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Reversal of P-glycoprotein–Mediated Multidrug Resistance by the Murine Double Minute 2 Antagonist Nutlin-3

¹ Institut für Medizinische Virologie, Klinikum der J.W. Goethe-Universität and ² Experimentelle Pädiatrische Onkologie und Hämatologie, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany; ³ Department of Neuropathology, Ruprecht-Karls-University Heidelberg and Deutsches Krebsforschungszentrum, Heidelberg, Germany; and ⁴ Institut für Pharmakologie und Toxikologie, Abteilung Klinische Pharmakologie, Klinikum der Eberhard-Karls-Universität, Tübingen, Germany

Requests for reprints: Jindrich Cinatl, Institut für Medizinische Virologie, Paul Ehrlich-Str. 40, Frankfurt/Main 60596, Germany. Phone: 49-69-6301-6409; Fax: 49-69-6301-4302; E-mail: Cinatl{at}em.uni-frankfurt.de.

Key Words: nutlin-3 • p53 • multidrug resistance • P-glycoprotein • ABC transporters

Murine double minute 2 (MDM2) negatively regulates the activity of the tumor suppressor protein p53. Nutlin-3 is a MDM2 inhibitor under preclinical investigation as nongenotoxic activator of the p53 pathway for cancer therapy. Here, nutlin-3 was evaluated for its activity alone or in combination with established chemotherapeutic drugs for antitumor action in chemosensitive and chemoresistant neuroblastoma and rhabdomyosarcoma cell lines. Effects of nutlin-3 single treatment were much more pronounced in p53 wild-type cell lines (IC₅₀s <3 µmol/L) than in p53-mutated cell lines (IC₅₀s >17 µmol/L). In sharp contrast to the expectations, nutlin-3 concentrations that did not affect viability of p53-mutated cell lines strongly increased the efficacy of vincristine in p53-mutated, P-glycoprotein (P-gp)–overexpressing cell lines (decrease in IC₅₀s 92- to 3,434-fold). Similar results were obtained for other P-gp substrates. Moreover, nutlin-3 reduced efflux of rhodamine 123 and other fluorescence dyes that are effluxed by P-gp. Investigation of Madin-Darby canine kidney (MDCK) II cells stably transfected with plasmids encoding for P-gp (MDCKII MDR1) or multidrug resistance protein 1 (MRP-1, MDCKII MRP1) revealed that nutlin-3 not only interferes with P-gp but also affects MRP-1–mediated efflux. Kinetic studies and investigation of P-gp-ATPase activity showed that nutlin-3 is likely to act as a P-gp transport substrate. Examination of the nutlin-3 enantiomers nutlin-3a and nutlin-3b revealed that, in contrast to MDM2-inhibitory activity that is limited to nutlin-3a, both enantiomers similarly interfere with P-gp–mediated drug efflux. In conclusion, nutlin-3–induced inhibition of P-gp and MRP-1 was discovered as a novel anticancer mechanism of the substance in this report. [Cancer Res 2009;69(2):416–21]