Inhibitory effects of ATO and etanercept (Enbrel) on NF-κB. 5 × 106 ML1 cells (A), BMMCs from a patient with RAEB (tested were marrows from patients 14, 15, 19, and 20; shown are results from patient 14) (B), and a patient with CMML (tested were patients 22 and 24; shown is patient 22) (C), and from a healthy donor (tested were 3) (D) were exposed to ATO in the presence of etanercept for 1 hour, after which TNF-α was added at the indicated concentration for 20 hours. Nuclear extracts were isolated, and 32P NF-κB gel shift was performed. A 50-fold molar excess of unlabeled probe (Cold) was used for competitive inhibition. (E) ML1 cells were exposed to ATO, etanercept, and BMS345541 (in this example at concentrations of 10 μM, 5 μg/mL, and 5 μM, respectively), for 1 hour, after which TNF-α (20 ng/mL) was added; cells were harvested 1 hour later. Phosphorylated IKBα and Bcl-XL were determined by Western blot (shown are results from 1 of 3 independent experiments). Actin served as protein control load. BMS345541, a specific inhibitor of IKBα kinase, was used here for comparison with the inhibitory effect of ATO on NF-κB. (F) ML1 cells were exposed to ATO (shown are results at 5 μM) for various time periods (4-24 hours). Results in untreated cells and cells exposed to TNF-α are shown for reference purpose (XIAP and Akt-P were not determined at 24 hours). Phosphorylated IKBα, Bcl-XL, Bcl-2, XIAP, and Akt-P were determined by Western blot. Results showed that ATO decreased NF-κB nuclear translocation and downstream antiapoptotic proteins under NF-κB control.
