Cell viability was also evaluated through the measurement of mitochondrial dehydrogenase activity using the colorimetric WST-1 assay (Figure 1B). Data confirmed that CF treatment Nutlin 3a induced cell viability PCI-32765 in vitro inhibition up-and-over 60% in U937 cells after 72 h of incubation. To investigate the selectivity of CF treatment towards tumor cells, human healthy lymphocytes were seeded in the presence of the same concentration of CF up to 96 h; data revealed no significant differences between untreated
and treated cells, confirming that CF did not affect healthy lymphocyte growth (Figure 2). Figure 1 Significant inhibition of leukemia cell proliferation (A) and viability (B) after 24, 48, and 72 h of incubation with CF in comparison with untreated cells (control), as evaluated by cell counting by Elacridar nmr trypan blue dye exclusion and WST-1 reagent, respectively. Data are expressed as mean ± SD of at least three independent experiments. *p < 0.05 vs. untreated cells. Figure 2 Lymphocyte cell growth in the presence of CF (5 μl/ml) in comparison with untreated cells (control). No effects were observed up to 96 h after CF administration to isolated lymphocytes as a non-tumor cell system Data are expressed as mean ± SD of at least three independent experiments. These results are in accordance with the growth-inhibitory properties
of Lithothamnion calcareum, the red algae from which the organic and inorganic components of CF are extracted [19, 20]. Indeed, the mineral-rich material derived from the algae has been shown to suppress the growth of a series of human colon cancer cell lines in vitro, as well as to protect mice against neoplastic and preneoplastic proliferative liver lesions . To clarify whether CF was able to reduce cancer cell viability by promoting apoptotic cell death, two classical
Thiamine-diphosphate kinase markers of apoptosis were determined. Caspase-3 is considered to be the most important effector of apoptosis and a marker for both intrinsic and extrinsic pathways . Noteworthy, we evidenced that CF treatment significantly stimulated caspase-3 activity in the three leukemia cell lines as compared to the respective untreated controls (Figure 3). Figure 3 Significant increment of caspase-3 activity in leukemia cells after 24, 48, and 72 h of incubation with CF (5 μl/ml) in comparison with untreated cells (control). Data are expressed as mean ± SD of at least three independent experiments. *p < 0.05 vs. untreated cells. On the other hand, the detection of the internucleosomal DNA cleavage (or DNA laddering) is a common hallmark of cells undergoing late-stage apoptosis . To verify if CF could induce DNA fragmentation and thus to confirm whether apoptosis occurred, leukemia cells exposed to CF treatment were assessed for DNA laddering by agarose gel electrophoresis (Figure 4).