The MTT viability assay showed that HU 100-V decreased the viability of most of the cell lines tested in a time- and dose-dependent manner for which they are achieved good values of IC50 (concentration inhibiting 50% of growth). Especially, prostate cancer DU-145, pancreas cancer BX-PC3 [26, 27], renal cancer RXF393 and glioblastoma cancer LN229 cells have proved to be the most

sensitive to this treatment, with IC50 values of less than 20 micromolar (Table 3 and Figure 2). Figure 2 Dose–response curves from the treatment of different cell lines with the molecule HU-100-V with an IC50 between Fludarabine supplier more less than 20 μM. Apoptotic cell death To ascertain whether loss of cell viability was mediated by effects on apoptosis we directly analyzed the effects of either V or HU-331 on apoptosis of M14 cells by using PI-staining of DNA fragmentation after cell permeabilization. Cells were treated with different concentrations (1–10 μM) of V and HU-331 for 24 and 72 hours and then the population of sub-G1 cells (hyplodiploid nuclei) was determined. Compound V induced apoptosis of M14 cells in a concentration-dependent manner with 40% of cell death at 10 μM after 72 h, whereas a small pro-apoptotic effect was observed with 10 μM HU-331 (Figure 3). These results showed that the cytotoxic effect check details of V is dependent

by an apoptotic mechanism that is more significant than HU-331 effect on M14 cells. Figure 3 Effects of HU compounds on apoptosis of human melanoma

M14 cells. Analysis of the % of apoptotic cells was performed using PI cell permeabilization staining. Rutecarpine M14 cells were treated with different concentrations of HU-331 and V (1–10 μM) for 24–72 h. Cells were then collected and % of hypodiploid nuclei was analyzed by flow cytometry (*** P < 0.001 vs 72 h control cells; ° P < 0.05, °°° P < 0.001 vs 24 h control cells). Results are expressed as mean ± SEM of three experiments performed in triplicate. Caspases involvement To investigate the involvement of caspases in the mechanism of apoptosis induced by compounds, we pretreated the cells with a pan-caspase inhibitor Z-VAD-fmk for 30 min before to add V and HU-331. Results in Figure 4 show that apoptosis induced by V in presence of the inhibitor was significantly reduced indicating the involvement of caspases in the apoptotic mechanism in M14 cells. Figure 4 Effects of the caspase inhibitor Z-VAD-FMK on apoptosis induced by HU331 and V in human melanoma M14 cells. Z-VAD-FMK (30 μM) was administered 30 min before incubation with HU-331 and V (10 μM) for 72 h and the % of apoptotic cell was evaluated by flow cytometry (mean ± SEM of three experiment performed in triplicate; ***P < 0.001 vs control cells, §§§ P < 0.001 HU331 vs V treated cells. Cell cycle analyses The cell cycle is divided into four phases, i.e. sub-G1, G1, S and G2.