Deguelin downregulates AKT/NFκB mediated prosurvival signals in CLL cells.

<p>(A) PBMCs from 3 CLL patients were treated for 24h with 0, 10, or 100 μM deguelin. Viability was evaluated by flow cytometry after Annexin V/IP staining. Figure shows immunoreactive bands for several deguelin target molecules and β-actin in western blots from a representative sample. Numbers indicate the signal intensity of β-actin-normalized bands from drug treated samples compared to the untreated ones (Control). (B) PBMCs from two CLL patients were treated with 0, 10, or 100 μM deguelin or 1 μg/ml fludarabine and co-cultured with <i>Ltk</i>^<i>-</i> 24h. Cell viability assays and western blot from a representative sample were done as in (A). (C) PBMCs from 2 CLL patients were co-cultured with <i>Ltk</i>^<i>-</i> or 3T3-CD40L cells and p-AKT expression was evaluated in CLL cells by intracellular staining and flow cytometry at different time points. Histograms show the stains in one of the samples (solid lines from left to right: p-AKT staining at 0, 24, 48 and 120h; dotted lines from left to right: unstained control (samples without primary antibody plus secondary antibody) and isotype control. The lower graph shows the differences in the Mean Fluorescence Intensity (MFI, in channel numbers) of p-AKT compared to the unstained control in both samples (bars show mean and SD). The dotted horizontal line indicates the mean difference in MFI (10 ± 5) between the isotype controls and corresponding unstained controls in the samples with both stains (n = 8), that represents an estimation of the limit for a clearly specific fluorescence signal. (D) PBMCs from 2 CLL patients were treated with 10 μM deguelin, 1 μg/ml fludarabine or the combination of both, and co-cultured with the 3T3-CD40L cell line up to 120h. Upper graphs show viability of CLL cells in both samples (black: control; green: deguelin; red: fludarabine; blue: deguelin plus fludarabine; dotted lines from left to right: unstained control and isotype control). FSC/SSC plots from samples treated with the combination of both drugs are also shown. Histograms show the expression of p-AKT, p-p65 and c-Myc in a representative sample evaluated by intracellular staining and flow cytometry and lower graphs show MFI differences in both samples as in (C).