Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Clinical studies have suggested that both MDR1 and MRP may play a significant role in the chemosensitivity and outcome of neuroblastoma. To clarify the nature of multidrug resistance (MDR) in this tumour a series of six neuroblastoma cell lines have been characterized with regard to P-glycoprotein, MRP and LRP expression using immunocytochemistry and expression of MDR1, MRP, LRP and topoisomerase II genes using reverse transcription polymerase chain reaction (RT-PCR). By RT-PCR, all lines expressed MRP, five expressed LRP and four expressed MDR1, but protein levels of each of these were variable. Chemosensitization to a range of MDR-associated drugs (vincristine, doxorubicin, etoposide, taxotere, topotecan) and non-MDR-associated drugs (cisplatin, melphalan) by three modulating agents, cyclosporin A, PSC 833 and the novel Biricodar (VX-710; Incel), was evaluated using a colourimetric cytotoxicity assay (MTS). Alteration of daunorubicin efflux by these agents was evaluated using FACS analysis. Clonogenic assay was used to study the influence of these chemosensitizers on vincristine cytotoxicity. Marked sensitization to vincristine was observed in MDR1-positive lines, and a similar but less consistent effect was seen with taxotere, doxorubicin and etoposide. With MRP-positive, MDR-negative lines, only VX-710 caused consistent sensitization. These data confirm MDR1 and MRP expression as contributory factors in chemoresistance in neuroblastoma and indicate that VX-710 may be a useful modulator of both mechanisms and worthy of clinical evaluation in this tumour.
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PMID:BIRICODAR (VX-710; Incel): an effective chemosensitizer in neuroblastoma. 1037 71

Assessment of the chemosensitivity of dendritic cells (DC) may allow more rational development of combined chemotherapy and immunotherapy protocols. Human monocyte-derived DC generated reproducible results in the MTS (Owen's reagent) assay, which was then used to study DC survival after treatment with four different chemotherapy agents. DC preparations from three different donors were used per drug. DC were sensitive to doxorubicin (concentration range 0.1-50 microM) with variation in sensitivity between donors (IC50 244-1100 nM). The most extreme variation was seen for vinblastine (concentration range 250-0.025 microM with IC50 0.15-17.25 microM). In contrast, there was relative resistance to etoposide (concentration range 0.2-200 microM) and 5-fluorouracil (concentration range 0.7-7700 microM) with no toxicity seen until 50 microM and 770 microM respectively. The function of DC in allogeneic mixed leucocyte reactions closely paralleled results from the MTS assays. The differential sensitivity to chemotherapy agents did not appear to be due to expression of P-glycoprotein. These results suggest that etoposide or 5-fluorouracil is less likely to reduce the immunotherapeutic potential of DC and may be valuable in the design of prodrug activation therapy.
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PMID:Human cultured dendritic cells show differential sensitivity to chemotherapy agents as assessed by the MTS assay. 1060 23

Defining the residues involved in the binding of a substrate provides insight into how the human multidrug resistance P-glycoprotein (P-gp) can transport a wide range of structurally diverse compounds out of the cell. Because verapamil is the most potent stimulator of P-gp ATPase activity, we synthesized a thiol-reactive analog of verapamil (MTS-verapamil) and used it with cysteine-scanning mutagenesis to identify the reactive residues within the drug-binding domain of P-gp. MTS-verapamil stimulated the ATPase activity of Cys-less P-gp and had a K(m) value (25 microM) that was similar to that of verapamil. 252 P-gp mutants containing a single cysteine within the predicted transmembrane (TM) segments were expressed in HEK 293 cells and purified by nickel-chelate chromatography and assayed for inhibition by MTS-verapamil. The activities of 15 mutants, Y118C (TM2), V125C (TM2), S222C (TM4), L339C (TM6), A342C (TM6), A729C (TM7), A841C (TM9), N842C (TM9), I868C (TM10), A871C (TM10), F942C (TM11), T945C (TM11), V982C (TM12), G984C (TM12), and A985C (TM12), were inhibited by MTS-verapamil. Four mutants, S222C (TM4), L339C (TM6), A342C (TM6), and G984C (TM12), were significantly protected from inhibition by MTS-verapamil by pretreatment with verapamil. Less protection was observed in mutants I868C (TM10), F942C (TM11) and T945C (TM11). These results indicate that residues in TMs 4, 6, 10, 11, and 12 must contribute to the binding of verapamil.
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PMID:Defining the drug-binding site in the human multidrug resistance P-glycoprotein using a methanethiosulfonate analog of verapamil, MTS-verapamil. 1127 63

Inappropriate expression of the multidrug resistance (MDR1) gene encoding the P-glycoprotein (Pgp) has been frequently implicated in resistance to different chemotherapeutic drugs. We have previously generated chronic myeloid leukemia (CML) cell lines resistant to the tyrosine kinase inhibitor imatinib mesylate (STI571), and one line (LAMA84-r) showed overexpression not only of the Bcr-Abl protein but also of Pgp. In the present study, we investigated this phenomenon in other cell lines overexpressing exclusively Pgp. Thus, cells from the K562/DOX line, described as resistant to doxorubicin due to MDR1 gene overexpression, grew continuously in the presence of 1 microM imatinib, but died in 4 to 5 days if the Pgp pump modulators verapamil or PSC833 were added to the imatinib-treated culture. Analysis of cell proliferation by the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay confirmed the differential sensitivity of K562/DOX to imatinib, which was also reversed by verapamil or PSC833. Flow cytometric analysis of the total phosphotyrosine content by intracytoplasmic staining after a 2-hour incubation with escalating doses of imatinib showed that the inhibitory concentrations of 50% (IC(50)) for inhibition of cellular protein tyrosine phosphorylation were 15, 10, and 5 microM for K562/DOX, K562/DOX plus verapamil, and K562, respectively. Retroviral-mediated transfection of the BCR-ABL(+) AR230 cell line with the MDR1 gene decreased its sensitivity to imatinib, an effect that was also reversed by verapamil. The possible role of MDR overexpression in clinical resistance to imatinib remains to be defined. We therefore confirm that imatinib should be added to the extensive list of drugs that can be affected by the MDR phenomenon.
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PMID:MDR1 gene overexpression confers resistance to imatinib mesylate in leukemia cell line models. 1286 89

The human multidrug resistance P-glycoprotein (P-gp, ABCB1) actively extrudes a broad range of potentially cytotoxic compounds out of the cell. Key steps in understanding the transport process are binding of drug substrates in the transmembrane domains, initiation of ATPase activity, and subsequent drug efflux. We used cysteine-scanning mutagenesis of the transmembrane segment residues and reaction with the thiol-reactive drug substrate analog of rhodamine, methane-thiosulfonate-rhodamine (MTS-rhodamine), to test whether P-gp could be trapped in an activated state with high levels of ATPase activity. The presence of such an activated P-gp could be used to further investigate P-gp-drug substrate interactions. Single cysteine mutants (149) were treated with MTS-rhodamine, and ATPase activities were determined after removal of unreacted MTS-rhodamine. One mutant, F343C(TM6), showed a 5.8-fold increase in activity after reaction with MTS-rhodamine. Pre-treatment of mutant F343C with rhodamine B protected it from activation by MTS-rhodamine, indicating that residue Cys-343 contributes to the rhodamine-binding site. The ATPase activity of MTS-rhodamine-treated mutant F343C, however, was not stimulated further by colchicine or calcein-AM. By contrast, verapamil and Hoechst 33342 stimulated and inhibited, respectively, the ATPase activity of the MTS-rhodamine-treated mutant F343C. These results indicate that the MTS-rhodamine binding site overlaps that of colchicine and calcein-AM but not that of verapamil and Hoechst 33342 within the common drug-binding pocket.
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PMID:Methanethiosulfonate derivatives of rhodamine and verapamil activate human P-glycoprotein at different sites. 1452 74

P-glycoprotein (P-gp; ABCB1) actively transports a broad range of structurally unrelated compounds out of the cell. An important step in the transport cycle is coupling of drug binding with ATP hydrolysis. Drug substrates such as verapamil bind in a common drug-binding pocket at the interface between the TM (transmembrane) domains of P-gp and stimulate ATPase activity. In the present study, we used cysteine-scanning mutagenesis and reaction with an MTS (methanethiosulphonate) thiol-reactive analogue of verapamil (MTS-verapamil) to test whether the first TM segment [TM1 (TM segment 1)] forms part of the drug-binding pocket. One mutant, L65C, showed elevated ATPase activity (10.7-fold higher than an untreated control) after removal of unchanged MTS-verapamil. The elevated ATPase activity was due to covalent attachment of MTS-verapamil to Cys65 because treatment with dithiothreitol returned the ATPase activity to basal levels. Verapamil covalently attached to Cys65 appears to occupy the drug-binding pocket because verapamil protected mutant L65C from modification by MTS-verapamil. The ATPase activity of the MTS-verapamil-modified mutant L65C could not be further stimulated with verapamil, calcein acetoxymethyl ester or demecolcine. The ATPase activity could be inhibited by cyclosporin A but not by trans-(E)-flupentixol. These results suggest that TM1 contributes to the drug-binding pocket.
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PMID:Transmembrane segment 1 of human P-glycoprotein contributes to the drug-binding pocket. 1649 38

P-gp (P-glycoprotein; ABCB1) protects us by transporting a broad range of structurally unrelated compounds out of the cell. Identifying the regions of P-gp that make up the drug-binding pocket is important for understanding the mechanism of transport. The common drug-binding pocket is at the interface between the transmembrane domains of the two homologous halves of P-gp. It has been shown in a previous study [Loo, Bartlett and Clarke (2006) Biochem. J. 396, 537-545] that the first transmembrane segment (TM1) contributed to the drug-binding pocket. In the present study, we used cysteine-scanning mutagenesis, reaction with an MTS (methanethiosulfonate) thiol-reactive analogue of verapamil (termed MTS-verapamil) and cross-linking analysis to test whether the equivalent transmembrane segment (TM7) in the C-terminal-half of P-gp also contributed to drug binding. Mutation of Phe728 to cysteine caused a 4-fold decrease in apparent affinity for the drug substrate verapamil. Mutant F728C also showed elevated ATPase activity (11.5-fold higher than untreated controls) after covalent modification with MTS-verapamil. The activity returned to basal levels after treatment with dithiothreitol. The substrates, verapamil and cyclosporin A, protected the mutant from labelling with MTS-verapamil. Mutant F728C could be cross-linked with a homobifunctional thiol-reactive cross-linker to cysteines I306C(TM5) and F343C(TM6) that are predicted to line the drug-binding pocket. Disulfide cross-linking was inhibited by some drug substrates such as Rhodamine B, calcein acetoxymethyl ester, cyclosporin, verapamil and vinblastine or by vanadate trapping of nucleotides. These results indicate that TM7 forms part of the drug-binding pocket of P-gp.
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PMID:Transmembrane segment 7 of human P-glycoprotein forms part of the drug-binding pocket. 1681 63

Given the widespread use of formulations combining anthelmintics which are possible P-glycoprotein interfering agents, the understanding of drug interactions with efflux ABC transporters is of concern for improving anthelmintic control. We determined the ability of 14 anthelmintics from different classes to interact with abcb1a (mdr1a, P-glycoprotein, Pgp) by following the intracellular accumulation of rhodamine 123 (Rho 123), a fluorescent Pgp substrate, in LLC-PK1 cells overexpressing Pgp. The cytotoxicity of the compounds that are able to interfere with Pgp activity was evaluated in cells overexpressing Pgp and compared with parental cells using the MTS viability assay. Among all the anthelmintics used, ivermectin (IVM), triclabendazole (TCZ), triclabendazole sulfoxide (TCZ-SO), closantel (CLOS) and rafoxanide (RAF) increased the intracellular Rho 123 in Pgp overexpressing cells, while triclabendazole sulfone, albendazole, mebendazole, oxfendazole, thiabendazole, nitroxynil, levamisole, praziquantel and clorsulon failed to have any effect. The concentration needed to reach the maximal Rho 123 accumulation (E(max)) was obtained with 10 microM for IVM, 80 microM for CLOS, 40 microM for TCZ and TCZ-SO, and 80 microM for RAF. We showed that for these five drugs parental cell line was more sensitive to drug toxicity compared with Pgp recombinant cell line. Such in vitro approach constitutes a powerful tool to predict Pgp-drug interactions when formulations combining several anthelmintics are administered and may contribute to the required optimization of efficacy of anthelmintics.
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PMID:Interaction of anthelmintic drugs with P-glycoprotein in recombinant LLC-PK1-mdr1a cells. 2051 41

Ultrasound treatment has been shown to enhance the uptake of both hydrophilic and hydrophobic compounds into PC3 and Huvec cell lines using an insonation regimen of a single 10-s burst of high-frequency (4 MHz), moderate intensity (32 W/cm(2)) ultrasound. The purpose of this work was to evaluate the effect of this ultrasound regimen on the cellular accumulation of paclitaxel (PTX) loaded in copolymer micellar of methoxy poly(ethylene glycol)-block-poly(D,L-lactide) (MePEG-b-PDLLA) in both drug-sensitive (MDCKII and MCF-7) and P-glycoprotein (Pgp)-expressing (MDCKII-MDR and NCI-ADR) cell lines. There were no effects of ultrasound on hydrodynamic diameters of micelles and the release of FRET pairs, indicating the integrity of micelles was maintained. There was a two-fold increase in intracellular PTX for all ultrasound-treated drug-sensitive cell lines and their respective drug-resistant counterparts compared with no ultrasound. Significant decreases in drug efflux rates were observed at 20, 40 and 60 min for both drug-sensitive and -resistant cell lines receiving ultrasound. The enhanced accumulation and retention of PTX by ultrasound resulted in greater cytotoxicity in both MDCKII and MDCKII-MDR cell lines, as indicated by the MTS assay. These data suggest that ultrasound may facilitate the uptake of intact paclitaxel-loaded micelles into cells, allowing greater retention of drug in both Pgp and non-Pgp-expressing cells.
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PMID:Increased accumulation and retention of micellar paclitaxel in drug-sensitive and P-glycoprotein-expressing cell lines following ultrasound exposure. 2242 83

The expression of ATP-dependent efflux pump P-glycoprotein (P-gp) in cancer cells generally results in multidrug resistance (MDR) to chemotherapeutic drugs, which is the main cause of chemotherapy failure in cancer treatment. The intracellular drug levels could be increased by some MDR reversal agents that inhibited the drug efflux activity of P-gp. The synthesized DNA nucleic acids of G-quadruplex represent a novel and unique class of anti-cancer agents. While there was no report on the roles of DNA G-quadruplex oligonucleotides (GQ-ODNs) in the MDR reversal, the present study was performed to investigate the ability of synthesized GQ-ODNs to reverse P-gp-mediated MDR and its mechanism in paclitaxel (PTX)-resistant SKOV3 (SKOV3/PTX) cells and their sensitive cell lines SKOV3. The ability of GQ-ODNs to reverse drug resistance was evaluated by MTS assay. The results showed that GQ-ODNs can reverse PTX resistance effectively. The potential of GQ-ODNs as reversal agents was evaluated with the nude mice tumor xenograft model and showed that the co-administration of the GQ-ODNs and PTX had better effects and was also more evident than treatment with only PTX. The P-gp expression was assessed by the Western blot; it showed that SKOV3/PTX cells showed highly expressed P-gp protein, while their sensitive cells scarcely showed P-gp. The presence of GQ-ODNs efficiently decreased the P-gp expression, showing that GQ-ODNs could reverse P-gp-mediated MDR by decreasing the expression of P-gp. This study indicated that GQ-ODNs could effectively reverse P-gp-mediated PTX resistance by inhibiting the expression of P-gp and by the co-administration of GQ-ODNs and PTX that could increase the apoptosis of SKOV3/PTX cells. Thus, the synthesized GQ-ODNs may be a potential inhibitor to overcome drug resistance.
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PMID:Inhibition of mdr1 by G-quadruplex oligonucleotides and reversal of paclitaxel resistance in human ovarian cancer cells. 2580 Dec 44


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