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Fig. 6 Changes in cell cycle progression in HL-60 (a) and K-562

(b) cells after 48 h treatment with ZKKs. Each bar represents the mean ± SD (n ≥ 4). The data obtained from FACSCalibur flow cytometer were analyzed using MacCycle software to determine the percentage of cells in each phase of the cell cycle Fig. 7 Exemplary DNA histograms of K-562 cells treated for 48 h with ZKK-3. The data obtained from FACSCalibur flow cytometer and analyzed using MacCycle software to determine the percentage of cells in each phase of the cell cycle. a: Control (no ZKK-3 added); b: 10 μM ZKK-3; c: 20 μM ZKK-3 Discussion We decided to synthesize modified pentabromobenzylisothioureas in a search for new inhibitors of the antiapoptotic enzyme casein kinase 2 (CK2), structurally similar to such known polyhalogenobenzimidazole CK2 inhibitors as 4,5,6,7-tetrabromobenzimidazole (TBI) or

4,5,6,7-tetrabromo-2-dimethylaminobenzimidazole Dibutyryl-cAMP (DMAT) (Szyszka et al., 1995; Pagano et al., 2004; Gianoncelli et al., 2009). We expected LY2874455 that the new compounds would show the advantage of increased water solubility while retaining high CK2 inhibitory activity. However, the novel compounds showed only moderate CK2 inhibitory NVP-BGJ398 activity (Ki ≈ 4 μM, Dr. F. Meggio, personal communication), whereas, surprisingly, they revealed a considerable antileukemic action in vitro. It should be noted that other known benzylisothioureas with substituents in the benzene part of the molecule (for example, 2,3,4,5,6-pentafluoro- and 3,4- and 2,4-dichlorobenzylisothioureas) showed only weak cytotoxic activity. Apparently, the introduction of a bulky substituent (e.g., phenyl or benzyl group) at one of the nitrogen atoms considerably reduces cytotoxicity of pentabromobenzylisothioureas (data not shown). As we previously reported, modified benzylisothioureas are also inhibitors of the Ca2+/calmodulin-dependent NO synthase (Kazimierczuk et al., 2010). The role of NO in cancer initation and progression is still debated and it is not yet decided whether

NO should be considered as a potential anticancer agent or instead a carcinogen (Mocellin, 2009). When comparing the NOS inhibitory Epothilone B (EPO906, Patupilone) activity and anticancer activity of other tested benzylisothioureas, we did not find a straightforward correlation between these attributes (data not shown). ZKKs showed considerable cytotoxic and cytostatic effects in both HL-60 (human promyleocytic leukemia) and K-562 (human chronic erythromyeloblastoid leukemia) cells. Proapoptotic effects were higher in HL-60 than in K-562 cells. Apoptotic death was associated with increased depolarization of the mitochondrial membrane and with increase in the level of 85 kDa fragments of PARP protein. The latter effect is an indirect measure of activation of the effector caspase-3 and caspase-7 that proteolytically cleave native 116 kDa PARP protein into 85 and 25 kDa fragments.