dimensional integral equation theory with rigid-body models in which the constituents interact only through hard-body potentials. Since the behavior of these models is purely entropic in origin, our analysis is

  5. Expression of the human multidrug transporter in insect cells by a recombinant baculovirus

    SciTech Connect

    Germann, U.A.; Willingham, M.C.; Pastan, I.; Gottesman, M.M. )

    1990-03-06

    The plasma membrane associated human multidrug resistance (MDR1) gene product, known as the 170-kDa P-glycoprotein or the multidrug transporter, acts as an ATP-dependent efflux pump for various cytotoxic agents. The authors expressed recombinant human multidrug transporter in a baculovirus expression system to obtain large quantities and further investigate its structure and mechanism of action. MDR1 cDNA was inserted into the genome of the Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. Spodoptera frugiperda insect cells synthesized high levels of recombinant multidrug transporter 2-3 days after infection. The transporter was localized by immunocytochemical methods on the external surface of the plasma membranes, in the Golgi apparatus, and within the nuclear envelope. The human multidrug transporter expressed in insect cells is not susceptible to endoglycosidase F treatment and has a lower apparent molecular weight of 140,000, corresponding to the nonglycosylated precursor of its authentic counterpart expressed in multidrug-resistant cells. Labeling experiments showed that the recombinant multidrug transporter is phosphorylated and can be photoaffinity labeled by ({sup 3}H)azidopine, presumably at the same two sites as the native protein. Various drugs and reversing agents compete with the ({sup 3}H)azidopine binding reaction when added in excess, indicating that the recombinant human multidrug transporter expressed in insect cells is functionally similar to its authentic counterpart.

  6. Use of Spatial Information to Predict Multidrug Resistance in Tuberculosis Patients, Peru

    PubMed Central

    Lin, Hsien-Ho; Shin, Sonya S.; Contreras, Carmen; Asencios, Luis; Paciorek, Christopher J.

    2012-01-01

    To determine whether spatiotemporal information could help predict multidrug resistance at the time of tuberculosis diagnosis, we investigated tuberculosis patients who underwent drug susceptibility testing in Lima, Peru, during 2005–2007. We found that crude representation of spatial location at the level of the health center improved prediction of multidrug resistance. PMID:22516236

  7. Worldwide Endemicity of a Multidrug-Resistant Staphylococcus capitis Clone Involved in Neonatal Sepsis.

    PubMed

    Butin, Marine; Martins-Simões, Patricia; Rasigade, Jean-Philippe; Picaud, Jean-Charles; Laurent, Frédéric

    2017-03-01

    A multidrug-resistant Staphylococcus capitis clone, NRCS-A, has been isolated from neonatal intensive care units in 17 countries throughout the world. S. capitis NRCS-A prevalence is high in some neonatal intensive care units in France. These data highlight the worldwide endemicity and epidemiologic relevance of this multidrug-resistant, coagulase-negative staphylococci clone.

  8. Worldwide Endemicity of a Multidrug-Resistant Staphylococcus capitis Clone Involved in Neonatal Sepsis

    PubMed Central

    Martins-Simões, Patricia; Rasigade, Jean-Philippe; Picaud, Jean-Charles; Laurent, Frédéric

    2017-01-01

    A multidrug-resistant Staphylococcus capitis clone, NRCS-A, has been isolated from neonatal intensive care units in 17 countries throughout the world. S. capitis NRCS-A prevalence is high in some neonatal intensive care units in France. These data highlight the worldwide endemicity and epidemiologic relevance of this multidrug-resistant, coagulase-negative staphylococci clone. PMID:28221122

  9. Human Multidrug-Resistant Salmonella Newport Infections, Wisconsin, 2003–2005

    PubMed Central

    Archer, John R.; Sotir, Mark J.; Monson, Timothy A.; Kazmierczak, James J.

    2007-01-01

    We conducted a retrospective study of Salmonella Newport infections among Wisconsin residents during 2003–2005. Multidrug resistance prevalence was substantially greater in Wisconsin than elsewhere in the United States. Persons with multidrug-resistant infections were more likely than persons with susceptible infections to report exposure to cattle, farms, and unpasteurized milk. PMID:18217570

  10. Undetected multidrug-resistant tuberculosis amplified by first-line therapy in mixed infection.

    PubMed

    Hingley-Wilson, Suzanne M; Casey, Rosalyn; Connell, David; Bremang, Samuel; Evans, Jason T; Hawkey, Peter M; Smith, Grace E; Jepson, Annette; Philip, Stuart; Kon, Onn Min; Lalvani, Ajit

    2013-07-01

    Infections with >1 Mycobacterium tuberculosis strain(s) are underrecognized. We show, in vitro and in vivo, how first-line treatment conferred a competitive growth advantage to amplify a multidrug-resistant M. tuberculosis strain in a patient with mixed infection. Diagnostic techniques that identify mixed tubercle bacilli populations are needed to curb the spread of multidrug resistance.

  11. Off-Label Use of Bedaquiline in Children and Adolescents with Multidrug-Resistant Tuberculosis.

    PubMed

    Achar, Jay; Hewison, Cathy; Cavalheiro, Ana P; Skrahina, Alena; Cajazeiro, Junia; Nargiza, Parpieva; Herboczek, Krzysztof; Rajabov, Assliddin S; Hughes, Jennifer; Ferlazzo, Gabriella; Seddon, James A; du Cros, Philipp

    2017-10-01

    We describe 27 children and adolescents <18 years of age who received bedaquiline during treatment for multidrug-resistant tuberculosis. We report good treatment responses and no cessation attributable to adverse effects. Bedaquiline could be considered for use with this age group for multidrug-resistant tuberculosis when treatment options are limited.

  12. In Silico Prediction of Inhibition of Promiscuous Breast Cancer Resistance Protein (BCRP/ABCG2)

    PubMed Central

    Ding, Yi-Lung; Shih, Yu-Hsuan; Tsai, Fu-Yuan; Leong, Max K.

    2014-01-01

    Background Breast cancer resistant protein has an essential role in active transport of endogenous substances and xenobiotics across extracellular and intracellular membranes along with P-glycoprotein. It also plays a major role in multiple drug resistance and permeation of blood-brain barrier. Therefore, it is of great importance to derive theoretical models to predict the inhibition of both transporters in the process of drug discovery and development. Hitherto, very limited BCRP inhibition predictive models have been proposed as compared with its P-gp counterpart. Methodology/Principal Findings An in silico BCRP inhibition model was developed in this study using the pharmacophore ensemble/support vector machine scheme to take into account the promiscuous nature of BCRP. The predictions by the PhE/SVM model were found to be in good agreement with the observed values for those molecules in the training set (n = 22, r2 = 0.82,  = 0.73, RMSE  =  0.40, s = 0.24), test set (n = 97, q2 = 0.75–0.89, RMSE  = 0.31, s = 0.21), and outlier set (n = 16, q2 = 0.72–0.91, RMSE  =  0.29, s = 0.17). When subjected to a variety of statistical validations, the developed PhE/SVM model consistently met the most stringent criteria. A mock test by HIV protease inhibitors also asserted its predictivity. Conclusions/Significance It was found that this accurate, fast, and robust PhE/SVM model can be employed to predict the BCRP inhibition of structurally diverse molecules that otherwise cannot be carried out by any other methods in a high-throughput fashion to design therapeutic agents with insignificant drug toxicity and unfavorable drug–drug interactions mediated by BCRP to enhance clinical efficacy and/or circumvent drug resistance. PMID:24614353

  13. The secondary resistome of multidrug-resistant Klebsiella pneumoniae

    PubMed Central

    Jana, Bimal; Cain, Amy K.; Doerrler, William T.; Boinett, Christine J.; Fookes, Maria C.; Parkhill, Julian; Guardabassi, Luca

    2017-01-01

    Klebsiella pneumoniae causes severe lung and bloodstream infections that are difficult to treat due to multidrug resistance. We hypothesized that antimicrobial resistance can be reversed by targeting chromosomal non-essential genes that are not responsible for acquired resistance but essential for resistant bacteria under therapeutic concentrations of antimicrobials. Conditional essentiality of individual genes to antimicrobial resistance was evaluated in an epidemic multidrug-resistant clone of K. pneumoniae (ST258). We constructed a high-density transposon mutant library of >430,000 unique Tn5 insertions and measured mutant depletion upon exposure to three clinically relevant antimicrobials (colistin, imipenem or ciprofloxacin) by Transposon Directed Insertion-site Sequencing (TraDIS). Using this high-throughput approach, we defined three sets of chromosomal non-essential genes essential for growth during exposure to colistin (n = 35), imipenem (n = 1) or ciprofloxacin (n = 1) in addition to known resistance determinants, collectively termed the “secondary resistome”. As proof of principle, we demonstrated that inactivation of a non-essential gene not previously found linked to colistin resistance (dedA) restored colistin susceptibility by reducing the minimum inhibitory concentration from 8 to 0.5 μg/ml, 4-fold below the susceptibility breakpoint (S ≤ 2 μg/ml). This finding suggests that the secondary resistome is a potential target for developing antimicrobial “helper” drugs that restore the efficacy of existing antimicrobials. PMID:28198411

  14. Therapeutic options and emerging alternatives for multidrug resistant staphylococcal infections.

    PubMed

    Magana, Maria; Ioannidis, Anastasios; Magiorkinis, Emmanouil; Ursu, Oleg; Bologa, Cristian G; Chatzipanagiotou, Stylianos; Hamblin, Michael R; Tegos, George P

    2015-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) remains the single biggest challenge in infectious disease in the civilized world. Moreover, vancomycin resistance is also spreading, leading to fears of untreatable infections as were common in ancient times. Molecular microbiology and bioinformatics have revealed many of the mechanisms involved in resistance development. Mobile genetic elements, up-regulated virulence factors and multi-drug efflux pumps have been implicated. A range of approved antibiotics from the glycopeptide, lipopeptide, pleuromutilin, macrolide, oxazolidinone, lincosamide, aminoglycoside, tetracycline, steptogramin, and cephalosporin classes has been employed to treat MRSA infections. The upcoming pipeline of drugs for MRSA includes some new compounds from the above classes, together with fluoroquinolones, antibacterial peptide mimetics, aminomethylciclines, porphyrins, peptide deformylase inhibitors, oxadiazoles, and diaminopyrimidines. A range of non-drug alternative approaches has emerged for MRSA treatment. Bacteriophage-therapy including purified lysins has made a comeback after being discovered in the 1930s. Quorum-sensing inhibitors are under investigation. Small molecule inhibitors of multi-drug efflux pumps may potentiate existing antibiotics. The relative failure of staphylococcal vaccines is being revisited by efforts with multi-valent vaccines and improved adjuvants. Photodynamic therapy uses non-toxic photosensitizers and harmless visible light to produce reactive oxygen species that can nonspecifically destroy bacteria while preserving host cells. Preparation of nanoparticles can kill bacteria themselves, as well as improve the delivery of anti-bacterial drugs. Anti-MRSA drug discovery remains an exciting field with great promise for the future.

  15. Repurposing ebselen for treatment of multidrug-resistant staphylococcal infections.

    PubMed

    Thangamani, Shankar; Younis, Waleed; Seleem, Mohamed N

    2015-06-26

    Novel antimicrobials and new approaches to developing them are urgently needed. Repurposing already-approved drugs with well-characterized toxicology and pharmacology is a novel way to reduce the time, cost, and risk associated with antibiotic innovation. Ebselen, an organoselenium compound, is known to be clinically safe and has a well-known pharmacology profile. It has shown potent bactericidal activity against multidrug-resistant clinical isolates of staphylococcus aureus, including methicillin- and vancomycin-resistant S. aureus (MRSA and VRSA). We demonstrated that ebselen acts through inhibition of protein synthesis and subsequently inhibited toxin production in MRSA. Additionally, ebselen was remarkably active and significantly reduced established staphylococcal biofilms. The therapeutic efficacy of ebselen was evaluated in a mouse model of staphylococcal skin infections. Ebselen 1% and 2% significantly reduced the bacterial load and the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and monocyte chemo attractant protein-1 (MCP-1) in MRSA USA300 skin lesions. Furthermore, it acts synergistically with traditional antimicrobials. This study provides evidence that ebselen has great potential for topical treatment of MRSA skin infections and lays the foundation for further analysis and development of ebselen as a potential treatment for multidrug-resistant staphylococcal infections.

  16. Repurposing ebselen for treatment of multidrug-resistant staphylococcal infections

    PubMed Central

    Thangamani, Shankar; Younis, Waleed; Seleem, Mohamed N.

    2015-01-01

    Novel antimicrobials and new approaches to developing them are urgently needed. Repurposing already-approved drugs with well-characterized toxicology and pharmacology is a novel way to reduce the time, cost, and risk associated with antibiotic innovation. Ebselen, an organoselenium compound, is known to be clinically safe and has a well-known pharmacology profile. It has shown potent bactericidal activity against multidrug-resistant clinical isolates of staphylococcus aureus, including methicillin- and vancomycin-resistant S. aureus (MRSA and VRSA). We demonstrated that ebselen acts through inhibition of protein synthesis and subsequently inhibited toxin production in MRSA. Additionally, ebselen was remarkably active and significantly reduced established staphylococcal biofilms. The therapeutic efficacy of ebselen was evaluated in a mouse model of staphylococcal skin infections. Ebselen 1% and 2% significantly reduced the bacterial load and the levels of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and monocyte chemo attractant protein-1 (MCP-1) in MRSA USA300 skin lesions. Furthermore, it acts synergistically with traditional antimicrobials. This study provides evidence that ebselen has great potential for topical treatment of MRSA skin infections and lays the foundation for further analysis and development of ebselen as a potential treatment for multidrug-resistant staphylococcal infections. PMID:26111644

  17. Photoexcited quantum dots for killing multidrug-resistant bacteria

    NASA Astrophysics Data System (ADS)

    Courtney, Colleen M.; Goodman, Samuel M.; McDaniel, Jessica A.; Madinger, Nancy E.; Chatterjee, Anushree; Nagpal, Prashant

    2016-05-01

    Multidrug-resistant bacterial infections are an ever-growing threat because of the shrinking arsenal of efficacious antibiotics. Metal nanoparticles can induce cell death, yet the toxicity effect is typically nonspecific. Here, we show that photoexcited quantum dots (QDs) can kill a wide range of multidrug-resistant bacterial clinical isolates, including methicillin-resistant Staphylococcus aureus, carbapenem-resistant Escherichia coli, and extended-spectrum β-lactamase-producing Klebsiella pneumoniae and Salmonella typhimurium. The killing effect is independent of material and controlled by the redox potentials of the photogenerated charge carriers, which selectively alter the cellular redox state. We also show that the QDs can be tailored to kill 92% of bacterial cells in a monoculture, and in a co-culture of E. coli and HEK 293T cells, while leaving the mammalian cells intact, or to increase bacterial proliferation. Photoexcited QDs could be used in the study of the effect of redox states on living systems, and lead to clinical phototherapy for the treatment of infections.

  18. Multidrug Efflux Pumps in Staphylococcus aureus: an Update.

    PubMed

    Costa, Sofia Santos; Viveiros, Miguel; Amaral, Leonard; Couto, Isabel

    2013-01-01

    The emergence of infections caused by multi- or pan-resistant bacteria in the hospital or in the community settings is an increasing health concern. Albeit there is no single resistance mechanism behind multiresistance, multidrug efflux pumps, proteins that cells use to detoxify from noxious compounds, seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria. During the last decades, experimental data has established their contribution to low level resistance to antimicrobials in bacteria and their potential role in the appearance of MDR phenotypes, by the extrusion of multiple, unrelated compounds. Recent studies suggest that efflux pumps may be used by the cell as a first-line defense mechanism, avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration occurs, that allows survival in the presence of that agent. In this paper we review the current knowledge on MDR efflux pumps and their intricate regulatory network in Staphylococcus aureus, a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that S. aureus MDR efflux pumps, either chromosomal or plasmid-encoded, have on resistance towards different antimicrobial agents and on the selection of drug - resistant strains. We will also discuss the many questions that still remain on the role of each specific efflux pump and the need to establish appropriate methodological approaches to address all these questions.

  19. Photoexcited quantum dots for killing multidrug-resistant bacteria.

    PubMed

    Courtney, Colleen M; Goodman, Samuel M; McDaniel, Jessica A; Madinger, Nancy E; Chatterjee, Anushree; Nagpal, Prashant

    2016-05-01

    Multidrug-resistant bacterial infections are an ever-growing threat because of the shrinking arsenal of efficacious antibiotics. Metal nanoparticles can induce cell death, yet the toxicity effect is typically nonspecific. Here, we show that photoexcited quantum dots (QDs) can kill a wide range of multidrug-resistant bacterial clinical isolates, including methicillin-resistant Staphylococcus aureus, carbapenem-resistant Escherichia coli, and extended-spectrum β-lactamase-producing Klebsiella pneumoniae and Salmonella typhimurium. The killing effect is independent of material and controlled by the redox potentials of the photogenerated charge carriers, which selectively alter the cellular redox state. We also show that the QDs can be tailored to kill 92% of bacterial cells in a monoculture, and in a co-culture of E. coli and HEK 293T cells, while leaving the mammalian cells intact, or to increase bacterial proliferation. Photoexcited QDs could be used in the study of the effect of redox states on living systems, and lead to clinical phototherapy for the treatment of infections.

  20. The secondary resistome of multidrug-resistant Klebsiella pneumoniae.

    PubMed

    Jana, Bimal; Cain, Amy K; Doerrler, William T; Boinett, Christine J; Fookes, Maria C; Parkhill, Julian; Guardabassi, Luca

    2017-02-15

    Klebsiella pneumoniae causes severe lung and bloodstream infections that are difficult to treat due to multidrug resistance. We hypothesized that antimicrobial resistance can be reversed by targeting chromosomal non-essential genes that are not responsible for acquired resistance but essential for resistant bacteria under therapeutic concentrations of antimicrobials. Conditional essentiality of individual genes to antimicrobial resistance was evaluated in an epidemic multidrug-resistant clone of K. pneumoniae (ST258). We constructed a high-density transposon mutant library of >430,000 unique Tn5 insertions and measured mutant depletion upon exposure to three clinically relevant antimicrobials (colistin, imipenem or ciprofloxacin) by Transposon Directed Insertion-site Sequencing (TraDIS). Using this high-throughput approach, we defined three sets of chromosomal non-essential genes essential for growth during exposure to colistin (n = 35), imipenem (n = 1) or ciprofloxacin (n = 1) in addition to known resistance determinants, collectively termed the "secondary resistome". As proof of principle, we demonstrated that inactivation of a non-essential gene not previously found linked to colistin resistance (dedA) restored colistin susceptibility by reducing the minimum inhibitory concentration from 8 to 0.5 μg/ml, 4-fold below the susceptibility breakpoint (S ≤ 2 μg/ml). This finding suggests that the secondary resistome is a potential target for developing antimicrobial "helper" drugs that restore the efficacy of existing antimicrobials.

  1. How multidrug resistance in typhoid fever affects treatment options.

    PubMed

    Tatavarthy, Aparna; Luna, Vicki A; Amuso, Philip T

    2014-09-01

    Salmonella enterica serotype Typhi (S. Typhi) is an enteric pathogen that causes typhoid fever. The infection can be severe, with significant morbidity and mortality, requiring antimicrobial therapy. Cases of S. Typhi infection in the United States and other developed countries are often associated with travel to endemic regions. The empirical use of first-line drugs for therapy, including ampicillin, chloramphenicol, and trimethoprim/sulfamethoxazole, has resulted in transmissible multidrug resistance. With the global increase in multidrug-resistant S. Typhi, use of ciprofloxacin, with excellent oral absorption, few side effects, and cost-effectiveness, has become popular for treatment. However, decreased ciprofloxacin susceptibility due to point mutations in the S. Typhi genes gyrA and/or parC has caused treatment failures, necessitating alternative therapeutic options. S. Typhi is typically genetically homogenous, with phylogenetic and epidemiological studies showing that identical clones and diverse S. Typhi types often coexist in the same geographic region. Studies investigating point mutations have demonstrated that selective pressure from empirical use of first-line drugs and fluoroquinolones has led to the global emergence of haplotype H-58. Antibiotic resistance is subject to high selective pressure in S. Typhi and thus demands careful use of antimicrobials. © 2014 New York Academy of Sciences.

  2. Paclitaxel Nanocrystals for Overcoming Multidrug Resistance in Cancer

    PubMed Central

    Liu, Yang; Huang, Leaf; Liu, Feng

    2013-01-01

    Here we described a paclitaxel (PTX) nanocrystals formulation using D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) as the sole excipient for overcoming multidrug resistance (MDR), a key challenge in current cancer therapy. To the best of our knowledge, it is the first report on PTX nanocrystals which can reverse MDR. TPGS serves as a surfactant to stabilize the nanocrystals and a P-gp inhibitor to reverse MDR. The size and morphology of the nanocrystals were studied by transmission electron microscopy and the crystalline structure was determined by powder X-ray diffraction. In vitro drug release profile showed that the nanocrystals exhibited sustained release kinetics compared to Taxol which is the clinical paclitaxel formulation. The cytotoxicity and antitumor efficacy in xenograft models were also investigated. It is demonstrated that PTX/TPGS nanocrystals have significant advantages over Taxol in achieving better therapeutic effect in Taxol-resistant cancer cells both in vitro and in vivo, which was also confirmed by apoptosis assays. We envision that further development of this type of nanocrystals will provide a novel strategy for drug delivery and multidrug resistance treatment. PMID:20420443

  3. Multidrug Efflux Pumps in Staphylococcus aureus: an Update

    PubMed Central

    Costa, Sofia Santos; Viveiros, Miguel; Amaral, Leonard; Couto, Isabel

    2013-01-01

    The emergence of infections caused by multi- or pan-resistant bacteria in the hospital or in the community settings is an increasing health concern. Albeit there is no single resistance mechanism behind multiresistance, multidrug efflux pumps, proteins that cells use to detoxify from noxious compounds, seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria. During the last decades, experimental data has established their contribution to low level resistance to antimicrobials in bacteria and their potential role in the appearance of MDR phenotypes, by the extrusion of multiple, unrelated compounds. Recent studies suggest that efflux pumps may be used by the cell as a first-line defense mechanism, avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration occurs, that allows survival in the presence of that agent. In this paper we review the current knowledge on MDR efflux pumps and their intricate regulatory network in Staphylococcus aureus, a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that S. aureus MDR efflux pumps, either chromosomal or plasmid-encoded, have on resistance towards different antimicrobial agents and on the selection of drug - resistant strains. We will also discuss the many questions that still remain on the role of each specific efflux pump and the need to establish appropriate methodological approaches to address all these questions. PMID:23569469

  4. Purification of a Multidrug Resistance Transporter for Crystallization Studies

    PubMed Central

    Alegre, Kamela O.; Law, Christopher J.

    2015-01-01

    Crystallization of integral membrane proteins is a challenging field and much effort has been invested in optimizing the overexpression and purification steps needed to obtain milligram amounts of pure, stable, monodisperse protein sample for crystallography studies. Our current work involves the structural and functional characterization of the Escherichia coli multidrug resistance transporter MdtM, a member of the major facilitator superfamily (MFS). Here we present a protocol for isolation of MdtM to increase yields of recombinant protein to the milligram quantities necessary for pursuit of structural studies using X-ray crystallography. Purification of MdtM was enhanced by introduction of an elongated His-tag, followed by identification and subsequent removal of chaperonin contamination. For crystallization trials of MdtM, detergent screening using size exclusion chromatography determined that decylmaltoside (DM) was the shortest-chain detergent that maintained the protein in a stable, monodispersed state. Crystallization trials of MdtM performed using the hanging-drop diffusion method with commercially available crystallization screens yielded 3D protein crystals under several different conditions. We contend that the purification protocol described here may be employed for production of high-quality protein of other multidrug efflux members of the MFS, a ubiquitous, physiologically and clinically important class of membrane transporters. PMID:27025617

  5. Characterization of a multidrug resistant C. difficile meat isolate.

    PubMed

    Mooyottu, Shankumar; Flock, Genevieve; Kollanoor-Johny, Anup; Upadhyaya, Indu; Jayarao, Bhushan; Venkitanarayanan, Kumar

    2015-01-02

    Clostridium difficile is a pathogen of significant public health concern causing a life-threatening, toxin-mediated enteric disease in humans. The incidence and severity of the disease associated with C. difficile have increased in the US with the emergence of hypervirulent strains and community associated outbreaks. The detection of genotypically similar and identical C. difficile strains implicated from human infections in foods and food animals indicates the potential role of food as a source of community associated C. difficile disease. One hundred samples each of ground beef, pork and chicken obtained from geographically distant grocery stores in Connecticut were tested for C. difficile. Positive isolates were characterized by ribotyping, antibiotic susceptibility, toxin production and whole genome sequencing. Of the 300 meat samples, only two pork samples tested positive for C. difficile indicating a very low prevalence of C. difficile in meat. The isolates were non toxigenic; however, genome characterization revealed the presence of several antibiotic resistance genes and mobile elements that can potentially contribute to generation of multidrug resistant toxigenic C. difficile by horizontal gene transfer. Further studies are warranted to investigate potential food-borne transmission of the meat isolates and development of multi-drug resistance in these strains. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Detection of multidrug-resistant tuberculosis gene using plasmonics nanoprobes

    NASA Astrophysics Data System (ADS)

    Wang, Hsin-Neng; Yan, Fei; Zhang, Yan; Vo-Dinh, Tuan

    2008-02-01

    This paper describes the use of plasmonics-based nanoprobes for detection of multidrug-resistant tuberculosis gene. The plasmonics nanoprobe is composed of a silver nanoparticle pre-coated with a stem-loop DNA probe that is tagged with a Raman label at one end of the stem region, while the other end of the probe is covalently conjugated to the nanoparticle via a thiol-silver bond. The loop region is designed to detect a specific target gene sequence. In the absence of target, the Raman label is in close proximity to the metal surface, resulting in an intense SERS signal upon laser excitation. In the presence of the target DNA sequence, hybridization between the target and probe disrupts the stem-loop configuration, separating the Raman label from the metal surface and quenching the SERS signal. In this study, we successfully demonstrated for the first time the feasibility of using plasmonics nanoprobes for the detection of multidrug-resistant tuberculosis gene.

  7. Multidrug resistance protein gene expression in Trichoplusia ni caterpillars.

    PubMed

    Simmons, Jason; D'Souza, Olivia; Rheault, Mark; Donly, Cam

    2013-02-01

    Many insect species exhibit pesticide-resistant phenotypes. One of the mechanisms capable of contributing to resistance is the overexpression of multidrug resistance (MDR) transporter proteins. Here we describe the cloning of three genes encoding MDR proteins from Trichoplusia ni: trnMDR1, trnMDR2 and trnMDR3. Real-time quantitative PCR (qPCR) detected trnMDR mRNA in the whole nervous system, midgut and Malpighian tubules of final instar T. ni caterpillars. To test whether these genes are upregulated in response to chemical challenge in this insect, qPCR was used to compare trnMDR mRNA levels in unchallenged insects with those of insects fed the synthetic pyrethroid, deltamethrin. Only limited increases were detected in a single gene, trnMDR2, which is the most weakly expressed of the three MDR genes, suggesting that increased multidrug resistance of this type is not a significant part of the response to deltamethrin exposure.

  8. Effect of multidrug resistance 1/P-glycoprotein on the hypoxia-induced multidrug resistance of human laryngeal cancer cells.

    PubMed

    Li, Dawei; Zhou, Liang; Huang, Jiameng; Xiao, Xiyan

    2016-08-01

    In a previous study, it was demonstrated that hypoxia upregulated the multidrug resistance (MDR) of laryngeal cancer cells to chemotherapeutic drugs, with multidrug resistance 1 (MDR1)/P-glycoprotein (P-gp) expression also being upregulated. The present study aimed to investigate the role and mechanism of MDR1/P-gp on hypoxia-induced MDR in human laryngeal carcinoma cells. The sensitivity of laryngeal cancer cells to multiple drugs and cisplatin-induced apoptosis was determined by CCK-8 assay and Annexin-V/propidium iodide staining analysis, respectively. The accumulation of rhodamine 123 (Rh123) in the cells served as an estimate of drug accumulation and was evaluated by flow cytometry (FCM). MDR1/P-gp expression was inhibited using interference RNA, and the expression of the MDR1 gene was analyzed using reverse transcription-quantitative polymerase chain reaction and western blotting. As a result, the sensitivity to multiple chemotherapeutic agents and the apoptosis rate of the hypoxic laryngeal carcinoma cells increased following a decrease in MDR1/P-gp expression (P<0.05). Additionally, FCM analysis of fluorescence intensity indicated that the downregulated expression of MDR1/P-gp markedly increased intracellular Rh123 accumulation (P<0.05). Such results suggest that MDR1/P-gp serves an important role in regulating hypoxia-induced MDR in human laryngeal carcinoma cells through a decrease in intracellular drug accumulation.

  9. Safety and effectiveness of home intravenous antibiotic therapy for multidrug-resistant bacterial infections.

    PubMed

    Mujal, A; Sola, J; Hernandez, M; Villarino, M-A; Machado, M-L; Baylina, M; Tajan, J; Oristrell, J

    2015-06-01

    Home intravenous antibiotic therapy is an alternative to hospital admission for moderately severe infections. However, few studies have analyzed its safety and effectiveness in the treatment of infections caused by multidrug-resistant bacteria. The purpose of this study is to analyze the safety and effectiveness of home intravenous antibiotic therapy in multidrug-resistant bacterial infections. We analyzed prospectively all patients admitted to our service who underwent home intravenous antibiotic therapy during the period 2008-2012. All the treatments were administered by caretakers or self-administered by patients, through elastomeric infusion devices. Effectiveness was evaluated by analyzing the readmission rate for poor infection control. Safety was evaluated by analyzing adverse events, catheter-related complications, and readmissions not related to poor infection control. There were 433 admissions (in 355 patients) for home intravenous antibiotic therapy during the study period. There were 226 (52.2 %) admissions due to multidrug-resistant bacterial infections and 207 (47.8 %) due to non-multidrug-resistant infections. Hospital readmissions in patients with multidrug-resistant infections were uncommon. Multidrug-resistant enterococcal infections, healthcare-associated infections, and carbapenem therapy were independent variables associated with increased readmissions due to poor infection control. Readmissions not related to poor infection control, adverse events, and catheter-related complications were similar in multidrug-resistant compared to non-multidrug-resistant bacterial infections. Home intravenous therapy, administered by patients or their caretakers using elastomeric infusion pumps, was safe and effective for the treatment of most multidrug-resistant bacterial infections.

  10. Susceptibility of Multidrug-Resistant Gram-Negative Urine Isolates to Oral Antibiotics

    PubMed Central

    Zucchi, Paola C.; Chen, Alice; Raux, Brian R.; Kirby, James E.; McCoy, Christopher; Eliopoulos, George M.

    2016-01-01

    Increasing resistance among Gram-negative uropathogens limits treatment options, and susceptibility data for multidrug-resistant isolates are limited. We assessed the activity of five oral agents against 91 multidrug-resistant Gram-negative urine isolates that were collected from emergency department/hospitalized patients. Fosfomycin and nitrofurantoin were most active (>75% susceptibility). Susceptibilities to sulfamethoxazole-trimethoprim, ciprofloxacin, and ampicillin were ≤40%; empirical use of these agents likely provides inadequate coverage in areas with a high prevalence of multidrug-resistant uropathogens. PMID:26883704