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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.

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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. Here, the dose-response curves of the UCC RT-112 are shown for compound 2, compound 5 and compound 6 after 72 h inhibitor treatment as measured by ATP-assay (■ compound 2; ● compound 5; ▲ compound 6). 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. 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.
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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. Here, the dose-response curves of the UCC RT-112 are shown for compound 2, compound 5 and compound 6 after 72 h inhibitor treatment as measured by ATP-assay (■ compound 2; ● compound 5; ▲ compound 6). 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. 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.

Mentions: 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.


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)

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. Here, the dose-response curves of the UCC RT-112 are shown for compound 2, compound 5 and compound 6 after 72 h inhibitor treatment as measured by ATP-assay (■ compound 2; ● compound 5; ▲ compound 6). 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. 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.
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Related In: Results  -  Collection

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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. Here, the dose-response curves of the UCC RT-112 are shown for compound 2, compound 5 and compound 6 after 72 h inhibitor treatment as measured by ATP-assay (■ compound 2; ● compound 5; ▲ compound 6). 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. 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.
Mentions: 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.

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