Accordingly, at least in urothelial cancer, PTEN deficiency does not seem to have a decisive Sorafenib impact on the efficacy of HDAC inhibitors. Effects of siRNA mediated downregulation and pharma cological inhibition on urothelial cancer cell lines were not thoroughly consistent. Differences might be explained by several factors. For example, knockdown depletes the protein thereby not only affecting enzymatic but also other protein functions for example complex assembly. Inhibitor treatment ideally only suppresses the enzymatic activity while further protein functions should not be affected. Accordingly, also compensatory mechanisms might be different in both conditions. Comparing expres sion levels of further class I HDACs after knockdown of HDAC8 as well as after pharmacological inhibition, only minor changes were observed.
Although upregulation of HDAC1 or HDAC2 was a little more consistently observed after HDAC8 knockdown, they can hardly ex plain the difference between knockdown and inhibition by c5 or c6. More likely, the stronger effects of the in hibitors may be due to inhibition of other targets in addition to HDAC8. Neither HDAC8 knockdown nor pharmacological treat ment with any compound led to a change in histone H3 or H4 acetylation, a widely used surrogate marker for intracellular HDAC inhibition. This finding suggests that HDAC8, as expected, does not substantially affect overall histone acetylation. In addition, this does also indicate that inhibitor treatment seems to be iso enzyme specific as other class I HDACs seemed to be not affected.
This was also observed in neuroblastoma cell lines after treatment of HDAC8. Global Histone H4 acetylation was not affected by HDAC8 knockdown or by selective inhibitor treatment. In contrast, HDAC8 knockdown in some cell lines and treatment with c5 or c6 resulted in a strong increase of acetylated tubulin. The latter finding is in accord with previous findings in HeLa and HEK293 cells. The cytoplasmic protein tubulin is especially a substrate of HDAC6 which is predominantly localized in the cyto plasm. HDAC6 influences the cytoskeleton and cell motility via deacetylation of tubulin and other cyto skeleton proteins. In vitro, c5 and c6 do not inhibit HDAC6 efficiently. Thus, the best explanation for these observations is that in vivo HDAC8 directly or indirectly influences tubulin acetylation.
Similar discrepancies be tween in vitro and in vivo activity of an isoenzyme selective HDAC inhibitor on tubulin acetylation have been observed by others for valproic acid. These effects on tubulin acetylation Anacetrapib may relate to the inhibition of cell migration by c5 and c6 we observed in UC cell lines. However, inhibition of HDAC6 as such does not inhibit migration of UCC as ef ficiently as the HDAC8 inhibitors c5 and c6.