Synergistic effects of clinically achievable concentrations of 12-O-tetradecanoylphorbol-13-acetate in combination with all-trans retinoic acid, 1α,25-dihydroxyvitamin D3, and sodium butyrate on differentiation in HL-60 cells

Our recent studies demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) has pharmacological activity for the treatment of acute myelocytic leukemia patients. In the present study, we investigated the potential synergistic effect of all-trans retinoic acid (RA), 1 alpha ,25-dihydroxyvitamin D-3 (VD3), and sodium butyrate (NaB) on TPA-induced differentiation in HL-60 human promyelocytic leukemia cells. The cells were treated once with these agents for 48 h or treated every 24 h for 96 h. Treatment of HL-60 cells once with TPA, RA, VD3, or NaB for 48 h resulted in concentration-dependent growth inhibition and cell differentiation. At clinically achievable concentrations, TPA (0.16 nM) increased the number of adherent cells and RA (0.1-1 muM) increased the number of nitroblue tetrazolium (NDT)-positive cells. The combinations of TPA (0.16 nM) with RA (0.1-1 muM), VD3 (1 nM), or NaB (100 muM) for 48 h synergistically increased differentiation as measured by the formation of adherent cells (P less than or equal to 0.01). Moreover, cells treated with various combinations of low concentrations of TPA, RA, VD3, and NaB every 24 h for 96 h resulted in a further decrease in cell growth and an increase in differentiation. At clinically achievable concentrations, the strongest stimulation of differentiation was achieved in cells treated with a "cocktail" that combined TPA, RA, VD3, and NaB. The synergistic effect of combinations of TPA with RA or NaB at clinically effective concentrations on HL-60 cell differentiation suggests that the combination of these agents may improve the therapeutic efficacy of TPA for the treatment of acute promyelocytic leukemia (APL) patients. A differentiation "cocktail" that combines TPA, RA, VD3, and NaB may provide an even more effective strategy for improving the therapeutic efficacy of TPA and RA.