P-glycoprotein (ABCB1) interacts directly with lipid-based anti-cancer drugs and platelet-activating factors.

The P-glycoprotein multidrug transporter (Pgp; ABCB1) is an ATP-binding cassette (ABC) protein that has been implicated in the multidrug resistance of human cancers. Pgp couples ATP hydrolysis to active extrusion from the cell of a broad array of amphipathic compounds via an ill-defined mechanism. Substrates are believed to interact with Pgp within the membrane. Reconstituted Pgp functions as an ATP-dependent flippase for a variety of fluorescently labelled membrane lipids. The protein may also function as a drug 'flippase', moving its substrates from the inner to the outer leaflet of the bilayer. We show that lipid-based anti-cancer drugs, such as miltefosine, and signaling molecules, such as platelet-activating factors, bind saturably to Pgp with Kd values in the low micromolar range, and modulate its ATPase activity. These compounds also inhibit Pgp-mediated flipping of fluorescent lipids and transport of Hoechst 33342 and tetramethylrosamine, which occupy different subsites in the drug-binding pocket. Bacterial lipid A modulates Pgp ATPase activity, and glycolipid flipping is inhibited by unlabelled glucosylceramide, suggesting that these lipids also interact with the transporter. These results indicate that Pgp treats a variety of lipid-based molecules as substrates, and likely interacts with lipids and drugs in the same manner.     

Transformation of Chlamydia muridarum Reveals a Role for Pgp5 in Suppression of Plasmid-Dependent Gene Expression

Transformation of Chlamydia trachomatis should greatly advance the chlamydial research. However, significant progress has been hindered by the failure of C. trachomatis to induce clinically relevant pathology in animal models. Chlamydia muridarum, which naturally infects mice, can induce hydrosalpinx in mice, a tubal pathology also seen in women infected with C. trachomatis. We have developed a C. muridarum transformation system and confirmed Pgp1, -2, -6, and -8 as plasmid maintenance factors, Pgp3, -5, and -7 as dispensable for in vitro growth, and Pgp4 as a positive regulator of genes that are dependent on plasmid for expression. More importantly, we have discovered that Pgp5 can negatively regulate the same plasmid-dependent genes. Deletion of Pgp5 led to a significant increase in expression of the plasmid-dependent genes, suggesting that Pgp5 can suppress the expression of these genes. Replacement of pgp5 with a mCherry gene, or premature termination of pgp5 translation, also increased expression of the plasmid-dependent genes, indicating that Pgp5 protein but not its DNA sequence is required for the inhibitory effect. Replacing C. muridarum pgp5 with a C. trachomatis pgp5 still inhibited the plasmid-dependent gene expression, indicating that the negative regulation of plasmid-dependent genes is a common feature of all Pgp5 regardless of its origin. Nevertheless, C. muridarum Pgp5 is more potent than C. trachomatis Pgp5 in suppressing gene expression. Thus, we have uncovered a novel function of Pgp5 and developed a C. muridarum transformation system for further mapping chlamydial pathogenic and protective determinants in animal models.     

Insights into the molecular mechanism of action of Celastraceae sesquiterpenes as specific, non-transported inhibitors of human P-glycoprotein

Dihydro-β-agarofuran sesquiterpenes from Celastraceae have been recently shown to bind to human P-glycoprotein (Pgp), functioning as specific, mixed-type inhibitors of its drug transport activity, as well as multidrug resistance (MDR) modulators in vitro. However, nothing is known about whether such compounds are themselves transported by Pgp, or whether they affect Pgp expression as well as its activity, or about the location of their binding site within the protein. We performed transport experiments with a newly synthesized fluorescent sesquiterpene derivative, which retains the anti-Pgp activity of its natural precursor. This probe was poorly transported by Pgp, MRP1, MRP2 and BCRP transporters, compared with classical MDR substrates. Moreover, Pgp did not confer cross-resistance to the most potent dihydro-β-agarofurans, which did not affect Pgp expression levels in several MDR cell lines. Finally, we observed competitive and non-competitive interactions between one of such dihydro-β-agarofurans (Mama12) and classical Pgp modulators such as cyclosporin A, verapamil, progesterone, vinblastine and GF120918. These findings suggest that multidrug ABC transporters do not confer resistance to dihydro-β-agarofurans and could not affect their absorption and biodistribution in the body. Moreover, we mapped their binding site(s) within Pgp, which may prove useful for the rational design of improved modulators based on the structure of dihydro-β-agarofurans.     

Co-translational effects of temperature on membrane insertion and orientation of P-glycoprotein sequences

P-glycoprotein (pgp) is a membrane transport protein that causes multidrug resistance (MDR) by actively extruding a wide variety of cytotoxic agents out of cells. It may also function as a peptide transporter, a volume-regulated chloride channel, and an ATP channel. Previously, it has been shown that hamster pgp1 Pgp is expressed in more than one topological form and that the generation of these structures is modulated by charged amino acids flanking the predicted transmembrane (TM) segments 3 and 4 and by soluble cytoplasmic factors. Different topological structures of Pgp may be related to its different functions. In this study, we examined the effects of translation temperature on the membrane insertion process and the topologies of Pgp. Using the rabbit reticulocyte lysate expression system, we showed that translation at different temperatures affects the membrane insertion and orientation of the putative TM3 and TM4 of hamster pgp1 Pgp in a co-translational manner. This observation suggests that the membrane insertion process of TM3 and TM4 of Pgp molecules may involve a protein conducting channel and/or the interaction between TM3 and TM4, which act in a temperature sensitive manner. We speculate that manipulating temperature may provide a way to understand the structure-function relationship of Pgp and help overcome Pgp-related multidrug resistance of cancer cells.Abbreviations Pgp P-glycoprotein - MDR multidrug resistance - ABC ATP-binding cassette - RRL rabbit reticulocyte lysate - TM transmembrane - RM rough microsomes - ER endoplasmic reticulum     

Drug-stimulated ATPase activity of the human P-glycoprotein

The human multidrug resistance protein, or P-glycoprotein (Pgp), exhibits a high-capacity drug-dependent ATP hydrolytic activity that is a direct reflection of its drug transport capability. This activity is readily measured in membranes isolated from cultured insect cells infected with a baculovirus carrying the humanmdrl cDNA. The drug-stimulated ATPase activity is a useful alternative to conventional screening systems for identifying high-affinity drug substrates of the Pgp with potential clinical value as chemosensitizers for tumor cells that have become drug resistant. Using this assay system, a variety of drugs have been directly shown to interact with the Pgp. Many of the drugs stimulate the Pgp ATPase activity, but certain drugs bind tightly to the drug-binding site of the Pgp without eliciting ATP hydrolysis. Either class of drugs may be useful as chemosensitizing agents. The baculovirus/insect cell Pgp ATPase assay system may also facilitate future studies of the molecular structure and mechanism of the Pgp.     

Shedding light on drug transport: structure and function of the P-glycoprotein multidrug transporter (ABCB1).

P-glycoprotein (Pgp; ABCB1), a member of the ATP-binding cassette (ABC) superfamily, exports structurally diverse hydrophobic compounds from the cell, driven by ATP hydrolysis. Pgp expression has been linked to the efflux of chemotherapeutic drugs in human cancers, leading to multidrug resistance (MDR). The protein also plays an important physiological role in limiting drug uptake in the gut and entry into the brain. Substrates partition into the lipid bilayer before interacting with Pgp, which has been proposed to function as a hydrophobic vacuum cleaner. Low- and medium-resolution structural models of Pgp suggest that the 2 nucleotide-binding domains are closely associated to form a nucleotide sandwich dimer. Pgp is an outwardly directed flippase for fluorescent phospholipid and glycosphingolipid derivatives, which suggests that it may also translocate drug molecules from the inner to the outer membrane leaflet. The ATPase catalytic cycle of the protein is thought to proceed via an alternating site mechanism, although the details are not understood. The lipid bilayer plays an important role in Pgp function, and may regulate both the binding and transport of drugs. This review focuses on the structure and function of Pgp, and highlights the importance of fluorescence spectroscopic techniques in exploring the molecular details of this enigmatic transporter.     

In archaebacteria,there is a doxorubicin efflux pump similar to mammalian P-glycoprotein

We selected for study an anthracycline-resistant mutant from the archaebacteria Haloferax volcanii. This resistance was reversed by a Ca²⁺-channel antagonist, nifedipine (NDP). This resistance and its reversal by NDP suggest P-glycoprotein (Pgp) to be responsible for maintaining an anticancer drug concentration below the cytotoxic level. Using rhodamine 123 (RH123) as a substrate for Pgp, we then examined whether the resistance to anthracyclines in this bacteria might involve a Pgp-like anthracycline efflux pump. RH123 accumulation by the bacteria was determined with flow cytometry. A steady-state RH123 accumulation by the resistant cells revealed approx. one-fifteenth of that by the wild-type cells, which could be remarkably enhanced by NDP. The other modulators of Pgp, diltiazem and verapamil, also enhanced RH123 accumulation in resistant cells. The uncoupler FCCP completely restored RH123 accumulation in resistant cells to the wild-type cell level. RH123 unidirectional efflux from resistant cells after its preloading revealed much greater than that from wild-type cells, which was remarkably inhibited by FCCP. These confirmed that RH123 low accumulation involves its active efflux mechanism. Taken together, the present study indicated that lower evolutionary archaebacteria might also express a Pgp-like protein very similar to mammalian Pgp.     

Structure of the Chlamydia trachomatis Immunodominant Antigen Pgp3

Chlamydia trachomatis infection is the most common sexually transmitted bacterial disease. Left untreated, it can lead to ectopic pregnancy, pelvic inflammatory disease, and infertility. Here we present the structure of the secreted C. trachomatis protein Pgp3, an immunodominant antigen and putative virulence factor. The ∼84-kDa Pgp3 homotrimer, encoded on a cryptic plasmid, consists of globular N- and C-terminal assemblies connected by a triple-helical coiled-coil. The C-terminal domains possess folds similar to members of the TNF family of cytokines. The closest Pgp3 C-terminal domain structural homologs include a lectin from Burkholderia cenocepacia, the C1q component of complement, and a portion of the Bacillus anthracis spore surface protein BclA, all of which play roles in bioadhesion. The N-terminal domain consists of a concatenation of structural motifs typically found in trimeric viral proteins. The central parallel triple-helical coiled-coil contains an unusual alternating pattern of apolar and polar residue pairs that generate a rare right-handed superhelical twist. The unique architecture of Pgp3 provides the basis for understanding its role in chlamydial pathogenesis and serves as the platform for its optimization as a potential vaccine antigen candidate.     

The influence of proteasome inhibitor bortezamib on ABC transporters’ expression and activity in tumor cells

The goal of this study was to investigate the role of the ABC transporters in the evolution of tumor cell populations treated with bortezomib. Bortezomib (PS-341, Velcade) is a proteasome inhibitor used for treatment of some malignancies. Several pairs of cell lines different in Pgp expression (P-glycoprotein transporter, ABCB1) have been used in the study. We showed that the influence of the Pgp hyperexpression on cell sensitivity to bortezomib was bidirectional and depended on the tissue type. Bortezomib changed the mRNA level of MDR1 (ABCB1 and MRP1 (ABCC1)) genes, suggesting that the proteasome inhibitor is able to decrease the activity of some regulators of genes/proteins implicated in MDR. Bortezomib treatment increased the levels of proteins (Pgp or MPR1) in 3 out of 4 cell populations studied. Pgp was shown to remain functionally active in the cells cultured in bortezamib-containing medium. The UIC2-shift assay has shown that bortezomib is able to activate Pgp. This means that bortezomib influences Pgp conformation, thus activating the protein (in K562/i-S9 cells). These experiments also demonstrate that bortezomib is Pgp substrate.     

Analysis of MDR1 P-glycoprotein conformational changes in permeabilized cells using differential immunoreactivity

The reactivity of the ATP-dependent multidrug transporter P-glycoprotein (Pgp) with the conformation-sensitive monoclonal antibody UIC2 is increased in the presence of Pgp transport substrates, ATP-depleting agents, or mutations that reduce the level of nucleotide binding by Pgp. We have investigated the effects of nucleotides and vinblastine, a Pgp transport substrate, on the UIC2 reactivity of Pgp in cells permeabilized by Staphylococcus aureus alpha-toxin. ATP, ADP, and nonhydrolyzable ATP analogues decreased the UIC2 reactivity; this effect was potentiated by vanadate, a nucleotide-trapping agent. The Hill number for the nucleotide-induced conformational transition was 2 for ATP and ADP but 1 for nonhydrolyzable ATP analogues. The Hill numbers for ATP and ADP were decreased to 1 by mutations in one of the two nucleotide binding sites of Pgp, whereas mutation of both sites greatly diminished the overall effect of nucleotides. Vinblastine reversed the decrease in the UIC2 reactivity brought about by all the nucleotides, including nonhydrolyzable analogues; this effect of vinblastine was blocked by vanadate. These data indicate that UIC2-detectable conformational changes of Pgp are driven by binding and debinding of nucleotides, that nucleotide hydrolysis affects the Hill number for its Pgp interactions, and that Pgp transport substrates promote nucleotide dissociation from Pgp. These findings are consistent with a conventional E1/E2 model that explains conformational transitions of a transporter protein through a series of linked equilibria.     

Multidrug Resistance-Related Transport Proteins in Isolated Human Brain Microvessels and in Cells Cultured from These Isolates

Abstract: The multidrug transporter, P-glycoprotein (Pgp), at the blood-brain barrier is thought to be important for limiting access of toxic agents to the brain, but controversy surrounds its cellular location, whether on endothelium or on adjacent astrocyte foot processes. In the present study, the distribution of protein and mRNA for Pgp and for another transporter, multidrug resistance-associated protein (MRP), is compared with that for the endothelial marker, platelet-endothelial cell adhesion molecule-1 (PECAM-1) and for the astrocyte-derived glial fibrillary acidic protein (GFAP) in microvessels isolated from human brain and in cells grown from these microvessels. Activities of the multidrug transporters are assessed in the cultured cells from the effects of transport inhibitors on intracellular [³H]vincristine accumulation. The isolated microvessels show strong immunocytochemical staining for Pgp and PECAM-1 and little or no staining for GFAP and MRP, and they contain mRNAs detectable by RT-PCR encoding only Pgp and PECAM-1, but not GFAP or MRP. Thus, Pgp may well be synthesised and expressed on cells within the microvessels rather than on adherent astrocyte foot processes. In cells grown from the microvessels, although PECAM-1 remains, Pgp expression decreases and MRP appears. Evidence suggests these multidrug transporters are functionally active in the cultured cells.     

Synthesis and evaluation of Strychnos alkaloids as MDR reversal agents for cancer cell eradication

Natural products represent the fourth generation of multidrug resistance (MDR) reversal agents that resensitize MDR cancer cells overexpressing P-glycoprotein (Pgp) to cytotoxic agents. We have developed an effective synthetic route to prepare various Strychnos alkaloids and their derivatives. Molecular modeling of these alkaloids docked to a homology model of Pgp was employed to optimize ligand–protein interactions and design analogues with increased affinity to Pgp. Moreover, the compounds were evaluated for their (1) binding affinity to Pgp by fluorescence quenching, and (2) MDR reversal activity using a panel of in vitro and cell-based assays and compared to verapamil, a known inhibitor of Pgp activity. Compound 7 revealed the highest affinity to Pgp of all Strychnos congeners (K_d = 4.4 μM), the strongest inhibition of Pgp ATPase activity, and the strongest MDR reversal effect in two Pgp-expressing cell lines. Altogether, our findings suggest the clinical potential of these synthesized compounds as viable Pgp modulators justifies further investigation.     

Can long range mechanical interaction between drugs and membrane proteins define the notion of molecular promiscuity? Application to P-glycoprotein-mediated multidrug resistance (MDR)

Background

Failure of treatment in over 90% of patients with metastatic cancer is due to acquired MDR. P-glycoprotein (Pgp) remains the archetypal drug membrane transporter expressed in many MDR cancer cells. Albeit the ATPase activity of Pgp is triggered by the presence of drug in the membrane, it is commonly assumed that when two drug molecules meet the same Pgp the protein cannot handle them efficiently due to steric effects and as a result the ATPase activity drops. However it is also possible that drug accumulating in the lipid-phase may affect the membrane in such a way that it imposes the mechanical closure of transporters by opposing the force mediated by ATP consumption. In this context, long range interactions between drug and membrane proteins could exist.

Methods

Recent data concerning Pgp structure have allowed us to formalize this hypothesis and we present a physico-mathematical model that is not based on predictive QSAR or other empirical methods applied to experimental data.

Results

Long range mechanical interactions between Pgp and drugs are predicted to occur at an external concentration of drug ~ 10–100 μM as previously determined experimentally at which concentration ~ 50% of transporters should be rendered inactive.

Conclusion

Distance interaction(s) between Pgp and drugs exist explaining an ill-defined effect concerning the ability of any drug to inhibit Pgp once a threshold concentration in the membrane has been reached.

General significance

Potential application of the theory in the field of pharmacology concentrating on the notion of molecular promiscuity and toxicity in drug discovery prediction is discussed.     

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.     

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.     

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.     

Endothelin-1 Reduces P-Glycoprotein Transport Activity in an In Vitro Model of Human Adult Blood–brain Barrier

Aims It is a huge challenge to understand the blood–brain barrier (BBB), which is a key element in neuroinflammation associated with many brain diseases. The BBB also regulates the passage of xenobiotics into the central nervous system (CNS), and therefore influences drug efficacy. This may be due to the presence of ATP binding cassette transporters such as P-glycoprotein (Pgp) on the BBB, which are efflux pumps known to transport many drugs. The peptide endothelin 1 (ET-1) is involved in different kinds of CNS diseases and neuroinflammation, and is known to modulate Pgp transport activity. Although there are data from animal models, data from human models are scarce. We evaluated Pgp expression and transport activity in adult human brain microvascular endothelial cells (HBMECs) when exposing an adult human in vitro BBB model to ET-1. Methods Adult HBMECs were cocultured with human adult glial cells on a Transwells^R to mimic blood and CNS compartments. These human in vitro BBBs were exposed for 24 h to 100 nM and 10 nM ET-1. Pgp expression was assessed by flow cytometry and its transport activity by measuring radiolabelled digoxin passage. Results After exposure to ET-1, flow cytometry showed no shift of fluorescence intensity for a Pgp specific antibody. The passage of digoxin increased with a significant decrease of Q ratio for 10 nM ET-1. Conclusion Our results show that ET-1 has no effect on Pgp expression of adult HBMECs, but does modulate Pgp transport activity.     

Effects of grapefruit juice on the multidrug transporter P-glycoprotein in the human proximal tubular cell line HK-2

The multidrug transporter MDR-1 P-glycoprotein (Pgp) has been recently pointed out as an important mechanism underlying chemical interaction between drugs and many commonly ingested substances, including grapefruit juice (GFJ). Modulation of intestinal Pgp dependent transport by GFJ may lead to changes in bioavailability of drugs that are substrates of Pgp itself, by affecting their presystemic clearance. Since other cellular sites expressing Pgp and devoted to drug disposition, like kidney proximal tubules, could be involved in these pharmacokinetic interactions, we investigated the effect of GFJ on the expression and activity of Pgp in the human immortalized tubular cell line HK-2. Two flavonoid compounds related to GFJ, kaempferol and naringenin, were also tested for their effects on HK-2 Pgp. HK-2 cells cultured for 4 days in the presence of GFJ, showed a dose-dependent decrease in Pgp immunoblottable amount as well as a decrease in MDR-1 mRNA level, as shown by western blot analysis and RT-PCR, respectively. Both kaempferol and naringenin were also able to significantly decrease Pgp immunoblottable amount. To test whether the downregulation of HK-2 Pgp due to GFJ exposition could influence the cell sensitivity to drugs that are transported by Pgp itself, HK-2 cells precultured with GFJ were exposed to scalar concentrations of Cyclosporin A or Vinblastine and cell viability examined 36 hours later. The cytotoxicity of both drugs was increased. The calcein-AM test in untreated cells showed that GFJ, kaempferol or naringenin inhibited Pgp activity. Downregulation of Pgp as well inhibition of its function by GFJ or its related components in tubular cells could have a role in changing disposition kinetics of some important therapeutic agents.