Curcumin induces DNA fragmentation and cell death in K562 cells.

<p><b>A)</b> A fraction of the arrested K562 cells treated with 20 μM of curcumin showed DNA damage as indicated by the presence of TUNEL-positive cells and FACS analysis. <b>B)</b> The percentage of TUNEL-positive cells is represented by graphic bars, and <b>C)</b> production of a typical apoptotic DNA ladder is shown. <b>D)</b> Cell death was confirmed by death assays; FACS analysis, and <b>E)</b> the results are also shown as graphic bars. As positive controls of DNA fragmentation, we used cells treated with 100 nM nocodazole for 24 h. As a positive control of cell death, K562 cells were exposed to UV (40 mJ/cm²) for 2 min in a cross-linker GS Gene linker-UV chamber (Bio-Rad, Hercules CA, USA) and were recovered 24 h post-irradiation.</p

Schematic representation of the mitotic catastrophe and P73-dependent apoptosis mechanisms triggered by curcumin treatment in the K562 cells.

<p>Schematic representation of the mitotic catastrophe and P73-dependent apoptosis mechanisms triggered by curcumin treatment in the K562 cells.</p

Mitotic spindles in K562 cells were altered by curcumin.

<p><b>A</b>) Expression levels of phosphorylsted-histone 3 in serine 10 (p-H3S10) were increased in K562 cells after curcumin treatment, as determined by western blot and <b>B</b>) FACS analysis. <b>C</b>) The relative numbers of p-H3S10-positive cells are presented as a histogram. K562 cells were stained with DAPI or immunostained using an antibody against α-tubulin (green fluorescence), and the cells with defects in the mitotic spindles were counted. <b>D</b>) The percentage of cells with monopolar (white bars), bipolar (black bars) and multipolar (gray bars) nuclei were determined in K562 cells. <b>E</b>) Representative fluorescence images of K562 cells bearing mitotic spindle alterations after 12 h, 18 h and 24 h of curcumin treatment are shown. <b>F</b>) Images of the most representative mitotic spindle alterations are shown. Actin was used as loading control.</p

Curcumin induces apoptotic cell death in mitotic K562 cells.

<p><b>A)</b> A fraction of the K562 cells arrested with 20 μM of curcumin underwent caspase-dependent apoptosis as revealed by the presence of active caspase-3 in the K562 cells positive for the mitosis p-H3S10 marker and FACS analysis. <b>B)</b> The relative numbers of p-H3S10 positive cells with caspase-3 activity are presented as a histogram. <b>C)</b> Representative images of the K562 curcumin-treated cells positive for caspase-3 activity using confocal microscopy. <b>D)</b> Processed caspases-8, -9 and -3 were detected by western blot analysis and <b>E)</b> pro-caspases quantification. Activity was confirmed by cleavage of the caspase-3 substrate PARP, and its protein fragment of 89 kDa was detected. As positive controls for p-H3S10 and caspase activities, K562 cells were exposed for 24h to 100 nM Nocodazole (Noc) or 1 μM Staurosporine (Staur), respectively; M = medium, D = DMSO.</p

K562 cells treated with curcumin were arrested in G2/M2 phase.

<p><b>A)</b> A subpopulation of the curcumin-treated K562 cells was arrested in G2/M phase, as determined by FACS analysis. Cells were stained with DAPI or immunostained with rhodamine-phalloidin (red fluorescence) to visualize the nuclei and actin fibers, respectively. <b>B</b>) Subsequently, they were analyzed by immunofluorescence microscopy, and the percentage of giant or multinucleated cells was determined. <b>C</b>) The amplification of these cells clearly showed the condensed chromatin and the actin cytoskeleton changes due to early mitotic events. Condensed nuclei were considered an indirect measure of mitotic cells. As a positive control for the enriched G2/M population, K562 cells were treated for 24 h with 100 nM nocodazole (Noc). IN = interphase nucleus; mN = multinucleated cell.</p

Apoptosis is produced by activation of P73.

<p><b>A)</b> Impairment of the expression of the anti-apoptotic proteins BCL-2 and XIAP is shown. <b>B</b>) The decreased expression of the anti-apoptotic proteins was caspase independent, as shown by the use of Z-VAD-FMK pancaspase inhibitor. Samples were treated with 2 pulses of 40 μM of pan-caspase inhibitor, Z-VAD-FMK (R&D, Systems), to 12 and 15 h of curcumin treatment. K562 cells were harvested after 18 h, lysed and total protein extracts were obtained for western blot analysis, using specific antibodies against of Caspase-3, PARP, BCL-2 and XIAP. <b>C</b>) The diminution of the expression of the antiapoptotic proteins was consistent with decreases in the IκBα and <b>D</b>) activation and nuclear translocation of the P73 protein, as consequence of the <b>E)</b> diminished expression of the P73 promoter repressor protein C/EBPα; M = medium, D = DMSO.</p