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)

We have established a quantitative flow cytometry system to elucidate the causal role of P-glycoprotein in the phenomenon of multidrug resistance. We have used this method to analyze the accumulation and release of adriamycin (ADM) in intact L5178Y and L5178Y/VMDR/C.06 (L5178Y/R) cells, by determining the effect of sodium orthovanadate (Na3VO4), verapamil, bovine serum albumin (BSA) and physiologically operative materials on the cells. Based on the experiments, we prepared a standard solution that contained NaCl, D-glucose, L-cysteine, HCO3- and BSA, which was sufficient to perform transport experiments. In particular, BSA caused a decrease in ADM accumulation and a facilitation of the rate of ADM release in both L5178Y and L5178Y/R cells, probably due to its relatively high affinity for ADM as compared to the cell membrane. In multidrug-resistant L5178Y/R cells, sodium orthovanadate, a strong ATP-binding inhibitor, caused a marked increase in the accumulation of ADM, whereas vanadate-treated drug-sensitive L5178Y cells showed little increase in ADM accumulation. In a release (0-trans exit) experiment, vanadate-treated L5178Y/R cells exhibited an apparent decrease in ADM release (increase in ADM retention), to a level which was almost the same as L5178Y cells. We thus confirmed that the P-glycoprotein-mediated efflux system is coupled with P-glycoprotein-associated ATP-hydrolysis. Further, verapamil, a potent inhibitor of P-glycoprotein-mediated transport, facilitated the ADM accumulation in L5178Y/R cells up to the level of L5178Y and vanadate-treated L5178Y/R cells. A more important finding is that, in the release experiment, verapamil-treated L5178Y/R cells exhibited a much greater ADM retention than drug-sensitive L5178Y and vanadate-treated L5178Y/R cells. These findings, in particular the potent effect of verapamil on drug-resistant cells, may afford new insight into the pathophysiology of the phenomenon of multidrug resistance and the mechanism of action of the multidrug transporter.
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PMID:Quantitative characterization of P-glycoprotein-mediated transport in mdr1-gene-transfected lymphoma cells. 874 16

Although multidrug resistance (mdr) may arise through a variety of mechanisms, the most widely studied and accepted form is associated with an increased concentration of P-glycoprotein (P-gp), a 170 kd protein found in the membrane fraction of a number of mammalian cells. Since mdr seems to be related to the ability of resistant cells to extrude drugs and the circumvention of mdr is supposed to be due to the restored ability to accumulate drugs, membrane has been regarded as the crucial site for such a regulation and an important role for membrane ion exchangers has been suggested. The aim of this work was to elucidate whether the Na+/H+ antiporter is involved in the mechanism of regulation and circumvention of mdr and if 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a selective inhibitor of the Na+/H+ exchanger, can modulate the functional expression of the mdr phenotype. The effect of EIPA on doxorubicin (DX) resistant cells (LoVo/DX) obtained from a human colon adenocarcinoma cell line (LoVo) was studied. EIPA at concentrations ranging from 10 to 50 mu M was able to increase the antibiotic cytotoxicity in the resistant Lovo/DX cells. The reversal of DX resistance paralleled an increase of the ability of the cells to accumulate the drug. Both drug loading and sensitivity to the inhibitory effect of DX on cell proliferation were restored by EIPA in a dose-dependent way. These results suggest a new mechanism of mdr reversal and indicate that amiloride and its derivatives may be useful in reversing DX resistance and in enhancing the clinical effectiveness of chemotherapeutics.
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PMID:Reversal of doxorubicin resistance by the amiloride analogue EIPA in multidrug resistant human colon carcinoma cells. 890 49

Plasma membrane P-glycoprotein is known as an ATP-dependent drug efflux pump that confers multidrug resistance to tumor cells. None of the reported purification procedures worked properly for our P-glycoprotein-overproducing cell lines, i.e. murine lymphoid leukemia P388/ADR25, rat hepatoma AS30-D/COL10, and human lymphoblastic leukemia CEM/VLB5 cells. We have thus developed a general procedure for efficient purification of P-glycoprotein by combining solubilization with sodium dodecyl sulfate and chromatography on ceramic hydroxyapatite. This procedure was successful for the three cell lines and yielded 70% of the P-glycoprotein present in the starting plasma membranes with more than 99% purity. After exchanging sodium dodecyl sulfate into dodecyl maltoside and reconstitution into liposomes, purified P-glycoprotein exhibited a specific ATPase activity of about 200 nmol/min/mg, which was very similar to that obtained for P-glycoprotein solubilized and purified with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. This ATPase activity was sensitive to orthovanadate inhibition and stimulated by verapamil and other drugs. More importantly, drug transport properties of the reconstituted P-glycoprotein were comparable with those of P-glycoprotein embedded in plasma membranes. Since it is virtually devoid of lipids, this preparation is suitable for both functional and structural investigations.
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PMID:Efficient purification and reconstitution of P-glycoprotein for functional and structural studies. 891 May 34

Murine and human cell lines overexpressing the multidrug-resistance protein (MRP) showed a marked decreased accumulation of the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In contrast, less altered accumulation was seen in the P-glycoprotein(P-gp)-overexpressing cell lines. The decreased drug accumulation was reversed by the energy inhibitors sodium azide/2-deoxyglucose and by the vinca alkaloid, vincristine, but not by the chemotherapeutic agents, etoposide and adriamycin. Decreased accumulation was linked to active efflux of the hydrophilic free acid form of BCECF from the MRP-overexpressing cell lines, indicating that dye extrusion occurs after the dye ester has been converted to the free acid form in the cytoplasm. The finding suggests that MRP mediates removal of substrates from a cytoplasmic location. Buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, decreased the vincristine and etoposide resistance displayed by the MRP-expressing murine cell lines, but did not affect the accumulation of BCECF. Thus, while glutathione may be involved in MRP-mediated resistance to some chemotherapeutic agents, it is not necessary for effiux of substrates such as BCECF.
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PMID:Active efflux of the free acid form of the fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in multidrug-resistance-protein-overexpressing murine and human leukemia cells. 903 Jul 42

Chemotherapy for adult T-cell leukemia (ATL) has been reported to fail to induce complete remission because of drug resistance in most patients. We have examined the expression of an ATL-derived factor (ADF)/thioredoxin in relation to resistance to adriamycin (ADM) in various T-cell leukemia cell lines including ATL cell lines. Immunoblot analysis demonstrated that ATL cell lines expressed ADF/thioredoxin at levels 2.8 to 12 times those of other T-cell acute lymphocytic leukemia (T-ALL) cell lines, and that ATL cell lines were 2 to 15 times more resistant to ADM than other T-ALL cell lines. Therefore, we established ADM-resistant cell lines from three different ATL cell lines, and examined the correlation between ADM resistance and expression of ADF/thioredoxin. ADM-resistant ATL cell lines were also found to be resistant to other drugs such as cisplatin and etoposide, and they expressed ADF/thioredoxin at levels 5 to 10 times those of parent ATL cell lines. Diamide and sodium selenite, which have been reported to inhibit ADF/thioredoxin, restored the sensitivity to ADM in ATL and ADM-resistant ATL cell lines. The MDR-1 gene product, a membrane P-glycoprotein (Pgp), was not expressed on ATL cell lines or ADM-resistant ATL cell lines. Topoisomerase II and glutathione peroxidase activities in T-cell leukemia cell lines were not correlated with ADM resistance. These results suggest that ADF/thioredoxin may play an important role in the drug resistance of ATL cells to ADM.
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PMID:Possible roles of an adult T-cell leukemia (ATL)-derived factor/thioredoxin in the drug resistance of ATL to adriamycin. 911 92

Up to now, removal of sodium dodecyl sulfate (SDS) from proteins in terms of restoration of their activity was an unsolved problem. A general procedure using ceramic hydroxyapatite (HAP) chromatography was developed for the complete removal of SDS bound to soluble or membrane proteins. This procedure involves (i) the binding of the SDS-protein complexes onto the ceramic hydroxyapatite column, (ii) extensive washing of bound proteins with phosphate buffer containing a mild detergent to exchange SDS, (iii) elution of the retained protein by increasing the phosphate concentration. Using this approach, complete exchange of [35S]SDS into a nonionic detergent such as dodecyl maltoside was achieved with a 90-100% protein recovery. The efficiency of protein-bound SDS removal is very likely due to the combined effect of phosphate ions and the hydrophobic tail of nonionic detergent: acting together, they are able to displace SDS molecules from their protein-binding sites. The advantages of this HAP-mediated SDS removal method include high efficiency, rapidity, simplicity and general applicability to a wide variety of detergents and soluble or membrane proteins. Of utmost importance, SDS-treated P-glycoprotein, glutamate dehydrogenase, and lysozyme fully recovered their enzymatic activities after HAP chromatography, including lysozyme electroeluted from SDS-polyacrylamide gel electrophoresis. This demonstrates that reactivation of SDS-treated protein can be achieved, provided that SDS is completely removed under mild conditions.
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PMID:Complete removal and exchange of sodium dodecyl sulfate bound to soluble and membrane proteins and restoration of their activities, using ceramic hydroxyapatite chromatography. 917 96

Iron (Fe) chelators of the pyridoxal isonicotinoyl hydrazone (PIH) class may be useful agents to treat Fe overload disease and also cancer. These ligands possess high activity at mobilizing 59Fe from neoplastic cells, and the present study has been designed to examine whether their marked activity may be related to an energy-dependent transport process across the cell membrane. Initial experiments examined the release of 59Fe from SK-N-MC neuroblastoma (NB) cells prelabelled for 3 h at 37 degrees C with 59Fe-transferrin (1.25 microM) and then reincubated in the presence and absence of the chelators for 3 h at 4 degrees C or 37 degrees C. Prelabelled cells released 4-5% of total cellular 59Fe when reincubated in minimum essential medium at 4 degrees C or 37 degrees C. When the chelators desferrioxamine (DFO; 0.1 mM) or PIH (0.1 mM) were reincubated with labelled cells at 4 degrees C, they mobilized only 4-5% of cellular 59Fe, whereas as 37 degrees C, these ligands mobilized 21% and 48% of cell 59Fe, respectively. The lipophilic PIH analogue, 311 (2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone; 0.1 mM), which exhibits high anti-proliferative activity, released 10% and 53% of cellular 59Fe when reincubated with prelabelled cells at 4 degrees C and 37 degrees C, respectively. Almost identical results were obtained using the SK-Mel-28 melanoma cell line. These data suggest that perhaps temperature-dependent mechanisms are essential for 59Fe mobilization from these cells. Interestingly, the metabolic inhibitors, 2,4-dinitrophenol, oligomycin, rotenone, and sodium azide, markedly decreased 59Fe mobilization mediated by PIH, but had either no effect or much less effect on 59Fe release by 311. Considering that an ATP-dependent process was involved in 59Fe release by PIH, further studies examined 4 widely used inhibitors of the multi-drug efflux pump P-glycoprotein (P-gp). All of these inhibitors, namely, verapamil (Ver), cyclosporin A (CsA), reserpine (Res) and quinine (Qui), decreased 59Fe mobilization by PIH but had little or no effect on 59Fe release mediated by analogue 311. Further, both CsA and Ver increased the proportion of ethanol-soluble 59Fe within cells in the presence of PIH, suggesting inhibited transport of the 59Fe complex from the cell. However, when PIH-mediated 59Fe release was compared between a well characterized Chinese hamster ovary cell line (CHRB30) expressing high levels of P-gp and the relevant control cell line (AuxB1), no appreciable difference in the kinetics of 59Fe release were found. In contrast, it was intriguing that the CHRB30 cells released more 59Fe into control medium (i.e., without PIH) than the AuxB1 control line (16.7% compared to 5.9%, respectively). In summary, the results suggest that a temperature- and energy-dependent process was involved in the efflux of the PIH-59Fe complex from the cells. In contrast, 59Fe release mediated by 311 was temperature-dependent but not energy-dependent, and could occur by simple diffusion or passive transport. Further studies investigating the membrane transport of Fe chelators may be useful in designing regimes that potentiate their anti-neoplastic effects.
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PMID:Mobilization of iron from neoplastic cells by some iron chelators is an energy-dependent process. 918 79

Drug accumulation studies with the anticancer agents adriamycin and vincristine were carried out on the MDR variant of the human lung cell lines DLKP, DLKP-A10 which overexpresses the MDR associated P-glycoprotein efflux pump. Reduced cellular accumulation of both agents was observed in the resistant variant. The subsequent addition of verapamil and cyclosporin A resulted in partial restoration of cellular accumulation of both drugs in the DLKP-A10 resistant variant while complete restoration of cellular drug levels was observed in the SKMES-1/ADR cell line. These results suggested that the accumulation defect observed in the SKMES-1/ADR cell line was P-glycoprotein mediated and that accordingly, the cells exhibited characteristics consistent with the classical MDR phenotype. In contrast, while P-glycoprotein also appears to mediate a reduction in cellular drug accumulation in the DLKP-A10 cells, an alternative transport mechanism may also be present. No significant increase in the expression of either the MRP or LRP transport proteins was observed in the resistant cells. Metabolic inhibition by antimycin A (but not sodium azide or 2-deoxy-D-glucose) resulted in complete restoration of drug accumulation suggesting the presence of an alternative energy dependent transport mechanism. Fluorescent microscopy studies indicated different cellular localisation of the drug within the parental and resistant cells despite equivalent intracellular concentrations. These studies also revealed the presence of an ATP-dependent, vesicular sequestration mechanism which may be involved in the reduction of nuclear adriamycin accumulation in the DLKP-A10 cell line. This was indicated by observation of the disruption of cytoplasmic vesicles by antimycin A and also inhibition of cytoplasmic drug sequestration by the carboxylic ionophores, monensin and nigericin, accompanied by increased adriamycin accumulation and redistribution of the drug from the cytoplasm to the nucleus.
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PMID:The multidrug-resistant human lung tumour cell line, DLKP-A10, expresses novel drug accumulation and sequestration systems. 926 Aug 77

99mTc-Tetrofosmin (TF) is a lipophilic diphosphine compound routinely used for myocardial scintigraphy. Extracardiac utilization has occurred in evaluation of patients with malignant neoplasms and in parathyroid adenomas. Although its uptake mechanisms are not completely understood, they appear similar to those of 99mTc Setamibi (MIBI). The importance of flow and the metabolic status of cells with an intracellular uptake depending on mitochondria and the Na+/K+ pump have been hypothesized. It has also been demonstrated that Tetrofosmin shares with MIBI the property of being a substrate for P-glycoprotein (P-gp), a multidrug resistance transporter. In this review the possible clinical role in breast cancer is analysed. First experiences suggest that scintimammography with TF is useful in patients with indeterminate Mammography and to obtain complementary data to avoid surgery and/or biopsy. TF is a reliable tracer for diagnosis of primary cancer, of local recurrence of axillary lymph node metastases. Preliminary data stimulate a possible role in functional imaging of chemoresistance and in differential diagnosis of distant metastases with main reference to the evaluation of single hot lesions at bone scan.
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PMID:Diagnostic and prognostic role of 99mTc-Tetrofosmin in breast cancer. 927 32

1. Human intestinal epithelial Caco-2 cells were used to investigate the mechanistic basis of transepithelial secretion of the fluoroquinolone antibiotic ciprofloxacin. 2. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to competitive inhibition by sulphate, thiosulphate, oxalate, succinate and para-amino hippurate, probenecid (10 mM), taurocholate (100 microM) or bromosulphophthalein (100 microM). Similarly tetraethylammonium and N-'methylnicotinamide (10 mM) were without effect. 3. Net secretion of ciprofloxacin was inhibited by the organic exchange inhibitor 4,4'-diisothiocyanostilbene-2-2'-disulphonic acid (DIDS, 400 microM). 4. Net secretion of ciprofloxacin was partially inhibited by 100 microM verapamil, whilst net secretion of the P-glycoprotein substrate vinblastine was totally abolished under these conditions. Ciprofloxacin secretion was unaltered after preincubation of cells with two anti-P-glycoprotein antibodies (UIC2 and MRK16), which both significantly reduced secretory vinblastine flux (measured in the same cell batch). Ciprofloxacin (3 mM) failed to inhibit vinblastine net secretin in Caco-2 epithelia, and was not itself secreted by the P-glycoprotein expressing and vinblastine secreting dog kidney cell line, MDCK. 5. Net secretion and cellular uptake of ciprofloxacin (at 0.1 mM) were not subject to alterations of either cytosolic or medium pH, or dependent on the presence of medium Na+, Cl- or K+ in the bathing media. 6. The substrate specificity of the ciprofloxacin secretory transport in Caco-2 epithelia is distinct from both the renal organic anion and cation transport. A role for P-glycoprotein in ciprofloxacin secretion may also be excluded. A novel transport mechanism, sensitive to both DIDS and verapamil mediates secretion of ciprofloxacin by human intestinal Caco-2 epithelia.
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PMID:Fluoroquinolone (ciprofloxacin) secretion by human intestinal epithelial (Caco-2) cells. 928 89


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