Influx and Efflux Transport as Determinants of Melphalan Cytotoxicity: Resistance to Melphalan in Overexpressing Tumor Cell Lines Annett Kühne, Mladen Vassilev Tzvetkov, Yohannes Hagos, Hermann Lage, Gerhard Burckhardt, Jürgen Brockmöller To cite this version: Annett Kühne, Mladen Vassilev Tzvetkov, Yohannes Hagos, Hermann Lage, Gerhard Burckhardt, et al.. Influx and Efflux Transport as Determinants of Melphalan Cytotoxicity: Resistance toMelpha- lan in Overexpressing Tumor Cell Lines. Biochemical Pharmacology, Elsevier, 2009, 78 (1), pp.45. 10.1016/j.bcp.2009.03.026. hal-00493510 HAL Id: hal-00493510 https://hal.archives-ouvertes.fr/hal-00493510 Submitted on 19 Jun 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. 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Accepted Manuscript Title: Influx and Efflux Transport as Determinants of Melphalan Cytotoxicity: Resistance to Melphalan in MDR1 Overexpressing Tumor Cell Lines Authors: Annett Kuhne,¨ Mladen Vassilev Tzvetkov, Yohannes Hagos, Hermann Lage, Gerhard Burckhardt, Jurgen¨ Brockmoller¨ PII: S0006-2952(09)00253-6 DOI: doi:10.1016/j.bcp.2009.03.026 Reference: BCP 10132 To appear in: BCP Received date: 13-2-2009 Revised date: 25-3-2009 Accepted date: 26-3-2009 Please cite this article as: Kuhne¨ A, Tzvetkov MV, Hagos Y, Lage H, Burckhardt G, Brockmoller¨ J, Influx and Efflux Transport as Determinants of Melphalan Cytotoxicity: Resistance to Melphalan in MDR1 Overexpressing Tumor Cell Lines, Biochemical Pharmacology (2008), doi:10.1016/j.bcp.2009.03.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. * Manuscript-clean Influx and Efflux Transport as Determinants of Melphalan 1 2 3 Cytotoxicity: Resistance to Melphalan in MDR1 Overexpressing 4 5 6 7 Tumor Cell Lines 8 9 10 11 a a b c 12 Annett Kühne , Mladen Vassilev Tzvetkov , Yohannes Hagos , Hermann Lage , Gerhard 13 14 Burckhardtb and Jürgen Brockmöllera 15 16 17 18 19 a Department of Clinical Pharmacology and 20 21 b 22 Department of Physiology and Pathophysiology University Medical Center, University of 23 24 Göttingen, Germany and 25 26 c 27 Charité Campus Mitte, Institute of Pathology, Berlin, Germany 28 29 30 31 AK and MVT contributed equally to this work 32 33 34 Running title: Transmembrane transport as determinant of melphalan toxicity 35 36 37 38 39 Address for correspondence: 40 41 Mladen Vassilev Tzvetkov, PhD 42 University Medical Center Göttingen 43 Department of Clinical Pharmacology 44 Robert-Koch-Str. 40 45 D-37075 Göttingen, Germany 46 47 Telephone: +49 - 551 – 39 13247 48 Fax: Accepted+49 - 551 – 39 12767 Manuscript 49 50 E-mail: [email protected] 51 52 53 54 Abbreviations: MM, multiple myeloma; MDR1, multidrug resistance protein 1 (also known 55 56 as P-glycoprotein, official symbol ABCB1); MRP1 multiple drug resistance-associated protein 57 58 59 1 (official symbol ABCC1); LAT, L-type amino acid transporter; TAT, T-type amino acid 60 61 transporter; BCRP, breast cancer resistance protein (official symbol ABCG2) 62 63 1 64 Page 1 of 36 65 Abstract 1 2 3 4 5 There is considerable variation in efficacy of melphalan therapy in multiple myeloma (MM) 6 7 and other hematopoietic tumors. We hypothesized that this may be due to variations in the 8 9 10 expression of influx and efflux transporters of melphalan. We measured the expression of the 11 12 influx transporters LAT1, LAT2, and TAT1 and the efflux transporters MDR1, MRP1 and 13 14 15 BCRP by quantitative RT-PCR and related their expression to the intracellular accumulation 16 17 and cytotoxicity of melphalan in 7 MM and 21 non-MM hematopoietic tumor cell lines. 18 19 20 Variation in the intracellular accumulation accounted for nearly half of the variation in the 21 22 cytotoxicity of melphalan in MM cell lines (r2=0.47, p=0.04). High expression of the efflux 23 24 25 transporter MDR1 was associated with low intracellular accumulation and low cytotoxicity of 26 27 melphalan (r2=0.56, p=0.03 and r2=0.62, p=0.02, respectively). The effect was reversed by the 28 29 MDR1 inhibitor cyclosporine. In addition, the MDR1-overexpressing HL-60 cell line showed 30 31 32 10-fold higher resistance to melphalan than the non-MDR1-expressing one. The resistance 33 34 was reversed again by cyclosporine and by MDR1-specific shRNA. 35 36 37 LAT1 was the major influx transporter in tumor cell lines with 4,000-fold higher expression 38 39 than LAT2. Down-regulation of LAT1 by siRNA reduced the melphalan uptake by 58% and 40 41 42 toxicity by 3.5-fold, but natural variation in expression between the tumor cell lines was not 43 44 associated with accumulation or cytotoxicity of melphalan. In conclusion, tumor-specific 45 46 variations in the expression of the efflux transporter MDR1, but not of the influx transporter 47 48 Accepted Manuscript 49 LAT1, affect the intracellular accumulation of melphalan and thus determine its cytotoxicity. 50 51 52 53 54 Keywords: melphalan transport, MDR1, P-glycoprotein, LAT1, multiple myeloma 55 56 57 58 59 60 61 62 63 2 64 Page 2 of 36 65 1 1. Introduction 2 3 4 5 Melphalan (L-phenylalanine mustard) is an alkylating cytotoxic drug that is used in a high 6 7 dose regimen followed by stem cell transplantation in therapy of multiple myeloma (MM; 8 9 10 [1]). In addition, melphalan is used in combination with prednisolone in a palliative regime by 11 12 patients not eligible for stem cell transplantation [2, 3] and in combination with novel 13 14 thalidomide or bortezomib based therapies [4, 5]. Variations in the response and acquired 15 16 17 resistance to melphalan are major problems in the treatment of MM [6, 7]. Increasing the dose 18 19 of melphalan may increase treatment efficacy [8], but at the cost of high toxicity, including 20 21 22 leucopenia, mucositis, and diarrhea [9, 10]. Therefore, predictors of melphalan efficacy are 23 24 necessary to optimize the therapy. 25 26 27 28 Low influx or excessive efflux transport may be limiting factors for melphalan efficacy. 29 30 Melphalan has been developed by coupling the bifunctional alkylating agent nitrogen mustard 31 32 33 to the amino acid phenylalanine [11, 12]. The conjugation with phenylalanine improved the 34 35 bioavailability of the nitrogen mustard by increasing its transporter-mediated cellular uptake. 36 37 Influx transporters from the SLC7 family, known as L-type amino-acid transporters (LATs), 38 39 40 were suggested to mediate the cellular uptake of melphalan [13]. Host-specific genetic 41 42 polymorphisms in the two human LAT isoforms, LAT1 and LAT2, do not affect melphalan 43 44 45 pharmacokinetics or toxicity [14]. However, the effects of tumor-specific variation in LAT1 46 47 and LAT2 on melphalan cytotoxicity and clinical efficacy are unknown. 48 Accepted Manuscript 49 50 51 On the other hand, overexpression of the efflux transporter MRP1 is known to mediate 52 53 resistance to melphalan [15, 16]. The mechanism of resistance includes initial conjugation 54 55 56 with glutathione by glutathione S-transferases alpha, mu and pi followed by excretion by the 57 58 ATP-dependent efflux transporter MRP1. However, it is not clear whether MRP1 is the only 59 60 efflux transporter that can cause resistance to melphalan. Overexpression of another efflux 61 62 63 3 64 Page 3 of 36 65 transporter MDR1 (P-glycoprotein, ABCB1) cause resistance to anthracycline-based therapies 1 2 in patients with MM [17, 18]. However, an overexpression of MDR1 has been less studied as 3 4 5 a cause of resistance in melphalan-based therapies of MM. 6 7 8 In this study, we asked whether tumor-specific variations in the efflux and the influx transport 9 10 11 of melphalan may cause variations in melphalan toxicity. To test this, we assessed the 12 13 expression of the efflux transporters MDR1, MRP1 and BCRP, and the influx transporters 14 15 LAT1, LAT2, their heavy chain 4F2hc, and TAT1 in 28 tumor cell lines and related them to the 16 17 18 intracellular accumulation and toxicity of melphalan. The observed correlations were 19 20 validated by selective down-regulation using RNAi and with small-molecular inhibitors of 21 22 23 drug transporters. 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Accepted Manuscript 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 4 64 Page 4 of 36 65 1 2. Materials and Methods 2 3 4 5 6 7 8 2.1. Materials 9 10 11 3 12 [ H]-melphalan was obtained from Jörg Kix Isotopes (Volxheim, Germany). Cyclosporine A 13 14 was obtained from Roche (Mannheim, Germany). Chemically synthesized siRNAs and 15 16 17 transfection reagents were obtained from Qiagen (Hilden, Germany) and all cell culture media 18 19 and supplements were obtained from Gibco-Invitrogen (Karlsruhe, Germany).