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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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Effects of HDAC8 knockdown and HDAC8 inhibitor treatment on cell cycle distribution. Changes in cell cycle distribution and amount of apoptotic cells (as sub-G1 fraction) after (A) siRNA mediated HDAC8 knockdown (72 h) and (B) HDAC8 inhibitor treatment (compound 2, compound 5, compound 6; IC50, 72 h) were measured by cell cycle analysis using flow cytometry. DMSO served as a solvent control. The relative distribution of the fractions is displayed on the y-axis.
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Figure 9: Effects of HDAC8 knockdown and HDAC8 inhibitor treatment on cell cycle distribution. Changes in cell cycle distribution and amount of apoptotic cells (as sub-G1 fraction) after (A) siRNA mediated HDAC8 knockdown (72 h) and (B) HDAC8 inhibitor treatment (compound 2, compound 5, compound 6; IC50, 72 h) were measured by cell cycle analysis using flow cytometry. DMSO served as a solvent control. The relative distribution of the fractions is displayed on the y-axis.

Mentions: To further characterize the impact of HDAC8 on cell cycle distribution UCCs were analyzed by flow cytometry after either knockdown or inhibitor treatment (Figure 9). Knockdown of HDAC8 resulted in a significant shift in cell cycle distribution only in SW-1710 cells, showing an S-phase-decrease. In the other UCCs no significant changes were observed (Figure 9A). In contrast, pharmacological inhibition of HDAC8 by c5 and c6 resulted in a significant increase of the sub-G1 fraction in the UCCs VM-CUB1 and SW-1710 and a significant decrease of the G1-fraction in VM-CUB1, SW-1710, 639-V and UM-UC-3 cells (Figure 9B). Further, indications of a G2/M-arrest were observed after c5 and c6 treatment in VM-CUB1, SW-1710, 639-V and UM-UC-3 cells.


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 HDAC8 knockdown and HDAC8 inhibitor treatment on cell cycle distribution. Changes in cell cycle distribution and amount of apoptotic cells (as sub-G1 fraction) after (A) siRNA mediated HDAC8 knockdown (72 h) and (B) HDAC8 inhibitor treatment (compound 2, compound 5, compound 6; IC50, 72 h) were measured by cell cycle analysis using flow cytometry. DMSO served as a solvent control. The relative distribution of the fractions is displayed on the y-axis.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230422&req=5

Figure 9: Effects of HDAC8 knockdown and HDAC8 inhibitor treatment on cell cycle distribution. Changes in cell cycle distribution and amount of apoptotic cells (as sub-G1 fraction) after (A) siRNA mediated HDAC8 knockdown (72 h) and (B) HDAC8 inhibitor treatment (compound 2, compound 5, compound 6; IC50, 72 h) were measured by cell cycle analysis using flow cytometry. DMSO served as a solvent control. The relative distribution of the fractions is displayed on the y-axis.
Mentions: To further characterize the impact of HDAC8 on cell cycle distribution UCCs were analyzed by flow cytometry after either knockdown or inhibitor treatment (Figure 9). Knockdown of HDAC8 resulted in a significant shift in cell cycle distribution only in SW-1710 cells, showing an S-phase-decrease. In the other UCCs no significant changes were observed (Figure 9A). In contrast, pharmacological inhibition of HDAC8 by c5 and c6 resulted in a significant increase of the sub-G1 fraction in the UCCs VM-CUB1 and SW-1710 and a significant decrease of the G1-fraction in VM-CUB1, SW-1710, 639-V and UM-UC-3 cells (Figure 9B). Further, indications of a G2/M-arrest were observed after c5 and c6 treatment in VM-CUB1, SW-1710, 639-V and UM-UC-3 cells.

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