Functional Expression of Multidrug-Resistance (MDR) Transporters in Developing Human Fetal Brain Endothelial Cells
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ABSTRACT:
SUBMITTER: Lye P
PROVIDER: S-EPMC9323234 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundP-glycoprotein (Pgp) and multidrug resistance-associated protein (MRP1) are membrane transporter proteins which function as efflux pumps at cell membranes and are considered to exert a protective function against the entry of xenobiotics. While evidence for Pgp and MRP transporter activity is reported for olfactory tissue, their possible interaction and participation in the olfactory response has not been investigated.Principal findingsFunctional activity of putative MDR transporters was assessed by means of the fluorometric calcein acetoxymethyl ester (calcein-AM) accumulation assay on acute rat and mouse olfactory tissue slices. Calcein-AM uptake was measured as fluorescence intensity changes in the presence of Pgp or MRP specific inhibitors. Epifluorescence microscopy measured time course analysis in the olfactory epithelium revealed significant inhibitor-dependent calcein uptake in the presence of each of the selected inhibitors. Furthermore, intracellular calcein accumulation in olfactory receptor neurons was also significantly increased in the presence of either one of the Pgp or MRP inhibitors. The presence of Pgp or MRP1 encoding genes in the olfactory mucosa of rat and mouse was confirmed by RT-PCR with appropriate pairs of species-specific primers. Both transporters were expressed in both newborn and adult olfactory mucosa of both species. To assess a possible involvement of MDR transporters in the olfactory response, we examined the electrophysiological response to odorants in the presence of the selected MDR inhibitors by recording electroolfactograms (EOG). In both animal species, MRPs inhibitors induced a marked reduction of the EOG magnitude, while Pgp inhibitors had only a minor or no measurable effect.ConclusionsThe findings suggest that both Pgp and MRP transporters are functional in the olfactory mucosa and in olfactory receptor neurons. Pgp and MRPs may be cellular constituents of olfactory receptor neurons and represent potential mechanisms for modulation of the olfactory response.
| S-EPMC3341370 | biostudies-literature
Project description:BackgroundThe multidrug resistance (MDR) transporters, P-glycoprotein (P-gp, encoded by ABCB1) and breast cancer resistance protein (BCRP/ABCG2) contribute to the blood-brain barrier (BBB), protecting the brain from drug exposure. The impact of infection on MDR in the developing human BBB remains to be determined. We hypothesized that exposure to bacterial and viral pathogen-associated molecular patterns (PAMPs) modify MDR expression and activity in human fetal brain endothelial cells (hfBECs) isolated from early and mid-gestation brain microvessels.MethodsWe modelled infection (4 h and 24 h) using the bacterial PAMP, lipopolysaccharide (LPS; a toll-like receptor [TLR]-4 ligand) or the viral PAMPs, polyinosinic polycytidylic acid (Poly I:C; TLR-3 ligand) and single-stranded RNA (ssRNA; TLR-7/8 ligand). mRNA expression was assessed by qPCR, whereas protein expression was assessed by Western blot or immunofluorescence. P-gp and BCRP activity was evaluated by Calcein-AM and Chlorin-6 assays.ResultsTLRs-3,4 and 8 were expressed by the isolated hfBECs. Infection mimics induced specific pro-inflammatory responses as well as changes in P-gp/ABCB1 or BCRP/ABCG2 expression (P < 0.05). LPS and ssRNA significantly decreased P-gp activity at 4 and 24 h in early and mid-gestation (P < 0.03-P < 0.001), but significantly increased BCRP activity in hfBECs in a dose-dependent pattern (P < 0.05-P < 0.002). In contrast, Poly-IC significantly decreased P-gp activity after 4 h in early (P < 0.01) and mid gestation (P < 0.04), but not 24 h, and had no overall effect on BCRP activity, though BCRP activity was increased with the highest dose at 24 h in mid-gestation (P < 0.05).ConclusionsInfectious PAMPs significantly modify the expression and function of MDR transporters in hfBECs, though effects are PAMP-, time- and dose-specific. In conclusion, bacterial and viral infections during pregnancy likely have profound effects on exposure of the fetal brain to physiological and pharmacological substrates of P-gp and BCRP, potentially leading to altered trajectories of fetal brain development.
| S-EPMC9887585 | biostudies-literature
Project description:P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) modulate the distribution of drugs and toxins across the blood-brain barrier (BBB). Animal studies reported that infection-induced disruption of these transporters in the developing BBB impairs fetal brain protection. However, the impact of infection mimics on P-gp/BCRP function in human brain endothelium is less well understood. We hypothesized that Toll-like receptor ligands mimicking bacterial and viral infection would modify the expression and function of P-gp and BCRP in human brain endothelial cells (BECs). Human cerebral microvascular endothelial cells (hCMEC/D3) were challenged with bacterial [Lipopolysaccharide (LPS)] and viral-mimics [polyinosinic:polycytidylic acid (PolyI:C) or single-stranded RNA (ssRNA)], or pro-inflammatory cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α and interferon gamma (IFN)-ɣ. P-gp and BCRP function was assessed after 4 or 24 h, using Calcein-AM and Chlorin-6 assays, respectively. Western blot and qPCR quantified P-gp/ABCB1 and BCRP/ABCG2 expression following treatments. Infection mimics are potent modulators of drug transporters in human BECs in vitro. LPS and PolyI:C increased, while ssRNA exposure reduced P-gp activity. In contrast, LPS and PolyI:C decreased, while ssRNA increased BCRP activity (P < .05). There was little correlation between drug transporter function, gene expression and total protein level. Altered plasma membrane BCRP may suggest modified intracellular trafficking induced by infection in human BECs. Bacterial and viral infection mimics modify P-gp and BCRP transport function in human BECs, in vitro. This knowledge may contribute and have important implications for human brain protection and possible altered biodistribution of drugs and xenobiotics in the brain following exposure to TLR agonists.
| S-EPMC8078541 | biostudies-literature
Project description:Brain edema is an important factor leading to morbidity and mortality associated with primary brain tumors. Dexamethasone, a synthetic glucocorticoid, is routinely prescribed with antineoplastic agents to alleviate pain associated with chemotherapy and reduce intracranial pressure. We investigated whether dexamethasone treatment increased the expression and activity of multidrug resistance (MDR) transporters at the blood-brain barrier. Treatment of primary rat brain microvascular endothelial cells with submicromolar concentrations of dexamethasone induced significantly higher levels of drug efflux transporters such as breast cancer resistance protein (abcg2), P-glycoprotein (P-gp; abcb1a/abcb1b), and MDR protein 2 (Mrp2; abcc2) as indicted by protein and mRNA levels as well as by functional activity. The effect of dexamethasone on transporter function was significant within 6 h of treatment, was dose dependent, and was reversible. Dexamethasone-induced upregulation of Bcrp and P-gp expression and function was partially abrogated by the glucocorticoid receptor (GR) antagonist RU486. In contrast, RU486 had no effect on the dexamethasone-induced upregulation of Mrp2, suggesting a GR-independent regulation of Mrp2, and a GR-dependent regulation of P-gp and Bcrp. In addition to the dexamethasone-induced upregulation of MDR transporters, we measured a dose-dependent and reversible increase in the expression of the nuclear transcription factor pregnane xenobiotic receptor (PXR). Administering dexamethasone to rats caused increased expression of PXR in brain microvessels within 24 h. These results suggest that adjuvant therapy with corticosteroids such as dexamethasone in the treatment of brain tumors may increase the expression of MDR transporters at the blood-brain barrier through pathways involving GR and PXR.
| S-EPMC2518425 | biostudies-literature
Project description:In this study, we cloned, expressed and functionally characterized Stronglycentrotus purpuratus (Sp) ATP-binding cassette (ABC) transporters. This screen identified three multidrug resistance (MDR) transporters with functional homology to the major types of MDR transporters found in humans. When overexpressed in embryos, the apical transporters Sp-ABCB1a, ABCB4a, and ABCG2a can account for as much as 87% of the observed efflux activity, providing a robust assay for their substrate selectivity. Using this assay, we found that sea urchin MDR transporters export canonical MDR susbtrates such as calcein-AM, bodipy-verapamil, bodipy-vinblastine, and mitoxantrone. In addition, we characterized the impact of nonconservative substitutions in the primary sequences of drug binding domains of sea urchin versus murine ABCB1 by mutation of Sp-ABCB1a and treatment of embryos with stereoisomeric cyclic peptide inhibitors (QZ59 compounds). The results indicated that two substitutions in transmembrane helix 6 reverse stereoselectivity of Sp-ABCB1a for QZ59 enantiomers compared with mouse ABCB1a. This suggests that subtle changes in the primary sequence of transporter drug binding domains could fine-tune substrate specificity through evolution.
| S-EPMC3527970 | biostudies-literature
Project description:Brain endothelial cells (BECs) form a major component of the blood-brain barrier (BBB). In late gestation, these cells express high levels of the multidrug transporter p-glycoprotein (P-gp; encoded by Abcb1), which prevents the passage of an array of endogenous factors and xenobiotics into the fetal brain. P-gp levels in the BECs increase dramatically in late gestation, coincident with astrocyte differentiation. However, the role of astrocytes in modulating P-gp in the developing BBB is unknown. We hypothesized that factors produced by astrocytes positively regulate P-gp in BECs. Astrocytes and BECs were isolated from fetal and postnatal guinea pigs. Levels of Abcb1 mRNA and P-gp were increased in BECs co-cultured with astrocytes compared to BECs in monoculture. Moreover, postnatal astrocytes enhanced P-gp function in fetal BECs but fetal astrocytes had no effect on postnatal BECs. These effects were dependent on secreted proteins with a molecular weight in the range of 3-100 kDa. LC/MS-MS revealed significant differences in proteins secreted by fetal and postnatal astrocytes. We propose that astrocytes are critical modulators of P-gp at the developing BBB. As such, aberrations in astrocyte maturation, observed in neurodevelopmental disorders, will likely decrease P-gp at the BBB. This would allow increased transfer of P-gp endogenous and exogenous substrates into the brain, many of which have neurodevelopmental consequences.
| S-EPMC5002904 | biostudies-literature
Project description:BackgroundThe major limitation to the success of chemotherapy in osteosarcoma is the development of multidrug resistance (MDR). Preventing the emergence of MDR during chemotherapy treatment has been a high priority of clinical and investigational oncology, but it remains an elusive goal. The NSC23925 has recently been identified as a novel and potent MDR reversal agent. However, whether NSC23925 can prevent the development of MDR in cancer is unknown. Therefore, this study aims to evaluate the effects of NSC23925 on prevention of the development of MDR in osteosarcoma.MethodsHuman osteosarcoma cell lines U-2OS and Saos were exposed to increasing concentrations of paclitaxel alone or in combination with NSC23925 for 6 months. Cell sublines selected at different time points were evaluated for their drug sensitivity, drug transporter P-glycoprotein (Pgp) expression and activity.ResultsWe observed that tumour cells selected with increasing concentrations of paclitaxel alone developed MDR with resistance to paclitaxel and other Pgp substrates, whereas cells cultured with paclitaxel-NSC23925 did not develop MDR and cells remained sensitive to chemotherapeutic agents. Paclitaxel-resistant cells showed high expression and activity of the Pgp, whereas paclitaxel-NSC23925-treated cells did not express Pgp. No changes in IC50 and Pgp expression and activity were observed in cells grown with the NSC23925 alone.ConclusionsOur findings suggest that NSC23925 may prevent the development of MDR by specifically preventing the overexpression of Pgp. Given the significant incidence of MDR in osteosarcoma and the lack of effective agents for prevention of MDR, NSC23925 and derivatives hold the potential to improve the outcome of cancer patients with poor prognosis due to drug resistance.
| S-EPMC4056062 | biostudies-literature
Project description:Current views of multidrug (MD) recognition focus on large drug-binding cavities with flexible elements. However, MD recognition in BmrR is supported by a small, rigid drug-binding pocket. Here, a detailed description of MD binding by the noncanonical BmrR protein is offered through the combined use of X-ray and solution studies. Low shape complementarity, suboptimal packing, and efficient burial of a diverse set of ligands is facilitated by an aromatic docking platform formed by a set of conformationally fixed aromatic residues, hydrophobic pincer pair that locks the different drug structures on the adaptable platform surface, and a trio of acidic residues that enables cation selectivity without much regard to ligand structure. Within the binding pocket is a set of BmrR-derived H-bonding donor and acceptors that solvate a wide range of ligand polar substituent arrangements in a manner analogous to aqueous solvent. Energetic analyses of MD binding by BmrR are consistent with structural data. A common binding orientation for the different BmrR ligands is in line with promiscuous allosteric regulation.
| S-EPMC3131311 | biostudies-literature
Project description:Candida dubliniensis is a recently described Candida species associated with oral candidosis in human immunodeficiency virus (HIV)-infected and AIDS patients, from whom fluconazole-resistant clinical isolates have been previously recovered. Furthermore, derivatives exhibiting a stable fluconazole-resistant phenotype have been readily generated in vitro from fluconazole-susceptible isolates following exposure to the drug. In this study, fluconazole-resistant isolates accumulated up to 80% less [3H] fluconazole than susceptible isolates and also exhibited reduced susceptibility to the metabolic inhibitors 4-nitroquinoline-N-oxide and methotrexate. These findings suggested that C. dubliniensis may encode multidrug transporters similar to those encoded by the C. albicans MDR1, CDR1, and CDR2 genes (CaMDR1, CaCDR1, and CaCDR2, respectively). A C. dubliniensis homolog of CaMDR1, termed CdMDR1, was cloned; its nucleotide sequence was found to be 92% identical to the corresponding CaMDR1 sequence, while the predicted CdMDR1 protein was found to be 96% identical to the corresponding CaMDR1 protein. By PCR, C. dubliniensis was also found to encode homologs of CDR1 and CDR2, termed CdCDR1 and CdCDR2, respectively. Expression of CdMDR1 in a fluconazole-susceptible delta pdr5 null mutant of Saccharomyces cerevisiae conferred a fluconazole-resistant phenotype and resulted in a 75% decrease in accumulation of [3H]fluconazole. Northern analysis of fluconazole-susceptible and -resistant isolates of C. dubliniensis revealed that fluconazole resistance was associated with increased expression of CdMDR1 mRNA. In contrast, most studies showed that overexpression of CaCDR1 was associated with fluconazole resistance in C. albicans. Increased levels of the CdMdr1p protein were also detected in fluconazole-resistant isolates. Similar results were obtained with fluconazole-resistant derivatives of C. dubliniensis generated in vitro, some of which also exhibited increased levels of CdCDR1 mRNA and CdCdr1p protein. These results demonstrate that C. dubliniensis encodes multidrug transporters which mediate fluconazole resistance in clinical isolates and which can be rapidly mobilized, at least in vitro, on exposure to fluconazole.
| S-EPMC105690 | biostudies-literature
Project description:Adenosine triphosphate binding cassette (ABC) transporters transfer lipid-soluble molecules across cellular interfaces either directly or after enzymatic metabolism. RNAseq analysis identified transcripts for ABC transporters and enzymes in rat E19, P5 and adult brain and choroid plexus and E19 placenta. Their functional capacity to efflux small molecules was studied by quantitative analysis of paracetamol (acetaminophen) and its metabolites using liquid scintillation counting, autoradiography and ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Animals were treated acutely (30 min) and chronically (5 days, twice daily) with paracetamol (15 mg/kg) to investigate ability of brain and placenta barriers to regulate ABC transport functionality during extended treatment. Results indicated that transcripts of many efflux-associated ABC transporters were higher in adult brain and choroid plexus than at earlier ages. Chronic treatment upregulated certain transcripts only in adult brain and altered concentrations of paracetamol metabolites in circulation of pregnant dams. Combination of changes to metabolites and transport system transcripts may explain observed changes in paracetamol entry into adult and fetal brains. Analysis of lower paracetamol dosing (3.75 mg/kg) indicated dose-dependent changes in paracetamol metabolism. Transcripts of ABC transporters and enzymes at key barriers responsible for molecular transport into the developing brain showed alterations in paracetamol pharmacokinetics in pregnancy following different treatment regimens.
| S-EPMC8494792 | biostudies-literature