Subsequently, A375DR cells were maintained in culture with 5 M dabrafenib for six months. effects, ONC reduced nuclear p65 NF-B amount and IKK phosphorylation level, as well as MMP2 activity in both cell subpopulations. ONC decreased cell colony formation, migration, and invasion capability. Notably, it induced apoptosis and inhibited colony formation and invasiveness more extensively in A375DR than in A375P cells. In conclusion, ONC successfully counteracts melanoma malignancy especially in BRAFi-resistant cells and could become a tool against melanoma recurrence. = 0.02, = 0.0002, = LY2157299 0.00008, = 0.0001, = 0.0004, and PPARG1 = 0.0001, for 1, 2, 5, 10, 20, and 50 nM dabrafenib, respectively). Open in a separate window Physique 1 Effect of dabrafenib or onconase (ONC) around the viability of melanoma A375 and of normal human epidermal melanocytes (NHEM) cells. (a) A375P (blue dots) and A375DR (red dots) cell viability detected after 72 h incubation with increasing concentrations of dabrafenib. For each dabrafenib concentration tested (panel a), all A375P versus A375DR comparisons are statistically significant (see text). (b) cell viability of A375P (blue dots), A375DR (red dots), and NHEM (cyan dots) after 72 h incubation with increasing concentrations of ONC. Statistically significant LY2157299 differences are present (< 0.0001) between NHEM versus A375P or A375DR cells, either at LY2157299 0.5 or 1 M ONC, while not between the two A375 cell subpopulations at all ONC concentrations tested. All values reported are the average of four to five impartial experiments, each performed in six replicates, S.D. In agreement with a recent paper published by our group [21], low ONC concentrations strongly reduced the viability of A375P cells (Physique 1b). In the present work we compare, instead, the effect of ONC registered on parental versus dabrafenib-resistant subpopulations of the same cell line. Figure 1b shows that the viability of both cell subpopulations is usually reduced to a similar extent, and in a dose-dependent manner, after a 72 h culture with ONC, with calculated IC50 values of 0.40 and 0.32 M for A375P and A375DR cells, respectively. No statistically significant differences in the sensitivity to ONC emerged within the two cell subpopulations, although the mean viability reduction of A375DR cells was lower than that of parental ones for each concentration tested (Physique 1b). 2.2. ONC Does not Affect Cell Viability of Normal Melanocytes To evaluate the specificity of ONC activity against melanoma cells, we also measured the sensitivity of normal human epidermal melanocytes (NHEM) to this RNase variant. LY2157299 NHEM cells were incubated for 72 h with the two ONC concentrations that were the most effective against malignant cells (0.5, 1 M), and also with 2 and 4 M ONC (Determine 1b). By the crystal violet assay, we found no reduction in cell viability either at 0.5 or 1 M ONC concentration (Determine 1b, cyan dots; NHEM versus A375P, = 0.00004 and = 0.00002 for 0.5 and 1 M ONC, respectively). Moreover, the maximal ONC dose (4 M), tested exclusively in the NHEM cells, reduced their viability only by 14%. Hence, we conclude that ONC displays quite high cytostatic and cytotoxic effects only in melanoma cells, while not doing so in normal melanocytes. 2.3. ONC Decreases the Proliferation Rate of both A375P and A375DR Cell Subpopulations We performed a 5-Br-2-deoxyuridine (BrdU) incorporation assay to identify whether in both cell subpopulations the viability reduction elicited by ONC might depend around the cell proliferation rate or, instead, on a cell mass decrease consequent to cell death. After 24, 48, and 72 h culture with ONC, an additional 4 h BrdU incubation showed a concentration-dependent reduction of its incorporation in both A375P and A375DR cells. Nevertheless, ONC-treated A375DR cells showed a smaller reduction of BrdU incorporation level than A375P ones, as is clearly visible in Physique 2aCc. In these panels, data have been normalized to each parental or dabrafenib-resistant ONC-free control. All time-point differences emerging by comparing the two ONC-treated cell subpopulations are statistically significant), except for 1 M ONC at 72 h (A375P versus A375DR for 0.5 and 1 M ONC, respectively: 24 h, = 0.05, = 0.04;.