Contribution of glutathione and MRP-mediated efflux to intracellular oxaliplatin accumulation* C. Mohn 1 , G.V. Kalayda 1 , H.-G. Häcker 2 , M. Gütschow 2 , S. Metzger 3 and U. Jaehde 1 1 Institute of Pharmacy, Department of Clinical Pharmacy, 2 Institute of Pharmacy, Department of Pharmaceutical Chemistry I, University of Bonn, and 3 Biological Medical Research Center (BMFZ), University of Düsseldorf, Germany Contribution of glutathione and MRP-mediated efflux to intracellular oxaliplatin accumula- tion Introduction Thirty years of experience using cisplatin in the treatment of cancer have resulted in a growing understanding of its mode of action and led to the successful development of sec- ond and third generation analogues. In contrast to the first clinically available compounds cisplatin and carboplatin which exhibit pri- mary resistance against colorectal cancer, oxaliplatin is also effective in this tumor en- tity. However, some mechanisms associated with acquired resistance against oxaliplatin resemble those reported for cisplatin, includ- ing lower intracellular platinum accumulation [1]. Detoxification of platinum complexes by intracellular formation of platinum-glutathione (GSH) adducts and their subsequent efflux via the ABC transporters MRP1 and MRP2 con- tribute to a decreased accumulation and have been repeatedly suggested as resistance mechanism for cisplatin [2] but have also been the subject of controversy [3]. The pres- ent study focuses on oxaliplatin and its detox- ification via GSH and MRP-mediated efflux. Electrospray ionization mass spectrometry (ESI-MS) was applied to detect oxaliplatin- GSH adducts formed after incubation of oxaliplatin with GSH. Gü83 (Figure 1), a 4- aminobenzoic acid derivative recently shown to inhibit MRP1 (IC 50 = 1.2 μM) and MRP2 (IC 50 = 21.5 μM) [4], was used to investigate the contribution of the transporters to oxaliplatin efflux in oxaliplatin-sensitive and oxaliplatin-resistant ileum carcinoma cells. Material and methods Identification of platinum-GSH adducts Oxaliplatin was incubated with GSH in a ratio of 1 : 10 (55 μM oxaliplatin and 550 μM GSH) in aqueous solution. A solution con- taining methanol (60%) and formic acid (1%) was added immediately or after a 12 h incuba- tion time at 37 °C. Subsequently, electrospray ionization-mass spectrometry measurements were performed using an ESI-Q-qTOF (QSTAR XL; Applied Biosystems, Darmstadt, Germany) equipped with a nanospray ion source. Cell culture The human ileocecal colorectal adenocar- cinoma cell line HCT8 and its oxaliplatin- resistant variant HCT8ox were kindly pro- vided by Dr. R.A. Hilger, University of Essen, Germany. Cells were cultivated in RPMI- 1640™ medium supplemented with 10% fe- tal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin (37 °C, 5% CO 2 ). Cellular platinum accumulation To investigate the cellular platinum accu- mulation, cells were allowed to attach in 6-well plates for 12 – 14 hours and incubated with oxaliplatin (100 μM) and in some exper- iments additionally with Gü83 (100 μM). At certain time points, cells were harvested and platinum was quantified using flameless atomic absorption spectrometry (SpectrAA™ Zeeman 220; Varian, Darmstadt, Germany). The plati- num concentration was related to the protein content determined by the bicinchonic acid (BCA) assay (Pierce, Rockford, IL, USA). Statistical analysis Statistical analysis was performed using Mann-Whitney test. P values of < 0.05 were considered statistically significant. Key words oxaliplatin – efflux – MRP1 – MRP2 – resistance – glutathione *This extended abstract summarizes a poster presented by C. Mohn during the Annual Meet- ing of the Central Euro- pean Society for Anti- cancer Drug Research- EWIV (CESAR) held in Heidelberg, Germany, October 29 – 31, 2009. Correspondence to Prof. Dr. U. Jaehde Department of Clinical Pharmacy, Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany u.jaehde@uni-bonn.de International Journal of Clinical Pharmacology and Therapeutics, Vol. 48 – No. 7/2010 (445-447) Extended Abstract ©2010 Dustri-Verlag Dr. K. Feistle ISSN 0946-1965