Abstract
BackgroundHistone deacetylase inhibitors (HDACi) are promising anti-cancer drugs that could also be employed for urothelial carcinoma (UC) therapy. It is unclear, however, whether inhibition of all 11 zinc-dependent HDACs or of individual enzymes is more efficacious and specific. Here, we investigated the novel HDACi 19i (LMK235) with presumed preferential activity against class IIA HDAC4/5 in comparison to the pan-HDACi vorinostat (SAHA) and the HDAC4-specific HDACi TMP269 in UC cell lines with basal expression of HDAC4 and characterized two HDAC4-overexpressing UC cell lines.MethodsCytotoxic concentrations 50% (CC50s) for HDACi were determined by MTT assay and high-content analysis-based fluorescent live/dead assay in UC cell lines with different expression of HDAC4 and as well as in normal urothelial cell cultures, HBLAK and HEK-293 cell lines. Effects of HDACis were analyzed by flow cytometry; molecular changes were followed by qRT-PCR and Western blots. UC lines overexpressing HDAC4 were established by lentiviral transduction. Inhibitor activity profiles of HDACi were obtained by current state in vitro assays, and docking analysis was performed using an updated crystal structure of HDAC4.ResultsIn UC cell lines, 19i CC50s ranged around 1 μM; control lines were similarly or less sensitive. Like SAHA, 19i increased the G2/M-fraction, disturbed mitosis, and elicited apoptosis or in some cells senescence. Thymidylate synthase expression was diminished, and p21CIP1 was induced; global histone acetylation and α-tubulin acetylation also increased. In most cell lines, 19i as well as SAHA induced HDAC5 and HDAC4 mRNAs while rather repressing HDAC7. UC cell lines overexpressing HDAC4 were not significantly less sensitive to 19i. Reevaluation of the in vitro HDAC isoenzyme activity inhibition profile of 19i and its docking to HDAC4 using current assays suggested rather low activity against class IIA HDACs. The specific class IIA HDAC inhibitor TMP269 impeded proliferation of UC cell lines only at concentrations > 10 μM.ConclusionsAnti-neoplastic effects of 19i on UC cells appear to be exerted by targeting class I HDACs. In fact, HDAC4 may rather impede UC growth. Our results suggest that targeting of class IIA HDACs 4/5 may not be optimal for UC therapy. Moreover, our investigation provides further evidence for cross-regulation of class IIA HDACs by class I HDACs.
Highlights
Histone deacetylase inhibitors (HDACi) are promising anti-cancer drugs that could be employed for urothelial carcinoma (UC) therapy
Proliferation and cell cycle following treatment with novel HDAC inhibitors Initially, the effects of the three inhibitors 19i, 19h, and 19e on cell viability were determined by MTT assay in three UC cell lines differing in HDAC4 expression (VM-CUB1—low, UM-UC-3—normal, 639-V—moderate, according to [28]), after 72 h of treatment. 19i was the most potent compound with cellular CC50s between 0.82 and 1.03 μM
Treatment with 2 μM 19i impaired the clonogenic potential of UC cells comparably to treatment with the pan-HDACi Suberoylanilide hydroxamic acid (SAHA)
Summary
Histone deacetylase inhibitors (HDACi) are promising anti-cancer drugs that could be employed for urothelial carcinoma (UC) therapy. It is unclear, whether inhibition of all 11 zinc-dependent HDACs or of individual enzymes is more efficacious and specific. Class I HDACs (HDACs 1, 2, 3, and 8) are essential for global acetylation patterns in the nucleus and the epigenetic regulation of gene expression [1]. Increased expression of these isoenzymes is observed in a variety of malignant tumors and often correlates with a worse patient outcome [2,3,4,5]. Though, more selective inhibitors may be superior for therapy and may elicit fewer adverse effects [8, 9]
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