Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.3.44 (P-glycoprotein)
 13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Microspectrofluorometry allows the analysis of fluorescent molecules such as anthracyclines in the nucleus of isolated living cells. Using this technique, we confirmed that the amount of doxorubicin or THP-doxorubicin incorporated into the nucleus was related to the resistant or sensitive character of K562 cells. It was then extended to the study of fresh leukemic cells and kinetic studies were performed allowing the calculation of the retention rate (RR) of anthracycline (THP-doxorubicin) into the cell nucleus. A reproducibility study confirmed the accuracy of the method. Blast cells collected in patients with acute myeloid (n = 22) or lymphoid (n = 8) leukemia, at diagnosis (n = 26), or in relapse (n = 4) have been studied. RR varied from 8 to 98% independently of the type of leukemia or the clinical status. RR did not correlate either with P-glycoprotein or with CD34 expression although this latter result should be confirmed on a higher number of subjects. Among 18 patients presenting with AML at diagnosis, 14 have been treated with intensive chemotherapy including anthracyclines; the only one who had resistant disease had the lowest RR value. In conclusion, the results obtained here show that microspectrofluorometry allows the performance of kinetic studies on fresh leukemic cells in order to quantify chemo-resistance phenomena related to drug transport.
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PMID:In vitro study of THP-doxorubicin retention in human leukemic cells using confocal laser microspectrofluorometry. 764 25

S9788 modulating resistance effect has been investigated on the activity of THP-DOX against multidrug-resistant K562R cells and compared to that of cyclosporin A and verapamil. Intracellular THP-DOX distribution and particulary its intranuclear concentration, with or without modulators, has been measured using confocal laser microspectrofluorometry. The kinetics of intranuclear accumulation of THP-DOX (1 microM in the medium), as a function of time, were rapid in K562S and K562R cells. Maximum accumulation of THP-DOX is reached in a few minutes (K562S, 400 microM; K562R, 40 microM). The addition of S9788, cyclosporin A and verapamil (5 microM) after one hour THP-DOX incubation, led to respectively 290, 250 and 114 microM. Uptake of THP-DOX was increased in K562R cells by a factor of 7 when S9788 was added. Results obtained on THP-DOX efflux from nuclei of K562S and K562R cells, after 3 hours of incubation without drug, showed a very short T1/2 (time corresponding to 50% decrease of intranuclear concentration of THP-DOX) in K562R cells (12 min) compared to that in K562S cells (150 min). The addition of S9788, cyclosporin A and verapamil (5 microM) led to a T1/2 of 90, 30 and 20 min respectively. The T1/2 of THP-DOX was increased in K562R cells by a factor of 7.5 when S9788 was added. We tried to correlate these results with those obtained in growth inhibition study. The IC50 (concentration which induces 50% growth inhibition) of THP-DOX, corresponding to one hour THP-DOX treatment and 3 days culture of K562S and K562R cells were respectively 230 and 7000 nM, and the RI (resistance index) value is 30. The addition of S9788, cyclosporin A and verapamil (5 microM), during the one hour treatment, led to an IC50 value of 350, 2000 and 5000 nM respectively. S9788 induced an IC50 value 20 times lower than without the modulator. Our study suggests that the higher activity of THP-DOX against K562R cells in the presence of S9788, compared to cyclosporin A and verapamil, is due to a higher intranuclear THP-DOX accumulation and to a strong decrease of drug efflux from K562R nuclei. This could be explained by a higher affinity of S9788 for membrane P-glycoprotein of K562R cells and/or an additional mechanism of action.
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PMID:Effect of S9788, cyclosporin A and verapamil on intracellular distribution of THP-doxorubicin in multidrug-resistant K562 tumor cells, as studied by laser confocal microspectrofluorometry. 782 78

The decrease of the intracellular concentration of drug in resistant cells as compared to sensitive cells is, in most of cases, correlated with the presence, in the membrane of resistant cells, of a 170-kDa P-glycoprotein (P-gp) responsible for an active efflux of the drug. The fluorescence emission spectra from anthracycline-treated cells suspended in buffer have been used to follow the P-gp-associated efflux of these drugs in the absence or presence of verapamil. In the present study, 4'-o-tetrahydro-pyranyladriamycin (THP-adriamycin) was used. Two different methods were used to determine the kinetics of active efflux of THP-adriamycin: (1) at the steady-state, (2) directly, after the addition of glucose to cells first incubated with THP-adriamycin in the presence of N3- and in the absence of glucose. Kinetic analysis indicates: (1) a saturation of the active efflux when the cytosolic free drug concentration increased (the Michaelis constant Km = 0.5 +/- 0.3 microM) and (2) that the inhibitory effect of verapamil on P-gp-associated efflux of THP-adriamycin in living cells is non-competitive.
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PMID:Non-competitive inhibition of P-glycoprotein-associated efflux of THP-adriamycin by verapamil in living K562 leukemia cells. 790 85

Confocal microspectrofluorometry allows the analysis of fluorescent molecules such as anthracylines in isolated living cells. An optical microscope fitted with a phase-contrast 100 X water-immersion objective enables simultaneous observation of the sample, focusing of the laser beam on the selected cell fraction (nucleus) and collection of the fluorescence emitted from the sample. The resulting intranuclear spectra are interpreted according to a quantitative model of the fluorescence spectra of both free and DNA-bound anthracycline. The intranuclear drug concentration can thus be determined. This technique has been applied to blast cells collected in patients with acute leukemia. Leukemic cells are aspirated from bone marrow, separated by Ficoll sedimentation and resuspended in RPMI-1640 containing 10% fetal calf serum and 200 nM tetrahydropyranyl-doxorubicin (THP-DOX). After one hour, 20 cells are analyzed and the mean nuclear drug content is determined (C1). Cells are then resuspended in the same medium but without anthracycline for 3 hours and the mean intranuclear drug concentration is then also determined (C3). From C1 and C3 the retention rate (RR) is calculated. Firstly, the accuracy of the method was checked. In 4 AML patients, two different samples aspirated on the same day were divided into two portions. Thus, two measurements were made on each one (4 values per patient). Coefficients of variation were satisfactory (4, 6, 12, and 12%). Secondly, blast cells collected in patients with AML and ALL at diagnosis or in relapse were studied. P-glycoprotein (P-gp) and CD34 expression was also studied using respectively immunohistochemistry land flow cytometry. Results obtained from the first 21 patients showed that there was no correlation between RR and either P-gp or CD34 expression. This could result from the efflux of THP-DOX by other mechanisms and/or low sensitivity of the staining technique.
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PMID:[Evaluation of multidrug resistance phenotype on medullary specimens from patients with acute leukemia by determination of nuclear efflux of tetrahydropyranyl-doxorubicin. Approach by confocal laser microspectrofluorometry]. 873 89

Effectiveness of chemotherapeutic treatment is limited by multidrug resistance (MDR) phenomenon mediated by the overexpression of P-glycoprotein 170 termed Pgp which serves as an efflux pump removing several types of cytostatic drugs from the MDR cells. Several small molecules, frequently lipophilic cations and weak bases, are able to reverse in vitro this resistance. Several studies have shown that MDR modulators interact with Pgp. However, some molecules do not interact with Pgp but are able to completely restore drug sensitivity (e.g., quinine). Bennis et al. (1995) have shown recently that in contrast to verapamil and S9788, quinine increases nuclear doxorubicin accumulation without modifying its intracellular concentration. From this work, the authors concluded that quinine has essentially intracellular targets involved in drug distribution (cytoplasm to nucleus) from sequestration compartments. Their results have been obtained using spectrofluorometry on cell populations and fluorescence microscopy. By using confocal laser microspectrofluorometry, we investigated restoration of nuclear THP-DOX accumulation and sensitivity by verapamil, S9788 and quinine in 2 variants of the Chinese hamster ovary cells LR73, selected for resistance to doxorubicin (LR73D) and transfected with the mdr1 gene (LR73R), as well as in the sensitive ones (LR73S). Results show that verapamil and S9788 were able to restore THP-DOX sensitivity in resistant cells by increasing nuclear THP-DOX accumulation. This restoration is the consequence of Pgp inhibition and redistribution of the anticancer drug from the cytoplasm to nucleus. Quinine, in contrast, restores the sensitivity of MDR cells to THP-DOX and decreased their resistance index, but has no effect on THP-DOX nuclear accumulation. This suggests that quinine modifies the molecular environment of anthracyclines and/or their binding to cytoplasmic targets involved in another mechanism of anthracycline action.
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PMID:[Effect of quinine on the multiple drug resistance and intracellular distribution of pirarubicin in LR73 tumor cells: a comparative study with verapamil and S9788 by confocal laser microspectrofluorometry]. 923 56

Confocal laser microspectrofluorometry was used to investigate restoration of nuclear pirarubicin (THP-DOX) accumulation and sensitivity by verapamil, quinine and S9788 in 2 variants of the Chinese hamster ovary cell lines LR73, selected for resistance to doxorubicin (LR73D) or transfected with the mdr1 gene (LR73R). The 2 resistant cell lines present a multidrug-resistance phenotype (MDR). Verapamil and S9788, which interact with P-glycoprotein (P-gp), were able to restore nuclear THP-DOX accumulation in LR73R and LR73D cells to a level equivalent to that in sensitive cells. On the other hand, quinine was unable to increase nuclear THP-DOX accumulation significantly even at a concentration of 50 microM. All modulators completely restored THP-DOX sensitivity in resistant cell lines. Our results also show that verapamil and S9788 allow high nuclear drug accumulation, whereas quinine did not affect nuclear accumulation. The effect of quinine on the mdr1 gene expression was determined by the use of reverse transcription coupled with polymerase chain reaction. After a 2 hr treatment with 20 microM of quinine, mdr1 gene expression increased slightly.
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PMID:Evidence for reversal of multidrug resistance by quinine in LR73 cells without alteration of nuclear pirarubicin uptake and down-regulation of mdr1 gene expression. 938 78

On the basis of the results obtained in previous research, three series of compounds (A-C), derived from verapamil, were designed and synthesized to obtain drugs able to revert multidrug resistance (MDR), an acquired resistance that frequently impairs cancer chemotherapy. The ability of the obtained compounds to revert MDR was evaluated on anthracycline-resistant erythroleukemia K 562 cells, measuring the uptake of THP-adriamycin (pirarubicin) by continuous spectrofluorometric monitoring of the decrease of the fluorescence signal of the anthracycline at 590 nm (lambdaex = 480 nm), after incubation with cells. Cardiovascular activity, which is responsible for unwanted side effects, was also evaluated. The results obtained show that many of the compounds studied are potent reverters of MDR and are endowed with reduced cardiovascular activity. One of the compounds (7, MM36) presents a pharmacological profile (unprecedented nanomolar potency, high reversal of MDR, low cardiovascular activity) that makes it a promising drug candidate to treat MDR and a useful tool for studying P-glycoprotein.
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PMID:Design, synthesis, and in vitro activity of catamphiphilic reverters of multidrug resistance: discovery of a selective, highly efficacious chemosensitizer with potency in the nanomolar range. 1034 21

P-glycoprotein (P-gp; MDR1), a major efflux transporter, recognizes various antibiotics and is present in macrophages. We have examined its effect on the modulation of the intracellular accumulation and activity of daptomycin towards phagocytized Staphylococcus aureus (ATCC 25923) in human THP-1 macrophages, in comparison with MDCK epithelial cells (wild type and MDCK-MDR1 overexpressing P-gp; the bulk of the protein was immunodetected at the surface of all three cell types). Daptomycin displayed concentration-dependent intracellular activity (Hill equation pattern) in THP-1 and MDCK cells with (i) 50% effective drug extracellular concentration (EC(50); relative potency) and static concentrations at 9 to 10 times the MIC and (ii) maximal efficacy (E(max); CFU decrease at infinite extracellular drug concentration) at 1.6 to 2 log compared to that of the postphagocytosis inoculum. Verapamil (100 microM) and elacridar (GF 120918; 0.5 microM), two known inhibitors of P-gp, decreased daptomycin EC(50) (about threefold) in THP-1 and MDCK cells without affecting E(max). Daptomycin EC(50) was about three- to fourfold higher and accumulation in MDCK-MDR1 commensurately lower than in wild-type cells. In THP-1 macrophages, (i) verapamil and ATP depletion increased, and ouabain (an inducer of mdr1 [the gene encoding P-gp] expression) decreased the accumulation of daptomycin in parallel with that of DiOC(2) (a known substrate of P-gp); (ii) silencing mdr1 with duplex human mdr1 siRNAs reduced the cell content in immunoreactive P-gp to 15 to 30% of controls and caused an eight- to 13-fold increase in daptomycin accumulation. We conclude that daptomycin is subject to efflux from THP-1 macrophages and MDCK cells by P-gp, which reduces its intracellular activity against phagocytized S. aureus.
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PMID:Modulation of the cellular accumulation and intracellular activity of daptomycin towards phagocytized Staphylococcus aureus by the P-glycoprotein (MDR1) efflux transporter in human THP-1 macrophages and madin-darby canine kidney cells. 1754 93

Some natural health products (NHPs) affect drug metabolism enzymes and transport proteins, potentially affecting the safety and efficacy of the drug or other NHPs. This study was undertaken to characterize the effect of uva-ursi (Arctostaphylos uva-ursi) on cytochrome P450 isozyme (3A4, 3A5, 3A7, 2C19, and 19)-mediated metabolism and P-glycoprotein (P-gp) transport. Three bulk and 2 capsulated uva-ursi samples were obtained from commercial outlets. The capsules were batched, and herbal samples were ground to a common consistency. Aqueous and methanol extracts were freshly prepared. Cytochrome P450 isozyme-mediated metabolism was determined by using in vitro bioassays. P-gp transport function was determined by using a rhodamine 123 (Rh123) uptake test in human (THP-1) monocytes and human Caco-2 cells. All products were analyzed by HPLC for arbutin, gallic acid, myricitrin, and isoquercetin. A large variation was observed in the biomarkers found between the bulk and capsulated samples. Our data indicate that both the aqueous and methanol extracts of all 5 uva-ursi products showed high cytochrome P450 isozyme inhibition, with the exception of the methanol extracts against cytochromes P3A4 and P19, which had low to moderate activity. The aqueous extracts of uva-ursi showed an inhibitory effect on Rh123 efflux by P-gp at 1 h and an inductive effect at 18 h for both cell lines. Our results show that the uva-ursi herbal products tested here have pharmacological properties, including the potential capacity to affect drug safety and efficacy. Further studies are warranted against a wider range of cytochrome P450 isozymes and to determine whether these effects are clinically significant.
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PMID:In vitro activity of uva-ursi against cytochrome P450 isoenzymes and P-glycoprotein. 1806 12

CEM-101 is a novel fluoroketolide with lower MICs than those of telithromycin and macrolides. Our aim was to assess the cellular accumulation and intracellular activity of CEM-101 using models developed for analyzing the pharmacokinetics and pharmacological properties of antibiotics against phagocytized bacteria. We used THP-1 macrophages and Staphylococcus aureus (ATCC 25923 [methicillin (meticillin) sensitive]), Listeria monocytogenes (strain EGD), and Legionella pneumophila (ATCC 33153). CEM-101 reached cellular-to-extracellular-concentration ratios of about 350 within 24 h (versus approximately 20, 30, and 160 for telithromycin, clarithromycin, and azithromycin, respectively). This intracellular accumulation was suppressed by incubation at a pH of < or = 6 and by monensin (proton ionophore) and was unaffected by verapamil (P-glycoprotein inhibitor; twofold accumulation increase for azithromycin) or gemfibrozil. While keeping with the general properties of the macrolide antibiotics in terms of maximal efficacy (Emax; approximately 1-log10-CFU decrease compared to the postphagocytosis inoculum after a 24-h incubation), CEM-101 showed significantly greater potency against phagocytized S. aureus than telithromycin, clarithromycin, and azithromycin (for which the 50% effective concentration [EC50] and static concentrations were about 3-, 6-, and 15-fold lower, respectively). CEM-101 was also about 50-fold and 100-fold more potent than azithromycin against phagocytized L. monocytogenes and L. pneumophila, respectively. These differences in EC50s and static concentrations between drugs were minimized when data were expressed as multiples of the MIC, demonstrating the critical role of intrinsic drug activity (MIC) in eliciting the antibacterial intracellular effects, whereas accumulation per se was unimportant. CEM-101 should show enhanced in vivo potency if used at doses similar to those of the comparators tested here.
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PMID:Cellular accumulation and pharmacodynamic evaluation of the intracellular activity of CEM-101, a novel fluoroketolide, against Staphylococcus aureus, Listeria monocytogenes, and Legionella pneumophila in human THP-1 macrophages. 1956 65


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