The

inhibitors c5 and c6 significantly reduced the viability of all UCCs, with half inhibitory concentrations between 9 and 20.8 μM. These differences follow the order of the affinity of the inhibitors for HDAC8 in vitro [41]. Though in vitro affinity of c5 and c6 is 20 – 50 fold higher compared to c2, in vivo effects on UCC were not as strong as expected. Focusing on morphological features of UCCs, the data suggested that cells Eltanexor datasheet with an epithelial phenotype and low HDAC8 expression are more sensitive towards pharmacological inhibition of HDAC8 with c5 and c6 compared to cells with a mesenchymal phenotype. Specifically, SW-1710 cells (mesenchymal, elevated HDAC8 expression) were least sensitive to the inhibitors c5 and c6 while RT112 cells (epithelial, lowest HDAC8 expression) responded to AZD1080 treatment with c5 and c6 already at low concentrations. As recently shown in endometrial stroma sarcoma cells, HDAC inhibition may be counteracted by increased activity of the PI3K pathway in PTEN-deficient cells [45]. In our cell line panel, UM-UC-3 are PTEN-deficient, resulting in increased PI3K activity. However, this cell line was not 3-MA price exceptionally resistant either in our previous study using pan-HDAC inhibition [39] or in the present study with HDAC8-specific inhibitors. Accordingly, at least in urothelial cancer, PTEN deficiency does not seem to

have a decisive impact on the efficacy of HDAC inhibitors. Effects of siRNA mediated downregulation and pharmacological inhibition on urothelial cancer cell lines were not thoroughly consistent. Differences might be explained by several factors. For example, knockdown depletes the protein thereby not only affecting enzymatic but also other protein functions for example complex assembly. Inhibitor treatment ideally only suppresses the enzymatic activity while further protein functions should not be affected. Accordingly, also compensatory

mechanisms might be different in both conditions. Comparing expression levels of further class I HDACs after knockdown of HDAC8 as well as after pharmacological inhibition, only minor changes were observed. Although upregulation of HDAC1 or HDAC2 was a little more consistently observed after HDAC8 Adenosine triphosphate knockdown, they can hardly explain the difference between knockdown and inhibition by c5 or c6. More likely, the stronger effects of the inhibitors may be due to inhibition of other targets in addition to HDAC8. Neither HDAC8 knockdown nor pharmacological treatment with any compound (except the SAHA control) led to a change in histone H3 or H4 acetylation, a widely used surrogate marker for intracellular HDAC inhibition. This finding suggests that HDAC8, as expected, does not substantially affect overall histone acetylation. In addition, this does also indicate that inhibitor treatment seems to be iso-enzyme specific as other class I HDACs seemed to be not affected.