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The molecular interaction of a copper chelate with human P-glycoprotein

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The molecular interaction of a copper chelate with human P-glycoprotein
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  The molecular interaction of a copper chelatewith human P-glycoprotein Ruma Dey Ghosh  • Paramita Chakraborty  • Kaushik Banerjee  • Arghya Adhikary  • Avijit Sarkar  • Mitali Chatterjee  • Tanya Das  • Soumitra Kumar Choudhuri Received: 23 October 2011/Accepted: 4 January 2012/Published online: 19 January 2012   Springer Science+Business Media, LLC. 2012 Abstract  One of the major reasons for multidrug resis-tance (MDR) in cancer is the overexpression of P-glyco-protein (P-gp, ABCB1), a drug efflux pump. A novelcopper complex, namely, copper (II) N-(2-hydroxyaceto-phenone) glycinate (CuNG) previously synthesized andcharacterized by the authors had been tested in this study.In a cell-based assay system with human MDR1 cDNAoverexpressed mouse fibroblast NIH MDR1-G185 cell line,we demonstrated that this metal complex can directlyinteract with this transporter. As CuNG increased cellularaccumulation of doxorubicin in P-gp-expressing cells, wepresumed that of CuNG may be potential to reverse P-gp-mediated drug resistance probably by lowering the P-gpexpression at the protein as well as mRNA level. Inter-estingly, our studies on UIC2 (a conformation sensitivemonoclonal antibody) binding assay indicated the directinteraction of CuNG with P-gp. However, CuNG did notcompete for the substrate binding as photoaffinity labelingof P-gp with a substrate analog [ 125 I] iodoarylazidoprazo-sin ([ 125 I] IAAP) showed approximately twofold increasein [ 125 I] IAAP binding in presence of CuNG. In vitro studyshowed that CuNG significantly stimulated P-gp-specificATPase activity in isolated membrane preparations fromNIH MDR1-G185 cells. This result further confirmed theCuNG–P-gp direct interaction. This study also demon-strated that CuNG has a drug interaction site different fromverapamil-, vinblastine- and progesterone-binding sites onP-gp. Taken together, this is the first report of molecularinteraction of any Schiff’s base metal chelate CuNG withhuman P-gp. This information may be useful to designmore efficacious nontoxic metal-based drugs as MDR-reversing agents. Keywords  Multidrug resistance    P-glycoprotein   Copper(II)N-(2-hydroxyacetophenone)glycinate(CuNG)  UIC2 antibody    [ 125 I] IAAP labeling    NIH MDR1-G185cells Abbreviations MDR Multidrug resistanceP-gp P-glycoproteinCuNG Copper(II)N-(2-hydroxyacetophenone)glycinateABC-transporter ATP-binding cassette transporterRT-PCR Reverse transcription-polymerase chainreaction[ 125 I] IAAP [ 125 I] iodoarylazidoprazosin Introduction Human P-glycoprotein (P-gp, ABCB1), the product of   MDR1  gene, belongs to ATP-binding cassette (ABC)transporter family that includes several clinically importantproteins such as cystic fibrosis transmembrane conductanceregulator (CFTR), sulfonylurea receptor (SUR) involved inmany genetic diseases [1, 2]. Physiologically, P-gp is also R. D. Ghosh    P. Chakraborty    K. Banerjee   S. K. Choudhuri ( & )Department of In Vitro Carcinogenesis and CellularChemotherapy, Chittaranjan National Cancer Institute,37, S.P. Mukherjee Road, Kolkata 700 026, Indiae-mail: soumitra01@yahoo.comA. Adhikary    T. DasDepartment of Molecular Medicine,Bose Institute, Kolkata, IndiaA. Sarkar    M. ChatterjeeDepartment of Pharmacology, Institute of Post GraduateMedical Education and Research, Kolkata, India  1 3 Mol Cell Biochem (2012) 364:309–320DOI 10.1007/s11010-012-1232-z  involved in absorption, distribution, and excretion of a largenumber of hydrophobic xenobiotics [1]. Cellular functionalexpression of human P-gp confers resistance to a broadvarietyofstructurallyunrelatedchemotherapeuticdrugsandrestricts their bioavailability in cancer cells [3, 4]. Recently, a number of chemical modulators of P-gptransport are known that are also called resistant-modifyingagents (RMA). These compounds inhibit the P-gp and cansensitize certain multidrug resistant (MDR) cancer cells[1]. The first-generation MDR modulators such as verap-amil and cyclosporine A have limited clinical use mainlybecause of their severe side effects and toxicity to hosts.Subsequently, the second- and third-generation modulatorssuch as R-verapamil, PSC833 (a cyclosporine analog),GF120918, XR9576, and LY335979 have been developedand studied for their clinical efficacy [5–9]. Cancer phar- macologists have been working for decades to developreversing agents with the explicit goal of inhibiting drugtransport by P-gp.The medicinal value of copper is well known, and var-ious copper compounds are used in treating differenthuman diseases [10]. A Schiff’s base copper chelate wasundertaken in this study. Copper compounds are nowattracting much attention for investigation and treatment of various neurodegenerative disorders and several types of cancers as anti-angiogenic molecules, proteasome inhibi-tors, and apoptosis inducers particularly in conjunctionwith chemotherapeutic agents in radiotherapy [10]. Copper  N  -(2-hydroxyacetophenone) glycinate (CuNG) was previ-ously synthesized and characterized by us and tested for itstoxicity [11]. CuNG generates ROS and depletes glutathi-one (GSH) and glutathione S-transferase (GST) in drug-resistant cell (EAC/Dox) bearing mice [11, 12]. Multiple roles of CuNG in various cellular pathways have beenreported [13–15]. Precise knowledge of the inhibitor interaction site(s) andits underlying mechanisms is required for the developmentof nontoxic and potent P-gp modulators. However, thestructural diversity among the RMAs presents a formidablechallenge in defining their modes of action as well as theirsites of interaction with P-gp. Although the three-dimen-sional structure of mouse P-gp has been resolved veryrecently [16], the structure of human P-gp (product of   MDR1  gene) is yet to be resolved. This information will beuseful in understanding the mode of its poly-specific drugbinding, which is important for the rational design of anticancer drugs and specific MDR inhibitors. Most of thereported studies have investigated the effect of the modu-lators on substrate binding in isolated membranes thatexclusively relied on the kinetic parameters of substrateinteraction with P-gp. At present, little is known about theP-gp-modulator interaction and mechanism of inhibition of its drug transport function.The purpose of this work is to characterize the molec-ular interaction of this copper compound, CuNG, with P-gpin a cell-based assay system. To this end, we made anattempt to find out the CuNG–P-gp interaction site bycomparing with three reference substrates, namely, vin-blastine, verapamil, and progesterone and subsequently wealso studied the photocross-linking of prazosine derivativephotoaffinity analog [ 125 I]IAAP. This study possibly pro-vides the first direct evidence of P-gp and a Schiff’s basemetal complex interaction in modulation of P-gp-mediateddrug transport. Our study may be helpful to develop anattractive candidate for chemo-sensitization of MDR can-cer cells. Materials and methods ChemicalsDoxorubicin, vinblastine, colchicine, ouabain, and MgATPwere purchased from Sigma (USA). Cyclosporin A wassupplied by Calbiochem (USA). Mouse monoclonal P-gpantibody C219 and UIC2 were purchased from Calbiochem(USA) and Santa Cruz Biotechnology (USA), respectively.Anti- b -actin, HRP-conjugated anti-mouse IgG, and FITC-conjugated anti-mouse IgG were purchased from Sigma(USA), and anti-rabbit IgG conjugated with horseradishperoxidase was obtained from Promega (USA). RNAqu-eous-4PCR kit for mRNA isolation and RETROscript RT-PCR kit were purchased from Ambion (USA). The cellculture media was purchased from Gibco (Invtrogen,USA). [ 125 I]IAAP (2,200 Ci/mmol) was supplied by PerkinElmer Life Sciences (USA). Polyclonal antibody B4007was a kind gift from Dr. Michael Gottesman (NationalInstitute of Health, Bethesda, MD, USA).Synthesis of CuNGCopper  N  -(2 Hydroxy acetophenone) glycinate was syn-thesized by the reaction of potassium  N  -(2-hydroxyaceto-phenone) glycinate with hydrated copper sulfate accordingto the previously described method; characterization andstructure determination were done by detailed spectro-scopic studies [11, 17]. The structure of CuNG (MW 308) is shown in Fig 1. As CuNG is insoluble in water, DMSOstock solution was prepared following serial dilution withgradual addition of water or media.Cell lines and culture conditionsMouse cell line NIH 3T3 fibroblast (drug-sensitive cellline) established from NIH Swiss mouse embryo cultures 310 Mol Cell Biochem (2012) 364:309–320  1 3  and the human P-gp (containing human  MDR1  cDNA)expressing NIH MDR1-G185 fibroblast (drug-resistant cellline; subsequently represented as NIH MDR1) having wild-type glycine at position 185 [18] were used for this study.Both the cell lines were kindly provided by Dr. Michael M.Gottesman (National Cancer Institute, Bethesda, MD) andwere maintained in DMEM containing 10% fetal bovineserum and antibiotics (penicillin and streptomycin) at 37  Cin 5% CO 2 . A mass of 60 ng/ml colchicine was added onlyto the medium of P-gp-expressing resistant NIH MDR1 cellline for further selection.Drug accumulation studyA FACSort flow cytometer equipped with Cell Questsoftware (Becton–Dickinson) was used for FACS analysis.The fluorescent property of doxorubicin, a good substratefor P-gp, was used for drug accumulation assay [19].Briefly, the drug-resistant NIH MDR1 cells and its parentalNIH 3T3 cells were harvested after trypsinization by cen-trifugation at 500 9 g , resuspended (1  9  10 6 cells) in pre-warmed DMEM with 10% FBS in culture tubes in presenceor absence of different concentrations of CuNG, andincubate for 15 min with 5% CO 2  at 37  C. Doxorubicin(10  l M) was added to each tube, gently mixed, and incu-bated at 37  C for 1 h. Then, the cells were pelleted bycentrifugation at 500 9 g  and the cell pellete was resus-pended in 500  l l of PBS containing 0.01% FBS andimmediately analyzed by using flow cytometer for intra-cellular accumulation of doxorubicin. A total of 10,000cells were counted. Mean fluorescence was recorded fromthe histogram and the data are expressed in the text asmean fluorescence channel numbers.NIH 3T3 and NIH MDR1 cells (0.5  9  10 6 cells) wereplatted on cover slips and cultured with 2 ml of completegrowth media and left overnight. Fresh media was added toeach cover slip 15 min before initiating experiments inpresence or absence of CuNG and incubated with doxo-rubicin (10  l M) as described for FACS analysis. The cellswere fixed with 4% paraformaldehyde, washed with PBScontaining 0.1% FBS, mounted with glycerol, and analyzedfor intracellular accumulation of doxorubicin by the help of confocal microscopy.Determination of MDR1 mRNA and P-gp expressionRT-PCR was performed to evaluate the mRNA expressionlevel of P-gp after CuNG treatment. Total RNA was iso-lated by using RNAqueous 4PCR kit (Ambion) accordingto manufacture’s protocol, and the quality of RNA waschecked by optical density measurement with A 260  / A 280 [ 1.8. One microgram total RNA was subjected to aRT reaction using random oligonucleotide primers andM-MLV reverse transcriptase (RETROscript kit, Ambion).RT reaction product was then amplified by PCR using TaqDNA polymerase under the following conditions: 30 cyclesof 94  C for 2 min, 60  C for 1 min, and 72  C for 1 min[20]. The PCR primers were 5 0 -GCCTGGCAGCTGGAAGACAAATACACAAAATT-3 0 and 5 0 -CAGACAGCAGCTGACAGTCCAAGAACAGGACT-3 0 for P-gp (285 bp);5 0 -GATGATATCGCCGCGCTCGTCGTCGAC-3 0 and 5 0 -AGCCAGGTCCAGACGCAGGATGGCATG-3 0 for  b -actin(538 bp). The PCR products were run on a 1% agarose geland visualized by ethidium bromide staining.The level of P-gp expression was analyzed by UIC2monoclonal anti-P-gp antibody (Santa Cruz). Briefly, NIH3T3 and NIH MDR1 cells (0.5  9  10 6 cells) were plattedon cover slips for confocal microscopy and culturedovernight. CuNG treatment was given and incubated for24, 48, and 72 h followed by fixing of the cells on coverslips with 4% paraformaldehyde. The cells were incubatedwith the monoclonal antibody UIC2 overnight. Followingincubation, the cells were washed with PBS containing0.1% FBS and re-incubated with FITC-conjugate second-ary antibody (anti-mouse FITC-conjugate) for another 1 h.Cells were then washed and mounted with mounting mediaand analyzed in confocal microscope for assessing the levelof P-gp expression. The control group was treated withCuNG for 15 min before the UIC2 incubation. The fluo-rescence intensity associated with cells was measured.Preparation of crude membranesCrude membrane from mouse fibroblasts NIH 3T3 andNIH MDR1 cells was prepared following the method of Dey et al .  [21]. In brief, cells (from 10  9  75 cm 2 flasks)were harvested and washed in ice-cold PBS containing 1%aprotinin, resuspended in hypotonic lysis buffer (50-mMTris–HCl [pH 7.5], 50-mM mannitol, 2-mM EGTA [eth-ylene glycol-bis(2-aminoethylether)-  N  ,  N  ,  N  0 ,  N  0 -tetraaceticacid], 2-mM DTT [dithiothreitol], 1-mM AEBSF [4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride], and1% aprotinin), and frozen at  - 80  C. Frozen cells werethawed and incubated on ice for 45 min and subsequentlydisrupted with a Dounce homogenizer. The undisruptedcells and nuclear debris were removed by centrifugation at500 9 g  for 10 min. The low-speed supernatant was diluted Fig. 1  Chemical structure of CuNGMol Cell Biochem (2012) 364:309–320 311  1 3  in 16-ml resuspension buffer containing 50-mM Tris–HCl(pH 7.5), 300-mM mannitol, 1-mM EGTA, 1-mM DTT,1-mM AEBSF, and 1% aprotinin. The membranes werecollected by centrifugation for 60 min at 100,000  g  andresuspended by passing through a bent hypodermic needle(gauge size 19 and then 23) in resuspension buffer con-taining 10% glycerol and stored in small aliquots at - 80  C. The protein content was determined by Bradfordmethod, using BSA as standard [22].Western blot analysisFollowing CuNG treatment for different time points, NIHMDR1 cells were harvested in ice-cold lysis buffer (50-mM Tris–HCl [pH 7.5], 50-mM mannitol, 2-mM EGTA,2-mM DTT, 1-mM AEBSF, and 1% aprotinin), membraneswere isolated by the method described in previous section,and stored in  - 80  C. Protein concentrations were deter-mined using Bradford method. A mass of 100- l g proteinwas loaded for each sample and separated by SDS-PAGE.Protein bands were transferred to PVDF (Millipore)membrane, blocked for 1 h at room temperature with 5%BSA, and then incubated with monoclonal mouse anti-P-gpantibody C219 (Calbiochem) at 4  C for overnight. Afterwashing in PBS containing 0.05% Tween-20, the mem-brane was incubated with HRP-conjugated secondaryantibody (Sigma), and protein bands were visualized usingchemiluminescence of Lumi Glow (Cell Signaling Tech-nology) and analyzed by using Bio-Rad Quantity1 soft-ware.  b -Actin was used as loading control.In case of photoaffinity-labeling experiment, electro-phoresis and immunoblot analyses were performed asdescribed above except human P-gp-specific polyclonalantibody 4007 was used and the protein band was detectedby chemiluminescence reaction (ECL Western blottingsystem) kit from Thermo scientific and analyzed by com-puter program ImageJ software.UIC2 reactivity shift assayThe UIC2 shift assay was performed as described [23] withminor modifications. In this work, the Schiff’s base metalchelate, CuNG, was also studied to verify specific con-formational change in P-gp. In brief, cells grown in mon-olayers were harvested by trypsinization, washed, andresuspended in 100  l l of prewarmed DMEM supplementedwith 10% FBS. A total of 10 6 cells were taken in each tubeand incubated for 15 min with occasional shaking with1-mM sodium orthovanadate or 5- l M vinblastine and25- l M CuNG at 37  C. Following incubation with drugs,the monoclonal antibody UIC2 was added and incubatedfor 30 min at 37  C. Then, 1 ml of ice-cold PBS with 1%FBS was added to stop the reaction, and washed,resuspended, and further incubated with FITC-conjugatedanti-mouse secondary antibody. Cells were washed andresuspended in 400  l l of ice-cold PBS and analyzed byfluorescence-assisted cell sorter (FACS). The fluorescenceintensity associated with cells was expressed on log scale.Measurement of ATPase activityP-gp associated ATPase activity was measured by deter-mining the level of sodium orthovanadate-sensitive releaseof inorganic phosphate from MgATP by spectrophoto-metric method [24]. In brief, membrane suspensions (15  l gof protein) were preincubated at 37  C for 5 min in areaction mixture containing 50-mM Tris–HCl (pH 7.5),5-mM sodium azide, 2-mM EGTA, 2-mM ouabain, 2-mMDTT, 50-mM KCl, and 10-mM MgCl 2 . Drugs were addedto the assay buffer and incubated for 3 min at 37  C. Fordrugs that were not water soluble, stock solutions weremade in dimethyl sulfoxide (DMSO) in such a way that thefinal concentration of DMSO in the assay never exceeded1%. This concentration of DMSO did not exhibit any effecton the P-gp ATPase activity. In the case of sequentialaddition of two drugs, the first drugs was added and pre-incubated for 5 min before the addition of second one. Theassay was initiated by the addition of 5-mM ATP (pH 7.0)to a total volume 100  l l and incubated at 37  C for 20 minas the rate of ATP hydrolysis remains linear up to 60 min[21]. Reactions were stopped by addition of 100  l l of 5%SDS, and the amount of inorganic phosphate released wasmeasured by a spectrophotometer [21]. The rates of ATPhydrolysis were expressed as nanomoles of ATP hydro-lyzed per mg of total membrane protein per minute (nmol/ mg/min). The vanadate-sensitive activities were calculatedas the differences between the ATPase activities obtainedin the absence and presence of 300- l M sodium orthovan-adate. The data of P-gp ATPase activity were subjected tononlinear regression analysis (GraphPad software). P-gpATPase activities were measured in one batch of mem-brane vesicle preparation in duplicates or triplicates.[ 125 I] IAAP photocross-linking of P-gp in intact cells[ 125 I] IAAP photocross-linking to intact cell was performedfollowing Maki et al. [23]. In brief, 0.5  9  10 6 cells/wellwere grown in 24-well culture plates. Cells were washedonce with 1 ml/well DMEM containing 10% FBS andincubated at 37  C for 60 min with 0.3 ml of IMEM with10% FBS containing 1.5-nM [ 125 I] IAAP either in presenceor absence of 5- l M cyclosporin A ( CsA ), 5- l M  cis -(Z)-flupentixol ( Cis -(  Z  )-flup), or various dilutions of CuNG(5, 25, 50, and 100  l M) for 1 h at 37  C. Following incuba-tion, culture medium was taken out, cells were washed withice-cold PBS, and exposed to UV light (SPECTROLINE, 312 Mol Cell Biochem (2012) 364:309–320  1 3  model XX-15A, 365 nm) for 5 min at room temperature.After photocross-linking, cells were resuspended in 100  l l/ well (0.5 9 10 6 cells/100  l l) of cell lysis buffer containing10-mM Tris, pH 8.0, 0.1% (v/v) Triton X-100, 10-mMMgSO 4 , 2-mM CaCl 2 , 1-mM dithiothreitol, 2-mM 4-(2-aminoethyl) benzenesulfonyl fluoride, and 50 units/mlmicrococcal nuclease ( Staphylococcus aureus ). Resus-pended cells were lysed by three cycles of freezing (on dryice) and thawing (in cold water), and resolved by SDS-PAGE. The gels were dried and exposed to an X-ray film orto a Phosphor-Imager screen.[ 125 I] IAAP photoaffinity labeling of P-gpin isolated membranesPhotoaffinity labeling of crude membrane was performedfollowing Maki et al. [23] in presence of 5-nM [ 125 I] IAAP.Quantification of radioactivity in associated with P-gpTo determine the amount of [ 125 I] IAAP photocross-linkedto P-gp, the radioactivity associated with each band cor-responds to P-gp was quantified from the dried gels byexposing to a Phosphor-Imager screen and analyzed using aSTORM 860 Phosphor-Imaging system (Amersham Bio-sciences). Values were expressed as the fold stimulation of the basal [ 125 I] IAAP binding.Statistical analysisData are the mean  ±  SD from triplicate samples of at leastthree independent experiments. Differences between themean values were analyzed by one way analysis of vari-ance and results were considered statistically significantwhen  P \ 0.05. Results CuNG increases the doxorubicin accumulationin NIH MDR1 cellsP-gp, an ATP-dependent active transporter, prevents theaccumulation of cytotoxic drugs inside cells, and the drugtransport function of P-gp is coupled with ATP hydrolysis.To understand the effect of CuNG on P-gp functions, westudied the intracellular accumulation of doxorubicin (avery well-known fluorescent P-gp substrate) in both NIH3T3 and NIH MDR1 cells in presence or absence of vari-ous dilutions of CuNG by using flow cytometry. Generally,at 37  C, NIHMDR1 cells have a lower level of intracellulardoxorubicin accumulation after 60 min compared with thatof the NIH3T3 cells. Presumably, this is because of activeefflux of the cytotoxic compound from the cells by P-gp inNIH MDR1 cells. CuNG can restore doxorubicin accu-mulation in resistant NIHMDR1 cells in a dose-dependentmanner (Fig. 2a), whereas in NIH 3T3, doxorubicin accu-mulation is remained unchanged in presence or absence of different dilutions of CuNG. In the presence of CuNG at25  l M concentration, the steady-state level of doxorubicinin NIHMDR1 cells was observed (Fig. 2a). When NIH 3T3and NIHMDR1 cells were preincubated either in presenceor absence of 25- l M CuNG, the doxorubicin accumulationwas examined to significantly increase in presence of CuNG ( thin line  in histogram: Fig. 2b;  third panel  inphotomicrographs: Fig. 2c) compared with the levelobserved in NIH MDR1 control cells ( thick line  in histo-gram: Fig. 2b and  second panel  in photomicrographs:Fig. 2c). This observation suggests that the outward effluxof doxorubicin by P-gp is inhibited by CuNG, resulting inhigher intracellular accumulation of the anticancer drug.CuNG downregulates intrinsic P-gp expressionP-gp-mediated MDR may be overcome by modulating theexpression of P-gp. The RT-PCR analysis and western blotanalysis were done to verify if CuNG could modulate theintrinsic P-gp expression at the mRNA or protein level. Inpresence of CuNG (25  l M), NIH MDR1 cells were incu-bated for 24, 48, and 72 h, and the values of MDR1 mRNAexpression (after normalization to  b -actin expression)decreased compared with untreated control by 1, 21, and29%, respectively in three independent experiments(Fig. 3a, b). Protein bands also showed lower expressionlevels by 12, 33, and 56% as compared with vehicle control(Fig. 3c, d). The expression of P-gp is undetectable atprotein and MDR1 mRNA level in NIH 3T3 cells by themethods employed here (data not shown). The confocalmicroscopic analysis for the level of P-gp in NIH MDR1cells and NIH 3T3 cells showed that the drug-resistant NIHMDR1 cells expressed high amounts of P-gp (Fig. 3e;photomicrograph in  second panel ), whereas in NIH 3T3cells showed that P-gp was undetectable (photomicrographin  first panel ) by the method used in our experiment. Theresults suggested that CuNG had potential ability todownregulate the P-gp expression probably by regulating  MDR1  gene expression.CuNG alters UIC2 monoclonal antibody bindingto P-gpWe further tried to understand whether CuNG has anyeffect in transport function of P-gp through direct interac-tion in the regulatory site(s) of P-gp. As NIH 3T3 cells donot contain any detectable amount of P-gp, this cell linewas not used in further CuNG–P-gp interaction study. Mol Cell Biochem (2012) 364:309–320 313  1 3
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