MDR3 P-glycoprotein, a phosphatidylcholine translocase, transports several cytotoxic drugs and directly interacts with drugs as judged by interference with nucleotide trapping

The human MDR3 gene is a member of the multidrug resistance (MDR) gene family. The MDR3 P-glycoprotein is a transmembrane protein that translocates phosphatidylcholine. The MDR1 P-glycoprotein related transports cytotoxic drugs. Its overexpression can make cells resistant to a variety of drugs. Attempts to show that MDR3 P-glycoprotein can cause MDR have been unsuccessful thus far. Here, we report an increased directional transport of several MDR1 P-glycoprotein substrates, such as digoxin, paclitaxel, and vinblastine, through polarized monolayers of MDR3-transfected cells. Transport of other good MDR1 P-glycoprotein substrates, including cyclosporin A and dexamethasone, was not detectably increased. MDR3 P-glycoprotein-dependent transport of a short-chain phosphatidylcholine analog and drugs was inhibited by several MDR reversal agents and other drugs, indicating an interaction between these compounds and MDR3 P-gp. Insect cell membranes from Sf9 cells overexpressing MDR3 showed specific MgATP binding and a vanadate-dependent, N-ethylmaleimide-sensitive nucleotide trapping activity, visualized by covalent binding with [alpha-(32)P]8-azido-ATP. Nucleotide trapping was (nearly) abolished by paclitaxel, vinblastine, and the MDR reversal agents verapamil, cyclosporin A, and PSC 833. We conclude that MDR3 P-glycoprotein can bind and transport a subset of MDR1 P-glycoprotein substrates. The rate of MDR3 P-glycoprotein-mediated transport is low for most drugs, explaining why this protein is not detectably involved in multidrug resistance. It remains possible, however, that drug binding to MDR3 P-glycoprotein could adversely affect phospholipid or toxin secretion under conditions of stress (e.g. in pregnant heterozygotes with one MDR3 null allele).     

A high-throughput screening microplate test for the interaction of drugs with P-glycoprotein

P-glycoprotein (P-gp) is a multidrug transporter responsible for resistance to anticancer chemotherapy and physiologically involved in absorption, distribution, and excretion of a large number of hydrophobic xenobiotics. P-gp exhibits both an ATPase activity correlated with its drug transport function and a basal ATPase activity in the absence of any drug. We have developed a high-throughput screening test to detect specific interactions between drugs and P-gp. We took into account the existence of multiple binding sites on P-gp to propose and validate an optimized strategy, based on the modulation of the basal ATPase activity of P-gp and of the ATPase activity stimulated by three reference substrates (verapamil, vinblastine, and progesterone). The ATPase activity measurements were performed on P-gp-containing membrane vesicles from actinomycin-D-selected hamster DC-3F lung fibroblasts by a spectrophotometric method based on continuous monitoring of ADP formation, regenerated in ATP by a coupled enzyme system. This assay may be performed on 96- or 384-well microtiter plates. When applying this ATPase assay to 41 compounds known from the literature for their interaction with P-gp, 95% of them were found to be positive, whereas only 78% were positive when considering solely the modulation of the basal activity.     

Specific reversal of multidrug resistance to colchicine in CEM/VLB100 cells by Gynostemma pentaphyllum extract

P-glycoprotein (P-gp)-mediated multiple drug resistance (MDR) is perhaps the most thoroughly studied cellular mechanism of cytotoxic drug resistance. Its efflux function can be circumvented by a wide range of pharmacological agents in vitro and in vivo. Most of these agents are pharmaceuticals used clinically for conditions other than cancer. However, their use in alleviating MDR is limited because the concentrations required for inhibition of the pump surpass their dose-limiting toxicity. The aim of this research is to study the role of gypenosides, isolated from Gynostemma pentaphyllum, as modulators of P-gp-mediated MDR in tumor cells, at both cellular and plasma membrane level. In the presence of total gypenoside preparation (0.1 mg/ml), an approximately 15-fold reversal of colchicine (COL) resistance was observed in P-gp-overexpressed CEM/VLB₁₀₀ cells. However, the gypenoside sample showed no reversal effect in cells treated with vinblastine and taxol. A purified gypenoside sample (gypenoside fraction 100) exhibited even more significant reversal of COL resistance (42-fold) in the CEM/VLB₁₀₀ cells. Further examination of the reversal effect of fraction 100 in membrane vesicles derived from CEM/VLB₁₀₀ cells using the continuous fluorescence method found that gypenoside fraction 100 at 0.1 mg/ml completely abolished the transport of fluorescein–COL.     

Modulators of the multidrug-transporter, P-glycoprotein, exist in the human plasma

P-glycoprotein (P-gp) is thought to mediate the transport of anticancer drugs and to be responsible for the multidrug-resistant (MDR) phenotype. P-gp is also expressed in normal human tissues, such as the adrenal gland, kidney, liver, colon and capillary endothelium of the brain. However, the function and transporting substrates of P-gp in normal tissues are still not understood. This paper explains that some compounds in the human plasma can modulate the transporting activity of P-gp. A partially purified fraction from the human plasma enhanced the accumulation of anti-cancer agents in MDR cells. This fraction inhibited the efflux of vinblastine from MDR cells, and also inhibited the photoaffinity labeling of P-gp with azidopine as effectively as vinblastine, quinidine and cepharanthine. The compounds in this purified fraction may be physiological substrates of P-gp and can probably overcome MDR.     

P-glycoprotein activity in human Caucasian male lymphocytes does not follow its increased expression during aging

P-glycoprotein (P-gp) is a transmembrane protein that mediates the efflux of innumerous structurally unrelated compounds. It was initially found over-expressed in tumor cells, associated to a multidrug resistance phenotype (MDR). Then, P-gp was found constitutively expressed in excretory cells/tissues and in circulating cells, such as lymphocytes. Considering the importance of this transporter in the establishment of therapeutic protocols and the existence of contradictory results, this study aimed at evaluating the influence of aging in the expression and function of P-gp in human lymphocytes, comparing two different methodologies to assess both parameters. P-gp activity and expression were evaluated in lymphocytes isolated from whole blood samples of 65 healthy caucasian male donors, divided into two groups according to age (group 1: under 30-years old; group 2: above 60-years old). P-gp expression was assessed using the anti-P-gp monoclonal antibody, UIC2, in the presence and in absence of vinblastine (Vbl). P-gp activity was evaluated measuring the efflux rate of the fluorescent P-gp substrate rhodamine 123 (Rho 123) and also using UIC2 shift assay. Flow cytometric analysis was performed to assess all the proceedings. Furthermore, P-gp expression and each of the P-gp activity determination methods were compared, through correlation analysis and linear regression models. We observed a significant age-dependent increase in mean P-gp expression (p = 0.029), which was not reflected in the transporter's activity (p > 0.050). Statistical analysis allowed selection of UIC2 shift assay over Rho 123 efflux assay as a more selective method to assess P-gp activity. Despite the significant correlation between P-gp expression and P-gp activity found in lymphocytes (Gp1(group 1)-r = 0.609, p < 0.001; Gp2-r = 0.461, p = 0.012), using UIC2 shift assay, these data reinforce the need for P-gp activity assessment, rather than P-gp expression determination alone, when starting new therapeutic regimens with P-gp substrates, especially in men older than 60 years of age.     

Functional expression of a multidrug P-glycoprotein transporter of Leishmania

P-glycoprotein (Pgp) transporters play an important role in multidrug resistance in eukaryotic cells and in protozoan parasites such as Leishmania. To search for new reversal agents of the Leishmania tropica Pgp, we developed a screening assay using the Baculovirus-insect cell expression system. We demonstrated a MgATP-dependent, vanadate-sensitive transport of Hoechst 33342 in membrane preparations of Sf9 insect cells expressing Pgp. We have found that dihydro-beta-agarofuran sesquiterpenes from Maytenus cuzcoina inhibited Hoechst 33342 transport that correlates with their reversal effect in a multidrug-resistant L. tropica line overexpressing Pgp. The results suggest that Sf9 cell membrane Hoechst 33342 transport system represents an efficient tool for examining the interactions of Leishmania Pgp with pharmacological agents.     

Expression and activity of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells.

Hematopoietic stem cells show reduced staining with a mitochondrial fluorescent dye, rhodamine 123 (Rh-123), which was supposed to indicate decreased mitochondrial activity in these cells. Rh123 and several other fluorescent dyes are substrates for transport mediated by P-glycoprotein (P-gp), an efflux pump responsible for multidrug resistance in tumor cells. We have found that staining of human bone marrow cells with fluorescent dyes is potentiated by P-gp inhibitors and inversely correlated with P-gp expression. P-gp is expressed in practically all hematopoietic progenitor cells, including long-term culture-initiating cells. The highest levels of P-gp among the progenitors are associated with cells displaying characteristics of pluripotent stem cells. These results have implications for stem cell purification and bone marrow resistance to cancer chemotherapy.     

Studies of Human MDR1-MDR2 Chimeras Demonstrate the Functional Exchangeability of a Major Transmembrane Segment of the Multidrug Transporter and Phosphatidylcholine Flippase

P-glycoprotein (P-gp), encoded by the MDR1 gene, is a plasma membrane transporter which effluxes a large number of structurally nonrelated hydrophobic compounds. The molecular basis of the broad substrate recognition of P-gp is not well understood. Despite the 78% amino acid sequence identity of the MDR1 and MDR2 transporter, MDR2, which has been identified as a phosphatidylcholine transporter, does not transport most MDR1 substrates. The structural and functional differences between MDR1 and MDR2 provide an opportunity to identify the residues essential for the broad substrate spectrum of MDR1. Using an approach involving exchanging homologous segments of MDR1 and MDR2 and site-directed mutagenesis, we have demonstrated that MDR1 residues Q330, V331, and L332 in transmembrane domain 6 are sufficient to allow an MDR2 backbone in the N-terminal half of P-gp to transport several MDR1 substrates, including bisantrene, colchicine, vinblastine, and rhodamine-123. These studies help define some residues important for multidrug transport and indicate the close functional relationship between the multidrug transporter (MDR1) and phosphatidylcholine flippase (MDR2).     

Role of P-glycoprotein in the renal transport of dideoxynucleoside analog drugs.

P-glycoprotein (P-gp), the MDR1 multidrug transporter, is known to be expressed in several human organs and tissues, including the apical membrane of the renal proximal tubular cells. It has been reported that human immunodeficiency virus 1 (HIV-1) can trigger the expression of P-gp in cultured cells (i.e., H9, a T-lymphocyte cell line, and U937, a monocyte cell line), which may render the cells resistant to antiretrovirals. Since multiple membrane transport systems (i.e., organic cation, organic anion, and nucleoside systems) can be involved in the renal tubular transport of dideoxynucleoside analog drugs (DADs) (i.e., zidovudine and zalcitabine), we have questioned if P-gp is involved in the renal transport of DADs. Chinese hamster ovary colchicine-resistant cells (CH(R)C5), a cell line that is well known to highly express P-gp, and continuous renal epithelial cell lines (LLC-PK1 and OK), which have also been shown to express P-gp, were used. The accumulation of [3H]vinblastine (20 nM), an established P-gp substrate, by the monolayer cells was significantly enhanced in the presence of two P-gp inhibitors (i.e., verapamil and cyclosporin A) and nucleoside transport inhibitors (i.e., dipyridamole and dilazep). In contrast, DADs (i.e., zidovudine, lamivudine, didanosine, and zalcitabine) did not significantly affect vinblastine accumulation by these cell lines. These data suggest that P-gp does not play a significant role in the renal tubular transport of DADs. Dipyridamole and dilazep, two nucleoside membrane transport inhibitors, appear to be P-gp inhibitors.     

Interactions of racemic mefloquine and its enantiomers with P-glycoprotein in an immortalised rat brain capillary endothelial cell line, GPNT

The brain distribution of the enantiomers of the antimalarial drug mefloquine is stereoselective according to the species. This stereoselectivity may be related to species-specific differences in the properties of some membrane-bound transport proteins, such as P-glycoprotein (P-gp). The interactions of racemic mefloquine and its individual enantiomers with the P-glycoprotein efflux transport system have been analysed in immortalised rat brain capillary endothelial GPNT cells. Parallel studies were carried out for comparison in human colon carcinoma Caco-2 cells. The cellular accumulation of the P-glycoprotein substrate, [(3)H]vinblastine, was significantly increased both in GPNT cells and in Caco-2 cells when treated with racemic mefloquine and the individual enantiomers. In GPNT cells, the (+)-stereoisomer of mefloquine was up to 8-fold more effective than its antipode in increasing cellular accumulation of [(3)H]vinblastine, while in Caco-2 cells, both enantiomers were equally effective. These results suggest that racemic mefloquine and its enantiomers are effective inhibitors of P-gp. Furthermore, a stereoselective P-glycoprotein inhibition is observed in rat cells but not in human cells. The efflux of [(14)C]mefloquine from GPNT cells was decreased when the cells were incubated with the P-gp modulators, verapamil, cyclosporin A or chlorpromazine, suggesting that MQ could be a P-gp substrate.     

Transport Inhibition of Digoxin Using Several Common P-gp Expressing Cell Lines Is Not Necessarily Reporting Only on Inhibitor Binding to P-gp

We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC₅₀ measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin''s cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable P-gp substrates such as amprenavir, quinidine, ketoconazole and verapamil do not, regardless of whether they actually use the basolateral transporter.     

Carotenoids reverse multidrug resistance in cancer cells by interfering with ABC-transporters

Proteins of the ATP-binding cassette superfamily, mainly P-glycoprotein (P-gp; MDR1), play an important role in the development of multidrug resistance (MDR) in cancer cells and thus in the potential failure of chemotherapy. A selection of carotenoids (β-carotene, crocin, retinoic acid, canthaxanthin, and fucoxanthin) was investigated whether they are substrates of P-gp, and if they can reverse MDR in resistant Caco-2 and CEM/ADR5000 cells as compared to the sensitive parent cell line CCRF-CEM. The activity of ABC transporter was determined in resistant and sensitive cells by spectrofluorometry and flow cytometry using the substrates doxorubicin, rhodamine 123, and calcein as fluorescent probes. The carotenoids increased accumulation of these P-gp substrates in a dose-dependent manner indicating that they themselves also function as substrates. Fucoxanthin and canthaxanthin (50-100μM) produced a 3-5-fold higher retention of the fluorescent probes than the known competitive inhibitor verapamil. Carotenoids showed a low cytotoxicity in cells with MDR with IC(50) values between 100 and 200μM. The combination of carotenoids with eight structurally different cytotoxic agents synergistically enhanced their cytotoxicity in Caco-2 cells, probably by inhibiting the function of the ABC transporters. For example, fucoxanthin synergistically enhanced the cytotoxicity of 5-FU 53.37-fold, of vinblastine 51.01-fold, and of etoposide 12.47-fold. RT-PCR was applied to evaluate the mRNA levels of P-gp in Caco-2 cells after treatment with carotenoids. Fucoxanthin and canthaxanthin significantly decreased P-gp levels to 12% and 24%, respectively as compared to untreated control levels (p<0.001). This study implies that carotenoids may be utilised as chemosensitisers, especially as adjuvants in chemotherapy.     

Hydrogel-assisted functional reconstitution of human P-glycoprotein (ABCB1) in giant liposomes

This paper describes the formation of giant proteoliposomes containing P-glycoprotein (P-gp) from a solution of small proteoliposomes that had been deposited and partially dried on a film of agarose. This preparation method generated a significant fraction of giant proteoliposomes that were free of internalized vesicles, making it possible to determine the accessible liposome volume. Measuring the intensity of the fluorescent substrate rhodamine 123 (Rho123) inside and outside these giant proteoliposomes determined the concentration of transported substrates of P-gp. Fitting a kinetic model to the fluorescence data revealed the rate of passive diffusion as well as active transport by reconstituted P-gp in the membrane. This approach determined estimates for the membrane permeability coefficient (P_s) of passive diffusion and rate constants of active transport (k_T) by P-gp as a result of different experimental conditions. The P_s value for Rho123 was larger in membranes containing P-gp under all assay conditions than in membranes without P-gp indicating increased leakiness in the presence of reconstituted transmembrane proteins. For P-gp liposomes, the k_T value was significantly higher in the presence of ATP than in its absence or in the presence of ATP and the competitive inhibitor verapamil. This difference in k_T values verified that P-gp was functionally active after reconstitution and quantified the rate of active transport. Lastly, patch clamp experiments on giant proteoliposomes showed ion channel activity consistent with a chloride ion channel protein that co-purified with P-gp. Together, these results demonstrate several advantages of using giant rather than small proteoliposomes to characterize transport properties of transport proteins and ion channels.     

The transmembrane domains of the human multidrug resistance P-glycoprotein are sufficient to mediate drug binding and trafficking to the cell surface.

The human multidrug resistance P-glycoprotein (P-gp) is organized in two tandem repeats with each repeat consisting of an N-terminal hydrophobic domain containing six potential transmembrane segments followed by a hydrophilic domain containing a nucleotide-binding fold. A series of deletion mutants together with an in vivo drug-binding assay were used to test whether the deletion mutants interacted with substrates or were transported to the cell surface. We found that a deletion mutant consisting of only the transmembrane domains (residues 1-379 plus 681-1025) retained the ability to interact with drug substrates. In the absence of drug substrates, the deletion mutant was sensitive to trypsin and endoglycosidase H. Expression in the presence of verapamil, vinblastine, capsaicin, or cyclosporin A, however, resulted in a mutant protein that was resistant to trypsin and endoglycosidase H. The mutant was then detected at the cell surface and was sensitive to digestion by endoglycosidase F. By contrast, the N-terminal transmembrane domain (residues 1-379) alone did not interact with drug substrates, since it was sensitive to only endoglycosidase H and was not detected at the cell surface. These results show that the nucleotide-binding domains are not required for interaction of P-gp with substrate or for trafficking of P-gp to the cell surface.     

Transepithelial transport of drugs by the multidrug transporter in cultured Madin-Darby canine kidney cell epithelia

We studied transepithelial transport of 3H-labeled hydrophobic cationic drugs in epithelia formed by wild-type and by drug-resistant Madin-Darby canine kidney (MDCk) cells that had been infected with a retrovirus carrying the multidrug-resistance (MDR1) cDNA which encodes the P-glycoprotein. P-glycoprotein is an ATP consuming plasma membrane multidrug transporter responsible for the efflux of cytotoxic chemotherapeutic drugs from resistant cancer cells. Wild-type MDCK cells have small amounts of P-glycoprotein detected by immunoprecipitation. Net transepithelial transport across wild-type MDCK epithelia was demonstrated. Basal to apical flux of 100 nM vinblastine was about six times higher than apical to basal flux. Addition of unlabeled vinblastine reduced basal to apical flux of tracer and increased apical to basal flux of tracer, a pattern expected if there is a saturable pump that extrudes vinblastine at the apical plasma membrane. Daunomycin, vincristine, and actinomycin D were also actively transported and at 20 microM these agents inhibited transport of vinblastine, suggesting that wild-type MDCK cells have a common transporter for all these drugs. Vinblastine transport was also inhibited by 20 microM verapamil, which inhibits the multidrug transporter and reverses multidrug-resistance in non-polarized cells. Net transepithelial transport of all these cytotoxic drugs and of verapamil was much higher in epithelia formed by MDCK cells infected with a human MDR1 virus (MDR-MDCK) which is expressed on the apical surface of MDR-MDCK monolayers. Because the transport of these cytotoxic drugs and verapamil is increased in MDR-MDCK epithelia compared to wild-type MDCK epithelia, transport in both these cell populations can be attributed to P-glycoprotein. These results are consistent with a role for P-glycoprotein in multidrug secretory transport across the epithelium of the proximal tubule since P-glycoprotein is normally expressed on the apical membrane of proximal tubule cells.     

Active transport of fluorescent P-glycoprotein substrates: evaluation as markers and interaction with inhibitors.

With P-glycoprotein (P-gp) continuing to have prominence among the ABC transporters for its ability to remove various xenobiotics from many cell types, accurate and robust methods for estimating the exposure of drug, carcinogen, toxicant, pesticide, and even some endobiotics to tissues and cells affected by P-gp are valuable. The inhibition of P-gp active transport of molecules, therefore, has often been quantified by concentration dependence of inhibitor effect on fluorescent substrate marker efflux mediated by this enzyme, with much evidence indicating two asymmetric yet interdependent substrate binding sites on P-gp. A uniqueness in the pair of binding sites could result in distinct effects of an inhibitor on the transport of certain substrates, thus leading to differences in fluorescent substrate responsiveness or sensitivity. Seven different fluorescent substrates of P-gp were quantitatively tested for their responsiveness to inhibition by a wide range of P-gp substrates/inhibitors. Interesting differences were observed in the IC(50) values caused by each of the inhibitors employed, in part exemplified by DNR and LDS being generally more sensitive to inhibition effects than any other fluorescent marker. However, no clear trend emerged to designate any fluorochrome marker as the most or least responsive to inhibition. Furthermore, LDS is more sensitive to some P-gp inhibitors than the substrate marker DNR, generally the most responsive. These results support the assertion of two unequal substrate binding sites that are allosterically dependent on each other. Therefore, an inhibitor that favors binding to the site opposite from that favored by a particular marker may have significant transduced effects through the protein between the two binding sites. Nevertheless, although either DNR or LDS is generally the fluorescent substrate most responsive to inhibition, there may be other substrates yet even more sensitive.     

Expression and function of P-glycoprotein and absence of multidrug resistance-related protein in rat and beige mouse peritoneal mast cells

To clarify the function of the multidrug transporter P-glycoprotein in mast cells we used the green fluorescent compound Bodipy-FL-verapamil, which is a substrate of P-glycoprotein. This compound is also transported by Multidrug Resistance-related Protein (MRP), another membrane transport protein expressed in many tumour resistant cells as well as in normal cells. When rat peritoneal mast cells were incubated with Bodipy-verapamil, a rapid uptake of this compound was observed. Pretreatment with modulators of P-glycoprotein activity, such as verapamil and vinblastine, increased Bodipy-verapamil intracellular concentrations. In addition, Bodipy-verapamil efflux from these cells was rapid and also inhibited by verapamil and vinblastine. In contrast, no effect was observed when cells were treated with agents, such as probenecid and indomethacin, that are known inhibitors of MRP. Methylamine and monensin, substances that modify the pH values in the granules, were able to lower the concentrations of Bodipy-verapamil. Microscopical observations, conducted in both rat and beige mouse mast cells, demonstrated that the fluorochrome accumulated in the cytoplasmic secretory granules. RT–PCR performed on rat peritoneal mast cells revealed the presence of MDR1a and MDR1b mRNAs; on the contrary, MRP mRNA was not expressed. Mast cells were further treated with the fluorescent probe LysoSensor Blue, a weak base that becomes fluorescent when inside acidic organelles. This substance accumulated in mast cell granular structures and its fluorescence was reduced either by treatment with P-glycoprotein modulators or with agents that disrupt pH gradients. In conclusion, these data further confirm the presence of an active P-glycoprotein, but not of MRP, in rat peritoneal mast cells. These findings, coupled with previous ultrastructural data, lend further support to the assumption that this protein is located on the mast cell perigranular membrane. The functional role of P-glycoprotein in these cells is at present unclear, but a possible involvement in the transport of molecules from the granules to the cytosol can be hypothesized. Alternatively, this protein might be indirectly implicated in changes of pH values inside secretory granules.     

Phospholipid flippase activity of the reconstituted P-glycoprotein multidrug transporter

The P-glycoprotein multidrug transporter acts as an ATP-powered efflux pump for a large variety of hydrophobic drugs, natural products, and peptides. The protein is proposed to interact with its substrates within the hydrophobic interior of the membrane. There is indirect evidence to suggest that P-glycoprotein can also transport, or "flip", short chain fluorescent lipids between leaflets of the membrane. In this study, we use a fluorescence quenching technique to directly show that P-glycoprotein reconstituted into proteoliposomes translocates a wide variety of NBD lipids from the outer to the inner leaflet of the bilayer. Flippase activity depended on ATP hydrolysis at the outer surface of the proteoliposome, and was inhibited by vanadate. P-Glycoprotein exhibited a broad specificity for phospholipids, and translocated phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. Lipid derivatives that were flipped included molecules with long, short, unsaturated, and saturated acyl chains and species with the NBD group covalently linked to either acyl chains or the headgroup. The extent of lipid translocation from the outer to the inner leaflet in a 20 min period at 37 degrees C was directly estimated, and fell in the range of 0.36-1.83 nmol/mg of protein. Phospholipid flipping was inhibited in a concentration-dependent, saturable fashion by various substrates and modulators, including vinblastine, verapamil, and cyclosporin A, and the efficiency of inhibition correlated well with the affinity of binding to Pgp. Taken together, these results suggest that P-glycoprotein carries out both lipid translocation and drug transport by the same path. The transporter may be a generic flippase for hydrophobic molecules with the correct steric attributes that are present within the membrane interior.     

Elevation of P-glycoprotein function by a catechin in green tea

The ABC transporter P-glycoprotein (P-gp) exerts a critical role in the systemic disposition of and exposure to lipophilic and amphipathic drugs, carcinogens, toxins, and other xenobiotics. The ability of P-gp to transfer a wide variety of structurally unrelated compounds from the cell interior across the membrane bilayer remains intriguing. Since dietary chemicals in green tea (and several other foods) appear to exert anticarcinogenic effects by an unknown mechanism, the constituents are frequently studied for interactions with various biomacromolecules as well as cytotoxins or isolated cells. We characterized several green tea catechins for their interaction with P-gp and their specific effects on P-gp export activity of several marker substrates. Some of these compounds inhibit the active efflux of the fluorescent markers LDS-751 (LDS) and rhodamine 123 (Rho) with low potency. Remarkably, others of these catechins facilitate the P-gp-mediated transport of LDS without affecting daunorubicin (DNR) transport or Rho. Moreover, (-)epicatechin, though an inhibitor of Rho transport, can significantly enhance the active net transport of another P-gp marker substrate, LDS. This result indicates that (-)epicatechin may bind to and activate an allosteric site that enhances P-gp overall function or efficiency. Such a mechanism of heterotropic allosteric enhancement of P-gp could serve as chemoprotective to many cells and contribute to the purported anticarcinogenic effect of green tea consumption.     

Inhibition of the multidrug resistance P-glycoprotein activity by green tea polyphenols

Many beneficial proprieties have been associated with polyphenols from green tea, such as chemopreventive, anticarcinogenic, antiatherogenic and antioxidant actions. In this study, we investigated the effects of green tea polyphenols (GTPs) and their principal catechins on the function of P-glycoprotein (P-gp), which is involved in the multidrug resistance phenotype of cancer cells. GTPs (30 microg/ml) inhibit the photolabeling of P-gp by 75% and increase the accumulation of rhodamine-123 (R-123) 3-fold in the multidrug-resistant cell line CH(R)C5, indicating that GTPs interact with P-gp and inhibit its transport activity. Moreover, the modulation of P-gp transport by GTPs was a reversible process. Among the catechins present in GTPs, EGCG, ECG and CG are responsible for inhibiting P-gp. In addition, EGCG potentiates the cytotoxicity of vinblastine (VBL) in CH(R)C5 cells. The inhibitory effect of EGCG on P-gp was also observed in human Caco-2 cells, which form an intestinal epithelial-like monolayer. Our results indicate that, in addition to their anti-cancer properties, GTPs and more particularly EGCG inhibit the binding and efflux of drugs by P-gp. Thus, GTPs or EGCG might be potential agents for modulating the bioavailability of P-gp substrates at the intestine and the multidrug resistance phenotype associated with expression of this transporter in cancer cells.