Non-canonical functions of the cellular transporter P-glycoprotein

P-glycoprotein/ABCB1 (Pgp) is a well known protein of cell defense system. It is localized in cell membrane and pumps different drugs out of various cells using ATP energy. Its overexpression is associated with the development of multidrug resistance (MDR) in cancer cells. The data showing that Pgp also has other functions appeared recently, and this review surveys these data. In particular, (1) Pgp can protect cells from apoptosis; it suppresses the expression of endogenous protein TRAIL and decreases the activity of caspases 8 and 3; (2) Pgp is able to act as an outwardly directed flippase; (3) Pgp participates in a proper development of the innate immune response to intracellular pathogens and in the development of inflammation; (4) functionally active Pgp can be transferred from drug-resistant to drug-sensitive cells by microvesicles (MV). This is a new way of the Pgp-mediated MDR emergence in populations of tumor cells. Thus, Pgp functions as a regulator of some cellular processes. Molecular mechanisms of the Pgp influence on tumor cell viability are related not only with the drug efflux but also with some other functions.     

P-glycoprotein: multidrug-resistance and a superfamily of membrane-associated transport proteins

The study of multidrug resistance (MDR) in tumor cell lines has led to the discovery of the plasma membrane P-glycoprotein (Pgp) molecule. This protein functions as an energy-dependent pump for the efflux of diverse anticancer drugs from MDR cells. It now appears that Pgp-mediated MDR tumor cells do occur in human cancers, and that they are likely to play a role in the ultimate response of patients to chemotherapy. Chemosensitizers, compounds able to reverse the MDR phenotype, have been identified and offer the exciting possibility of improving efficacy for some nonresponsive malignancies. Surprisingly, Pgp-like molecules can be found in evolutionarily distant species among both eukaryotes and prokaryotes. As a group, these proteins form a superfamily of ATP-dependent transport proteins. This finding has broad implications and provides new insights into how living organisms use this fundamental transport system to regulate the trafficking of diverse molecules across biological membranes.     

P-glycoprotein Mediates Drug Resistance via a Novel Mechanism Involving Lysosomal Sequestration

Localization of the drug transporter P-glycoprotein (Pgp) to the plasma membrane is thought to be the only contributor of Pgp-mediated multidrug resistance (MDR). However, very little work has focused on the contribution of Pgp expressed in intracellular organelles to drug resistance. This investigation describes an additional mechanism for understanding how lysosomal Pgp contributes to MDR. These studies were performed using Pgp-expressing MDR cells and their non-resistant counterparts. Using confocal microscopy and lysosomal fractionation, we demonstrated that intracellular Pgp was localized to LAMP2-stained lysosomes. In Pgp-expressing cells, the Pgp substrate doxorubicin (DOX) became sequestered in LAMP2-stained lysosomes, but this was not observed in non-Pgp-expressing cells. Moreover, lysosomal Pgp was demonstrated to be functional because DOX accumulation in this organelle was prevented upon incubation with the established Pgp inhibitors valspodar or elacridar or by silencing Pgp expression with siRNA. Importantly, to elicit drug resistance via lysosomes, the cytotoxic chemotherapeutics (e.g. DOX, daunorubicin, or vinblastine) were required to be Pgp substrates and also ionized at lysosomal pH (pH 5), resulting in them being sequestered and trapped in lysosomes. This property was demonstrated using lysosomotropic weak bases (NH₄Cl, chloroquine, or methylamine) that increased lysosomal pH and sensitized only Pgp-expressing cells to such cytotoxic drugs. Consequently, a lysosomal Pgp-mediated mechanism of MDR was not found for non-ionizable Pgp substrates (e.g. colchicine or paclitaxel) or ionizable non-Pgp substrates (e.g. cisplatin or carboplatin). Together, these studies reveal a new mechanism where Pgp-mediated lysosomal sequestration of chemotherapeutics leads to MDR that is amenable to therapeutic exploitation.     

MDR1 P-glycoprotein reduces influx of substrates without affecting membrane potential

MDR1 (multidrug resistance) P-glycoprotein (Pgp; ABCB1) decreases intracellular concentrations of structurally diverse drugs. Although Pgp is generally thought to be an efflux transporter, the mechanism of action remains elusive. To determine whether Pgp confers drug resistance through changes in transmembrane potential (E(m)) or ion conductance, we studied electrical currents and drug transport in Pgp-negative MCF-7 cells and MCF-7/MDR1 stable transfectants that were established and maintained without chemotherapeutic drugs. Although E(m) and total membrane conductance did not differ between MCF-7 and MCF-7/MDR1 cells, Pgp reduced unidirectional influx and steady-state cellular content of Tc-Sestamibi, a substrate for MDR1 Pgp, without affecting unidirectional efflux of substrate from cells. Depolarization of membrane potentials with various concentrations of extracellular K(+) in the presence of valinomycin did not inhibit the ability of Pgp to reduce intracellular concentration of Tc-Sestamibi, strongly suggesting that the drug transport activity of MDR1 Pgp is independent of changes in E(m) or total ion conductance. Tetraphenyl borate, a lipophilic anion, enhanced unidirectional influx of Tc-Sestamibi to a greater extent in MCF-7/MDR1 cells than in control cells, suggesting that Pgp may, directly or indirectly, increase the positive dipole potential within the plasma membrane bilayer. Overall, these data demonstrate that changes in E(m) or macroscopic conductance are not coupled with function of Pgp in multidrug resistance. The dominant effect of MDR1 Pgp in this system is reduction of drug influx, possibly through an increase in intramembranous dipole potential.     

MDR1 P-glycoprotein transports endogenous opioid peptides

MDR1 P-glycoprotein is generally regarded as an efflux pump for amphipathic toxic compounds. The question remains, however, whether certain endogenous compounds are also substrates for this transporter. Certain peptides have been shown to interact with MDR1 Pgp as well and we have therefore investigated whether endogenous bioactive peptides are substrates. We demonstrate here that the synthetic μ-opioid peptide DAMGO is a good substrate for MDR1 Pgp. In view of its low interaction with the membrane it is an attractive ligand for measurement of MDR1 Pgp-mediated transport activity in membrane vesicles. Various linear peptides with amidated C-termini were found to inhibit MDR1 Pgp-mediated DAMGO transport. This group includes endogenous opioid peptides such as adrenorphin and endomorphin 1 and 2, as well as the neurokinin, Substance P. The latter bioactive peptides have a relatively high affinity for the transporter. Transport of endomorphin 1 and 2 could be directly demonstrated by the uptake of the radiolabeled opioid peptides in membrane vesicles from MDR1-transfected cells with a K_m of 15 and 12 μM, respectively. This opens the possibility that MDR1 Pgp is involved in the elimination and/or tissue distribution of these bioactive peptides.     

Biochemical basis of polyvalency as a strategy for enhancing the efficacy of P-glycoprotein (ABCB1) modulators: stipiamide homodimers separated with defined-length spacers reverse drug efflux with greater efficacy

Human P-glycoprotein (Pgp) is as an ATP-dependent efflux pump for a variety of chemotherapeutic drugs. The aim of this study is to evaluate whether Pgp modulators can be engineered to exhibit high-affinity binding using polyvalency. Five bivalent homodimeric polyenes based on stipiamide linked with polyethylene glycol ethers in the range of 3-50 A were synthesized and quantitatively characterized for their effect on Pgp function. The stipiamide homodimers displaced [(125)I]iodoarylazidoprazoin (IAAP), an analogue of the Pgp substrate prazosin. A minimal spacer of 11 A is necessary for inhibition of IAAP labeling, beyond which there is an inverse correlation between the length of the spacer and the IC(50) for the displacement of IAAP. ATP hydrolysis by Pgp on the other hand is stimulated by the dimers with spacers of up to 22 A, whereas dimers with longer spacers inhibit ATP hydrolysis. Finally, the homodimers reverse Pgp-mediated drug efflux in intact cells overexpressing Pgp, and 11 A is a threshold beyond which the effectiveness of the homodimers increases exponentially and levels off at 33 A. We demonstrate that dimerization and identification of an optimal spacer length increase by 11-fold the affinity of stipiamide, and this is reflected in the efficacy with which Pgp-mediated drug efflux is reversed. These results suggest that polyvalency could be a useful strategy for the development of more potent Pgp modulators.     

The “Multi” of Drug Resistance Explained by Oscillating Drug Transporters,Drug–Membrane Physical Interactions and Spatial Dimensionality

Multi-drug resistance (MDR) can be explained by a drug handling-type activity. In this context it is also necessary to consider the multi-specificity between drugs and drug transporters in order to explain MDR. Accordingly, the efficiency of drug efflux in MDR has always been a conceptual problem in biochemistry. Indeed, how one protein can expel, from cells, hundreds of compounds with high specificity is still controversial today. To safeguard the notion of biochemical specificity, many studies have suggested alternative mechanisms to Pgp-mediated drug resistance, which do not involve the handling of drugs. However, none of these studies have definitively ruled out the possibility concept of drug handling. Thus, until now it was not possible to imagine MDR without drug-transporter affinity or specificity. However, drug-transporter affinity is not always needed to generate what appears to be a very efficient chemical reaction. Indeed, based on the fact that bi-dimensional diffusion properties (i.e. diffusion in the membrane) are paramount to explain drug pumping-mediated MDR, it is possible to suggest how specific mathematical properties of random motions can be used to construct a model of Pgp-MDR, providing that Pgp oscillates between open/drug-accepting and closed/drug-expelling conformations. This different viewpoint highlights the fact that the multi-specificity of drug transporters and the “vacuum cleaner” hypothesis may actually be two sides of the same coin, both explained by the diffusion properties of drugs in the membrane. After retrieving basic results, predictions will be highlighted. Finally, the coherence of this model in the context of cancer biology will be discussed further.     

Effects of cholesterol and enantiomeric cholesterol on P-glycoprotein localization and function in low-density membrane domains

Multidrug resistance P-glycoprotein (Pgp) has been reported to localize in low-density, cholesterol-enriched membranes. However, effects of low-density membrane domains on function of Pgp remain unexplored in whole cell systems. In cells that express modest levels of the protein endogenously or through drug selection, Pgp predominantly localized to low-density membranes following separation on a sucrose gradient. When highly overexpressed in NIH 3T3 cells, a prominent amount of Pgp also was detected in high-density membranes. Removing cholesterol from cells with beta-methylcyclodextrin (CD), a sterol acceptor molecule, shifted fractions that contained Pgp from low toward high density, and this effect was reversed to a similar extent by restoring sterols with either cholesterol or enantiomeric cholesterol. However, function of human MDR1 Pgp as probed with Tc-Sestamibi, a transport substrate for Pgp, was not dependent on localization of Pgp in cholesterol-enriched membranes. Specific inhibition of MDR1 Pgp with GF120918 or LY335979 also was independent of cholesterol. Cell-type-specific effects of cholesterol content on function of human Pgp were detected by use of daunomycin, another substrate for Pgp, although efficacy of inhibitors remained independent of cholesterol. Conversely, both function and inhibition of hamster Pgp as measured with Tc-Sestamibi and daunomycin were in part dependent on normal cell content of cholesterol. These data show that Pgp preferentially localizes to low-density, cholesterol-enriched membrane domains, but acute depletion of cholesterol impacts Pgp-mediated drug transport in a substrate- and cell-type-specific manner.     

Mechanisms of altered sequestration and efflux of chemotherapeutic drugs by multidrug-resistant cells

This review considers the mechanisms associated with the pleiotropic resistance of cancer cells to chemotherapeutic drugs, and more particularly those related to intracellular pH (pHi). The multidrug resistance (MDR) phenomenon responsible for the decreased accumulation and increased efflux of cytotoxic drugs is generally associated with excess levels of P-glycoproteins (Pgps) encoded by MDR genes and/or the multidrug resistance-associated protein (MRP). MDR cell lines, derived from normal or tumor cells, frequently exhibit abnormally elevated pHi and changes in the production of various proteins. Recent studies have suggested that, in addition to the impact of the ATP-dependent membrane transporters Pgp and MRP on drug transport, other mechanisms linked to pHi changes in MDR cells may play an important role in drug resistance. We have shown that alkalinization of the acidic compartments (endosomes and lysosomes) by lysosomotropic agents could stimulate the efflux of vinblastine from drug-resistant mouse renal proximal tubule cells. The fact that weak base chemotherapeutic drugs can be sequestered within the acidic organelles of MDR cells sheds new light on the cellular mechanisms of drug resistance. This revised version was published online in July 2006 with corrections to the Cover Date.     

(+/-)-3'-O, 4'-O-dicynnamoyl-cis-khellactone, a derivative of (+/-)-praeruptorin A, reverses P-glycoprotein mediated multidrug resistance in cancer cells

P-glycoprotein (Pgp) is an ATP-driven membrane exporter for a broad spectrum of hydrophobic xenobiotics. Pgp-overexpression is a common cause of multidrug resistance (MDR) in cancer cells and could lead to chemotherapeutic failure. Through an extensive herbal drug screening program we previously showed that (+/-)-praeruptorin A (PA), a naturally existing pyranocumarin isolated from the dried root of Peucedanum praeruptorum Dunn., re-sensitizes Pgp-mediated MDR (Pgp-MDR) cancer cells to cancer drugs. A number of PA derivatives were synthesized and one of these, (+/-)-3'-O, 4'-O-dicynnamoyl-cis-khellactone (DCK), was more potent than PA or verapamil in the reversal of Pgp-MDR. In Pgp-MDR cells DCK increased cellular accumulation of doxorubicin without affecting the expression level of Pgp. In Pgp-enriched membrane fractions DCK moderately stimulated basal Pgp-ATPase activity, suggesting some transport substrate-like function. However, DCK also inhibited Pgp-ATPase activity stimulated by the standard substrates verapamil or progesterone with decreased V(max)s but K(m)s were relatively unchanged, suggesting a primarily non-competitive mode of inhibition. While the binding of substrates to active Pgp would increase the reactivity of the Pgp-specific antibody UIC2, DCK decreased UIC2 reactivity. These results suggest that DCK could bind simultaneously with substrates to Pgp but perhaps at an allosteric site and thus affect Pgp-substrate interactions.     

Di-2-pyridylketone 4,4-Dimethyl-3-thiosemicarbazone (Dp44mT) Overcomes Multidrug Resistance by a Novel Mechanism Involving the Hijacking of Lysosomal P-Glycoprotein (Pgp)

Multidrug resistance (MDR) is a major obstacle in cancer treatment. More than half of human cancers express multidrug-resistant P-glycoprotein (Pgp), which correlates with a poor prognosis. Intriguingly, through an unknown mechanism, some drugs have greater activity in drug-resistant tumor cells than their drug-sensitive counterparts. Herein, we investigate how the novel anti-tumor agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes MDR. Four different cell types were utilized to evaluate the effect of Pgp-potentiated lysosomal targeting of drugs to overcome MDR. To assess the mechanism of how Dp44mT overcomes drug resistance, cellular studies utilized Pgp inhibitors, Pgp silencing, lysosomotropic agents, proliferation assays, immunoblotting, a Pgp-ATPase activity assay, radiolabeled drug uptake/efflux, a rhodamine 123 retention assay, lysosomal membrane permeability assessment, and DCF (2′,7′-dichlorofluorescin) redox studies. Anti-tumor activity and selectivity of Dp44mT in Pgp-expressing, MDR cells versus drug-sensitive cells were studied using a BALB/c nu/nu xenograft mouse model. We demonstrate that Dp44mT is transported by the lysosomal Pgp drug pump, causing lysosomal targeting of Dp44mT and resulting in enhanced cytotoxicity in MDR cells. Lysosomal Pgp and pH were shown to be crucial for increasing Dp44mT-mediated lysosomal damage and subsequent cytotoxicity in drug-resistant cells, with Dp44mT being demonstrated to be a Pgp substrate. Indeed, Pgp-dependent lysosomal damage and cytotoxicity of Dp44mT were abrogated by Pgp inhibitors, Pgp silencing, or increasing lysosomal pH using lysosomotropic bases. In vivo, Dp44mT potently targeted chemotherapy-resistant human Pgp-expressing xenografted tumors relative to non-Pgp-expressing tumors in mice. This study highlights a novel Pgp hijacking strategy of the unique dipyridylthiosemicarbazone series of thiosemicarbazones that overcome MDR via utilization of lysosomal Pgp transport activity.     

Synthesis and evaluation of nitric oxide-releasing DDB derivatives as potential Pgp-mediated MDR reversal agents in MCF-7/Adr cells

Novel furoxan-based nitric oxide (NO)-releasing DDB derivatives (7a-j) were synthesized. Compounds 7i and 7j significantly reversed the resistance of MCF-7/Adr cells to doxorubicin in the combination treatment, and markedly increased the intracellular accumulation of doxorubicin probably via inhibiting Pgp-mediated intracellular drug efflux as well as down-regulating doxorubicin-induced Pgp expression. It was demonstrated that NO released by 7i and 7j played an important role in increasing intracellular doxorubicin accumulation and chemo-sensitizing MCF-7/Adr cells to doxorubicin, and the synergic effects of DDB and NO-donor moieties in 7i and 7j may contribute to reversing Pgp-mediated MDR in MCF-7/Adr cells to doxorubicin.     

Kinetics of daunorubicin transport by P-glycoprotein of intact cancer cells.

Drug permeation across the plasma membrane of multidrug-resistant cells depends on the kinetics of the P-glycoprotein-mediated pump activity as well as on the passive permeation of the drug. We here demonstrate a method to characterize kinetically the pump in intact cells. To this purpose, we examined the membrane-transport properties of daunorubicin in various sensitive cancer cell lines and in their multidrug resistant (MDR) counterparts. First, we determined the passive permeability coefficient for daunorubicin. Then, using a flow-through system, the drug flux into the cell was measured after inhibition of the P-glycoprotein-mediated efflux pump. Combining the two results allowed us to calculate the intracellular free concentration of the drug. In the steady-state, the pump rate must equal the net rate of passive diffusion of the drug and, therefore, the same experiments gave us the pumping rate of daunorubicin. These experiments were then repeated at various extracellular drug concentrations. By plotting the pumping rate versus the intracellular drug concentration, we then characterized the P-glycoprotein kinetically. Four independent methods were used to measure the passive permeability coefficient for the cell line A2780. Similar values were obtained. Maximal pump rates (Vmax) showed a good correlation with the amount of P-glycoprotein in the cell lines used. We obtained saturation curves for the variation of the pump rates with the intracellular daunorubicin concentrations. These curves were typical for positive cooperativity, which provides evidence that at least two binding sites for daunorubicin are present on the active transport system of daunorubicin. The apparent Km values for P-glycoprotein-mediated transport, the intracellular free cytosolic daunorubicin concentrations at half-maximal velocity for the cell lines used, were approximately 1.5 microM. Except for the cell lines with the highest amount of P-glycoprotein, the passive efflux rate of daunorubicin proved to be a substantial part of the total daunorubicin efflux rate for the cell lines used. In cell lines with relatively low levels of P-glycoprotein, passive daunorubicin efflux was even the main route of daunorubicin transport from the cells, determining the intracellular steady-state concentrations of daunorubicin.     

Membrane homoeostasis and multidrug resistance in yeast

The development of MDR (multidrug resistance) in yeast is due to a number of mechanisms. The most documented mechanism is enhanced extrusion of drugs mediated by efflux pump proteins belonging to either the ABC (ATP-binding cassette) superfamily or MFS (major facilitator superfamily). These drug-efflux pump proteins are localized on the plasma membrane, and the milieu therein affects their proper functioning. Several recent studies demonstrate that fluctuations in membrane lipid composition affect the localization and proper functioning of the MDR efflux pump proteins. Interestingly, the efflux pumps of the ABC superfamily are particularly susceptible to imbalances in membrane-raft lipid constituents. This review focuses on the importance of the membrane environment in functioning of the drug-efflux pumps and explores a correlation between MDR and membrane lipid homoeostasis.     

Analysis of the tangled relationships between P-glycoprotein-mediated multidrug resistance and the lipid phase of the cell membrane.

P-glycoprotein (Pgp), the so-called multidrug transporter, is a plasma membrane glycoprotein often involved in the resistance of cancer cells towards multiple anticancer agents in the multidrug-resistant (MDR) phenotype. It has long been recognized that the lipid phase of the plasma membrane plays an important role with respect to multidrug resistance and Pgp because: the compounds involved in the MDR phenotype are hydrophobic and diffuse passively through the membrane; Pgp domains involved in drug binding are located within the putative transmembrane segments; Pgp activity is highly sensitive to its lipid environment; and Pgp may be involved in lipid trafficking and metabolism. Unraveling the different roles played by the membrane lipid phase in MDR is relevant, not only to the evaluation of the precise role of Pgp, but also to the understanding of the mechanism of action and function of Pgp. With this aim, I review the data from different fields (cancer research, medicinal chemistry, membrane biophysics, pharmaceutical research) concerning drug-membrane, as well as Pgp-membrane, interactions. It is emphasized that the lipid phase of the membrane cannot be overlooked while investigating the MDR phenotype. Taking into account these aspects should be useful in the search of ways to obviate MDR and could also be relevant to the study of other multidrug transporters.     

Coordinate changes in drug resistance and drug-induced conformational transitions in altered-function mutants of the multidrug transporter P-glycoprotein

The MDR1 P-glycoprotein (Pgp), responsible for a clinically important form of multidrug resistance in cancer, is an ATPase efflux pump for multiple lipophilic drugs. The G185V mutation near transmembrane domain 3 of human Pgp increases its relative ability to transport several drugs, including etoposide, but decreases the transport of other substrates. MDR1 cDNA with the G185V substitution was used in a function-based selection to identify mutations that would further increase Pgp-mediated resistance to etoposide. This selection yielded the I186N substitution, adjacent to G185V. Pgps with G185V, I186N, or both mutations were compared to the wild-type Pgp for their ability to confer resistance to different drugs in NIH 3T3 cells. In contrast to the differential effects of G185V, I186N mutation increased resistance to all the tested drugs and augmented the effect of G185V on etoposide resistance. The effects of the mutations on conformational transitions of Pgp induced by different drugs were investigated using a conformation-sensitive antibody UIC2. Ligand-binding analysis of the drug-induced increase in UIC2 reactivity was used to determine the K(m) value that reflects the apparent affinity of drugs for Pgp, and the Hill number reflecting the apparent number of drug-binding sites. Both mutations altered the magnitude of drug-induced increases in UIC2 immunoreactivity, the K(m) values, and the Hill numbers for individual drugs. Mutation-induced changes in the magnitude of UIC2 reactivity shift did not correlate with the effects of the mutations on resistance to the corresponding drugs. In contrast, an increase or a decrease in drug resistance relative to that of the wild type was accompanied by a corresponding increase or decrease in the K(m) or in both the K(m) and the Hill number. These results suggest that mutations that alter the ability of Pgp to transport individual drugs change the apparent affinity and the apparent number of drug-binding sites in Pgp.     

Importance of the Difference in Surface Pressures of the Cell Membrane in Doxorubicin Resistant Cells That do not Express Pgp and ABCG2

P-glycoprotein (Pgp) represents the archetypal mechanism of drug resistance. But Pgp alone cannot expel drugs. A small but growing body of works has demonstrated that the membrane biophysical properties are central to Pgp-mediated drug resistance. For example, a change in the membrane surface pressure is expected to support drug–Pgp interaction. An interesting aspect from these models is that under specific conditions, the membrane is predicted to take over Pgp concerning the mechanism of drug resistance especially when the surface pressure is high enough, at which point drugs remain physically blocked at the membrane level. However it remains to be determined experimentally whether the membrane itself could, on its own, affect drug entry into cells that have been selected by a low concentration of drug and that do not express transporters. We demonstrate here that in the case of the drug doxorubicin, alteration of the surface pressure of membrane leaflets drive drug resistance.     

Radiopharmaceuticals for assessment of multidrug resistance P-glycoprotein-mediated drug transport activity

Multidrug resistance (MDR) mediated by overexpression of MDR1 P-glycoprotein (Pgp) is one of the best characterized transporter-mediated barriers to successful chemotherapy in cancer patients. Thus, noninvasive interrogation of Pgp-mediated transport activity in vivo would be beneficial in guiding therapeutic choices. Both small organic medicinals as well as metal complexes characterized as transport substrates for Pgp are amenable to incorporation of PET or SPECT radionuclides and may enable noninvasive imaging of Pgp in cancer patients. Toward this objective, clinically approved agents, exemplified by (99m)Tc-Sestamibi and (99m)Tetrofosmin, have already shown promise for the functional evaluation of Pgp-mediated transport activity in human tumors in vivo. In addition, selected agents from an upcoming class of substituted Schiff-base gallium(III) complexes containing an N(4)O(2) donor core in their organic scaffold and capable of generating both SPECT and PET radiopharmaceuticals have also been shown to be promising for noninvasive assessment of Pgp activity in vitro and in vivo.     

In situ biochemical demonstration that P-glycoprotein is a drug efflux pump with broad specificity

While P-glycoprotein (Pgp) is the most studied protein involved in resistance to anti-cancer drugs, its mechanism of action is still under debate. Studies of Pgp have used cell lines selected with chemotherapeutics which may have developed many mechanisms of resistance. To eliminate the confounding effects of drug selection on understanding the action of Pgp, we studied cells transiently transfected with a Pgp-green fluorescent protein (GFP) fusion protein. This method generated a mixed population of unselected cells with a wide range of Pgp-GFP expression levels and allowed simultaneous measurements of Pgp level and drug accumulation in living cells. The results showed that Pgp-GFP expression was inversely related to the accumulation of chemotherapeutic drugs. The reduction in drug concentration was reversed by agents that block multiple drug resistance (MDR) and by the UIC2 anti-Pgp antibody. Quantitative analysis revealed an inverse linear relationship between the fluorescence of Pgp-GFP and MDR dyes. This suggests that Pgp levels alone limit drug accumulation by active efflux; cooperativity between enzyme, substrate, or inhibitor molecules is not required. Additionally, Pgp-GFP expression did not change cellular pH. Our study demonstrates the value of using GFP fusion proteins for quantitative biochemistry in living cells.     

Allosteric modulation of human P-glycoprotein. Inhibition of transport by preventing substrate translocation and dissociation

The human multidrug transporter P-glycoprotein (Pgp, ABCB1) contributes to the poor bioavailability of many anticancer and antimicrobial agents as well as to drug resistance at the cellular level. For rational design of effective Pgp inhibitors, a clear understanding of its mechanism of action and functional regulation is essential. In this study, we demonstrate that inhibition of Pgp-mediated drug transport by cis-(Z)-flupentixol, a thioxanthene derivative, occurs through an allosteric mechanism. Unlike competitive inhibitors, such as cyclosporin A and verapamil, cis-(Z)-flupentixol does not interfere with substrate ([(125)I]iodoarylazidoprazosin) recognition by Pgp, instead it prevents substrate translocation and dissociation, resulting in a stable but reversible Pgp-substrate complex. cis-(Z)-Flupentixol-induced complex formation requires involvement of the Pgp substrate site, because agents that either physically compete (cyclosporin A) for or indirectly occlude (vanadate) the substrate-binding site prevent formation of the complex. Allosteric modulation by cis-(Z)-flupentixol involves a conformational change in Pgp detectable by monoclonal antibody UIC2 binding to a conformation-sensitive external epitope of Pgp. The conformational change observed is distinct from that induced by Pgp substrates or competitive inhibitors. A single amino acid substitution (F983A) in TM12 of Pgp that impairs inhibition by cis-(Z)-flupentixol of Pgp-mediated drug transport also affects stabilization of the Pgp-substrate complex as well as the characteristic conformational change. Taken together, our results describe the molecular mechanism by which the Pgp modulator cis-(Z)-flupentixol allosterically inhibits drug transport.