Synergistic Inhibitory Effect of Quercetin and Cyanidin-3O-Sophoroside on ABCB1.

The human ABCB1 (P-glycoprotein, Pgp) protein is an active exporter expressed in the plasma membrane of cells forming biological barriers. In accordance with its broad substrate spectrum and tissue expression pattern, it affects the pharmacokinetics of numerous chemotherapeutic drugs and it is involved in unwanted drug-drug interactions leading to side effects or toxicities. When expressed in tumor tissues, it contributes to the development of chemotherapy resistance in malignancies. Therefore, the understanding of the molecular details of the ligand-ABCB1 interactions is of crucial importance. In a previous study, we found that quercetin (QUR) hampers both the transport and ATPase activity of ABCB1, while cyandin-3O-sophroside (C3S) stimulates the ATPase activity and causes only a weak inhibition of substrate transport. In the current study, when QUR and C3S were applied together, both a stronger ATPase inhibition and a robust decrease in substrate transport were observed, supporting their synergistic ABCB1 inhibitory effect. Similar to cyclosporine A, a potent ABCB1 inhibitor, co-treatment with QUR and C3S shifted the conformational equilibrium to the "inward-facing" conformer of ABCB1, as it was detected by the conformation-selective UIC2 mAb. To gain deeper insight into the molecular details of ligand-ABCB1 interactions, molecular docking experiments and MD simulations were also carried out. Our in silico studies support that QUR and C3S can bind simultaneously to ABCB1. The most favourable ligand-ABCB1 interaction is obtained when C3S binds to the central substrate binding site and QUR occupies the "access tunnel". Our results also highlight that the strong ABCB1 inhibitory effect of the combined treatment with QUR and C3S may be exploited in chemotherapy protocols for the treatment of multidrug-resistant tumors or for improving drug delivery through pharmacological barriers.

Nucleotide binding is the critical regulator of ABCG2 conformational transitions.

ABCG2 is an exporter-type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here, we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.

Effects of Polyphenols on P-Glycoprotein (ABCB1) Activity.

P-glycoprotein (Pgp, ABCB1) is a member of one of the largest families of active transporter proteins called ABC transporters. Thanks to its expression in tissues with barrier functions and its broad substrate spectrum, it is an important determinant of the absorption, metabolism and excretion of many drugs. Pgp and/or some other drug transporting ABC proteins (e.g., ABCG2, MRP1) are overexpressed in nearly all cancers and cancer stem cells by which cancer cells become resistant against many drugs. Thus, Pgp inhibition might be a strategy for fighting against drug-resistant cancer cells. Previous studies have shown that certain polyphenols interact with human Pgp. We tested the effect of 15 polyphenols of sour cherry origin on the basal and verapamil-stimulated ATPase activity of Pgp, calcein-AM and daunorubicin transport as well as on the conformation of Pgp using the conformation sensitive UIC2 mAb. We found that quercetin, quercetin-3-glucoside, narcissoside and ellagic acid inhibited the ATPase activity of Pgp and increased the accumulation of calcein and daunorubicin by Pgp-positive cells. Cyanidin-3O-sophoroside, catechin, naringenin, kuromanin and caffeic acid increased the ATPase activity of Pgp, while they had only a weaker effect on the intracellular accumulation of fluorescent Pgp substrates. Several tested polyphenols including epicatechin, trans-ferulic acid, oenin, malvin and chlorogenic acid were ineffective in all assays applied. Interestingly, catechin and epicatechin behave differently, although they are stereoisomers. We also investigated the effect of quercetin, naringenin and ellagic acid added in combination with verapamil on the transport activity of Pgp. In these experiments, we found that the transport inhibitory effect of the tested polyphenols and verapamil was additive or synergistic. Generally, our data demonstrate diverse interactions of the tested polyphenols with Pgp. Our results also call attention to the potential risks of drug-drug interactions (DDIs) associated with the consumption of dietary polyphenols concurrently with chemotherapy treatment involving Pgp substrate/inhibitor drugs.

Human ABCB1 with an ABCB11-like degenerate nucleotide binding site maintains transport activity by avoiding nucleotide occlusion.

Several ABC exporters carry a degenerate nucleotide binding site (NBS) that is unable to hydrolyze ATP at a rate sufficient for sustaining transport activity. A hallmark of a degenerate NBS is the lack of the catalytic glutamate in the Walker B motif in the nucleotide binding domain (NBD). The multidrug resistance transporter ABCB1 (P-glycoprotein) has two canonical NBSs, and mutation of the catalytic glutamate E556 in NBS1 renders ABCB1 transport-incompetent. In contrast, the closely related bile salt export pump ABCB11 (BSEP), which shares 49% sequence identity with ABCB1, naturally contains a methionine in place of the catalytic glutamate. The NBD-NBD interfaces of ABCB1 and ABCB11 differ only in four residues, all within NBS1. Mutation of the catalytic glutamate in ABCB1 results in the occlusion of ATP in NBS1, leading to the arrest of the transport cycle. Here we show that despite the catalytic glutamate mutation (E556M), ABCB1 regains its ATP-dependent transport activity, when three additional diverging residues are also replaced. Molecular dynamics simulations revealed that the rescue of ATPase activity is due to the modified geometry of NBS1, resulting in a weaker interaction with ATP, which allows the quadruple mutant to evade the conformationally locked pre-hydrolytic state to proceed to ATP-driven transport. In summary, we show that ABCB1 can be transformed into an active transporter with only one functional catalytic site by preventing the formation of the ATP-locked pre-hydrolytic state in the non-canonical site.

Interactions of retinoids with the ABC transporters P-glycoprotein and Breast Cancer Resistance Protein.

Retinoids - derivatives of vitamin A - are important cell permeant signaling molecules that regulate gene expression through activation of nuclear receptors. P-glycoprotein (Pgp) and ABCG2 are plasma membrane efflux transporters affecting the tissue distribution of numerous structurally unrelated lipophilic compounds. In the present work we aimed to study the interaction of the above ABC transporters with retinoid derivatives. We have found that 13-cis-retinoic acid, retinol and retinyl-acetate inhibited the Pgp and ABCG2 mediated substrate transport as well as the substrate stimulated ATPase activity of these transporters. Interestingly, 9-cis-retinoic acid and ATRA (all-trans retinoic acid), both are stereoisomers of 13-cis-retinoic acid, did not have any effect on the transporters' activity. Our fluorescence anisotropy measurements revealed that 13-cis-retinoic acid, retinol and retinyl-acetate selectively increase the viscosity and packing density of the membrane. Thus, the mixed-type inhibition of both transporters by retinol and ABCG2 by 13-cis-retinoic acid may be the collective result of direct interactions of these retinoids with the substrate binding site(s) and of indirect interactions mediated by their membrane rigidifying effects.

A single active catalytic site is sufficient to promote transport in P-glycoprotein.

P-glycoprotein (Pgp) is an ABC transporter responsible for the ATP-dependent efflux of chemotherapeutic compounds from multidrug resistant cancer cells. Better understanding of the molecular mechanism of Pgp-mediated transport could promote rational drug design to circumvent multidrug resistance. By measuring drug binding affinity and reactivity to a conformation-sensitive antibody we show here that nucleotide binding drives Pgp from a high to a low substrate-affinity state and this switch coincides with the flip from the inward- to the outward-facing conformation. Furthermore, the outward-facing conformation survives ATP hydrolysis: the post-hydrolytic complex is stabilized by vanadate, and the slow recovery from this state requires two functional catalytic sites. The catalytically inactive double Walker A mutant is stabilized in a high substrate affinity inward-open conformation, but mutants with one intact catalytic center preserve their ability to hydrolyze ATP and to promote drug transport, suggesting that the two catalytic sites are randomly recruited for ATP hydrolysis.

2'[(18)F]-fluoroethylrhodamine B is a promising radiotracer to measure P-glycoprotein function.

In vivo detection of the emergence of P-glycoprotein (Pgp) mediated multidrug resistance in tumors could be beneficial for patients treated with anticancer drugs. PET technique in combination with appropriate radiotracers could be the most convenient method for detection of Pgp function. Rhodamine derivatives are validated fluorescent probes for measurement of mitochondrial membrane potential and also Pgp function. The aim of this study was to investigate whether 2'[(18)F]-fluoroethylrhodamine B ((18)FRB) a halogenated rhodamine derivative previously synthesized for PET assessment of myocardial perfusion preserved its Pgp substrate character. ATPase assay as well as accumulation experiments carried out using Pgp(+) and Pgp(-) human gynecologic (A2780/A2780(AD) and KB-3-1/KB-V1) and a mouse fibroblast cell pairs (NIH 3T3 and NIH 3T3 MDR1) were applied to study the interaction of (18)FRB with Pgp. ATPase assay proved that (18)FRB is a high affinity substrate of Pgp. Pgp(-) cells accumulated the (18)FRB rapidly in accordance with its lipophilic character. Dissipation of the mitochondrial proton gradient by a proton ionophore CCCP decreased the accumulation of rhodamine 123 (R123) and (18)FRB into Pgp(-) cells. Pgp(+) cells exhibited very low R123 and (18)FRB accumulation (around 1-8% of the Pgp(-) cell lines) which was not sensitive to the mitochondrial proton gradient; rather it was increased by the Pgp inhibitor cyclosporine A (CsA). Based on the above data we conclude that (18)FRB is a high affinity Pgp substrate and consequently a potential PET tracer to detect multidrug resistant tumors as well as the function of physiological barriers expressing Pgp.

18FDG, [18F]FLT, [18F]FAZA, and 11C-methionine are suitable tracers for the diagnosis and in vivo follow-up of the efficacy of chemotherapy by miniPET in both multidrug resistant and sensitive human gynecologic tumor xenografts.

Expression of multidrug pumps including P-glycoprotein (MDR1, ABCB1) in the plasma membrane of tumor cells often results in decreased intracellular accumulation of anticancer drugs causing serious impediment to successful chemotherapy. It has been shown earlier that combined treatment with UIC2 anti-Pgp monoclonal antibody (mAb) and cyclosporine A (CSA) is an effective way of blocking Pgp function. In the present work we investigated the suitability of four PET tumor diagnostic radiotracers including 2-[(18)F]fluoro-2-deoxy-D-glucose ((18)FDG), (11)C-methionine, 3'-deoxy-3'-[(18)F]fluorothymidine ((18)F-FLT), and [(18)F]fluoroazomycin-arabinofuranoside ((18)FAZA) for in vivo follow-up of the efficacy of chemotherapy in both Pgp positive (Pgp(+)) and negative (Pgp(-)) human tumor xenograft pairs raised in CB-17 SCID mice. Pgp(+) and Pgp(-) A2780AD/A2780 human ovarian carcinoma and KB-V1/KB-3-1 human epidermoid adenocarcinoma tumor xenografts were used to study the effect of the treatment with an anticancer drug doxorubicin combined with UIC2 and CSA. The combined treatment resulted in a significant decrease of both the tumor size and the accumulation of the tumor diagnostic tracers in the Pgp(+) tumors. Our results demonstrate that (18)FDG, (18)F-FLT, (18)FAZA, and (11)C-methionine are suitable PET tracers for the diagnosis and in vivo follow-up of the efficacy of tumor chemotherapy in both Pgp(+) and Pgp(-) human tumor xenografts by miniPET.

The strong in vivo anti-tumor effect of the UIC2 monoclonal antibody is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity.

P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10-40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC50 of doxorubicin (DOX) in KB-V1 (Pgp+) cells in vitro almost to the level of KB-3-1 (Pgp-) cells. At the same time, UIC2 alone did not affect the EC50 value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp+ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp+ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased 18FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC).

¹8FDG a PET tumor diagnostic tracer is not a substrate of the ABC transporter P-glycoprotein.

2-[(18)F]fluoro-2-deoxy-d-glucose ((18)FDG) is a tumor diagnostic radiotracer of great importance in both diagnosing primary and metastatic tumors and in monitoring the efficacy of the treatment. P-glycoprotein (Pgp) is an active transporter that is often expressed in various malignancies either intrinsically or appears later upon disease progression or in response to chemotherapy. Several authors reported that the accumulation of (18)FDG in P-glycoprotein (Pgp) expressing cancer cells (Pgp(+)) and tumors is different from the accumulation of the tracer in Pgp nonexpressing (Pgp(-)) ones, therefore we investigated whether (18)FDG is a substrate or modulator of Pgp pump. Rhodamine 123 (R123) accumulation experiments and ATPase assay were used to detect whether (18)FDG is substrate for Pgp. The accumulation and efflux kinetics of (18)FDG were examined in two different human gynecologic (A2780/A2780AD and KB-3-1/KB-V1) and a mouse fibroblast (3T3 and 3T3MDR1) Pgp(+) and Pgp(-) cancer cell line pairs both in cell suspension and monolayer cultures. We found that (18)FDG and its derivatives did not affect either the R123 accumulation in Pgp(+) cells or the basal and the substrate stimulated ATPase activity of Pgp supporting that they are not substrates or modulators of the pump. Measuring the accumulation and efflux kinetics of (18)FDG in different Pgp(+) and Pgp(-) cell line pairs, we have found that the Pgp(+) cells exhibited significantly higher (p⩽0.01) (18)FDG accumulation and slightly faster (18)FDG efflux kinetics compared to their Pgp(-) counterparts. The above data support the idea that expression of Pgp may increase the energy demand of cells resulting in higher (18)FDG accumulation and faster efflux. We concluded that (18)FDG and its metabolites are not substrates of Pgp.

Antibody binding shift assay for rapid screening of drug interactions with the human ABCG2 multidrug transporter.

The ABCG2 multidrug transporter protein has been identified as a key player in cancer drug resistance and xenobiotic elimination, as its actively transported substrates include anticancer drugs, intermediates of heme metabolism, xenobiotics, and also drug conjugates. Several transported substrates at higher concentrations, and some anticancer agents even at low concentrations directly inhibit the ABCG2 transporter, thus it is difficult to provide estimation for pharmacologically important ABCG2-dependent interactions. In addition, as documented here, in mutant variants of the transporter, inhibitors of the wild-type ABCG2 may become actively transported substrates. In this paper we describe a rapid in vitro assay to identify transport modulation by measuring the cell surface interaction of a conformation sensitive monoclonal antibody (5D3) with ABCG2 in intact cells. As documented, in conjunction with membrane ATPase, transport and cytotoxicity measurements, this assay provides a reliable estimate of concentration-dependent modulation of ABCG2 by newly emerging pharmacophores. A high-throughput, 96-well plate assay platform is also provided.

Pgp inhibition by UIC2 antibody can be followed in vitro by using tumor-diagnostic radiotracers, 99mTc-MIBI and 18FDG.

P-glycoprotein (Pgp, ABCB1) is one of the active efflux pumps that are able to extrude a large variety of chemotherapeutic drugs from the cells, causing the phenomenon of multidrug resistance. It has been shown earlier that the combined application of a class of Pgp modulators (e.g. cyclosporine A and SDZ PSC 833) used at low concentrations and UIC2 antibody is a novel, specific, and effective way of blocking Pgp function (Goda et al., 2007). In the present work we study the UIC2 antibody mediated Pgp inhibition in more detail measuring the accumulation of tumor diagnostic radiotracers, 2-[(18)F]fluoro-2-deoxy-d-glucose ((18)FDG) and [(99m)Tc]hexakis-2-methoxybutyl isonitrile ((99m)Tc-MIBI), into Pgp(+) (A2780AD) and Pgp(-) (A2780) human ovarian carcinoma cells. Co-incubation of cells with UIC2 and cyclosporine A (CSA, 2µM) increased the binding of UIC2 more than 3-fold and reverted the rhodamine 123 (R123), daunorubicin (DNR) and (99m)Tc-MIBI accumulation of the Pgp(+) 2780AD cells to approx. the same level as observed in Pgp(-) cells. Similarly, 50µM paclitaxel (Pacl) increased UIC2 binding, and consequently reinstated the uptake of R123, DNR and (99m)Tc-MIBI into the Pgp(+) cells. Blocking Pgp by combined treatments with CSA+UIC2 or Pacl+UIC2 also decreased the glucose metabolic rate of the A2780AD Pgp(+) cells measured in (18)FDG accumulation experiments suggesting that the maintenance of Pgp activity requires a considerable amount of energy. Similar treatments of the A2780 Pgp(-) cells did not result in significant change in the R123, DNR, (99m)Tc-MIBI and (18)FDG accumulation demonstrating that the above effects are Pgp-specific. Thus, combined treatment with the UIC2 antibody and Pgp modulators can completely block the function of Pgp in human ovarian carcinoma cells and this effect can be followed in vitro by using tumor-diagnostic radiotracers, (99m)Tc-MIBI and (18)FDG.

Daunorubicin and doxorubicin inhibit the [(11)C]choline accumulation in cancer cells.

We studied how very short (10-40min) incubation with anthracycline derivatives modifies the accumulation of PET tumor-diagnostic radiotracers in cancer cells. The human ovarian A2780 and A2780AD, human B lymphoid JY, human epidermoid KB-3-1 and KB-V-1, and smooth muscle DDT1 MF-2 cells were pre-incubated with daunorubicin and doxorubicin, and the uptake of [(18)F]FDG and [(11)C]choline was measured. Anthracycline treatment decreased remarkably the [(11)C]choline accumulation in a concentration dependent manner, while it did not modify significantly the [(18)F]FDG uptake of the cells.

Multidrug resistance through the spectacle of P-glycoprotein.

P-glycoprotein (Pgp), coded for by the mdr1 gene, is one of the ABC transporters held responsible for the phenomenon of multidrug resistance (mdr), which is reflected by a rapidly escalating failure of chemotherapy with different classes of cytotoxic agents: anthracyclins, vinca alkaloids, taxanes, epipodophylotoxins. Although overcoming resistance conveyed by Pgp alone may not be sufficient for reaching effective treatment, the abundance of observations available for this paradigmatic multidrug transporter at both in vitro and in vivo setting is a tempting ground for an updated assessment of the main currents of mdr research. In this review we attempt to help keep track of the features of Pgp-mediated drug transport that serve as the major starting points for ongoing efforts of mdr reversal. We will analyze the slowly narrowing gaps that prevail between our ever increasing understanding at the protein, cell and organism level, focusing on the molecular interactions involving Pgp.

Interaction with the 5D3 monoclonal antibody is regulated by intramolecular rearrangements but not by covalent dimer formation of the human ABCG2 multidrug transporter.

Human ABCG2 is a plasma membrane glycoprotein working as a homodimer or homo-oligomer. The protein plays an important role in the protection/detoxification of various tissues and may also be responsible for the multidrug-resistant phenotype of cancer cells. In our previous study we found that the 5D3 monoclonal antibody shows a function-dependent reactivity to an extracellular epitope of the ABCG2 transporter. In the current experiments we have further characterized the 5D3-ABCG2 interaction. The effect of chemical cross-linking and the modulation of extracellular S-S bridges on the transporter function and 5D3 reactivity of ABCG2 were investigated in depth. We found that several protein cross-linkers greatly increased 5D3 labeling in ABCG2 expressing HEK cells; however, there was no correlation between covalent dimer formation, the inhibition of transport activity, and the increase in 5D3 binding. Dithiothreitol treatment, which reduced the extracellular S-S bridge-forming cysteines of ABCG2, had no effect on transport function but caused a significant decrease in 5D3 binding. When analyzing ABCG2 mutants carrying Cys-to-Ala changes in the extracellular loop, we found that the mutant C603A (lacking the intermolecular S-S bond) showed comparable transport activity and 5D3 reactivity to the wild-type ABCG2. However, disruption of the intramolecular S-S bridge (in C592A, C608A, or C592A/C608A mutants) in this loop abolished 5D3 binding, whereas the function of the protein was preserved. Based on these results and ab initio folding simulations, we propose a model for the large extracellular loop of the ABCG2 protein.

P-glycoprotein inhibition by membrane cholesterol modulation.

P-glycoprotein (Pgp) is a transmembrane protein that actively exports lipophilic chemotherapeutics from the cells causing multidrug resistance. Pgp molecules are partially localized in TX-100-resistant rafts, and the activity of the transporter is highly sensitive to the presence of cholesterol. To better understand these relationships, the influence of membrane cholesterol content on Pgp function, as measured via calcein accumulation, was studied in correlation with changes elicited in membrane structure. Membrane cholesterol was modulated by heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DIMEB) and the cholesterol inclusion complex of DIMEB (Chol-DIMEB). Changes in membrane cholesterol level were reflected by alterations in the overall lipid packing as measured by Merocyanine 540 (MC540) staining and were also accompanied by changes in the raft association of the pump. DIMEB and Chol-DIMEB treatments have also lead to increased permeability of the cell membrane in both directions, raising the possibility that the effects on pumping efficiency reflect leakage of ATP also from the non-permeabilized cells. However, the treatments did not influence the intracellular ATP levels of the non-permeabilized cells. Our data suggest that Pgp inhibition by cyclodextrin treatments arises through modulation of its membrane microenvironment, rather than as a result of concomitant cytotoxicity.

Ribonucleoprotein-masked nicks at 50-kbp intervals in the eukaryotic genomic DNA.

By using a microscopic approach, field inversion single-cell gel electrophoresis, we show that preformed single-strand discontinuities are present in the chromatin of resting and proliferating mammalian and yeast cells. These single-strand breaks are primarily nicks positioned at approximately 50-kbp intervals throughout the entire genome that could be efficiently labeled in situ by DNA polymerase I holoenzyme but not by Klenow fragment and terminal transferase unless after ribonucleolytic treatments. The RNA molecules involved appear to comprise R-loops, recognized by the S9.6 RNA/DNA hybrid-specific antibody. By using the breakpoint cluster region of the Mixed Lineage Leukemia (MLL) gene as a model, we have found that the number of manifest nicks detected by FISH performed after field inversion single-cell gel electrophoresis depends on epigenetic context, but the difference between germ-line and translocated MLL alleles is abolished by protease treatment. Our data imply that the double-stranded genomic DNA is composed of contiguous rather than continuous single strands and reveal an aspect of higher-order chromatin organization with ribonucleoprotein-associated persistent nicks defining approximately 50-kbp domains.

Complete inhibition of P-glycoprotein by simultaneous treatment with a distinct class of modulators and the UIC2 monoclonal antibody.

P-glycoprotein (Pgp) is one of the active efflux pumps that are able to extrude a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance. The conformation-sensitive UIC2 monoclonal antibody potentially inhibits Pgp-mediated substrate transport. However, this inhibition is usually partial, and its extent is variable because UIC2 binds only to 10 to 40% Pgp present in the cell membrane. The rest of the Pgp molecules become recognized by this antibody only in the presence of certain substrates or modulators, including vinblastine, cyclosporine A (CsA), and SDZ PSC 833 (valspodar). Simultaneous application of any of these modulators and UIC2, followed by the removal of the modulator, results in a completely restored steady-state accumulation of various Pgp substrates (calcein-AM, daunorubicin, and 99mTc-hexakis-2-methoxybutylisonitrile), indicating near 100% inhibition of pump activity. Remarkably, the inhibitory binding of the antibody is brought about by coincubation with concentrations of CsA or SDZ PSC 833 approximately 20 times lower than what is necessary for Pgp inhibition when the modulators are applied alone. The feasibility of such a combinative treatment for in vivo multidrug resistance reversal was substantiated by the dramatic increase of daunorubicin accumulation in xenotransplanted Pgp+ tumors in response to a combined treatment with UIC2 and CsA, both administered at doses ineffective when applied alone. These observations establish the combined application of a class of modulators used at low concentrations and of the UIC2 antibody as a novel, specific, and effective way of blocking Pgp function in vivo.