The calculated IC50 value in this cell line for compound 2 is greater than 50 μM, for compound 5 it is 9.7 μM
and for compound 6 it is 9.1 μM. (B) Sensitivity of urothelial cancer cell lines and one representative normal uroepithelial control to compound 5 and compound 6 after 72 h of treatment. Y-27632 chemical structure The IC50 of compound 2 was only reached at concentrations near 50 μM. The cell lines outlined by bold letters were used for the functional experiments. While c5 and c6 significantly reduced the viability of all UCCs, their effect varied among the cell lines. It is noticeable that cells with an epithelial phenotype e.g. RT-112 were more sensitive than cells with a mesenchymal phenotype (SW-1710 and UM-UC-3; Figure 5B). The influence of the inhibitors on clonogenic growth after a 72 h treatment at the determined IC50 concentrations is illustrated in Figure 6. Compound 2
inhibited clonogenicity only in VM-CUB1 cells. Treatment with compound 5 resulted in a moderate reduction of colony numbers in RT-112, UM-UC-3 and 639-V cells, whereas in VM-CUB1 cells, clonogenic growth was completely abolished. In contrast, c5 had no effect on SW-1710 cells. Compound 6 was active in all cell lines, being most efficient in VM-CUB1, UM-UC-3 and 639-V cells. Figure 6 Effect of HDAC8 specific Raf phosphorylation inhibitor treatment on clonogenic growth of urothelial cancer cells. Giemsa-staining of grown colonies from
Acyl CoA dehydrogenase inhibitor treated RT-112, VM-CUB1, SW-1710, 639-V and UM-UC-3 cells is compared to DMSO solvent control (compound 2, compound 5, compound 6; IC50, 72 h). As the effect of pharmacological HDAC8 inhibition was stronger than the effect of HDAC8 knock-down, wound healing assays of UCCs after HDAC8 inhibitor treatment were additionally performed (Figure 7A). A clear difference was observed in VM-CUB1 and UM-UC-3 cells, respectively, comparing DMSO controls to cells treated with c5 and c6, especially after 6 – 12 h (Figure 7B). Figure 7 Migration assay of urothelial cancer cells after HDAC8 inhibitor treatment. (A) Representative photographs of wound healing assay at 0 and 12 hours from inhibitor treated RT-112, VM-CUB1, SW-1710, 639-V and UM-UC-3 cells (compound 2, compound 5, compound 6; IC50, 72 h) in comparison to a DMSO solvent control (co). (B) Relative scratch size after 3, 6, 9 and 12 h of migration in comparison to the starting point 0 h. The relative scratch size is displayed on the y-axis. p < 0.05 was regarded as significant and marked as *, whereas p < 0.01 and p < 0.001 were defined as highly significant and marked as ** and ***. The calculated significances refer to the DMSO solvent control. The impact of the HDAC8 inhibitor treatment was further analyzed by western blot analysis of different target proteins (Figure 8).