2) The observed

2). The observed apoptotic effect was dose-and time-dependent. ZKK-3 [(N,N′-dimethyl-S-2,3,4,5,6-pentabromobenzyl)isothiouronium bromide] was the most effective in HL-60 cell line, whereas ZKK-2 [N-methyl-S-(2,3,4,5,6-pentabromobenzyl)isothiouronium bromide] showed the most potent cytotoxic apoptotic effect in K-562 cells. Fluorescence microscopy showed typical concentrating chromatin and apoptotic bodies’ formation (Fig. 3). Fig. 2 Induction of apoptosis by ZKKs in HL-60 cells (a) and K-562 cells (b). The data were determined by FACS cytometer after

24 and 48 h treatment. Veliparib research buy Cells were stained with annexin V-FITC and PI. Each bar represents the mean ± SD (n ≥ 4) Fig. 3 Morphology (fluorescence microscopy employing DAPI/sulforhodamine 101 labeling) of HL-60 cells cultured for 48 h in the absence (control, a)

and presence of ZKK-3 (8 μM, b). Arrows indicate apoptotic nuclei Changes in mitochondrial FRAX597 purchase membrane potential (ΔΨm) Analysis of the respective cytograms (for a representative cytogram see Fig. 4) showed that the tested compounds caused mitochondrial membrane depolarization (as evidenced by increased green-to-red fluorescence intensity ratio) in both cell lines studied. Fig. 4 Representative flow cytograms demonstrating changes in mitochondrial membrane potential (ΔΨm) of HL-60 cells (upper panels) and K-562 cells (lower panels) induced by 48 h culturing with various ZKKs compounds. Anlotinib mouse The cells were stained with JC-1 dye. The cells in the lower right (R3) quadrant showed increased red-to-green fluorescence ratio (apoptotic cells) ZKKs-induced cleavage of PARP protein The enhancement of apoptosis was confirmed by detecting PARP cleavage after 48 h incubation with the tested

compounds. During ZKKs-induced Ureohydrolase apoptosis, the presence of 85 kDa PARP fragments was revealed in both cell lines with the use of specific antibody (Fig. 5). Fig. 5 Effect of ZKKs on proteolytic cleavage of PARP protein in cells were exposed for 48 h to ZKKs. Representative histograms showing increased level of 85 kDa fragment of PARP protein indicating induction of apoptosis after ZKKs treatment. a: Histogram of HL-60 control cells and overlay histogram of treated cells at 8 μM ZKK-3. b: Histogram of K-562 control cells and overlay histogram of treated cells at 10 μM ZKK-2. Marker M1 designates negative cell populations whereas M2 designates positive cell populations (indicate apoptosis). Thin line control cells, thick line ZKK-treated cells Effect of ZKKs on cell cycle progression Figures 6a, b, and 7 demonstrate changes in the cell cycle progression of HL-60 and K-562 cells after 48 h incubation with the tested compounds.

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