Statistical analysis was done by one way analysis of variance (ANOVA) followed by a comparative LSD

test (Least Significant Difference). Results were considered significant when p < 0.05. Results Cytotoxicity of PD166866 on HeLa cells in culture We explored the dose/response effect of HeLa cells exposed to Pifithrin�� a relatively broad range of PD166866 concentrations (0.1 – 50 μM). Cells were treated for 24 hours with the drug and their vitality assessed by the MTT assay [12]. A significant reduction of vital cells can be monitored already at 2.5 μM concentration (Figure 1, left panel). The loss of viability seems to stabilize at 25 μM (about 25% survival) with no further decrease at a 50 μM concentration of drug. This result may indicate the presence of a cell subpopulation, intrinsically resistant to the drug. This result was confirmed by vital cell count with trypan blue (only the data obtained at 2.5 μM of drug is shown; Figure 1, right panel). check details The negative effect of PD166866 on the cell growth was already observed in a previous works performed on 3T6 cells: a stabilized murine fibroblast line [10, 11]. The results presented here validate those already published and, as far as cell survival

is concerning, no difference can be monitored on HeLa in comparison to 3T6 cells in matching experiments also run in this work (not shown). Interestingly, as observed in a former study, HeLa cells showed a significantly higher sensitivity than murine cells towards resveratrol, a natural product showing both cytotoxic and antiviral properties [16]. One way to rationalize this data is that the cellular/molecular target of the two drugs could be different. Figure 1 Assessment of cell survival after treatment with PD166866. Cells were treated with PD166866 for 24 hours at the indicated concentrations. At the end of the treatment, the samples were subjected to the Mossman assay (right 3-oxoacyl-(acyl-carrier-protein) reductase panel). Alternatively after treatment cells were stained with trypan blue according to standard laboratory procedures (left panel). In this latter case only the survival at 2.5

μM is reported. The Mosmann assay [12] indicates membrane damage, essentially at mitochondrion level. Therefore, we investigated the possibility that PD166866 may be detrimental to the membrane integrity by lipoperoxidation assays [13]. Lipoperoxidation shows that PD166866 causes membrane damage The lipoperoxidation assay is a very powerful tool to evaluate in a quantitative manner the membrane damage deriving from phenomena of oxidative stress. The formation of poly-unsaturated acids, consequent to this stress, causes the formation malonyl-dihaldeyde (MDA) and of 4-hydroxyhalkenals. The concentration of intracellular MDA, a compound normally not found in the cytoplasm, is correlated directly to the extent of the membrane damage [13].