Imatinib mesylate (STI571), a potent tyrosine kinase inhibitor, is
      successfully used in the treatment of chronic myelogenous leukemia and
      gastrointestinal stromal tumors. However, the intended chronic oral
      administration of imatinib may lead to development of cellular resistance
      and subsequent treatment failure. Indeed, several molecular mechanisms
      leading to imatinib resistance have already been reported, including
      overexpression of the MDR1/ABCB1 drug pump. We examined whether imatinib
      is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug
      pump that is frequently overexpressed in human tumors. Using a panel of
      well-defined BCRP-overexpressing cell lines, we provide the first evidence
      that imatinib is a substrate for BCRP, that it competes with mitoxantrone
      for drug export, and that BCRP-mediated efflux can be reversed by the
      fumitremorgin C analog Ko-143. Since BCRP is highly expressed in the
      gastrointestinal tract, BCRP might not only play a role in cellular
      resistance of tumor cells but also influence the gastrointestinal
      absorption of imatinib

Boersma, A.W.M. (Anton)

Brok, M. (Mariël)

Burger, H. (Herman)

Nooter, K. (Kees)

Stoter, G. (Gerrit)

Tol, H. van

Wiemer, E.A.C. (Erik)

Erasmus University Digital Repository

NEOPLASIABrief reportImatinib mesylate (STI571) is a substrate for the breast cancer resistance protein(BCRP)/ABCG2 drug pumpHerman Burger, Hans van Tol, Antonius W. M. Boersma, Marie¨l Brok, Erik A. C. Wiemer, Gerrit Stoter, and Kees NooterImatinib mesylate (STI571), a potent ty-rosine kinase inhibitor, is successfullyused in the treatment of chronic myelog-enous leukemia and gastrointestinal stro-mal tumors. However, the intendedchronic oral administration of imatinibmay lead to development of cellular resis-tance and subsequent treatment failure.Indeed, several molecular mechanismsleading to imatinib resistance have al-ready been reported, including overex-pression of the MDR1/ABCB1 drug pump.We examined whether imatinib is a sub-strate for the breast cancer resistanceprotein (BCRP)/ABCG2 drug pump that isfrequently overexpressed in human tu-mors. Using a panel of well-defined BCRP-overexpressing cell lines, we provide thefirst evidence that imatinib is a substratefor BCRP, that it competes with mitox-antrone for drug export, and that BCRP-mediated efflux can be reversed by thefumitremorgin C analog Ko-143. SinceBCRP is highly expressed in the gastroin-testinal tract, BCRP might not only play arole in cellular resistance of tumor cellsbut also influence the gastrointestinalabsorption of imatinib. (Blood. 2004;104:2940-2942)© 2004 by The American Society of HematologyIntroductionImatinib mesylate (STI571) selectively inhibits the tyrosine kinaseactivity of BCR-ABL, c-KIT, and PDGFR and is successfully usedfor treatment of BCR-ABL–dependent chronic myelogenous andacute lymphoblastic leukemia, and c-KIT–dependent gastrointesti-nal stromal tumors.1-6 Clinical trials with imatinib in other tumortypes such as glioblastoma and lung and prostate cancer are inprogress.7 However, the intended chronic use of this oral tyrosinekinase inhibitor over a prolonged time period may support thedevelopment of cellular resistance. Several molecular mechanismsleading to imatinib resistance have been reported, ranging fromimpaired binding of the drug due to mutations, decreased efficacydue to amplification of the target gene, and decreased imatinibuptake in the tumor cells due to overexpression of the MDR1/ABCB1 gene.8-12Since imatinib is orally administered, systemic imatinib levelsmight depend largely on gastrointestinal absorption and metabolicinactivation and on clearance by intestinal ABC transporters.13,14Breast cancer resistance protein (BCRP)/ABCG2 is abundantlyexpressed in the gut and, based on its cellular localization in theapical membrane of the small intestine and colon epithelium, it isvery likely that this ABC transporter is involved in active backtransport of drugs entering from the gut, and in that way may play asignificant role in the systemic bioavailability of oral drugs such asimatinib.15,16 In addition, BCRP expressed in tumor cell types thatare potential targets for imatinib treatment may cause drugresistance at the level of tumor cells.16 Therefore, we investigatedwhether imatinib is a substrate for the BCRP drug pump using apanel of well-defined BCRP-overexpressing cell lines. We studiedaccumulation of 14C-labeled imatinib and show that the level ofintracellular imatinib is significantly decreased in BCRP-overex-pressing cells, which could be reversed by the addition of thefumitremorgin C analog Ko-143. Here, we provide for the first timeevidence that imatinib is a substrate for the BCRP efflux pump.Study designReagents and cell linesImatinib and [14C]STI571 mesylate were obtained from Novartis (Basel,Switzerland). Ko-134 was used as a specific BCRP inhibitor.17 Mitox-antrone and doxorubicin were obtained from Pharmachemie (Haarlem, TheNetherlands). MCF7 (ATCC, HTB-22); MCF7/MR, a mitoxantrone-resistant and BCRP-overexpressing subline; MCF7/AdVp3000 overexpress-ing the R482T BCRP variant; HEK293 cells transfected with pcDNA3(HEK293/Neo); wild-type BCRP/ABCG2-R482R (HEK293/R); BCRP/ABCG2-R482G (HEK293/G); and BCRP/ABCG2-R482T (HEK293/T)were used.Real-time RT-PCRRelative mRNA expression for BCRP, MRP1, MRP2, and MDR1 wasdetermined by real-time reverse transcriptase–polymerase chain reaction(RT-PCR) using an ABI PRISM 7700 (Applied Biosystems, Foster City,CA) and the comparative cycle threshold (CT) method as described.18Analysis of protein expressionBCRP protein levels were determined by Western blot analysis using BCRP-specific monoclonal antibody BXP-21 (1:200) as described previously.19From the Department of Medical Oncology, Erasmus Medical CenterRotterdam, Daniel den Hoed Kliniek/Josephine Nefkens Institute, Rotterdam,The Netherlands.Submitted April 13, 2004; accepted June 13, 2004. Prepublished online asBlood First Edition Paper, July 13, 2004; DOI 10.1182/blood-2004-04-1398.Reprints: Herman Burger, Department of Medical Oncology, ErasmusMC/Josephine Nefkens Institute (Rm Be420), PO Box 1738, 3000 DRRotterdam, The Netherlands; e-mail: h.burger@erasmusmc.nl.The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.© 2004 by The American Society of Hematology2940 BLOOD, 1 NOVEMBER 2004  VOLUME 104, NUMBER 9Accumulation of intracellular [14C]STI571 mesylateCells (106/mL) were exposed to [14C]STI571 (range, 0.1 Ci [3.7 kBq]-0.001 Ci [0.037 kBq]; 2.14 M-0.021 M imatinib) for 2 hours at 37°Cand then extensively washed with ice-cold phosphate-buffered saline(PBS). Imatinib accumulation was calculated from the radioactivitymeasured in the cell pellet over that retained in the supernatant asdetermined by liquid scintillation counting. Relative accumulation(mean  standard deviation [SD]), based on at least 3 independentexperiments, is expressed as a fraction of that found in parental cells(% control).Flow cytometryMitoxantrone and doxorubicin accumulation (fluorescence intensity) wasdetermined by flow cytometry as described previously.20Results and discussionTo determine whether imatinib is a substrate for the BCRP effluxpump, we examined 14C-labeled imatinib accumulation inMCF7/MR and MCF7/AdVp3000, 2 cell lines known to overex-press BCRP.16 The relative intracellular imatinib accumulation,measured after a 2-hour exposure to [14C]STI571 in both MCF7/MR(59.5% 9.5% [mean  SD]) and MCF7/AdVp (3.6% 1.1%)was significantly lower as compared with MCF7 (Figure 1A). Thisdecreased accumulation is indicative of BCRP-mediated efflux;however, in these drug-selected MCF7 sublines other drug transport-ers may have been activated. Quantitative real-time RT-PCR (datanot shown) and protein data confirmed that only BCRP (Figure 1A,insert), and not MDR1, MRP1, or MRP2 (data not shown), wasoverexpressed in these MCF7 sublines. Although the difference inimatinib accumulation between MCF7/MR and MCF/AdVp maybe explained by the expression data, it should be noted thatMCF7/MR overexpresses wild-type (wt)–BCRP whereas MCF/AdVp overexpresses the 482Thr BCRP variant. Notably, mutationsat 482Arg of wt-BCRP have been implicated in substrate specific-ity.17 Therefore, BCRP-mediated transport of imatinib was studiedin HEK293 cells transfected with BCRP variants, that is, 482Arg(HEK293/R), 482Gly (HEK293/G), 482Thr (HEK293/T), or emptyvector (HEK293/Neo). Similar to MCF7, overexpression of BCRPin HEK293 cell lines resulted in a markedly decreased imatinibuptake (Figure 1B), confirming that imatinib is a substrate forwt-BCRP and both mutant (mt)–BCRP variants. However, theexistence of small differences in imatinib accumulation betweenwt- and mt-BCRP cannot be excluded. Such subtle differenceswere also observed for mitoxantrone.21 Regarding BCRP-mediatedimatinib efflux, our data suggest that codon 482 of BCRP does notplay such a pivotal role in substrate specificity as has been reportedfor methotrexate (MTX) and doxorubicin.17,22-24 Accordingly, wefound that doxorubicin is not transported by wt-BCRP, whereas asignificant decrease in doxorubicin accumulation (40%) was seenin both mt-BCRP variants (Figure 1C). Furthermore, we showedthat mitoxantrone is a substrate for both wt- and mt-BCRP (Figure1D). With respect to this mutational hot-spot at codon 482 ofBCRP, imatinib parallels the substrate specificity pattern of mitox-antrone and not that of doxorubicin or MTX.17,23,24Next, we determined whether the decreased imatinib accumula-tion could be reversed by Ko-143, a specific inhibitor of BCRP-mediated transport.17 Evidently, Ko-143 could almost completelyrestore the decreased imatinib accumulation in cells overexpressingwt-BCRP (HEK293/R: 1.7-fold increase in the presence of Ko-143,P  .01 Student t test) as well as in the 2 mt-BCRP variants(HEK293/G: 4.1-fold increase, P  .001; HEK293/T: 2.1-foldincrease, P  .01), whereas this specific inhibitor had no effect onthe control HEK293/Neo cells (Figure 1E).Since mitoxantrone is one of the key substrates for BCRP, wedetermined whether imatinib is a competitive substrate for thesame binding site. Mitoxantrone accumulation in the BCRP-positive MCF sublines was clearly increased by addition of 15 Mimatinib as a competitor, whereas accumulation in the BCRP-negative MCF7 parental line was hardly affected (Figure 2A).Similar results were found in BCRP-overexpressing HEK293 cells,that is, mitoxantrone steady-state levels were markedly increasedby the addition of imatinib (Figure 2B). Moreover, mitoxantroneefflux was almost completely inhibited by 15 M imatinib. Thesecompetition studies suggest that cotreatment of imatinib with otherBCRP substrate drugs may have a significant impact on the oralbioavailability of these drugs.It was recently shown that imatinib can potently reverseBCRP-mediated resistance to topotecan and SN-38 in vitro;however, in contrast to our results presented here, it was concludedthat imatinib directly inhibits BCRP-mediated transport withoutbeing a competitive substrate.25 The investigators reported that theFigure 1. Intracellular accumulation of [14C]STI571 mesylate, doxorubicin, andmitoxantrone. (A) The intracellular [14C]STI571 accumulation (mean  SD) inBCRP-overexpressing MCF7 sublines and (B) BCRP-transfected HEK293 cells. Therelative accumulation of [14C]STI571, after a 2-hour exposure to this radioactivecompound, is based on the mean of at least 3 independent experiments and isexpressed as a fraction of that found in parental BCRP-negative cells (% control). (C)Relative accumulation of doxorubicin and (D) mitoxantrone in HEK293 cells,transfected with pcDNA3 (HEK293/Neo), wt-BCRP (HEK293/R), or mt-BCRP vari-ants at residue 482 (HEK293/G and HEK293/T). Intracellular accumulation wasmeasured by flow cytometry. Relative accumulation of these drugs, shown asfluorescence intensity (peak value) of at least 20 000 events, is shown as fractionrelative to that found in control HEK293/Neo cells (% control). (E) The effect of 200nM Ko-143 on the steady-state accumulation of [14C]STI571 in the panel of HEK293cells. HEK293 BCRP variants were preincubated with Ko143 (200 nM) for 1 hour andthen exposed to 14C-labeled imatinib (0.21 M) for an additional 2 hours. Beta-radiation (dpm) was used as a measure of the intracellular [14C]STI571 accumulation.IMATINIB MESYLATE IS A BCRP SUBSTRATE 2941BLOOD, 1 NOVEMBER 2004  VOLUME 104, NUMBER 9accumulation and subsequent efflux of 14C-labeled imatinib wassimilar in cells expressing functional or nonfunctional BCRP. Theapparent discrepancy with our findings may be ascribed tomethodologic differences, among which are substantial dissimi-larities in incubation time and temperature. Evidently, ouraccumulation data (Figure 1A-B) and in particular the results ofthe BCRP-specific inhibitor Ko-143 (Figure 1E) are consistentwith the fact that imatinib is a substrate of BCRP. Together, ourresults indicate that BCRP can function as an active outwardtransport mechanism for imatinib and this drug efflux pump maytherefore play an important role in drug-drug interaction andcellular resistance to imatinib.AcknowledgmentsWe thank Dr E. Buchdunger (Novartis, Basel, Switzerland) forthe generous gift of imatinib mesylate and [14C]STI571 mesy-late, Dr A.H. Schinkel (The Netherlands Cancer Institute,Amsterdam, The Netherlands) for providing Ko-143, Dr R.J.Scheper (Free University Medical Center, Amsterdam, TheNetherlands) for MCF/MR and the BXP-21 antibody, and DrR. Robey and Dr S.E. Bates (National Cancer Institute, Be-thesda, MD) for providing the BCRP-transfected HEK293 cellsand MCF7/AdVp.References1. Buchdunger E, Cioffi CL, Law N, et al. Abl proteintyrosine kinase inhibitor STI571 inhibits in vitrosignal transduction mediated by c-kit and plateletderived growth factor receptors. J Pharmacol ExpTher. 2000;295:139-145.2. Druker BJ, Sawyers CL, Kantarjian H, et al. Activ-ity of a specific inhibitor of the BCR-Abl tyrosinekinase in the blast crisis of chronic myeloid leuke-mia and acute lymphoblastic leukemia with thePhiladelphia chromosome. N Engl J Med. 2001;344:1038-1042.3. Goldman JM, Druker B. Chronic myeloid leuke-mia: current treatment options. Blood. 2001;98:2039-2042.4. Van Oosterom AT, Judson I, Verweij J, et al.Safety and efficacy of imatinib (STI571) in meta-static gastrointestinal stromal tumours: a phase Istudy. Lancet. 2001;358:1421-1423.5. Verweij J, Judson I, van Oosterom A. STI571: amagic bullet? Eur J Cancer. 2001;37:1816-1819.6. Demetri GD, von Mehren M, Blanke CD, et al.Efficacy and safety of imatinib mesylate in ad-vanced gastrointestinal stromal tumors. N EnglJ Med. 2002;347:472-480.7. Radford IR. Imatinib. Novartis. Curr Opin InvestigDrugs. 2002;3:492-499.8. Weisberg E, Griffin JD. Mechanisms of resistanceimatinib (STI571) in preclinical models and in leu-kemia patients. Drug Resistance Updates. 2001;4:22-28.9. Gambacorti-Passerini CB, Gunby RH, Piazza R,Galietta A, Rostagno R, Scapozza L. Molecularmechanisms of resistance to imatinib in Philadel-phia-chromosome-positive leukemias. Lancet.2003;4:75-85.10. Shah NP, Sawyers CL. Mechanisms of resistanceto STI571 in Philadelphia chromosome-associ-ated leukemias. Oncogene. 2003;22:7389-7395.11. Mahon FX, Belloc F, Lagarde V, et al. MDR1 geneoverexpression confers resistance to imatinibmesylate in leukemia cell line models. Blood.2003;101:2368-2373.12. Widmer N, Colombo S, Buclin T, Decosterd LA.Functional consequence of MDR1 expression onimatinib intracellular concentrations [letter].Blood. 2003;102:1142.13. Kruijtzer CMF, Beijnen JH, Schellens JHM. Im-provement of oral drug treatment by temporaryinhibition of drug transporters and/or cytochromeP450 in the gastrointestinal tract and liver: anoverview. The Oncologist. 2002;7:516-530.14. Lindell M, Karlsson MO, Lennernas H, PahlmanL, Lang A. Variable expression of CYP and Pgpgenes in the human small intestine. Eur J ClinInvest. 2003;33:493-499.15. Maliepaard M, Scheffer GL, Faneyte IF, et al.Subcellular localization and distribution of thebreast cancer resistance protein transporter innormal human tissues. Cancer Res. 2001;8:3458-3464.16. Doyle LA, Ross DD. Multidrug resistance medi-ated by the breast cancer resistance proteinBCRP (ABCG2). Oncogene. 2003;22:7340-7358.17. Allen JD, Jackson SC, Schinkel AH. A mutationhot spot in the Bcrp1 (Abcg2) multidrug trans-porter in mouse cell lines selected for doxorubicinresistance. Cancer Res. 2002;62:2294-2299.18. Burger H, Foekens JA, Look MP, et al. RNA ex-pression of breast cancer resistance protein, lungresistance protein, multidrug resistance-associ-ated proteins 1 and 2, and multidrug resistancegene 1 in breast cancer: correlation with chemo-therapeutic response. Clin Cancer Res. 2003;9:827-836.19. Burger H, Nooter K, Boersma AWM, et al. Distinctp53-independent apoptotic cell death signallingpathways in testicular germ cell tumour cell lines.Int J Cancer. 1999;81:620-628.20. Boersma AWM, Nooter K, Oostrum RG, Stoter G.Quantification of apoptotic cells with fluoresceinisothiocyanate-labeled annexin V in Chinesehamster ovary cell cultures treated with cisplatin.Cytometry. 1996;24:123-130.21. Robey RW, Honjo Y, Morisaki K, et al. Mutationsat amino-acid 482 in the ABCG2 gene affect sub-strate and antagonist specificity. Brit J Cancer.2003;89:1971-1978.22. Honjo Y, Hrycyna CA, Yan Q-W, et al. Acquiredmutations in the MXR/BCRP/ABCP gene altersubstrate specificity in MXR/BCRP/ABCP-over-expressing cells. Cancer Res. 2001;61:6635-6639.23. Chen Z-S, Robey RW, Belinsky MG, et al. Trans-port of methotrexate, methotrexate polygluta-mates, and 17beta-estradiol 17-(beta-D-glucuro-nide) by ABCG2: effects of acquired mutations atR482 on methotrexate transport. Cancer Res.2003;63:4048-4054.24. Volk EL, Schneider E. Wild-type breast cancerresistance protein (BCRP/ABCG2) is a metho-trexate polyglutamate transporter. Cancer Res.2003;63:5538-5543.25. Houghton PJ, Germain GS, Harwood FC, et al.Imatinib mesylate is a potent inhibitor of theABCG2 (BCRP) transporter and reverses resis-tance to topotecan and SN-38 in vitro. CancerRes. 2004;64:2333-2337.Figure 2. Effect of imatinib as a competitor for BCRP-mediated efflux of mitoxantrone. (A) Effect of imatinib (15 M) on mitoxantrone steady-state accumulation (3 M) inMCF7 parental (left panel), MCF7/MR (middle panel), and MCF7/AdVp (right panel). MCF7 sublines were simultaneously incubated for 2 hours with mitoxantrone (3 M), withor without imatinib as a competitor (1 M-15 M). The fluorescence data of at least 20 000 events per time point are shown and fluorescence intensity (peak signal) ofmitoxantrone is expressed in arbitrary units (au). (B) Mitoxantrone steady-state accumulation in the presence of 15 M imatinib in HEK293 cells. Representative results from 3independent experiments are shown. Relative accumulation data (mean  SD) are given as a fraction (% control) relative to the accumulation found in HEK293 control cellswithout imatinib.2942 BURGER et al BLOOD, 1 NOVEMBER 2004  VOLUME 104, NUMBER 9

Imatinib mesylate (STI571) is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump

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Imatinib mesylate (STI571) is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump

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