(B) Giemsa-staining of colonies from irrelevant siRNA and HDAC8 siRNA transfected RT-112, VM-CUB1, SW-1710, 639-V and UM-UC-3 cells compared to an untreated control (72 h). To characterize the effect of the HDAC8 knockdown on UCCs, we investigated downstream targets of HDAC8 known

from other cancers: the proliferation marker thymidylate synthase (TS), cleavage of PARP and expression of p21. In addition, we examined the acetylation status of α-tubulin to estimate the specificity of the HDAC8 treatment (Figure 4). The expression of TS 72 h after HDAC8 knockdown was only slightly reduced in SW-1710, 639-V and UM-UC-3 cells. In RT-112 and VM-CUB1 cells no effects were observed. Effects on cleavage of PARP could only be detected in UM-UC-3 cells after HDAC8 knockdown. There a decrease can be observed. The expression level of p21 indicates a decreased expression in comparison to irrelevant control in the cell lines RT-112, VM-CUB1,

Kinase Inhibitor Library 639-V and UM-UC-3 after HDAC8 knockdown. In the cell line SW-1710 no altered p21 expression could anti-PD-1 antibody be observed. An increase of acetylated α-tubulin could be detected in all cell lines after HDAC8 siRNA transfection (Figure 4). Figure 4 Effects of siRNA mediated HDAC8 knockdown on target proteins. PARP, p21, acetylated α-tubulin and thymidylate synthase (TS) protein expression levels subsequent to HDAC8 knockdown were determined by western blot analysis in comparison to a irrelevant control in the UCCs RT-112, VM-CUB1, SW-1710, 639-V and UM-UC-3 (72 h). As a loading control α-tubulin 3-mercaptopyruvate sulfurtransferase was stained on each blot. Effects of HDAC8 specific hydroxamic acid inhibitors on urothelial carcinoma cells Based on the observation that the HDAC8 knockdown inhibited proliferation of urothelial carcinoma cells we investigated the sensitivity of several UCCs to three different HDAC8 inhibitors [41]. The treatment with the HDAC8 selective small molecule inhibitors c2, c5 and c6 inhibited the cell proliferation of all UCCs in a concentration dependent manner, with stronger effects of the higher affinity compounds c5

and c6 (Table 1). The three dose response curves for the cell line RT-112 in Figure 5A show a low sensitivity for c2 with a calculated IC50 value greater than 50 μM and a higher sensitivity for c5 and c6 with an IC50 value of about 9.7 μM and 9.1 μM. Table 1 Stated are IC 50 values after 72 h of HDAC8 inhibitor treatment in eight urothelial cancer cell lines and a representative normal uroepithelial control   IC 50 [μM] Compound 2 Compound 5 Compound 6 VM-CUB1   ≥ 50 18.7 16 SW-1710   ≥ 50 20.8 18.8 RT-112   ≥ 50 9.7 9.1 639-V   ≥ 50 12.6 18.6 UM-UC-3   ≥ 50 18.9 18.2 Normal Uroepithelial Control   ≥ 50 24.2 10.2 Figure 5 Dose-dependent effects of three different HDAC8 specific inhibitors on viability of urothelial cancer cell lines. (A) Several urothelial cancer cell lines were treated with different concentrations of HDAC8 inhibitors.