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)

P-glycoprotein, a plasma membrane protein overexpressed in multidrug-resistant (MDR) cells, exhibits in vitro an ATPase activity and is responsible for the energy-dependent efflux of structurally unrelated cytotoxic drugs (like vinblastine) and various MDR-reversing agents (like verapamil and progesterone) from these MDR cells. To investigate the mechanism of P-glycoprotein interaction with various compounds, we measured the P-glycoprotein ATPase activity on membrane vesicles prepared from the MDR cell line DC-3F/ADX, and we studied the effects of vinblastine, verapamil and progesterone on this ATPase activity. The basal P-glycoprotein ATPase activity is increased by verapamil and progesterone, with respective half-maximal activating concentrations of approximately 1.5 microM and approximately 25 microM, and activation factors of approximately 1.7 and approximately 2.2. Vinblastine inhibits the activation of P-glycoprotein ATPase induced by verapamil or progesterone with an inhibition constant approximately 0.5 microM in both cases. This demonstrates that vinblastine has a specific modulating site on P-glycoprotein. The combined modulation of P-glycoprotein ATPase by vinblastine and verapamil reveals that these two drugs are mutually exclusive. Since these two molecules have different effects both on the basal P-glycoprotein ATPase activity and on the MgATP concentration dependence of P-glycoprotein ATPase activity, they could bind P-glycoprotein either on different and overlapping sites, or on distant but interacting sites. In contrast, the combined modulation of P-glycoprotein ATPase by vinblastine and progesterone reveals a non-competitive relationship between these two drugs, and hence shows that they can independently and simultaneously bind P-glycoprotein on distinct sites. Since verapamil and progesterone are mutual inhibitors of P-glycoprotein ATPase stimulation in a non-competitive manner, these two molecules can also bind independently P-glycoprotein on separated sites. This is confirmed here by the observation of a synergistic effect when mixtures of verapamil and progesterone are tested for the modulation of P-glycoprotein ATPase. Three MDR-related molecules, taken as models for interaction with P-glycoprotein, appear thus to bind on at least two different separated specific sites. These results favor a multisite model rather than a universal site model to describe the broad substrate specificity characterizing P-glycoprotein function.
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PMID:Competitive and non-competitive inhibition of the multidrug-resistance-associated P-glycoprotein ATPase--further experimental evidence for a multisite model. 911 38

To gather further insight into the interaction between P-glycoprotein (Pgp) and its substrates, 167 compounds were analyzed in multidrug resistant human colon carcinoma cells. These compounds were selected from the National Cancer Institute Drug Screen repository using computer-generated correlations with known Pgp substrates and antagonists. The compounds were prospectively defined as Pgp substrates if cytotoxicity was increased > or =4-fold by the addition of cyclosporin A (CsA) and as Pgp antagonists if inhibition of efflux increased rhodamine accumulation by 4-fold. Among the 84 agents that met either criterion, 35 met only the criterion for substrates, 42 met only the criterion for antagonists, and only seven met both criteria. Thus, compounds interacting with Pgp form two distinct groups: one comprising cytotoxic compounds that are transported and have poor or no antagonistic activity and a second comprising compounds with antagonistic activity and no evidence of significant transport. Vinblastine accumulation and kinetic studies performed on a subset of 18 compounds similarly differentiated substrates and antagonists, but inhibition of 3H-azidopine labeling and induction of ATPase activity did not. These data support an emerging concept of Pgp in which multiple regions instead of specific sites are involved in drug transport.
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PMID:P-glycoprotein substrates and antagonists cluster into two distinct groups. 918 69

Tumor necrosis factor-alpha (TNF-alpha) has been shown to enhance the cytotoxicity of a variety of antineoplastic agents. To examine whether multidrug-resistant cells are targets of TNF-alpha, and whether TNF-alpha is capable of modulating chemoresistance of these cells, a pleural mesothelioma cell line (PXF1118L) and two multidrug-resistant sublines thereof were used as experimental models. Drug resistance of these cells was due to P-glycoprotein expression, as confirmed by (1) staining with a monoclonal antibody (MRK16) specific for human P-glycoprotein, (2) decreased accumulation of [3H]vinblastine that was reversed by verapamil, and (3) enhanced cytotoxicity of vindesine in the presence of verapamil. Parental and multidrug-resistant cells exhibited little but comparable sensitivity to TNF-alpha alone. Combining TNF-alpha with vindesine or, to a lesser extent, with doxorubicin, but not with cisplatin, resulted in greater cytotoxicity towards multidrug-resistant cells than seen for each compound alone, indicating a synergism. In contrast, TNF-alpha failed to modulate vindesine or doxorubicin cytotoxicity in parental cells. [3H]Vinblastine accumulation was unaffected by TNF-alpha, and chemoresistance was reduced by TNF-alpha also in the presence of verapamil (10 microM), indicating that TNF-alpha was acting in a way different from calcium-channel blockers. Though the molecular mechanism by which TNF-alpha was enhancing vindesine and doxorubicin cytotoxicity remained undefined in this study, the numbers of TNF-alpha binding sites on parental and on multidrug-resistant cells were similar, and P-glycoprotein expression was unmodulated during the entire 48 h incubation period. In conclusion, we show that TNF-alpha increases the cytotoxicity of anticancer drugs in multidrug-resistant tumor cells by a mechanism that differs from most chemosensitizing agents, including verapamil. Further studies will be needed to clarify the mechanism by which TNF-alpha synergizes with anticancer drugs.
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PMID:Modulation of vindesine and doxorubicin resistance in multidrug-resistant pleural mesothelioma cells by tumor necrosis factor-alpha. 938 69

In this paper, we show that P-glycoprotein contains two distinct sites for drug binding and transport, and that, unexpectedly, these sites interact in a positively cooperative manner. The kinetics of transport of rhodamine 123 and Hoechst 33342 in isolated P-glycoprotein-rich plasma membrane vesicles from Chinese hamster ovary CH(R)B30 cells were followed by continuous fluorescence monitoring. Each substrate stimulated P-glycoprotein-mediated transport of the other. Colchicine and quercetin stimulated rhodamine 123 transport and inhibited Hoechst 33342 transport. In contrast, anthracyclines such as daunorubicin and doxorubicin stimulated Hoechst 33342 transport and inhibited rhodamine 123 transport. Vinblastine, actinomycin D, and etoposide inhibited transport of both dyes. The results are consistent with a functional model of P-glycoprotein containing at least two positively cooperative sites (H site and R site) for drug binding and transport. This model is consistent with earlier observations of competitive and non-competitive effects of P-glycoprotein substrates and chemosensitizers. Such a two-site model may be fundamental to multidrug transport by P-glycoprotein, and it may be a feature common to other ATP-dependent transporters belonging to the ATP-binding cassette superfamily.
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PMID:Positively cooperative sites for drug transport by P-glycoprotein with distinct drug specificities. 943

We studied the interaction between the multidrug transporter, P-glycoprotein, and two compounds that interact with it: vinblastine, a classical substrate of the pump, and verapamil, a classical reverser. Steady-state levels of accumulation of these two drugs were determined in a multidrug resistant P388 leukemia cell line, P388/ADR. The time course of accumulation of these drugs, and the effect of energy starvation and the presence of chloroquine on the level of their steady-state accumulation were quite disparate. Vinblastine inhibited the accumulation of verapamil whereas it enhanced the accumulation of daunomycin, another classic substrate of P-glycoprotein. Verapamil did not compete with the intracellular binding sites of vinblastine. In all these aspects, vinblastine behaved as a typical substrate of P-glycoprotein but verapamil did not. Our data suggest that verapamil is a reverser of P-glycoprotein but that its intracellular accumulation is not affected by this membrane-bound transporter.
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PMID:Drug accumulation in the presence of the multidrug resistance pump: dissociation between verapamil accumulation and the action of P-glycoprotein. 960 21

1. The liver has an important role in the elimination of circulating catecholamines. Adrenaline and noradrenaline are avidly taken up and metabolized by rat hepatocytes, but the nature of the mechanism(s) involved remains partially unknown. 2. The aim of this work was to further characterize the uptake of catecholamines by isolated rat hepatocytes. For that purpose, the effects of a series of chemically unrelated compounds, including substrates/inhibitors of P-glycoprotein, on [3H]-adrenaline removal was investigated. 3. Freshly isolated rat hepatocytes were incubated in Krebs-Henseleit solution at 37 degrees C with 50 nM [3H]-adrenaline for 5 min. Removal of [3H]-adrenaline was calculated as the sum of [3H]-adrenaline present in cells, and its [3H]-metabolites present both in cells and in the incubation medium. Radioactivity was determined by liquid scintillation counting. 4. Verapamil, quinidine, 1-methyl-4-phenylpyridinium, cimetidine, tetraethylammonium, d-tubocurarine, taurocholate, daunomycin and vinblastine (100 microM), progesterone, bilirubin (200 microM), vecuronium (45 microM), and amiloride (1 mM) significantly reduced [3H]-adrenaline removal. On the other hand, cyclosporine A (100 microM) apparently had no effect. The O-methylated metabolite of adrenaline, metanephrine (30 microM), produced a 40% reduction of [3H]-adrenaline removal. 5. Vinblastine and corticosterone produced concentration-dependent decreases of [3H]-adrenaline removal, with IC50 values of 23.3 and 116.0 microM, respectively. 6. In the presence of verapamil (100 microM), desipramine (1 microM) was devoid of significant effect on [3H]-adrenaline removal. Corticosterone (40 microM) produced a further decrease (+/- 50%) on removal of the [3H]-amine. 7. Removal of [3H]-adrenaline by isolated cells did not show pH-dependence since an increase or a decrease in the pH of incubation medium (to 8.2 or 6.2, respectively) caused no alteration of that parameter. 8. In conclusion, [3H]-adrenaline is efficiently removed and subsequently metabolized by isolated rat hepatocytes. The results are compatible with the involvement of multiple mechanisms in the hepatic uptake of this amine including the type I and the type II hepatic transporters for organic cations, uptake2 and P-glycoprotein.
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PMID:Uptake of [3H]-adrenaline by freshly isolated rat hepatocytes: putative involvement of P-glycoprotein. 972 26

Experiments with purified P-glycoprotein (Pgp) reconstituted into proteoliposomes have conclusively demonstrated that Pgp is an ATP-dependent drug transporter. Detailed biochemical analyses of drug transport by Pgp are beginning to yield a clearer picture of its mechanism. Working with Pgp-rich plasma membrane vesicles from CHRB30 cells, we have recently clarified several aspects of the drug transport mechanism. A major question about drug transport by Pgp is how it can recognize a vast array of unrelated chemical compounds as substrates. The substrate Hoechst 33342 is fluorescent in the lipid bilayer but not in aqueous solution. This property enabled us to show that Pgp transports Hoechst 33342 out of the lipid bilayer, not the aqueous phase. Because Hoechst 33342, like all Pgp substrates, is lipophilic its concentration in the bilayer greatly exceeds its concentration in the aqueous medium. High local substrate concentrations may allow for broad substrate recognition by one or more relatively low affinity binding site(s) within the lipid bilayer. Another fundamental question about Pgp is the number of drug binding sites it possesses. We have found evidence for at least two sites for drug binding and transport that interact in a positively cooperative manner. Initial rates of transport of two Pgp substrates, Hoechst 33342 and Rhodamine 123 by ChRB30 plasma membrane vesicles were measured. Each dye stimulated transport of the other. Additionally, colchicine stimulated Rhodamine 123 transport and inhibited Hoechst 33342 transport. Anthracyclines such as daunorubicin and doxorubicin had the reverse effect. Vinblastine, etoposide, and actinomycin D inhibited transport of both dyes. We interpret these results as follows. One site (R) preferentially recognizes Rhodamine 123, doxorubicin and daunorubicin. The other site (H) preferentially recognizes Hoechst 33342 and colchicine. Vinblastine, actinomycin D, and etoposide interact equally with both sites. Binding of drug at the R site stimulates transport of Hoechst 33342 by the H site and binding of drug at the H site stimulates transport of Rhodamine 123 by the R site. The existence of two drug binding sites on Pgp with different specificities is another way in which Pgp may expand the range of substrates it can transport. A third essential detail of the drug transport mechanism of Pgp is the ratio of substrate molecules transported per ATP hydrolyzed. By comparing the initial rate of Rhodamine 123 transport with the rate of ATP hydrolysis at saturating Rhodamine 123 concentration, we found that, under suitable conditions, Pgp is capable of transporting one Rhodamine 123 molecule per ATP molecule hydrolyzed.
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PMID:The mechanism of ATP-dependent multidrug transport by P-glycoprotein. 978 65

Resistance to chemotherapy is the major cause of cancer treatment failure. Insight into the mechanism of action of agents that modulate multidrug resistance (MDR) is instrumental for the design of more effective treatment modalities. Here we show, using KB-V-1 MDR human epidermoid carcinoma cells and [3H]palmitic acid as metabolic tracer, that the MDR modulator SDZ PSC 833 (PSC 833) activates ceramide synthesis. In a short time course experiment, ceramide was generated as early as 15 min (40% increase) after the addition of PSC 833 (5.0 microM), and by 3 h, [3H]ceramide was >3-fold that of control cells. A 24-h dose-response experiment showed that at 1.0 and 10 microM PSC 833, ceramide levels were 2.5- and 13.6-fold higher, respectively, than in untreated cells. Concomitant with the increase in cellular ceramide was a progressive decrease in cell survival, suggesting that ceramide elicited a cytotoxic response. Analysis of DNA in cells treated with PSC 833 showed oligonucleosomal DNA fragmentation, characteristic of apoptosis. The inclusion of fumonisin B1, a ceramide synthase inhibitor, blocked PSC 833-induced ceramide generation. Assessment of ceramide mass by TLC lipid charring confirmed that PSC 833 markedly enhanced ceramide synthesis, not only in KB-V-1 cells but also in wild-type KB-3-1 cells. The capacity of PSC 833 to reverse drug resistance was demonstrated with vinblastine. Whereas each agent at a concentration of 1.0 microM reduced cell survival by approximately 20%, when PSC 833 and vinblastine were coadministered, cell viability fell to zero. In parallel experiments measuring ceramide metabolism, it was shown that the PSC 833/vinblastine combination synergistically increased cellular ceramide levels. Vinblastine toxicity, also intensified by PSC 833 in wild-type KB-3-1 cells, was as well accompanied by enhanced ceramide formation. These data demonstrate that PSC 833 has mechanisms of action in addition to P-glycoprotein chemotherapy efflux pumping.
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PMID:SDZ PSC 833, the cyclosporine A analogue and multidrug resistance modulator, activates ceramide synthesis and increases vinblastine sensitivity in drug-sensitive and drug-resistant cancer cells. 1002 79

To determine the tissue-specific impact of P-glycoprotein on the accumulation of a substrate drug, we have studied the tissue distribution of vinblastine in mdr1a(-/-) and wild-type mice at approximately similar, relatively low plasma levels. Vinblastine was administered as a 96-h continuous infusion at dose rates of 1 to 10 microgram/h, which were delivered by a s.c.-implanted osmotic pump. Drug concentrations were determined in plasma and tissues by HPLC. In comparison to wild-type mice, 4.4- to 9.6-fold higher drug concentrations were observed in the brains of mdr1a(-/-) mice (p </=. 014), whereas a 2-fold increase was found in the heart (p =.014) and the intestinal tissues (p </=.028). No or only slight differences were observed in all other tissues. These results indicate that, except for the brain and, to a lesser extent, the heart and the intestinal tissues, P-glycoprotein does not protect individual organs against vinblastine. Given its polarized cell-specific and organ-specific distribution and its affinity for a broad range of compounds, it is suggested that P-glycoprotein has mainly evolved to provide a general protection of the complete organism against potentially toxic substrates by inhibiting their uptake and by mediating their transport from the internal to the external environment. For the clinical application of reversal agents, these data indicate that, in general, a blockade of endogenous P-glycoprotein will probably not result in an increased accumulation of the coadministered anticancer drug in complete organs, but, possibly, only in classes of cells making up a fraction of an organ.
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PMID:Comparative pharmacokinetics of vinblastine after a 96-hour continuous infusion in wild-type mice and mice lacking mdr1a P-glycoprotein. 1008 21

Ro 32-2241 is a bisindolylmaleimide that selectively inhibits protein kinase C (PKC) as compared with other protein kinases. Experiments were carried out to examine its potential as a multidrug resistance-reversing agent. Ro 32-2241 inhibited efflux, and increased accumulation, of [3H]-daunomycin in multidrug-resistant (MDR) KB-8-5 and KB-8-5-11 cells and had no effect on drug-sensitive KB-3-1 cells. Ro 32-2241 completely reversed the doxorubicin resistance of KB-8-5 and KB-8-5-11 cells, showing no effect on the sensitivity of drug-sensitive KB-3-1 cells. The potency of Ro 32-2241 was comparable with that of cyclosporin A and better than that of verapamil, known modulators of multidrug resistance. Ro 32-2241 also completely reversed the taxol resistance of KB-8-5 cells and partially reversed the resistance of KB-8-5-11 cells. Vinblastine resistance was also partially reversed. Mechanistic experiments were carried out to determine whether Ro 32-2241 interacted with P-glycoprotein (Pgp) directly. Increased efflux of [14C]-Ro 32-2241 was seen with the more resistant KB-8-5-11 cells (although the percentage effluxed was very low as compared with [3H]-daunomycin), suggesting that Ro 32-2241 can act as a substrate for Pgp. Direct interaction of Ro 32-2241 with Pgp was confirmed by demonstration that it inhibited binding of [3H]-azidopine to Pgp in KB-8-5-11 membranes. In conclusion, Ro 32-2241, acting directly on Pgp (rather than, or in addition to, an effect on PKC), is effective in reducing or reversing resistance to doxorubicin, taxol and vinblastine in human tumour cells with a clinically relevant degree of MDR. However, results of in vivo experiments conducted to investigate the effects of Ro 32-2241 on resistance to doxorubicin suggest that it may not be possible to achieve sufficiently high levels of Ro 32-2241 in vivo to modulate MDR.
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PMID:The bisindolylmaleimide protein kinase C inhibitor, Ro 32-2241, reverses multidrug resistance in KB tumour cells. 1010 May 91


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