Abstract 4529: Cellular pharmacokinetic and activity studies with the MDM2-p53 inhibitor Nutlin-3

Abstract

Abstract The tumor suppressor p53 plays a central role in the cell cycle, apoptosis, DNA repair and senescence. Murine double minute 2 (MDM2) negatively regulates the activity of the tumor suppressor protein p53. Nutlin-3, a small molecule inhibitor of MDM2 which can block the interaction of p53 and MDM2, is under preclinical investigation as a nongenotoxic activator of the p53 pathway. In order to understand the cellular pharmacology of nutlin-3 in cells with different p53 status, Nutlin-3 activity and its cellular uptake were studied in p53 wild type and p53 non-functional cancer cell lines. Human colon cancer cell lines HCT116 (p53wt), HCT116 p53−/− and HCT116 N7 (p53 non-functional due to E6 expression), and the human ovarian cancer cell lines A2780, A2780CP70 (p53 mutated) were treated with Nutlin-3 alone for up to 96 hours. Nutlin-3 is stable in tissue culture medium for up to 120 hours and can be readily detected in both HCT116 and A2780 cells. Although intracellular concentrations do not exceed extracellular levels, at 10 µM Nutlin-3 96 hours cellular uptake in p53 wild type cells is two to three times higher (HCT116 25 µg/1×106 cells, A2780 12 µg/1×106 cells) than in p53 non-functional cells (HCT116 p53−/− 9.1 µg/1×106 cells, HCT116 N7 8.2 µg/1×106 cells, A2780 CP70 5.3 µg/1×106 cells). Up to a concentration of 50 μM Nutlin-3, there is no clear evidence of saturation of uptake in HCT116 cells; however, in A2780 cells the intracellular concentrations are the same following exposure to 30μM or 50μM Nutlin-3. Nutlin-3 is 10 to 20-fold more growth-inhibitory and 3 to 9-fold more cytotoxic (by SRB and clonogenic assay) in p53 wild type cells: HCT116 (GI50 = 2.4 ± 0.3 µM, LD50 = 6.8 ± 2.9 µM) and A2780 (GI50 = 0.9 ± 0.3 µM, LD50 = 5.5 ± 0.5 µM), versus matched p53-deficient cells: HCT116 p53−/− (GI50 = 24 ± 2.6 µM, LD50 = 48 ± 1.4 µM), HCT116 N7 (GI50 = 17 ± 2 µM, LD50 ≥ 50 µM) and A2780 CP70 (GI50 = 23 ± 1.7 µM, LD50 = 31 ± 13 µM). Following rernoval of extracellular Nutlin-3 differential cytotoxicity is lower in cells remaining after 24 or 96 hours treatment. Overall, these studies demonstrate that both Nutlin-3 cellular uptake and growth inhibition/cytoxicity is dependent upon p53 status. Our results suggest that functional p53 promotes the intracellular accumulation of Nutlin-3. Potential mechanisms are: a) Direct binding to p53 or MDM2, b) Increased uptake - possibly as a result of expression of a p53-regulated influx transporter, c) Decreased efflux - possibly due to the reduced activity of an efflux pump secondary to Nutlin-3 induced cellular toxicity. These experiments demonstrate that both the cellular uptake and growth inhibition/cytotoxicity of Nutlin-3 is more pronounced in p53 wild type cell lines than in p53 mutated or deficient cell lines. Furthermore, maximal differential activity of p53/MDM2 antagonists in p53 wild type and p53 non-functional cells requires continuous exposure. (This work was supported by the CRUK) Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4529.