Abstract 5385: Fenretinide cytotoxicity is independent of both constitutive and pharmacologically modulated intracellular glutathione levels in acute lymphoblastic leukemia cells

Abstract

Abstract Background: Fenretinide (4-HPR) is a synthetic retinoid that has shown clinical activity in adult lymphoid malignancies and its in vitro cytotoxicity in acute lymphoblastic leukemia (ALL) cell lines was shown to be dependent on ROS generation. Previous studies suggested that the sensitivity of malignant cells to 4-HPR was inversely associated with intracellular glutathione (GSH) levels in cell lines cultured in 20% O2. In our panel of 10 ALL cell lines cultured at physiologic, bone marrow hypoxia (5% O2), we examined whether constitutive levels of GSH or pharmacologic depletion of GSH affects 4-HPR cytotoxicity. Methods: Cell line panel included T, Pre-B, and Pro-B lineages as well as lines established at diagnosis and relapse. Cytotoxicity was determined using a fluorescence-based digital imaging assay (DIMSCAN), ROS by flow cytometry using 2′,7′-dichlorofluorescein diacetate, GSH levels via HPLC, and RNA levels by RT-PCR. Results: 4-HPR concentration cytotoxic for 90% of cells (IC90) after 72 hours ranged from 0.7 – 29.2 µM for the 10 lines (median: 2.2 µM). At 10 µM, 4-HPR increased ROS levels in all 10 lines within 6 hours (range: 8.9 – 1.5 fold over control; median: 3.5 ± 0.2, P < 0.001). IC90 values of 4-HPR inversely correlated with the level of ROS increase (Spearman rank correlation coefficient: -0.90, P < 0.001), but there were no significant correlations with basal intracellular GSH levels or mRNA levels of γ-glutamylcysteine synthase (GCS) and glutathione peroxidase. The GCS inhibitor buthionine sulfoximine (BSO) at 50 µM reduced basal GSH levels by an average of 78.7% ± 13.9 after 24 hours (P < 0.001) in the 10 lines and increased 4-HPR-induced ROS generation at 6 hours by an average of 75.5% (range: 13.1% ± 3.4 – 163.1% ± 2.6, P < 0.001 for each cell line). However, 4-HPR cytotoxicity was only minimally enhanced when combined with BSO across the 10 lines. Pretreatment with 300 µM N-acetylcysteine (NAC) increased GSH levels in 2 T-ALL cell lines (CCRF-CEM and COG-LL-317) under both basal conditions and after incubation with 10 µM 4-HPR for 12 hours (P < 0.01). Despite these GSH increases, NAC did not attenuate 4-HPR cytotoxicity in either line, while ascorbic acid (AA) and α-tocopherol (E) each individually attenuated 4-HPR cytotoxicity in the two lines (P < 0.01). 4-HPR-induced ROS generation was reduced by AA and E in both lines (P < 0.01), but NAC only reduced 4-HPR ROS generation in COG-LL-317 (P < 0.01) and not in CCRF-CEM. Conclusion: ROS generation is an important component of 4-HPR cytotoxicity in ALL cells, but the depletion of intracellular GSH levels minimally enhances sensitivity to 4-HPR. Our in vitro results suggest that BSO may not enhance 4-HPR activity in vivo or in patients with ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5385. doi:10.1158/1538-7445.AM2011-5385