血脑屏障上P-糖蛋白与耐药性癫痫关系的研究进展

耐药性癫痫是癫痫治疗的瓶颈。P-糖蛋白通过跨膜外排泵作用阻止抗癫痫药物由血脑屏障入脑发挥期望效应是耐药性癫痫产生的重要原因。本文介绍了耐药性癫痫与血脑屏障上P-糖蛋白的相互关系,对目前提出的调节P-糖蛋白功能,改善耐药性癫痫预后应注意的问题进行说明。安全地调节P-糖蛋白功能到适度可能为耐药性癫痫的治疗带来希望。     

P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity

We compared the P-glycoprotein ATPase activity in inside-out plasma membrane vesicles and living NIH-MDR1-G185 cells with the aim to detect substrate transport. To this purpose we used six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because their concentrations in the cytosolic leaflets were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles. Their concentration was significantly lower in the cytosolic leaflet of cells than in the cytosolic leaflet of inside-out membrane vesicles, indicating that P-glycoprotein could cope with passive influx. P-glycoprotein thus transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). However, it prevented substrate entry into the cytosol only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux.     

The nature of amino acid 482 of human ABCG2 affects substrate transport and ATP hydrolysis but not substrate binding

Several members of the ATP-binding cassette (ABC) transporter superfamily, including P-glycoprotein and the half-transporter ABCG2, can confer multidrug resistance to cancer cells in culture by functioning as ATP-dependent efflux pumps. ABCG2 variants harboring a mutation at arginine 482 have been cloned from several drug-resistant cell lines, and these variants differ in their substrate transport phenotype. In this study, we changed the wild-type arginine 482 in human ABCG2 to each one of the 19 other standard amino acids and expressed each one transiently in HeLa cells. Using the 5D3 antibody that recognizes a cell surface epitope of ABCG2, we observed that all the mutants were expressed at the cell surface. However, the mutant ABCG2 proteins differed markedly in transport activity. All of the variants were capable of transporting one or more of the substrates used in this study, with the exception of the R482K mutant, which is completely devoid of transport ability. Six of the mutants (R482G, R482H, R482K, R482P, R482T, and R482Y) and the wild-type protein (R482wt) were selected for studies of basal and stimulated ATPase activity and photoaffinity labeling with the substrate analog [125I]iodoarylazidoprazosin. Whereas these seven ABCG2 variants differed markedly in ATPase activity, all were able to specifically bind the substrate analog [125I]iodoarylazidoprazosin. These data suggest that residue 482 plays an important role in substrate transport and ATP turnover, but that the nature of this amino acid may not be important for substrate recognition and binding.     

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.     

Accumulation of Eu chelates in cells expressing or not P-glycoprotein: Implications for blood-brain barrier crossing

Alzheimer’s disease (AD) is the most commonly form of dementia in the elderly. The development of molecules able to detect biomarkers characteristic of AD is critical to its understanding and treatment. However, such molecules must be able to pass blood-brain barrier (BBB) which is a major impediment to the entry of many therapeutic drugs into the brain. Such a limitation applies to the development of magnetic resonance imaging molecular neuroimaging agents using biomarkers of AD-like β-amyloid deposits, as the common extracellular contrast agents (CAs) are not able to cross an intact BBB. In this work, we have studied the ability of a series of simple Eu³⁺ complexes to enter cells overexpressing or not the ABCB1 (P-gp or P-glycoprotein) protein, which is expressed at the BBB and in human embryonic astrocytes. The intracellular uptake of the Eu³⁺ complexes of linear and macrocyclic polyaminocarboxylate ligands with different charges and lipophilicities was followed by atomic absorption spectrometry. Based on biochemical argument, we propose that lipophilic contrast agents can be efficiently taken up by cells and accumulate inside mitochondria when they are positively charged. The important point is that they are not P-gp substrates, which is one of the major obstacles for them to cross the BBB.     

Mechanism of thioflavin T accumulation inside cells overexpressing P-glycoprotein or multidrug resistance-associated protein: role of lipophilicity and positive charge

Alzheimer's disease is characterized by the presence of amyloid deposition. Thioflavin T (ThT) has been one of the molecules of choice to attempt the detection of these amyloid deposits. However, it has been reported that ThT was unable to cross blood-brain barrier (BBB). Our aim was to understand the mechanism according to which it has been said that ThT is not able to cross the BBB. For this purpose we have used cellular models overexpressing P-glycoprotein (P-gp) or multidrug resistance-associated protein (MRP1), two proteins overexpressed in BBB. Our results show that: (i) ThT is able to cross membranes and to penetrate inside the cells; (ii) ThT is a P-gp substrate; (iii) ThT is poor MRP1 substrate. In conclusion, our results suggest that two factors could be involved in the low accumulation of ThT in the brain: ThT is a P-gp substrate and its lipophilicity is low.     

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.     

Functional expression of human P-glycoprotein in Schizosaccharomyces pombe

Human MDR1 cDNA was introduced into the human cultured cells KB-3-1 and Schizosaccharomyces pombe pmdI null mutant KN3. The drug sensitivity of KB-G2 and KN3/pgp, expressing human P-glycoprotein, was examined. KB-G2 was resistant to the peptide antibiotics valinomycin and gramicidin D as well as having a typical multidrug resistance (MDR) phenotype. KN3/pgp was resistant to valinomycin and actinomycin D, but not to adriamycin. The ATP-hydrolysis-deficient mutant did not confer KN3 resistance to these antibiotics. Human P-glycoprotein expressed in S. pombe seemed to lack N-glycosylation. The N-glycosylation-deficient mutant, however, conferred a typical MDR phenotype on KB-3-1. These results suggest that human P-glycoprotein functions as an efflux pump of valinomycin and actinomycin D in the membrane of S. pombe.     

Purification and reconstitution of functional human P-glycoprotein

The overexpression of the P-glycoprotein, theMDR1 gene product, has been linked to the development of resistance to multiple cytotoxic natural product anticancer drugs in certain cancers and cell lines derived from tumors. P-glycoprotein, a member of the ATP-binding cassette (ABC) superfamily of transporters, is believed to function as an ATP-dependent drug efflux pump with broad specificity for chemically unrelated hydrophobic compounds. We review here recent studies on the purification and reconstitution of P-glycoprotein to elucidate the mechanism of drug transport. P-glycoprotein from the human carcinoma multidrug resistant cell line, KB-V1, was purified by sequential chromatography on anion exchange followed by a lectin (wheat germ agglutinin) column. Proteoliposomes reconstituted with pure protein exhibited high levels of drug-stimulated ATPase activity as well as ATP-dependent [³H]vinblastine accumulation. Both the ATPase and vinblastine transport activities of the reconstituted P-glycoprotein were inhibited by vanadate. In addition, the vinblastine transport was inhibited by verapamil and daunorubicin. These studies provide strong evidence that the human P-glycoprotein functions as an ATP-dependent drug transporter. The development of the reconstitution system and the availability of recombinant protein in large amounts due to recent advances in overexpression of P-glycoprotein in a heterologous expression system should facilitate a better understanding of the function of this novel protein.     

Sensitivity of P-glycoprotein tryptophan residues to benzodiazepines and ATP interaction

Plasma membrane P-glycoprotein is a member of the ATP-binding cassette family of membrane transporters. In the present study tryptophan intrinsic fluorescence was used to understand the P-glycoprotein response to three benzodiazepines (bromazepam, chlordiazepoxide and flurazepam) in the presence and absence of ATP. Fluorescence emission spectra showed a red shift on the maximal emission wavelength upon interaction of P-glycoprotein with all benzodiazepines. Benzodiazepine association with nucleotide-bound P-glycoprotein also showed this trend and the quenching profile was attributed to a sphere-of-action model, for static fluorescence. Furthermore, quenching data of benzodiazepine-bound P-glycoprotein with ATP were concentration dependent and saturable, indicating that nucleotide binds to P-glycoprotein whether drug is present or not. These results seems in agreement with the proposal of the ATP-switch model by Higgins and Linton, where substrate binding to the transporters initiates the transport cycle by increasing the ATP binding affinity.     

Modulation of function of three ABC drug transporters, P-glycoprotein (ABCB1), mitoxantrone resistance protein (ABCG2) and multidrug resistance protein 1 (ABCC1) by tetrahydrocurcumin, a major metabolite of curcumin

Many studies have been performed with the aim of developing effective resistance modulators to overcome the multidrug resistance (MDR) of human cancers. Potent MDR modulators are being investigated in clinical trials. Many current studies are focused on dietary herbs due to the fact that these have been used for centuries without producing any harmful side effects. In this study, the effect of tetrahydrocurcumin (THC) on three ABC drug transporter proteins, P-glycoprotein (P-gp or ABCB1), mitoxantrone resistance protein (MXR or ABCG2) and multidrug resistance protein 1 (MRP1 or ABCC1) was investigated, to assess whether an ultimate metabolite form of curcuminoids (THC) is able to modulate MDR in cancer cells. Two different types of cell lines were used for P-gp study, human cervical carcinoma KB-3-1 (wild type) and KB-V-1 and human breast cancer MCF-7 (wild type) and MCF-7 MDR, whereas, pcDNA3.1 and pcDNA3.1-MRP1 transfected HEK 293 and MXR overexpressing MCF7AdrVp3000 or MCF7FL1000 and its parental MCF-7 were used for MRP1 and MXR study, respectively. We report here for the first time that THC is able to inhibit the function of P-gp, MXR and MRP1. The results of flow cytometry assay indicated that THC is able to inhibit the function of P-gp and thereby significantly increase the accumulation of rhodamine and calcein AM in KB-V-1 cells. The result was confirmed by the effect of THC on [³H]-vinblastine accumulation and efflux in MCF-7 and MCF-7MDR. THC significantly increased the accumulation and inhibited the efflux of [³H]-vinblastine in MCF-7 MDR in a concentration-dependent manner. This effect was not found in wild type MCF-7 cell line. The interaction of THC with the P-gp molecule was clearly indicated by ATPase assay and photoaffinity labeling of P-gp with transport substrate. THC stimulated P-gp ATPase activity and inhibited the incorporation of [¹²⁵I]-iodoarylazidoprazosin (IAAP) into P-gp in a concentration-dependent manner. The binding of [¹²⁵I]-IAAP to MXR was also inhibited by THC suggesting that THC interacted with drug binding site of the transporter. THC dose dependently inhibited the efflux of mitoxantrone and pheophorbide A from MXR expressing cells (MCF7AdrVp3000 and MCF7FL1000). Similarly with MRP1, the efflux of a fluorescent substrate calcein AM was inhibited effectively by THC thereby the accumulation of calcein was increased in MRP1-HEK 293 and not its parental pcDNA3.1-HEK 293 cells. The MDR reversing properties of THC on P-gp, MRP1, and MXR were determined by MTT assay. THC significantly increased the sensitivity of vinblastine, mitoxantrone and etoposide in drug resistance KB-V-1, MCF7AdrVp3000 and MRP1-HEK 293 cells, respectively. This effect was not found in respective drug sensitive parental cell lines. Taken together, this study clearly showed that THC inhibits the efflux function of P-gp, MXR and MRP1 and it is able to extend the MDR reversing activity of curcuminoids in vivo.     

Characterization of the system L amino acid transporter in T24 human bladder carcinoma cells

System L is a major nutrient transport system responsible for the Na⁺-independent transport of large neutral amino acids including several essential amino acids. In malignant tumors, a system L transporter L-type amino acid transporter 1 (LAT1) is up-regulated to support tumor cell growth. LAT1 is also essential for the permeation of amino acids and amino acid-related drugs through the blood-brain barrier. To search for in vitro assay systems to examine the interaction of chemical compounds with LAT1, we have investigated the expression of system L transporters and the properties of [¹⁴C]l-leucine transport in T24 human bladder carcinoma cells. Northern blot, real-time quantitative PCR and immunofluorescence analyses have reveled that T24 cells express LAT1 in the plasma membrane together with its associating protein 4F2hc, whereas T24 cells do not express the other system L isoform LAT2. The uptake of [¹⁴C]l-leucine by T24 cells is Na⁺-independent and almost completely inhibited by system L selective inhibitor BCH. The profiles of the inhibition of [¹⁴C]l-leucine uptake by amino acids and amino acid-related compounds in T24 cells are comparable with those for the LAT1 expressed in Xenopus oocytes. The majority of [¹⁴C]l-leucine uptake is, therefore, mediated by LAT1 in T24 cells. Consistent with LAT1 in Xenopus oocytes, the efflux of preloaded [¹⁴C]l-leucine is induced by extracellularly applied substrates of LAT1 in T24 cells. This efflux measurement has been proven to be more sensitive than that in Xenopus oocytes, because triiodothyronine, thyroxine and melphalan were able to induce the efflux of preloaded [¹⁴C]l-leucine in T24 cells, which was not detected for Xenopus oocyte expression system. T24 cell is, therefore, proposed to be an excellent tool to examine the interaction of chemical compounds with LAT1.     

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.     

Brain microvascular P-glycoprotein and a revised model of multidrug resistance in brain

1. P-Glycoprotein is a 170-kDa transmembrane glycoprotein active efflux system that confers multidrug resistance in tumors, as well as normal tissues including brain.2. The classical model of multidrug resistance in brain places the expression of P-glycoprotein at the luminal membrane of the brain microvascular endothelial cell. However, recent studies have been performed with human brain microvessels and double-labeling confocal microscopy using (a) the MRK16 antibody to human P-glycoprotein, (b) an antiserum to glial fibrillary acidic protein (GFAP), an astrocyte foot process marker, or (c) an antiserum to the GLUT1 glucose transporter, a brain endothelial plasma membrane marker. These results provide evidence for a revised model of P-glycoprotein function at the brain microvasculature. In human brain capillaries, there is colocalization of immunoreactive P-glycoprotein with astrocytic GFAP but not with endothelial GLUT1 glucose transporter.3. In the revised model of multidrug resistance in brain, P-glycoprotein is hypothesized to function at the plasma membrane of astrocyte foot processes. These astrocyte foot processes invest the brain microvascular endothelium but are located behind the blood–brain barrier in vivo, which is formed by the brain capillary endothelial plasma membrane.4. In the classical model, an inhibition of endothelial P-glycoprotein would result in both an increase in the blood–brain barrier permeability to a given drug substrate of P-glycoprotein and an increase in the brain volume of distribution (V _D) of the drug. However, in the revised model of P-glycoprotein function in brain, which positions this protein transporter at the astrocyte foot process, an inhibition of P-glycoprotein would result in no increase in blood–brain barrier permeability, per se, but only an increase in the V _D in brain of P-glycoprotein substrates.     

Nitensidine A,a guanidine alkaloid from Pterogyne nitens,is a novel substrate for human ABC transporter ABCB1

The Pterogyne nitens (Fabaceae) tree, native to South America, has been found to produce guanidine alkaloids as well as bioactive flavonols such as kaempferol, quercetin, and rutin. In the present study, we examined the possibility of interaction between human ATP-binding cassette (ABC) transporter ABCB1 and four guanidine alkaloids isolated from P. nitens (i.e., galegine, nitensidine A, pterogynidine, and pterogynine) using human T cell lymphoblast-like leukemia cell line CCRF-CEM and its multi-drug resistant (MDR) counterpart CEM/ADR5000. In XTT assays, CEM/ADR5000 cells were resistant to the four guanidine alkaloids compared to CCRF-CEM cells, although the four guanidine alkaloids exhibited some level of cytotoxicity against both CCRF-CEM and CEM/ADR5000 cells. In ATPase assays, three of the four guanidine alkaloids were found to stimulate the ATPase activity of ABCB1. Notably, nitensidine A was clearly found to stimulate the ATPase activity of ABCB1 as strongly as the control drug, verapamil. Furthermore, the cytotoxic effect of nitensidine A on CEM/ADR5000 cells was synergistically enhanced by verapamil. Nitensidine A inhibited the extrusion of calcein by ABCB1. In the present study, the possibility of interaction between ABCB1 and two synthetic nitensidine A analogs (nitensidine AT and AU) were examined to gain insight into the mechanism by which nitensidine A stimulates the ATPase activity of ABCB1. The ABCB1-dependent ATPase activity stimulated by nitensidine A was greatly reduced by substituting sulfur (S) or oxygen (O) for the imino nitrogen atom (N) in nitensidine A. Molecular docking studies on human ABCB1 showed that, guanidine alkaloids from P. nitens dock to the same binding pocket as verapamil. Nitensidine A and its analogs exhibit similar binding energies to verapamil. Taken together, this research clearly indicates that nitensidine A is a novel substrate for ABCB1. The present results also suggest that the number, binding site, and polymerization degree of the isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their stimulation of ABCB1's ATPase activity.     

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.     

ATP binding cassette transporter A1 - key roles in cellular lipid transport and atherosclerosis

ATP-binding cassette transporter A1 (ABCA1) was recently recognized as the mutant molecule responsible for Tangier disease with low HDL levels, accumulation of cholesteryl esters in tissues, and increased risk of cardiovascular disease. Extensive studies for the past 2 years have recognized the critical role of ABCA1 in cholesterol and phospholipid trafficking. Since the removal of cholesterol from tissues is a key step in the prevention of atherosclerosis, significant attention has been focused on this molecule. Natural ABCA1 mutations in Tangier disease (TD) patients and WHAM chickens together with induced mutation in ABCA1 knock-out mice unequivocally established the important role of ABCA1 in maintaining circulating HDL levels and promoting cholesterol efflux from the arterial wall. Mice lacking ABCA1 showed similar phenotypes observed in Tangier disease patients with low levels of HDL. Further understanding of the roles of ABCA1 in lipid transport and atherosclerosis became clear from studies with ABCA1 transgenic mice. These mice showed enhanced cholesterol efflux from macrophages and reduced atherosclerotic lesion formation. The promoter of the ABCA1 gene has been mapped to a large extent, with the exception of cAMP response element. The present review summarizes recent developments on the role of ABCA1 in cholesterol efflux and prevention of atherosclerosis. Given the antiatherogenic properties of ABCA1, this molecule can serve as an appropriate target for developing drugs to treat individuals with low levels of HDL.