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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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Migration assay of urothelial cancer cells after HDAC8 inhibitor treatment. (A) Representative photographs of wound healing assay at 0 and 12 hours from inhibitor treated RT-112, VM-CUB1, SW-1710, 639-V and UM-UC-3 cells (compound 2, compound 5, compound 6; IC50, 72 h) in comparison to a DMSO solvent control (co). (B) Relative scratch size after 3, 6, 9 and 12 h of migration in comparison to the starting point 0 h. The relative scratch size is displayed on the y-axis. p < 0.05 was regarded as significant and marked as *, whereas p < 0.01 and p < 0.001 were defined as highly significant and marked as ** and ***. The calculated significances refer to the DMSO solvent control.
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Figure 7: Migration assay of urothelial cancer cells after HDAC8 inhibitor treatment. (A) Representative photographs of wound healing assay at 0 and 12 hours from inhibitor treated RT-112, VM-CUB1, SW-1710, 639-V and UM-UC-3 cells (compound 2, compound 5, compound 6; IC50, 72 h) in comparison to a DMSO solvent control (co). (B) Relative scratch size after 3, 6, 9 and 12 h of migration in comparison to the starting point 0 h. The relative scratch size is displayed on the y-axis. p < 0.05 was regarded as significant and marked as *, whereas p < 0.01 and p < 0.001 were defined as highly significant and marked as ** and ***. The calculated significances refer to the DMSO solvent control.

Mentions: As the effect of pharmacological HDAC8 inhibition was stronger than the effect of HDAC8 knock-down, wound healing assays of UCCs after HDAC8 inhibitor treatment were additionally performed (Figure 7A). A clear difference was observed in VM-CUB1 and UM-UC-3 cells, respectively, comparing DMSO controls to cells treated with c5 and c6, especially after 6 - 12 h (Figure 7B).


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)

Migration assay of urothelial cancer cells after HDAC8 inhibitor treatment. (A) Representative photographs of wound healing assay at 0 and 12 hours from inhibitor treated RT-112, VM-CUB1, SW-1710, 639-V and UM-UC-3 cells (compound 2, compound 5, compound 6; IC50, 72 h) in comparison to a DMSO solvent control (co). (B) Relative scratch size after 3, 6, 9 and 12 h of migration in comparison to the starting point 0 h. The relative scratch size is displayed on the y-axis. p < 0.05 was regarded as significant and marked as *, whereas p < 0.01 and p < 0.001 were defined as highly significant and marked as ** and ***. The calculated significances refer to the DMSO solvent control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4230422&req=5

Figure 7: Migration assay of urothelial cancer cells after HDAC8 inhibitor treatment. (A) Representative photographs of wound healing assay at 0 and 12 hours from inhibitor treated RT-112, VM-CUB1, SW-1710, 639-V and UM-UC-3 cells (compound 2, compound 5, compound 6; IC50, 72 h) in comparison to a DMSO solvent control (co). (B) Relative scratch size after 3, 6, 9 and 12 h of migration in comparison to the starting point 0 h. The relative scratch size is displayed on the y-axis. p < 0.05 was regarded as significant and marked as *, whereas p < 0.01 and p < 0.001 were defined as highly significant and marked as ** and ***. The calculated significances refer to the DMSO solvent control.
Mentions: As the effect of pharmacological HDAC8 inhibition was stronger than the effect of HDAC8 knock-down, wound healing assays of UCCs after HDAC8 inhibitor treatment were additionally performed (Figure 7A). A clear difference was observed in VM-CUB1 and UM-UC-3 cells, respectively, comparing DMSO controls to cells treated with c5 and c6, especially after 6 - 12 h (Figure 7B).

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