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)

To determine the number of drug binding sites that exist on the multidrug transporter, P-glycoprotein, we used azidopine, a dihydropyridine photoaffinity compound that reverses multidrug resistance and labels P-glycoprotein. Azidopine labels P-glycoprotein in two distinct locations: one labeled site is within the amino half of P-glycoprotein between amino acid residues 198 and 440, and the other site is within the carboxy half of the protein. Vinblastine is a cytotoxic drug that is used in cancer chemotherapy and is a substrate for transport by P-glycoprotein. We found that vinblastine inhibits azidopine labeling to approximately the same extent at each labeled site on P-glycoprotein. Because several studies have shown that amino acid residue 185 of P-glycoprotein plays a critical role in some aspects of drug binding and transport, we also studied the effect that amino acid residue 185 has on azidopine labeling. These studies show that azidopine labels both sites equivalently in both wild-type (G185) and mutant (V185) P-glycoproteins. We conclude from our results that the two halves of P-glycoprotein approach each other to form a single binding site for these drugs.
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PMID:Characterization of the azidopine and vinblastine binding site of P-glycoprotein. 135 86

Resistance of human cancer cells to multiple cytotoxic hydrophobic agents (multidrug resistance) is due to overexpression of the MDR1 gene whose product is the ATP-dependent multidrug transporter, P-glycoprotein. We have previously reported that plasma membrane vesicles partially purified from multidrug-resistant human KB carcinoma cells, but not from drug-sensitive cells, accumulated [3H]vinblastine in an ATP-dependent manner (Horio, M., Gottesman, M.M. and Pastan, I. (1988) Proc. Natl. Acad. Sci. USA 85, 3580-3584). Certain calcium-channel blockers, quinidine, and phenothiazines are able to overcome multidrug resistance in cultured cells. In this work, the effect of these reversing agents on ATP-dependent vinblastine (VBL) transport by vesicles from drug-resistant KB cells has been characterized. Azidopine was the most potent inhibitor of ATP-dependent VBL uptake tested (ID50: concentration of inhibitor such that the transport of vinblastine is inhibited by 50%, less than 1 microM). Verapamil, quinidine, and the tiapamil analogue RO-11-2933 were potent but less effective inhibitors (ID50 less than 5 microM). Diltiazem, nifedipine and trifluoperazine were even less effective. These agents had no effect on Na(+)-dependent and Na(+)-independent L-leucine uptake by the vesicles, indicating that the inhibition of ATP dependent VBL transport by these agents is not a non-specific effect, as might result from leaks in the vesicle membrane. Verapamil, quinidine, azidopine and trifluoperazine increased the apparent Km value of vinblastine transport, suggesting that these agents may be competitive inhibitors of vinblastine transport.
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PMID:Agents which reverse multidrug-resistance are inhibitors of [3H]vinblastine transport by isolated vesicles. 167 42

Verapamil has been proposed to modulate the multidrug resistance phenotype by competitive inhibition of an energy dependent efflux of cytotoxic drug. However, the accumulation of both 14C-verapamil and 3H-verapamil was similar in wild type EHR2 and multidrug resistant EHR2/DNR+ Ehrlich ascites cells, and was much less in both cell lines in energy deprived medium than in medium containing glucose. Azidopine accumulation was also similar in both EHR2 and EHR2/DNR+ cells but, in contrast to verapamil, did not differ significantly with changes in cellular energy levels. Azidopine photolabelled a 170 kDa protein in EHR2/DHR+ plasma membrane vesicles which was immunoprecipitated by monoclonal antibody towards P-glycoprotein. Azidopine increased daunorubicin accumulation and modulated vincristine resistance in EHR2/DNR+ cells in a similar fashion to verapamil. Azidopine photolabelling was inhibited by vincristine and verapamil, but not by daunorubicin. Vincristine, but not daunorubicin, was able to increase both azidopine and verapamil accumulation in EHR2/DNR+ cells only. Finally, though both verapamil and azidopine are a substrate for P-glycoprotein in EHR2/DNR+ cells, they do not themselves appear to be transported by the multidrug resistance efflux mechanism to any significant extent in these cells.
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PMID:Transport of the multidrug resistance modulators verapamil and azidopine in wild type and daunorubicin resistant Ehrlich ascites tumour cells. 197 2

Cremophor EL (polyoxyethylene castor oil) and Tween 80, used as solvents for cyclosporin A and VP-16, respectively, were found to reverse the multidrug resistant (MDR) phenotype. In daunorubicin (DNR) resistant Ehrlich ascites tumor cells (EHR2/DNR+), both solvents at percentages of 0.01% (v/v) enhanced DNR accumulation to sensitive levels. Cremophor EL and Tween 80 did not influence DNR accumulation in drug-sensitive cells (EHR2). The concentration of cyclosporin A alone that enhanced DNR accumulation in EHR2/DNR+ cells to sensitive levels was 5 micrograms/mL whereas 0.2 micrograms/mL of cyclosporin A dissolved in 0.001% (v/v) Cremophor EL enhanced DNR accumulation to sensitive levels, thus indicating synergy between Cremophor EL and cyclosporin A. Cyclosporin A had a negligible effect on DNR accumulation in the drug-sensitive cells. In clonogenic assays, the LD10 of DNR was 1 microM in EHR2/DNR+ cells. Combining 1 microM DNR with non-toxic amounts of Cremophor EL (0.001% and 0.002%, v/v) potentiated the cytotoxicity of DNR and resulted in a cell kill of 77% and 86%, respectively, in the resistant cells. In non-toxic amounts, CrEL and Tween 80 acted synergistically with reduced concentrations of verapamil, resulting in DNR accumulation approaching close to the sensitive level. Azidopine photoaffinity labeling of P-glycoprotein in plasma membrane vesicles from EHR2/DNR+ cells was inhibited 100% and 80%, by 0.003% (v/v) Cremophor EL or Tween 80, respectively. These data permit the conclusion that non-toxic amounts of CrEL and Tween 80 modulated DNR resistance by raising intracellular DNR levels, due to their abilities to bind to the plasma membrane P-glycoprotein.
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PMID:The solvents cremophor EL and Tween 80 modulate daunorubicin resistance in the multidrug resistant Ehrlich ascites tumor. 197 41

Cells that express P-glycoprotein are resistant to many unrelated anticancer drugs. All evidence suggests that P-glycoprotein is a plasma membrane protein that confers multidrug resistance by actively transporting these cytotoxic drugs out of cells. The objective of our work is to locate drug binding sites on P-glycoprotein. Azidopine is a photoaffinity drug analog that specifically labels P-glycoprotein. To determine the region of P-glycoprotein that binds azidopine, we labeled P-glycoprotein with azidopine and digested the labeled protein into fragments. We then identified the labeled fragments with specific antibodies. We have determined that azidopine labels two different regions of P-glycoprotein: one region is in the amino half of P-glycoprotein, and the other is in the carboxyl half of the protein. Our results suggest that P-glycoprotein contains either two binding sites for azidopine or a single site formed by the two homologous halves of the protein.
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PMID:Two different regions of P-glycoprotein [corrected] are photoaffinity-labeled by azidopine. 247

A radioactive photoactive dihydropyridine calcium channel blocker, [3H]azidopine, was used to photoaffinity label plasma membranes of multidrug-resistant Chinese hamster lung cells selected for resistance to vincristine (DC-3F/VCRd-5L) or actinomycin D (DC-3F/ADX). Sodium dodecyl sulfate-polyacrylamide gel electrophoretic fluorograms revealed the presence of an intensely radiolabeled 150-180-kDa doublet in the membranes from drug-resistant but not from the drug-sensitive parental (DC-3F) cells. A similar radiolabeled doublet was barely detected in a drug-sensitive partial revertant (DC-3F/ADX-U) cell line. The 150-180-kDa doublet exhibited a specific half-maximal saturable photolabeling at 1.07 X 10(-7) M [3H]azidopine. The dihydropyridine binding specificity was established by competitive blocking of specific photolabeling with nonradioactive azidopine as well as with nonphotoactive calcium channel blockers nimodipine, nitrendipine, and nifedipine. In addition, [3H]azidopine photolabeling was blocked by verapamil and diltiazem but was stimulated by excess prenylamine and bepridil suggesting a cross-specificity for up to four different classes of calcium channel blockers. The 150-180-kDa calcium channel blocker acceptor co-electrophoresed exactly with the 150-180-kDa surface membrane glycoprotein (gp150-180 or P-glycoprotein) Vinca alkaloid acceptor from multidrug-resistant cells and was immunoprecipitated by polyclonal antibody recognizing gp150-180. [3H]Azidopine photolabeling of the 150-180-kDa component in the presence of excess vinblastine was reduced over 90%, confirming the identity or close relationship of the calcium channel blocker acceptor and the gp150-180 Vinca alkaloid acceptor. The [3H]azidopine photolabeling of gp150-180 also was reduced by excess actinomycin D, adriamycin, or colchicine, demonstrating a broad gp150-180 drug recognition capacity. The ability of gp150-180 to recognize multiple natural product cytotoxic drugs as well as calcium channel blockers suggests a direct function for gp150-180 in the multidrug resistance phenomenon and a role in the circumvention of that resistance by calcium channel blockers.
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PMID:Identification of the multidrug resistance-related membrane glycoprotein as an acceptor for calcium channel blockers. 303 8

Chinese hamster P-glycoprotein ("multidrug-resistance protein") was purified and reconstituted in proteoliposomes by the procedure of I. L. Urbatsch, M. K. al-Shawi, and A. E. Senior (1994, Biochemistry 33, 7069-7076). The presence of lipid during the octylglucoside solubilization and Reactive Red 120 chromatography steps was found to be mandatory for retention of ATPase activity. Sheep brain or bovine liver lipid extracts could be substituted for the Escherichia coli lipids used previously. Stimulation of ATPase activity of purified, reconstituted P-glycoprotein by vinblastine, colchicine, and daunomycin was seen with sheep brain and bovine liver lipids, but not with E. coli lipids. Basal (i.e., not drug-stimulated) ATPase activity was different in the three lipids. Azidopine labeling of the drug binding sites in purified, reconstituted P-glycoprotein was carried out; vinblastine, colchicine, and daunomycin competed for labeling in all three lipids. It is therefore evident that the lipid environment can significantly influence the characteristics of purified, reconstituted P-glycoprotein ATPase activity and the apparent coupling between drug-binding and catalytic sites.
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PMID:Effects of lipids on ATPase activity of purified Chinese hamster P-glycoprotein. 784 Jun 7

Since P-glycoprotein (P-gp) in normal tissues may serve as a cellular defense mechanism against naturally occurring xenobiotics, we considered whether physiologically active components of commonly ingested plant foods could influence P-gp function. To examine this possibility, a series of flavonoids commonly found in plant foods was tested for their ability to modulate [14C]Adriamycin ([14C]ADR) accumulation and efflux in P-gp-expressing HCT-15 colon cells. Many flavonoids, in the micromolar range, inhibited the accumulation of [14C]ADR. Detailed experiments utilizing flavonoids with the greatest activity in reducing [14C]ADR accumulation, i.e. galangin, kaempferol, and quercetin, revealed that the efflux of [14C]ADR is increased markedly in the presence of these compounds. Flavonoid-induced stimulation of efflux was rapid and was blocked by the multidrug-resistant (MDR) reversal agents verapamil, vinblastine, and quinidine. The magnitude of flavonoid-stimulated efflux in sodium butyrate-treated cells with a 4-fold induction of P-gp protein was similar to that in uninduced cells. [3H]Azidopine photoaffinity labeling of P-gp in crude membrane preparations revealed mild to no competition for binding by flavonoids possessing either activity or inactivity in reducing ADR accumulation. Although flavonoid hydrophobicity was found to be unrelated to flavonoid activity in altering [14C]ADR accumulation, certain structural features were associated with enhancement or diminution of activity. Finally, the significance of flavonoid-related reduction of [14C]ADR accumulation was underscored in cell growth studies, showing partial protection by quercetin against ADR-induced growth inhibition. It is concluded that certain naturally occurring plant flavonoids may acutely upregulate the apparent activity of P-gp.
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PMID:Modulation of adriamycin accumulation and efflux by flavonoids in HCT-15 colon cells. Activation of P-glycoprotein as a putative mechanism. 794 44

Two multidrug-resistant human leukemic CCRF-CEM sublines (CEM/VCR R and CEM/VLB100) were significantly more resistant to tetracycline, a hydrophilic antibiotic, than parental cells (P < 0.001). Verapamil and cyclosporin A completely reversed tetracycline resistance in CEM/VCR R cells, which also accumulated and retained significantly less [3H]tetracycline than CCRF-CEM cells. Like verapamil, addition of tetracycline to CEM/VCR R cells which had achieved steady-state vincristine levels resulted in augmented vincristine accumulation. [3H]Azidopine photoaffinity labelling of CEM/VCR R membrane proteins was inhibited by tetracycline in a dose-dependent manner. Although drugs associated with the multidrug-resistance phenotype are typically hydrophobic compounds, these data suggest that resistance to tetracycline, despite its hydrophilic nature, is mediated by P-glycoprotein in these cell lines.
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PMID:Resistance to tetracycline, a hydrophilic antibiotic, is mediated by P-glycoprotein in human multidrug-resistant cells. 809 60

Inhibitors of P-glycoprotein (P-gp) or chemosensitizers, such as verapamil, are used to reverse multi-drug resistance (MDR) in cancer patients. Clinical studies in patients with myeloma have shown that some patients with P-gp-positive cancer cells respond to the chemosensitizing effect of verapamil. However, this response is short-lived and tumor cells ultimately become resistant to chemosensitizers. To study mechanisms of resistance to chemosensitizers, a human myeloma cell line, 8226/MDR10V, was selected from a P-gp-positive cell line, 8226/Dox40, in the continuous presence of doxorubicin and verapamil. MDR10V cells are consistently more resistant to MDR drugs than parent cells, Dox40. Chemosensitizers, including verapamil and cyclosporin A, were less effective in reversing resistance in MDR10V compared with Dox40 cells. Verapamil and cyclosporin A were only partially effective in blocking P-gp drug efflux in MDR10V compared to Dox40 cells. Despite higher resistance to cytotoxic agents, MDR10V cells express less P-gp in the plasma membrane than do its parent cells, Dox40. [3H]Azidopine photoaffinity labeling of P-gp and its binding competition with unlabeled verapamil showed similar affinity for P-gp between Dox40 and MDR10V cell lines. Non-P-gp-mediated mechanisms of drug resistance, including over-expression of MRP and alterations in topoisomerase II, were not different for MDR10V cells compared with Dox40 cells.
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PMID:Resistance to the chemosensitizer verapamil in a multi-drug-resistant (MDR) human multiple myeloma cell line. 863 66


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