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 studied the relationship between the chemical structure and multidrug resistance (MDR) reversal activity of racemic verapamil (VER) and 14 VER analogs (VAs). The LoVo-R human colon carcinoma cell line was used as an experimental model. This cell line exhibited a typical MDR phenotype and overexpressed the MDR1 gene products. Key structural features were identified as being related to MDR reversal and cytotoxic activity. In particular, we demonstrated that the methoxy groups in the VER molecule structure [1.7-Bis-(3.4-dimethoxyphenyl)-3-methylaza-7-cyan-8-methyl-n onane] prevented cytotoxicity when the VAs were used alone, whereas the 7-cyan-8-methyl groups were important for MDR reversal activity and interaction with P-glycoprotein (P-gp). Among the VAs tested, the most active compounds were gallopamil, R-isomer of VER (R-VER), and nor-VER, which potentiated doxorubicin (DOX) cytotoxicity by 52.3 +/- 7.2 (n = 3 +/- SD), 38.9 +/- 6.4 (n = 4 +/- SD), and 35.4 +/- 4.3 (n = 3 +/- SD) times, respectively. The reversal activity of these compounds was similar to that of VER, which enhanced DOX cytotoxicity by 41.3 +/- 5.0 (n = 3 +/- SD) times. The potentiation of DOX cytotoxicity was associated with an increase in DOX uptake in LoVo-R cells and with an increased [3H]azidopine P-gp photolabeling inhibition. Some compounds that had a high reversal potency (i.e. R-VER and nor-VER) showed a lower calcium antagonist activity than VER, and seem useful candidates for the treatment of MDR in cancer patients.
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PMID:Structure-activity relationship of verapamil analogs and reversal of multidrug resistance. 748 41

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

Chinese hamster ovary (CHO) 10B2 cells do not stain well with indo-1 and thus cannot be used for experiments to measure intracellular calcium using this dye. We have isolated a mutant CHO cell line (CHO IS1) that stains quite well with indo-1 and that has virtually identical growth characteristics and heat sensitivity as the parent line. The mutant was isolated by sorting individual mutagenized cells with high indo-1 fluorescence and cloning them. Since it has been reported that cells with multiple drug resistance (MDR+) can pump out various fluorescent dyes, the mutant and parent lines were characterized for Hoechst 33342 staining, Adriamycin toxicity, and P-glycoprotein expression, which are markers of the MDR phenotype. P-Glycoprotein was measured with the C219 antibody using flow cytometry. Multidrug-resistant cells (CHRC5) were used as positive controls. The IS1 cells stained as well with Hoechst 33342 as fixed 10B2 cells, and much better than unfixed 10B2 cells. The IS1 cells were 10- to 30-fold more sensitive to Adriamycin than the 10B2 cells, and both cell lines were much more sensitive than the CHRC5 cells. The amount of P-glycoprotein was similar in both 10B2 and IS1 cell lines, but was about fivefold lower than the CHRC5 cells. Thus, the poor staining for indo-1 in the 10B2 cells may not be caused by the P-glycoprotein MDR pump, but by a different efflux pathway. Alternatively, the P-glycoprotein may be altered and less efficient in the CHO IS1 cells.
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PMID:Isolation and characterization of a Chinese hamster ovary cell mutant with improved staining for indo-1. 752 22

It is well documented that the Ca2+ channel antagonist verapamil can reverse multidrug resistance in cancer cells by decreasing P-glycoprotein mediated drug efflux. However, less information is available about effects of verapamil on drug-phospholipid interactions and on passive diffusion of drugs across the membrane, which both may play an important role in resensitizing cells to anti-cancer drugs. Therefore we studied the binding of verapamil to model membranes (large unilamellar vesicles) composed of various phospholipids and biological membranes. An increase of the amount of anionic phospholipids resulted in an enhanced binding of verapamil. Competition between verapamil and the anti-cancer drug and P-glycoprotein substrate doxorubicin for binding to anionic phospholipids was observed in model membranes composed of synthetic lipids, or composed of native Escherichia coli phospholipid mixtures, and in cytoplasmic membrane vesicles of this organism. Furthermore, verapamil specifically increased the rate of passive diffusion of doxorubicin across model membranes containing anionic phospholipids. It can be concluded that besides the decrease of P-glycoprotein mediated efflux at least two other effects may account for an increase of the internal (free and DNA-bound) doxorubicin concentration in the presence of verapamil; (i) a decrease of binding to anionic phospholipids in plasma-and intracellular membranes and (ii) an increase of the rate of passive import of doxorubicin across the plasma membrane.
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PMID:Verapamil competes with doxorubicin for binding to anionic phospholipids resulting in increased internal concentrations and rates of passive transport of doxorubicin. 754 28

Enterocytes are the major epithelial cell type of the small intestine. Their capacity to secret, absorb and digest specific ions and nutrients is dependent on their position along the length of the small intestine as well as their stage of development as they migrate and differentiate along the crypt-villus axis. In order to further understand the molecular processes that regulate enterocyte differentiation and function, this study has compared the levels of six mRNA species produced by genes expressed in rabbit enterocytes; specifically, the multidrug resistance (MDR1) gene encoding the 170-kDa P-glycoprotein, CaBP 9k, which encodes a putative intracellular calcium buffer, calbindin, LPH, APN, and AP which encode the brush-border hydrolases lactase-phlorizin hydrolase, aminopeptidase N and alkaline phosphatase, respectively, and SGLT1, encoding the brush border Na(+)-glucose cotransporter. The level of each mRNA species has been mapped along the small intestine using quantitative in situ hybridisation. This has revealed characteristic regional variations in the abundance of each of the mRNAs, supporting the opinion that there is a strong genetic component to the maintenance of gradients in epithelial function along the length of the small intestine. Analysis of the cellular accumulation of mRNA during enterocyte migration along the crypt-villus axis, over gut-associated lymphoid tissue, and at epithelial boundaries, has, by contrast, established a clear correlation in the expression of these genes. These data illustrate the dynamics of enterocyte gene expression, thereby providing an insight into the molecular mechanisms which co-ordinate the events of cell transformation that underlie functional differences between the epithelial populations of the small intestine.
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PMID:Parallel patterns of cell-specific gene expression during enterocyte differentiation and maturation in the small intestine of the rabbit. 758 2

Fluorescence image analysis of isolated rat hepatocyte couplets, which retain a bile canaliculus between them, has shown the presence of transport systems for the bile acid and non-bile acid organic anion in the canalicular membrane. The cells also transported Fura-2 and BCECF, which are fluorescent indicators of intracellular Ca2+ and H+ concentrations, respectively, into the canaliculus. Both Fura-2 and BCECF transports were inhibited by progesterone, verapamil, vinblastine, and daunomycin, indicating that the transports are due to a multidrug efflux pump (P-glycoprotein) in the canalicular membrane. Interestingly, the transport by the multidrug efflux pump was inhibited by immunosuppressants FK506 (tacrolimus) and cyclosporine, their half-maximal inhibitory concentrations in the cell suspension being 10 microM and 0.6 microM, respectively. In contrast, the reported immunosuppressive potency of FK506 is 10- to 100-fold that of cyclosporine. Inhibition by immunosuppressants of the multidrug efflux pump, which is a transporter of cytotoxic and other drugs and present in normal human tissues--such as kidney, liver, the blood-brain barrier, and colon--may, at least partly, explain side effects caused by cyclosporine in these tissues of transplant recipients. FK506 at its clinical concentrations may not inhibit the multidrug efflux pump.
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PMID:Inhibition of the multidrug efflux pump in isolated hepatocyte couplets by immunosuppressants FK506 and cyclosporine. 768 Dec 29

Multidrug resistant cells may become acutely sensitive to the calcium channel blocker verapamil, in spite of the fact that its accumulation by these cells is negligible. We selected verapamil-resistant mutants from multidrug resistant Chinese hamster ovary cells. Levels of P-glycoprotein expression and cross-resistance profiles remained unaltered in the verapamil-resistant multidrug resistant cells. As well, a photoactive verapamil analog specifically bound to P-glycoprotein in these cells. We had previously used a photoactive anthracycline to show that calcium antagonists and several anticancer drugs bind to P-glycoprotein at overlapping or interacting sites. Verapamil and its analogues no longer inhibit the binding of either anticancer drugs or calcium channel blockers to P-glycoprotein. Sequencing of P-glycoprotein revealed that no change had occurred in the coding sequence as a result of the selection procedure.
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PMID:Selection and characterization of verapamil-resistant multidrug resistant cells. 773 17

P-glycoprotein (Pgp), the multidrug resistance (mdr) gene product, has been described in normal tissues with diverse physiologic functions. A broad role as a transporter protein for toxins, hormones, and physiologic metabolites has been provisionally deduced, based on structural analysis and immunoanatomic localization. Recently, significant levels of Pgp have been demonstrated in endocrine and hormonally responsive tissues and tumors. We examined calcium-regulated, clonal parathyroid epithelial (PT-r) and endothelial cells (BPE-1) and frozen parathyroid tissue from normal human parathyroid, parathyroid hyperplasia, parathyroid adenoma, and parathyroid carcinoma for expression of the multidrug resistance gene (Mdr1) and Pgp utilizing Northern and Western analysis and immunohistochemistry. We also investigated the effect of extracellular calcium (eCa) on Pgp expression in PT-r cells at the molecular/cellular level. Immunohistochemistry, utilizing three murine monoclonal antibodies (MAbs)--C494, JSB-1, and C219--which recognize spatially distinct cytoplasmic epitopes of Pgp, revealed strong immunoreactivity in PT-r cells, normal parathyroid, and parathyroid hyperplasia, and weak immunostaining in parathyroid adenomas. BPE-1 cells, endothelial cells, and parathyroid carcinoma were negative. PT-r cells showed a single 130 kDa band (120 KDa after glycosidase treatment) on Western blot and a 4.6 kb transcript on Northern analysis, consistent with Pgp. Western and Northern blot analysis of PTr cells cultured in different eCa concentrations showed that eCa up-regulated Pgp expression.
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PMID:P-glycoprotein is expressed in parathyroid epithelium and is regulated by calcium. 773 28

Studies with inside-out plasma membrane vesicles from multidrug-resistant (MDR 3) murine erythroleukemia (MEL/VCR-6) cells have provided evidence for down-modulation of P-glycoprotein (P-gp) function by Ca(2+)-calmodulin (CLM). These studies showed that CLM in the presence or absence of Ca2+ had no effect on binding of [3H]vinblastine (VBL) by P-gp in inside-out plasma membrane vesicles. However, profound inhibition of ATP-dependent [3H]VBL efflux by these vesicles was demonstrated by the addition of subnanomolar concentrations of CLM (IC50 = 0.15 +/- 0.02 nM). The addition of 1 microM Ca2+ reduced the inhibition of [3H]VBL efflux by CLM, shifting the concentration required for inhibition to the nM range (IC50 = 2.55 +/- 0.35 nM). The inhibition of as 0.01 mM Ca2+, and no inhibition occurred with concentrations greater than 0.2 mM Ca2+. Binding of CLM, itself, to P-gp was demonstrated in two ways. The P-gp content of detergent-solubilized plasma membrane from MEL/VCR-6 cells could be appreciably depleted by treating this material with CLM-Sepharose beads as shown by SDS-polyacrylamide gel electrophoresis (PAGE) and Western blotting with anti-P-gp antibody (C219) before and after CLM-Sepharose treatment. Also, depletion of P-gp from solution by CLM was less in the presence of 1 mM Ca2+. Blotting of P-gp after SDS-PAGE of plasma membrane from MEL/VCR-6 cells was also obtained using 125I-CLM as a probe. These results strongly suggest that the MDR 3 homolog of P-gp is a CLM-binding protein and that direct interaction of Ca(2+)-CLM with P-gp, while not affecting its binding of [3H]VBL, down-modulates the translocation of this agent in the presence of ATP.
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PMID:Functional modulation of multidrug resistance-related P-glycoprotein by Ca(2+)-calmodulin. 774 32

Calcium channel inhibitors, such as verapamil, have been identified as having the ability to modulate the multidrug-resistant (MDR) phenotype due to overexpression of P-glycoprotein (Pgp). We have studied the effect of verapamil on Pgp expression levels in a cell line originating from acute myeloblastic leukemia and resistant to adriamycin, K562/ADR. In this line, the addition of 15 microM verapamil in the culture medium gives a 3-fold decrease of Pgp expression after 72 hours of treatment. Similar results have been obtained for two other MDR cell lines, which suggest that this phenomenon is not specific of a single model. The level of mdr1 mRNAs is decreased in the presence of verapamil (with a maximum effect obtained at the 24th hour), which suggests that the mechanism of action of verapamil is transcriptional and/or post-transcriptional. We have also studied the effect of verapamil on the level of expression of mdr1 mRNAs in non-drug selected cells such as the HEL line (human acute myeloblastic leukemia) and the parental K562 line, which present a very low level of expression of Pgp, detectable only by PCR. In these lines, verapamil treatment has no effect on the level of expression of mdr1 mRNAs. The effect of verapamil is therefore restricted to drug-selected lines presenting high levels of Pgp expression. The impact of the negative regulation of Pgp expression on the MDR phenotype has been studied in the K562/ADR line. When the cells are treated for 72 h by verapamil, there is a decrease of resistance and an increase of intracellular accumulation of anticancer agents such as daunorubicin or vinblastine. Negative regulation of Pgp expression appears therefore as a possible strategy for MDR phenotype reversal. The effect of verapamil, whose molecular mechanism of action is being studied, could constitute a basis for this strategy.
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PMID:[Pharmacological control of P-glycoprotein expression]. 774 15


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