Biphasic accumulation kinetics of [99mTc]-hexakis-2-methoxyisobutyl isonitrile in tumour cells and its modulation by lipophilic P-glycoprotein ligands

Biphasic accumulation kinetics of [99mTc]-hexakis-2-methoxyisobutyl isonitrile in tumour cells and its modulation by lipophilic P-glycoprotein ligands
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  European Journal of Pharmaceutical Sciences 25 (2005) 201–209 Biphasic accumulation kinetics of [ 99m Tc]-hexakis-2-methoxyisobutylisonitrile in tumour cells and its modulation bylipophilic P-glycoprotein ligands Ter´ez M´ari´an a , ∗ , L´aszl´o Balkay a , G´abor Szab´o b , Zo´ard T. Krasznai c , Zolt´an Hern´adi c ,L´aszl´o Galuska d , Judit Szab´o-P´eli e , Olga ´Esik  f  , Lajos Tr´on a , e , Zolt´an Krasznai b a PET Center, University of Debrecen, Medical and Health Science Centre, 4012 Debrecen, Nagyerdei krt 98, Hungary b  Department of Biophysics and Cell Biology, University of Debrecen, Medical and Health Science Centre, Debrecen, Hungary c  Department of Obstetrics and Gynaecology, University of Debrecen, Medical and Health Science Centre, Debrecen, Hungary d Center of Nuclear Medicine, University of Debrecen, Medical and Health Science Centre, Debrecen, Hungary e PET Study Group of Hungarian Academy of Sciences, University of Debrecen, Medical and Health Science Centre, Debrecen, Hungary f   Department of Oncology, University of P´ ecs, Hungary Received 2 August 2004; received in revised form 12 January 2005; accepted 16 February 2005Available online 16 March 2005 Abstract  Aim:  To study the accumulation and washout kinetics of [ 99m Tc]-hexakis-2-methoxyisobutyl isonitrile ( 99m Tc-MIBI) in MDR positive andMDR negative tumour cells and how this is modified by lipophilic P-glycoprotein ligands.  Methods:  The tumour cells were incubated inthe presence and absence of the ligands and the uptakes of   99m Tc-MIBI, rhodamine 123 and 2-[ 18 F]fluoro-2-deoxy- d -glucose ( 18 FDG) weremeasured.  Results:  The accumulation of   99m Tc-MIBI in the tumour cells followed biphasic kinetics. Verapamil and cyclosporin A increasedthe membrane fluidity and significantly enhanced the  99m Tc-MIBI uptake of the MDR negative cells, while the rhodamine 123 uptake wasnot affected. Verapamil significantly increased the uptake of rhodamine 123 and  18 FDG but did not modify that of   99m Tc-MIBI in the MDRpositive cells. Cyclosporin A significantly increased the  18 FDG uptake of the MDR positive and negative tumour cells; these effects wereouabain-sensitive. Depolarization of the cytoplasmic membrane, acidification of the extracellular medium and the administration of CCCPdecreased the accumulation of   99m Tc-MIBI and rhodamine 123 uptake in the tumour cells.  Conclusions:  Lipophilic P-glycoprotein ligandsmodified the biphasic accumulation kinetics of the  99m Tc-MIBI uptakes of MDR negative and positive tumour cells in different and complexways and could therefore mask the P-glycoprotein pump-dependent changes in tracer accumulation.© 2005 Elsevier B.V. All rights reserved. Keywords:  Biphasic  99m Tc-MIBI uptake;  18 FDG; Rhodamine 123; Lipophilic ligand; Multidrug resistance  Abbreviations:  Pgp; P-glycoprotein; MDR; multidrug resistance;MRP1; multidrug resistance-associated protein;  18 FDG; 2-[ 18 F]fluoro-2-deoxy- d -glucose;  99m Tc-MIBI; [ 99m Tc]-hexakis-2-methoxyisobutyl isoni-trile; R123; rhodamine 123; VER; verapamil; CSA; cyclosporin A; CCCP;carbonylcyanide m -chlorophenylhydrazone;PET;positronemissiontomog-raphy; SPET; single photon emission computed tomography ∗ Corresponding author. Tel.: +36 52 431 958; fax: +36 52 431 958.  E-mail address: (T. M´ari´an). 1. Introduction Earlier biophysical studies established that  99m Tc-hexakis-2-methoxyisobutyl isonitrile ( 99m Tc-MIBI) is aNernstian probe of the membrane potential (Piwnica-Wormset al., 1990; Pauwels et al., 1998). The net accumulationandunidirectionaluptakeratesof  99m Tc-MIBIarethermody-namically driven by negative mitochondrial inner matrix andplasma membrane potentials (Piwnica-Worms et al., 1995; 0928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.ejps.2005.02.010  202  T. M´ ari´ an et al. / European Journal of Pharmaceutical Sciences 25 (2005) 201–209 Pauwels et al., 1998) thereby concentrating the agent withincellsinasimilarmannerasforotherlipophiliccationicprobesof the membrane potential.However, little is known about the possible distributionof   99m Tc-MIBI between its intracellular free state and statesboundtointracellularstructuresandorganiccellcomponents,aphenomenonaffectingtheintracellularaccumulationofthetracer.It is well documented that malignant tumours display anincreasedaccumulationofthisradiopharmaceutical(Pauwelsetal.,1998;Buraketal.,2003). 99m Tc-MIBIhasbeenshownto be a substrate for the MDR1 gene coded P-glycoprotein(Pgp) and multidrug resistance-associated protein (MRP1)pumps(Piwnica-Wormsetal.,1995;Kostakogluetal.,1998;Utsunomiya et al., 2000). Consequently, the accumulationof   99m Tc-MIBI is reduced in cancer cells expressing Pgp orMRP1. These findings lend support to the hypothesis thatthe  99m Tc-MIBI accumulation assay may be an appropriatein vivo tool for exploration of the Pgp status of tumours.However, certain of the experimental results are somewhatcontroversial, and thus do not provide firm evidence of ageneral and straightforward relationship between the extentof Pgp expression and the  99m Tc-MIBI accumulation levelsin the individual tumours (Kostakoglu et al., 1998; Kinuya etal., 2003; Del Vecchio et al., 2003).Discordantresultscanbeduetoavarietyofreasons.Manyof the  99m Tc-MIBI accumulation studies involved the use of Pgpsubstratesandmodulators(e.g.verapamil(VER)andcy-closporin A (CSA)) that are transported by and/or influencethe function of the Pgp pump. These molecules are often of highlyhydrophobicnature(KrishnaandMayer,2000;Takaraetal.,2002),whichmayhaveeffectsonthe 99m Tc-MIBIaccu-mulationandwashoutkineticsofbothMDRpositive(MDR + )and MDR negative (MDR − ) cancer cells. Some of the mostrecent publications have drawn attention to the complexityof the reverting and other effects of VER when  99m Tc-MIBIis used (Cayre et al., 1999; Rodrigues et al., 2001; Arbabet al., 2003; M´ari´an et al., 2003). In tumours, MDR does notusuallyappearinanallornonefashion;thereisextensivehet-erogeneity in the different cell types and even within a singlecell type (Kostakoglu et al., 1998).  99m Tc-MIBI images pro-vide information on a summed tracer accumulation in whichmultidirectional effects are averaged. It is therefore essentialto examine the effects of different ligands in the uptake of  99m Tc-MIBI in both MDR + and MDR − tumor cells. 18 FDG is the most commonly used PET radiotracer in tu-mour diagnostics, which visualizes the changes of glucosemetabolic rate in tissues (Wahl, 1996; Mankoff and Bellon,2001). The high metabolic rate is usually characteristic of rapidly dividing tumour cells and is generally manifestedin an increased  18 FDG uptake (Wahl, 1996). It was shown earlier that the MDR + cells and the tumour xenografts of the same line exhibit higher  18 FDG uptake than the MDR − cells and the tumours. Activation of the Pgp pump by VERis also manifested in an increased glucose metabolism whileCSAtreatmentdidnotshowPgpdependent 18 FDGelevation(M´ari´an et al., 2003). The increase of the  18 FDG uptake of VER treated MDR + cells is thought to be the result of an ele-vated the ATP-ase activity (M´ari´an et al., 2003). Broxtermanetal.(1990)havealsoshownthattheeffectsofCSAandVERon energy metabolism in multidrug resistant tumour cells isdifferent.Detailed studies of the transport and accumulation pro-cesses of   99m Tc-MIBI may be instrumental in the devel-opment of a measuring protocol for the assessment of thePgp status of malignant cells, a parameter of utmost impor-tance, influencing the management of tumour patients. For abetter understanding of   99m Tc-MIBI accumulation phenom-ena, we set out to examine the accumulation and washoutkinetics of   99m Tc-MIBI and the accumulation of   18 FDG intumour cells under different extracellular and intracellularconditions. These effects were compared with the changesin mitochondrial membrane potential accompanying thesetreatments, as measured using the validated mitochondrialmembrane potential-sensitive dye rhodamine 123 (R123). 2. Materials and methods 2.1. Reagents All of the chemicals applied were of analytical orspectroscopic grade. Propidium iodide (PI), rhodamine123, carbonyl cyanide  m -chlorophenylhydrazone (CCCP),trimethylammonio-diphenyl-hexatriene (TMA-DPH), wereobtained from Molecular Probes (Eugene, OR).  d -Glucose,bovine serum albumin (BSA), digitonin (DIG), verapamil,cyclosporin A, ouabain and inorganic chemicals were pur-chasedfromSigmaChemicalCo.(St.Louis,MO).Phosphatebuffered saline (PBS) contained: 140mM NaCl, 5mM KCl,8mMNa 2 HPO 4 ,3mMNaH 2 PO 4  and5mM d -glucoseatpH7.4. Buffers of different potassium concentrations contained5mM KCl, 135mM NaCl or 120mM KCl, 20mM NaCl andwere buffered with 10mM HEPES (pH 7.4). 2.2. Cells Human epidermoid carcinoma cell line KB-3-1 andits vinblastine-selected Pgp-expressing counterpart KB-V1,mouse fibroblast cell line NIH 3T3, human lymphoid B-cellline JY and hamster vas deferens smooth muscle cell lineDDT1 MF-2 were used.The KB-3-1, KB-V1 and NIH 3T3 cells were grownas monolayer cultures at 37 ◦ C in an incubator contain-ing 5% CO 2  and maintained by regular passage in Dul-becco’s minimal essential medium (supplemented with10% heat-inactivated fetal calf serum, 2mM  l -glutamine,100unitsml − 1 penicillin and 100  gml − 1 streptomycin).TheJYcellsweremaintainedinRPMI-1640mediumsup-plemented with 10% heat-inactivated fetal calf serum, 2mM l -glutamine, 100unitsml − 1 penicillin and 100  gml − 1 streptomycin at 37 ◦ C in a 5% CO 2  atmosphere.  T. M´ ari´ an et al. / European Journal of Pharmaceutical Sciences 25 (2005) 201–209  203 DDT1 MF-2 cells were cultured at 37 ◦ C in a humidi-fied atmosphere of 5% CO 2.  The growth medium was Dul-becco’s modified Eagle’s medium supplemented with 2mM l -glutamine and 10% (v/v) fetal calf serum.Thecellviabilitywasalwayshigherthan90%,asassessedby the trypan blue exclusion test. 2.3. Radiotracers The glucose analogue  18 FDG was synthesized and la-beled with the positron decaying isotope  18 F according toHamacher et al. (1986). MIBI (purchased from the F.J. CurieRadiobiological Research Institute, Budapest, Hungary) waslabeled with  99m Tc according to the kit instructions. 2.4. Flow cytometric measurements A modified Becton Dickinson FACStar plus flow cytome-ter (Becton Dickinson, Mountain View, CA) equipped withan argon ion laser was used to determine fluorescence in-tensities. The small angle forward-scattered light was usedfor electronic gating of the data collection, allowing the ex-clusion of dead cells from the analysis. The argon-ion laserwas tuned to 488nm and used at a power of 500mW. R123emission was detected through a 540nm broadband interfer-ence filter, while PI emission was detected through a 620nmlong-pass filter. 2.5. Mitochondrial membrane potential measurements Cells were resuspended in the appropriate buffer contain-ing1  MR123andincubatedfor30minat37 ◦ Cinthepres-enceorabsenceofdifferentligandsandionophores.Afterin-cubations,thecellswerewashedtwicewithice-coldPBSandkeptoniceuntilthemeasurement.Themeanfluorescencein-tensities were evaluated by flow cytometry. The viability of the cells after incubation was checked by PI staining. 2.6. Intracellular pH measurement  Intracellular pH was measured by flow cytometry, usingthe fluorescent dye BCECF, as described previously (Balkayet al., 1992). 2.7. Flow cytometric Pgp detection Formaldehyde (1% in PBS) prefixed cells were cen-trifuged at 500 × g  for 5min and washed twice with 1%BSA–PBS.For immunofluorescent labeling, Pgp specific antibodiesUIC2 (Mechetner and Roninson, 1992) prepared from hy- bridoma purchased from ATCC were used. Cells were incu-bated with a saturating concentration of UIC2 antibodies inPBS and 1% BSA at 1 × 10 7 cells/ml for 40min. Labelingwas carried out on ice in the dark. Unbound mAb was re-moved by washing the cells twice with ice-cold PBS. For vi-sualization,fluoresceinisothiocyanateconjugatedrabbitanti-mouse IgG 2a  secondary antibody (RAMIG-FITC,  F   /  P =4.3fromSigma)wasused.ThesaturatingconcentrationofUIC2antibodywasdeterminedinadilutionseriesandwasfoundtobe10  g/ml.IrrelevantmouseIgG 2a  appliedatthesamecon-centration was used for isotypic control.  R UIC2  values werecalculated as the means of the fluorescence histograms forspecificallylabeledcellsnormalizedtothemeansofthenon-specific isotypic control. 2.8. Membrane fluidity assessment  1 × 10 − 4 M stock solution of TMA-DPH dissolved indimethylformamide was used. The cells were resuspendedat a concentration of 0.7 × 10 6  /ml in PBS containing8 × 10 − 7 M TMA-DPH and incubated for 5min in the pres-ence or absence of CSA or VER in a quartz cuvette withthe temperature of the sample set to 25 ◦ C. Steady stateanisotropy ( r  f   value) was determined using a Perkin-Elmerspectrofluorimeter by measuring the vertically and horizon-tallypolarizedintensitycomponents.Thesamplewasexcitedwithverticallypolarizedlight(340nm)andtheemissionwasmeasured above 430nm. 2.9. Thin layer chromatography (TLC) TLC was performed on C18 silica gel (Nanosil C18:Macherey-Nagel) with two different solvent systems asmobile phase: methanol/sodium hydrogencarbonate 0.01M(80/20, v/v; pH 7.5), or methanoltrifluoroacetic acid 0.01M(80/20, v/v; pH 7.5) (Bouquillon et al., 1995). 2.10. logP (octanol/water) determination One millilitre of octanol was added to a centrifugetube containing 1ml of PBS+50  Ci  99m Tc-MIBI andthe lipophilic ligands investigated, and the mixture wasshaken intensively for 10min. The tubes were centrifugedat 1000 × g  for 10min and the radioactivities of the octanoland water phases were measured with a calibrated gammacounter. The log ratio of the  99m Tc-MIBI concentrations inthe octanol and water phases were calculated. 2.11. Radiotracer uptake studies The cells were washed with and resuspended in PBS or indifferentKClcontainingsolutionsbufferedwithHEPES.Thesamples were preincubated at 36 ◦ C for 10min at a cell con-centration of 1 × 10 6  /ml in PBS containing 5mM  d -glucoseand other agents (VER, CSA, CCCP, etc.), and 5  Ci/ml 18 FDG or 5–10  Ci/ml  99m Tc-MIBI was then added to eachsample. In certain multitracer analysis experiments, both ra-diopharmaceuticals and R123 were co-administered at thesame time to the cells. After the addition of the radioli-gands, the cells were further incubated at 36 ◦ C and the up-take was terminated by the addition of ice-cold PBS. In the  204  T. M´ ari´ an et al. / European Journal of Pharmaceutical Sciences 25 (2005) 201–209 washout experiments, the cells were centrifuged at 1000 × g for 2min, the supernatant was removed and the cells wereresuspended in radiotracer free PBS and further incubatedat 36 ◦ C. The cells were then washed three times with coldPBS, and resuspended in 1ml cold PBS, and the radioactiv-ity was measured. The tubes were measured in a CanberraPackard gamma-counter for 1min within the  18 F sensitiveenergy window and also for 1min within the  99m Tc sensitiveenergy window. Decay corrected radiotracer uptake was ex-pressedascounts/min/10 6 cells(cpm).Thedisplayeddataarethe mean ± S.D. of the results of at least three independentexperiments each performed in triplicate. 2.12. Data analysis Thedatawereexpressedasmean ± S.D.Traceraccumula-tion data were compared with those measured under controlconditions using Student’s  t  -test (two-tailed). The level of significance was set at  p =0.01 unless otherwise stated. 2.13. Comparison of fitting models The generated time-activity curves were fittedwith three model equations, relating to the follow-ing models:  A exp(  Bt  )+  Z   as single-exponential model,  A exp(  Bt  )+ C  exp(  Dt  )+  Z   as double-exponential model and  A exp(  Bt  )+ C  exp(  Dt  )+  E  exp( Ft  )+  Z   as triple-exponentialkinetic model, where  t   is time,  A ,  B ,  C  ,  D ,  E  ,  F   and  Z   werethe fitted parameters. The kinetic model giving the best fit tothe experimental data was selected according to the AkaikeInformation Criteria (AIC) (Akaike, 1974; Motulsky andChristopoulos, 2004), calculated as:AIC = N   ln(SS) + 2 K where  N   is the number of measurements, SS is the sum of thesquares of the vertical distances of the points from the fittedcurve and  K   is the number of parameters fitted by the regres-sionplusone.Inaddition, F  testswereperformedtocomparethe models, and a level  p <0.05 was considered to indicate asignificant difference (Motulsky and Christopoulos, 2004). 3. Results 3.1. Effects of lipophilic ligands on the  99m Tc-MIBI uptake of tumour cells The  99m Tc-MIBI uptake of JY cells at different extracel-lular  99m Tc-MIBI concentrations is shown in Fig. 1. The intracellular  99m Tc-MIBI accumulation increased in directproportion to the extracellular activity concentration in therange 0.05–100  Ci/ml. The dose dependent effects of VERand CSA on the uptakes of   99m Tc-MIBI by JY and DDT1MF-2 cells are shown in Fig. 2. Both lipophilic ligands sig- nificantly increased the  99m Tc-MIBI accumulation in bothkinds of cells. Fig. 1. Dependence of   99m Tc-MIBI accumulation by JY cells on the extra-cellular activity concentration. The cells were incubated at a concentrationof 1 × 10 6  /ml in PBS, containing 0.05–100  Ci  99m Tc-MIBI in 1ml, for30min. Data are presented as mean ± S.D. of the results of three indepen-dent experiments. Theuptakeandwashoutkineticsof  99m Tc-MIBIintheJYtransformedcellsaredisplayedinFig.3A.Theaccumulationandwashoutarerapidprocesses,60–70%ofthetotalchangesoccurring during the first 10min. The pattern of the kineticsmeasured on the control samples remained very much thesamewhenVERorCSAwasaddedtotheincubationmixture.VER (50  M) and CSA (20  M) significantly increasedthe  99m Tc-MIBI uptake of the JY, DDT1 MF-2, KB-3-1 andNIH 3T3 tumour cells (Fig. 4), but the uptake of the dif-ferent cell lines was different. The least  99m Tc-MIBI uptakewas observed in the DDT1 MF-2 cell line in the case of ei-ther VER or CSA treatment (130% and 140% of the control,respectively) while the most pronounced increase was ob-served in the NIH 3T3 cell line (230% and 240% of the con-trol, respectively). The  99m Tc-MIBI uptake of the cell lineswas increased after VER treatment in the following order: Fig.2. EffectsofVERandCSAonthe 99m Tc-MIBIuptakesofJYandDDT1M-F2 cells. The cells were preincubated with VER and CSA at differentconcentrations for 10min, 10  Ci/ml  99m Tc-MIBI was then added and themixturewasincubatedforafurther30min.Theradiopharmaceuticaluptakewas expressed a percentage of the extracellular 10  Ci  99m Tc-MIBI activ-ity. Data are presented as mean ± S.D. of the results of three independentexperiments.  T. M´ ari´ an et al. / European Journal of Pharmaceutical Sciences 25 (2005) 201–209  205Fig. 3. Accumulation and washout kinetics of   99m Tc-MIBI on JY cells inthe absence and presence of 20  M CSA or 50  M VER. Data are presentedas mean ± S.D. of the results of three independent experiments (A). Rep-resentative curves of single-, double- and triple-exponential fittings of the 99m Tc-MIBI accumulation kinetics (B). DDT1MF2<JY<KB-3-1<NIH3T3,whileafterCSAtreat-ment the order was DDT1 MF2<KB-3-1<JY<NIH 3T3.CCCP (10  M) a drug eliminating the mitochondrial mem-branepotential,significantlyreducedthe 99m Tc-MIBIuptakeofthecells,to30–50%ofthatofthecontrol.Whenthemem-brane was permeabilized by 20  M DIG, a channel formingionophore,  99m Tc-MIBI bound to intracellular componentsand structures could be detected. This component amountedto approximately 10–17% of the control when DIG was ad- Fig. 4. Lipophilic ligands modify the  99m Tc-MIBI uptake of the tumourcells. For the experimental details see Section 2. Data are presented as mean ± S.D. of the results of four independent experiments.  *  p <0.01, **  p <0.001. ministeredtogether(at t  =0)withtheradiotracer.Theamountof bound  99m Tc-MIBI increased to 32% if 20  M digitoninwas added to the incubation mixture only after 25min. R123uptake was also dramatically reduced (to 4% of the control)upon late DIG treatment (administration at  t  =25min), indi-cating that in the latter protocol DIG permeabilized also themitochondrial membrane. 3.2.  99m Tc-MIBI,  18  FDG and R123 uptakes of tumour cells To study the transport mechanism of   99m Tc-MIBI, KB-3-1, KB-V1 and JY cells were used. The radiotracer and themitochondrial membrane potential probe R123 were addedsimultaneouslytothecellsuspensions.Theenergyconsump-tion of the cells was measured by using  18 FDG. Significantincreases in the  99m Tc-MIBI accumulation in MDR − cellswereinducedbyVERandCSA,whereastheR123uptakebythese cells was slightly decreased indicating the depolariza-tionofthemitochondrialmembranepotential.(Table1).VER decreasedthe 18 FDGuptakeoftheMDR − cells,butCSAsig-nificantly increased  18 FDG uptake in all three kinds of cells. Table 1Parallel study of   99m Tc-MIBI, R123 and  18 FDG uptake of transformed cells in different extracellular and intracellular conditionsTreatment  99m Tc-MIBI uptake (% of the control) R-123 uptake (% of the control)  18 FDG uptake (% of the control)KB-3-1 KB-V1 JY KB-3-1 KB-V1 JY KB-3-1 KB-V1 JY50  M VER 175  ±  11 * 87  ±  9 209  ±  12 * 81  ±  6 1200  ±  32 b 90  ±  2 72±7 # 175±12 * 82±920  M CSA 140  ±  15 # 1500  ±  12 ** 219  ±  4 ** 85  ±  6 2500  ±  132 ** 92  ±  4 128±8 # 126±8 # 157±8 * 10  M CCCP 34  ±  6 * 112  ±  7 37  ±  3 * 33  ±  5 * 120  ±  7 38  ±  3 * 102±12 94±5 110±15pH e  5.5 65  ±  10 # 95  ±  6 73  ±  6 # 36  ±  3 * 110  ±  11 60  ±  6 * – – –pH e  8 108  ±  5 90  ±  5 126  ±  3 # 125  ±  6 # 100  ±  9 133  ±  2 * – – –pH e  7.4 [120mM  K + e  ] 75  ±  5 # 97  ±  4 59  ±  2 * 76  ±  4 # 77  ±  5 # 51  ±  2 * – – –Data presented as mean ± S.D. of three independent experiments. #  p <0.05. ∗  p <0.01. ∗∗  p <0.001.
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