Limits...
Histone deacetylase 8 is deregulated in urothelial cancer but not a target for efficient treatment.

Lehmann M, Hoffmann MJ, Koch A, Ulrich SM, Schulz WA, Niegisch G - J. Exp. Clin. Cancer Res. (2014)

Bottom Line: Efficient siRNA-mediated knockdown of HDAC8 reduced proliferation up to 45%.Expression of thymidylate synthase was partly reduced; PARP-cleavage was not detected.The influence of the pharmacological inhibition on clonogenic growth and migration show a cell line- and inhibitor-dependent reduction with the strongest effects after treatment with compound 5 and compound 6.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Previous studies have shown that class-I histone deacetylase (HDAC) 8 mRNA is upregulated in urothelial cancer tissues and urothelial cancer cell lines compared to benign controls. Using urothelial cancer cell lines we evaluated whether specific targeting of HDAC8 might be a therapeutic option in bladder cancer treatment.

Methods: We conducted siRNA-mediated knockdown and specific pharmacological inhibition of HDAC8 with the three different inhibitors compound 2, compound 5, and compound 6 in several urothelial carcinoma cell lines with distinct HDAC8 expression profiles. Levels of HDAC and marker proteins were determined by western blot analysis and mRNA levels were measured by quantitative real-time PCR. Cellular effects of HDAC8 suppression were analyzed by ATP assay, flow cytometry, colony forming assay and migration assay.

Results: Efficient siRNA-mediated knockdown of HDAC8 reduced proliferation up to 45%. The HDAC8 specific inhibitors compound 5 and compound 6 significantly reduced viability of all urothelial cancer cell lines (IC₅₀ 9 - 21 μM). Flow cytometry revealed only a slight increase in the sub-G1 fraction indicating a limited induction of apoptosis. Expression of thymidylate synthase was partly reduced; PARP-cleavage was not detected. The influence of the pharmacological inhibition on clonogenic growth and migration show a cell line- and inhibitor-dependent reduction with the strongest effects after treatment with compound 5 and compound 6.

Conclusions: Deregulation of HDAC8 is frequent in urothelial cancer, but neither specific pharmacological inhibition nor siRNA-mediated knockdown of HDAC8 impaired viability of urothelial cancer cell lines in a therapeutic useful manner. Accordingly, HDAC8 on its own is not a promising drug target in bladder cancer.

Show MeSH

Related in: MedlinePlus

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 was stained on each blot.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230422&req=5

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 was stained on each blot.

Mentions: 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, 639-V and UM-UC-3 after HDAC8 knockdown. In the cell line SW-1710 no altered p21 expression could be observed. An increase of acetylated α-tubulin could be detected in all cell lines after HDAC8 siRNA transfection (Figure 4).


Histone deacetylase 8 is deregulated in urothelial cancer but not a target for efficient treatment.

Lehmann M, Hoffmann MJ, Koch A, Ulrich SM, Schulz WA, Niegisch G - J. Exp. Clin. Cancer Res. (2014)

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 was stained on each blot.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230422&req=5

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 was stained on each blot.
Mentions: 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, 639-V and UM-UC-3 after HDAC8 knockdown. In the cell line SW-1710 no altered p21 expression could be observed. An increase of acetylated α-tubulin could be detected in all cell lines after HDAC8 siRNA transfection (Figure 4).

Bottom Line: Efficient siRNA-mediated knockdown of HDAC8 reduced proliferation up to 45%.Expression of thymidylate synthase was partly reduced; PARP-cleavage was not detected.The influence of the pharmacological inhibition on clonogenic growth and migration show a cell line- and inhibitor-dependent reduction with the strongest effects after treatment with compound 5 and compound 6.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Previous studies have shown that class-I histone deacetylase (HDAC) 8 mRNA is upregulated in urothelial cancer tissues and urothelial cancer cell lines compared to benign controls. Using urothelial cancer cell lines we evaluated whether specific targeting of HDAC8 might be a therapeutic option in bladder cancer treatment.

Methods: We conducted siRNA-mediated knockdown and specific pharmacological inhibition of HDAC8 with the three different inhibitors compound 2, compound 5, and compound 6 in several urothelial carcinoma cell lines with distinct HDAC8 expression profiles. Levels of HDAC and marker proteins were determined by western blot analysis and mRNA levels were measured by quantitative real-time PCR. Cellular effects of HDAC8 suppression were analyzed by ATP assay, flow cytometry, colony forming assay and migration assay.

Results: Efficient siRNA-mediated knockdown of HDAC8 reduced proliferation up to 45%. The HDAC8 specific inhibitors compound 5 and compound 6 significantly reduced viability of all urothelial cancer cell lines (IC₅₀ 9 - 21 μM). Flow cytometry revealed only a slight increase in the sub-G1 fraction indicating a limited induction of apoptosis. Expression of thymidylate synthase was partly reduced; PARP-cleavage was not detected. The influence of the pharmacological inhibition on clonogenic growth and migration show a cell line- and inhibitor-dependent reduction with the strongest effects after treatment with compound 5 and compound 6.

Conclusions: Deregulation of HDAC8 is frequent in urothelial cancer, but neither specific pharmacological inhibition nor siRNA-mediated knockdown of HDAC8 impaired viability of urothelial cancer cell lines in a therapeutic useful manner. Accordingly, HDAC8 on its own is not a promising drug target in bladder cancer.

Show MeSH
Related in: MedlinePlus