NO induces apoptosis in Acute Myelogenous Leukemia (AML) cells. Glutathione S-Transferases (GST) play an important role in multidrug resistance and are upregulated in 90% of AML cells. We have designed a prodrug class that releases NO on metabolism by GST. O2-(2,4-Dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent antileukemic activity (Molecular Cancer Therapeutics 2:409–417,2003). We sought to identify synergistic interactions between JS-K and other antileukemic agents. The human HL-60 myeloid leukemia cell line was used for in vitro studies of the combination of JS-K with Daunorubicin (Dauno), Cytarabine (Ara-C) or Etoposide (Etop). Drugs were added to cells in logarithmic growth. Cell viability was determined 3 days later using the CellTiter 96 assay (Promega, Madison, WI). The median effect method of Chou and Talalay was used to determine synergistic, antagonistic, or additive effects with the CalcuSyn software (Biosoft, Ferguson, MO). Combinations of JS-K added simultaneously, 2 hours before or 2 hours after the other compounds were used. Drug combinations were added in increments at a fixed concentration ratio. The 50% growth inhibitory concentrations (IC50) for Dauno, Ara-C, Etop, and JS-K alone were 0.07, 0.18, 0.79, and 0.30 μM, respectively. JS-K and Dauno (Dauno:JS-K fixed concentration ratio = 1:5) were antagonistic in all 3 drug sequences. The Combination Index (CI) at the 50% fraction affected was 12.5 ± 1.6, 10.7 ± 0.41, and 7.2 ± 0.46 for simultaneous, JS-K first and Dauno first additions, respectively. JS-K and Etop (Etop:JS-K fixed concentration ratio = 2:1) were antagonistic but to a lesser degree. The CI at the 50% fraction affected was 3.03 ± 0.26, 2.2 ± 0.33, and 1.7 ± 0.082 for simultaneous, JS-K first and Etop first additions, respectively. JS-K and Ara-C (Ara-C:JS-K fixed concentration ratio = 1:1) showed strong synergy. The CI at the 50% fraction affected was 0.37 ± 0.23, 0.24 ± 0.27, and 0.15 ± 0.11 for simultaneous, JS-K first and Ara-C first additions, respectively. We conclude that Ara-C and JS-K have synergistic anti-leukemic activity and warrant further exploration in combination.

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