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)

Cyclosporin A (CsA) has been shown to increase the sensitivity of multidrug resistant (MDR) cells to chemotherapeutic agents. Although the concentration of drug required to produce this effect is clinically achievable, the use of this drug would be hampered by significant immunosuppression. We report a comparison of the effects of 11-methyl-leucine cyclosporin (11-met-leu CsA), a non-immunosuppressive homolog to the parent drug, on MDR cell lines. Both cyclosporins sensitized resistant cell lines to doxorubicin, including P388 murine leukemia and GM 3639 human T-cell leukemia. The action of the cyclosporins was more pronounced with resistant cells than with sensitive ones. 11-Met-leu CsA was less potent than, but equally effective as, the parent drug. Both agents increased the intracellular accumulation and retention of doxorubicin in MDR cells. The sensitization caused by the cyclosporins was independent of their effects on cyclophilin, calmodulin, and protein kinase C. Furthermore, there were no differences in the binding of labelled CsA to MDR cells compared to the binding to sensitive cells, suggesting that P-glycoprotein was also not the molecular site of action. These studies demonstrate that a non-immunosuppressive cyclosporin can modulate multidrug resistance and suggest its further evaluation for use in clinical trials.
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PMID:Activity of cyclosporin A and a non-immunosuppressive cyclosporin against multidrug resistant leukemic cell lines. 264 Jan 54

To determine whether endogenous P-glycoprotein, the MDR1 gene product that functions as a drug transport pump, is a volume-sensitive Cl- channel molecule or a protein kinase C-mediated regulator of the Cl- channel, whole-cell patch-clamp and molecular biological experiments were carried out in a human small intestinal epithelial cell line. Endogenous expression of P-glycoprotein was confirmed by Northern blot analysis, reverse transcription-polymerase chain reaction, Western blot analysis, and immunostaining. The P-glycoprotein expression was abolished by the antisense (but not sense) oligonucleotide for the MDR1 gene, whereas the magnitude of the Cl- current activated by osmotic swelling was not distinguishable between both antisense- and sense-treated cells. The volume-sensitive Cl- currents were not specifically affected by the anti-P-glycoprotein monoclonal antibodies, MRK16, C219, and UIC2. An inhibitor of P-glycoprotein-mediated pump activity, verapamil, was found to never affect the Cl- current. A substrate for the P-glycoprotein-mediated drug pump, vincristine or daunomycin, did not prevent swelling-induced activation of the Cl- current. Furthermore, the Cl- current was not affected by an activator of protein kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol). Thus, it is concluded that the endogenous P-glycoprotein molecule is not itself a volume-sensitive Cl- channel nor a protein kinase C-mediated regulator of the channel in the human epithelial cells.
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PMID:Volume-sensitive chloride channel activity does not depend on endogenous P-glycoprotein. 749 63

Expression of P-glycoprotein by tumor cells confers resistance to multiple natural product drugs because of its ability to export these compounds. This transporter is a substrate for several protein kinases; however, the functional significance of its phosphorylation is not defined. We examined the effects of many activators and inhibitors of protein kinases on the activity of P-glycoprotein in drug-resistant human breast carcinoma cells (MCF-7/ADR). Several phorbol esters sensitized these cells to P-glycoprotein substrate drugs; however, there was no correlation with activation of protein kinase C. The 4 alpha- and 4 beta-isomers of phorbol 12-myristate 13-acetate were equally potent in sensitizing the cells to actinomycin D and daunomycin and in increasing the intracellular accumulation of [3H]vinblastine. These effects of 4 beta-phorbol myristate acetate required much higher concentrations than were needed to increase P-glycoprotein phosphorylation and were not antagonized by staurosporine. Similar to verapamil, the phorbol esters did not sensitize MCF-7/ADR cells to cisplatin, nor parental MCF-7 cells to any of the anticancer drugs. Mezerein, K-252a, and H-89 sensitized MCF-7/ADR cells, increased intracellular accumulation of [3H]vinblastine, and antagonized photolabeling of P-glycoprotein by [3H]azidopine. Therefore, phosphorylation does not appear to play a significant role in regulating P-glycoprotein activity in MCF-7/ADR cells.
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PMID:Circumvention of P-glycoprotein-mediated multiple drug resistance by phosphorylation modulators is independent of protein kinases. 749 4

Calphostin C is a potent and specific inhibitor of protein kinase C (PKC). In this investigation we examined the effect of Calphostin C (without prior exposure to light) on daunorubicin (DNR) accumulation and sensitivity to DNR in multidrug-resistant (MDR) murine leukemia P388/ADR and human myeloid leukemia HL60/AR cells. P388/ADR cells overexpress P-glycoprotein, whereas HL60/AR cells lack any expression of P-glycoprotein (both at mRNA and protein levels). Calphostin C, in a concentration-dependent manner, increased the accumulation of DNR in P388/ADR cells and partially reversed (threefold) the DNR resistance in P388/ADR cells but had no effect on either of the parameters in HL60/AR cells. Calphostin C-induced increased accumulation of DNR in P388/ADR cells was due to increased uptake and decreased efflux of DNR. Furthermore, Calphostin C increased the uptake and decreased the efflux of rhodamine 123 (a substrate for P-gp) in P388/ADR cells but had no such effect in P388 cells. In addition, Calphostin C without exposure to light did not inhibit PKC activity in any of the cell lines studied. Taken together, these data suggest that Calphostin C may reverse drug resistance via P-glycoprotein independently of its effect on PKC activity. Therefore, any data regarding the effect of Calphostin C on the reversal of MDR should be interpreted in the light of these findings.
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PMID:Effect of Calphostin C (PKC inhibitor) on daunorubicin resistance in P388/ADR and HL60/AR cells: reversal of drug resistance possibly via P-glycoprotein. 751 83

A human leukemia K562 cell mutant (K562/OA200) selected for resistance to okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A (PP1/PP2A), has been established. In wild type cells, the cytotoxicity of OA was associated with mitotic arrest and concentration- and time-dependent DNA fragmentation, a hallmark of apoptosis. The mutant was 100-fold more resistant to OA in terms of effects on these parameters. Although the synthesis of several proteins was altered, enzyme assay and immunoblot analysis indicated that the levels of PP1 and PP2A were unchanged in the mutant. Protein kinase C (PKC) assays and immunoblot analysis of calcium-dependent (cPKC) and calcium-independent (nPKC) isoforms revealed that nPKC-epsilon was strikingly absent in the mutant, which otherwise expressed in comparable amounts all other isotypes (cPKC-alpha, cPKC-beta, and nPKC-zeta) also present in the wild type. Northern blot analysis confirmed an absence of PKC-epsilon mRNA in the mutant cells. The OA200 cells were cross-resistant not only to another PP1/PP2A inhibitor, calyculin A, but also to structurally unrelated anticancer drugs (such as vinblastine and taxol) and furthermore, overexpressed the verapamil-sensitive drug pump P-glycoprotein at both the protein and mRNA levels. The mutant, however, was not cross-resistant to several PKC inhibitors tested including cardiotoxin, mastoparan, staurosporine, and an alkylphospholipid. Cardiotoxin, at a subtoxic concentration, enhanced by 6-fold vinblastine cytotoxicity in OA200 cells. These findings indicate that the multidrug resistance phenotype can be induced by cytotoxic agents other than conventional anticancer drugs, show that the development of multidrug resistance is not necessarily associated with increased cPKC activity, and identify certain PKC inhibitors that have potential as resistance modulators.
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PMID:Human leukemia K562 cell mutant (K562/OA200) selected for resistance to okadaic acid (protein phosphatase inhibitor) lacks protein kinase C-epsilon, exhibits multidrug resistance phenotype, and expresses drug pump P-glycoprotein. 751 66

To identify the role of protein kinase C (PKC) in multidrug resistance, the effects of phorbol-12-myristate-13-acetate (PMA), a PKC activator, or calphostin C, a PKC inhibitor, on intracellular vincristine accumulation and expression of P-glycoprotein phosphorylation were studied in one multidrug-resistant and three multidrug-sensitive human glioma cell lines. Basal PKC activities and immunoreactivities of PKC-alpha and -zeta were higher in multidrug-resistant cells than in multidrug-sensitive cells. There was no significant difference in the immunoreactivity of PKC-delta between multidrug-resistant and -sensitive cells, and immunoreactive PKC-beta, -gamma, and -epsilon were not detected in either multidrug-resistant or -sensitive cells. The treatment of multidrug-resistant cells with 100 nM PMA for 2 hours resulted in the activation not of PKC-zeta but of PKC-alpha, with concomitant decrease in vincristine accumulation and increase in P-glycoprotein phosphorylation. The exposure of multidrug-resistant cells to 100 nM PMA for 24 hours induced down-regulation not of PKC-zeta but of PKC-alpha, with concurrent decrease in vincristine accumulation, and reduced but still increased P-glycoprotein phosphorylation. The treatment of multidrug-resistant cells with 100 nM calphostin C for 2 hours decreased immunoreactive PKC-zeta and not immunoreactive PKC-alpha, inducing increase in vincristine accumulation, with concomitant decrease in P-glycoprotein phosphorylation. There was no evidence of significant change in vincristine accumulation in multidrug-sensitive cells treated with PMA or calphostin C. This may suggest that at least two isozymes of PKC, PKC-alpha and -zeta, are involved in P-glycoprotein phosphorylation and that vincristine efflux function in multidrug-resistant human glioma cells is closely associated with P-glycoprotein phosphorylation and is decreased by PKC inhibitor.
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PMID:Effects of protein kinase C modulators on multidrug resistance in human glioma cells. 753 36

Multidrug resistance (MDR) in mammalian cells and tumors is associated with overexpression of an approximately 170 kDa integral membrane efflux transporter, the MDR1 P-glycoprotein. Hexakis (2-methoxyisobutyl isonitrile)technetium(I) (Tc-SESTAMIBI), a gamma-emitting lipophilic cationic metallopharmaceutical, has recently been shown to be a P-glycoprotein transport substrate. Exploiting the negligible lipid membrane adsorption properties of this organometallic substrate, we studied the transport kinetics, pharmacology, drug binding, and modulation of P-glycoprotein in cell preparations derived from a variety of species and selection strategies, including SW-1573, V79, Alex, and CHO drug-sensitive cells and in 77A, LZ-8, and Alex/A.5 MDR cells. Rapid cell accumulation (t1/2 approximately 6 min) of the agent to a steady state was observed which was inversely proportional to immunodetectable levels of P-glycoprotein. Many MDR cytotoxic agents inhibited P-glycoprotein-mediated Tc-SESTAMIBI efflux, thereby enhancing organometallic cation accumulation. Median effective concentrations (EC50; microM) were as follows: vinblastine, 13; daunomycin, 55; idarubicin, 65; actinomycin D, 235; colchicine, minimal inhibition; adriamycin, no effect. P-glycoprotein modulators generally demonstrated significantly greater potency (EC50; microM): SDZ PSC 833, 0.08; cyclosporin A, 1.3; verapamil, 4.1; quinidine, 6.4; prazosin, > 300. Modulator-induced enhancement up to 100-fold was observed with Hill coefficients approximately 1, consistent with simple Michaelis-Menten kinetics. Vanadate was an efficacious transport inhibitor, while agents usually not included in the MDR phenotype were without effect. Scatchard analysis showed quinidine to be a noncompetitive inhibitor of P-glycoprotein-mediated Tc-SESTAMIBI transport, indicating allosteric effector sites on P-glycoprotein. The lipid bilayer adsorbing agents tetraphenyl borate and phloretin induced large increases in final Tc-SESTAMIBI accumulation, showing maximal accumulations 2-fold greater than classic MDR modulators and Hill coefficients >> 2. In V79 and 77A cells, modulators of PKC activity altered Tc-SESTAMIBI accumulation, while there was no indication of modulation of P-glycoprotein-mediated Tc-SESTAMIBI transport by hypotonic buffer, extracellular ATP, Cl-, or K+ (membrane potential). While recognized and avidly transported by the P-glycoprotein at buffer concentrations as low as 7 pM, Tc-SESTAMIBI at up to 100 microM only minimally modulated the cytotoxic action of colchicine, doxorubicin, or vinblastine in MDR cells. In conclusion, transport analysis with Tc-SESTAMIBI is a sensitive assay for detecting functional expression of low levels of P-glycoprotein and for the quantitative characterization of transporter modulation and regulation. The biochemical data favor a high Km, high capacity allosterically modulated translocation mechanism for P-glycoprotein-mediated transport of this organometallic cation.
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PMID:Characterization of multidrug resistance P-glycoprotein transport function with an organotechnetium cation. 754 62

Phosphorylation may play a role in modulating multidrug resistance by P-glycoprotein (P-gp). The linker region between the two homologous halves of human P-gp harbors several serine residues which are phosphorylated by protein kinase C (PKC) in vitro. We used the glutathione S-transferase gene fusion system to express and purify a series of fusion proteins containing the relevant portion (residues 644-689) of the linker region of the human MDR1 gene product. The fusion proteins were subjected to in vitro phosphorylation and phosphopeptide mapping analysis to identify specific phosphorylation sites. On the basis of a mutational strategy in which individual serine residues were systematically replaced with nonphosphorylatable alanine residues, Ser-661 and Ser-667 were identified as major PKC sites and Ser-683 was identified as a minor PKC site. Ser-661 and Ser-667 were also found to be the primary sites of phosphorylation for a novel membrane-associated P-gp specific kinase isolated from the multidrug-resistant KB-V1 cell line. Individual phosphorylation sites were recognized independently of each other. These data show that the linker region of P-gp represents a target for multisite phosphorylation not only for PKC but also for the P-gp specific V1 kinase. Specific serine phosphorylation sites are identified, and evidence is presented that the V1 kinase has a specificity which overlaps, but is more restricted than, that of PKC. In addition, these studies also suggest that the use of GST fusion peptides may be applicable for the analysis of multisite and ordered protein phosphorylation in other systems.
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PMID:Bacterial expression of the linker region of human MDR1 P-glycoprotein and mutational analysis of phosphorylation sites. 757 13

Safingol is a lysosphingolipid protein kinase C (PKC) inhibitor that competitively interacts at the regulatory phorbol binding domain of PKC. We investigated the effects of safingol on antineoplastic drug sensitivity and PKC activity of MCF-7 tumor cell lines. Safingol treatment of 32P-labeled MCF-7 WT and MCF-7 DOXR cells inhibited phosphorylation of the myristoylated alanine-rich protein kinase C substrate in both cell lines, suggesting inhibition of cellular PKC. However, only in MCF-7 DOXR cells did safingol treatment increase accumulation of [3H]vinblastine and enhance toxicity of Vinca alkaloids and anthracyclines. Drug accumulation changes in MCF-7 DOXR cells treated with safingol were accompanied by inhibition of basal and phorbol 12,13-dibutyrate-stimulated phosphorylation of P-glycoprotein (P-gp). Expression of P-gp and levels of mdr1 message in MCF-7 DOXR cells were not altered by safingol treatment alone or in combination with vinblastine. Treatment of MCF-7 DOXR cell membranes with safingol did not inhibit [3H]vinblastine binding or [3H]azidopine photoaffinity labeling of P-gp. Furthermore, safingol did not stimulate P-gp ATPase activity in membranes prepared from MCF-7 DOXR cells. We conclude that enhanced drug accumulation and sensitivity in MCF-7 DOXR cells treated with safingol are correlated with inhibition of PKC rather than competitive interference with P-gp drug binding through direct interaction with P-glycoprotein.
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PMID:Partial inhibition of multidrug resistance by safingol is independent of modulation of P-glycoprotein substrate activities and correlated with inhibition of protein kinase C. 759 89

We compared the influence of exogenous N-ras oncogene and treatment with PKC agonist 12-O-tetradecanoylphorbol-13-acetate (TPA) on P-glycoprotein (Pgp) function in various human, rat and dog cell lines. Two approaches were used: (a) flow cytometry analysis of Rhodamine 123 (Rh123) exclusion; and (b) sensitivity to cytotoxic action of colchicine. We have found that in Rat1 fibroblasts, rat IAR2 epithelial cells and rat McA RH 7777 (hepatoma), ras activates Pgp function, while in MDCK (dog kidney), K562 (human chronic myelogenous leukaemia) and LIM1215 (human colon carcinoma) cells it either has no effect or even acts in opposite direction. TPA-induced Pgp function shows dissimilar pattern of cell specificity. It is assumed that PKC and ras oncogene regulate mdr1 gene expression through at least partially distinct signalling pathways.
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PMID:Cell-specific effects of RAS oncogene and protein kinase C agonist TPA on P-glycoprotein function. 762 41


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