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. 2021 Dec 17;14(12):1319.
doi: 10.3390/ph14121319.

Improved Anticancer Activities of a New Pentafluorothio-Substituted Vorinostat-Type Histone Deacetylase Inhibitor

Nils Goehringer  1 , Yayi Peng  2 , Bianca Nitzsche  1 , Hannah Biermann  1 , Rohan Pradhan  3 , Rainer Schobert  4 , Marco Herling  2   5 , Michael Höpfner  1 , Bernhard Biersack  4
Affiliations

Improved Anticancer Activities of a New Pentafluorothio-Substituted Vorinostat-Type Histone Deacetylase Inhibitor

Nils Goehringer et al. Pharmaceuticals (Basel). .

Abstract

The development of new anticancer drugs is necessary in order deal with the disease and with the drawbacks of currently applied drugs. Epigenetic dysregulations are a central hallmark of cancerogenesis and histone deacetylases (HDACs) emerged as promising anticancer targets. HDAC inhibitors are promising epigenetic anticancer drugs and new HDAC inhibitors are sought for in order to obtain potent drug candidates. The new HDAC inhibitor SF5-SAHA was synthesized and analyzed for its anticancer properties. The new compound SF5-SAHA showed strong inhibition of tumor cell growth with IC50 values similar to or lower than that of the clinically applied reference compound vorinostat/SAHA (suberoylanilide hydroxamic acid). Target specific HDAC inhibition was demonstrated by Western blot analyses. Unspecific cytotoxic effects were not observed in LDH-release measurements. Pro-apoptotic formation of reactive oxygen species (ROS) and caspase-3 activity induction in prostate carcinoma and hepatocellular carcinoma cell lines DU145 and Hep-G2 seem to be further aspects of the mode of action. Antiangiogenic activity of SF5-SAHA was observed on chorioallantoic membranes of fertilized chicken eggs (CAM assay). The presence of the pentafluorothio-substituent of SF5-SAHA increased the antiproliferative effects in both solid tumor and leukemia/lymphoma cell models when compared with its parent compound vorinostat. Based on this preliminary study, SF5-SAHA has the prerequisites to be further developed as a new HDAC inhibitory anticancer drug candidate.

Keywords: anticancer drugs; fluorine; histone deacetylase inhibitor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Structures of SAHA (vorinostat) and the new analog SF5-SAHA (ZBG = zinc-binding group).
Scheme 1
Scheme 1
Synthesis of SF5-SAHA.
Figure 2
Figure 2
Unspecific cytotoxic effects of SF5-SAHA in DU145 prostate cancer and Hep-G2 hepatoblastoma cells. LDH release of cells was measured after 3 and 24 h of incubation with 1, 5 or 10 µM of SF5-SAHA or SAHA. Results show changes in LDH release relative to untreated controls. Data are given as percentage changes relative to basal LDH release of controls. Means ± SEM of n = 3 independent experiments.
Figure 3
Figure 3
Apoptosis induction by SF5-SAHA. (A) Caspase-3 induction in DU145 or Hep-G2 cells after 24 h treatment with SF5-SAHA and SAHA. Means ± SEM of n = 3 independent experiments. (B) Representative Western blot out of n = 3 experiments, showing changes in the expression of PARP and PARP after cleavage (cl. PARP) in DU145 cells after 24 h treatment with test compounds. (C) From these Western blots gray intensities, of PARP and cleaved PARP specific bands were quantified adjusted to protein loading and normalized to untreated controls. (D) Representative Western blot out of n = 3 experiments, showing changes in the expression of Apaf-1, PARP, and Bcl-2 in Hep-G2 cells after 24 h treatment with SF5-SAHA. Data are given as means ± SEM of n = 3 independent experiments. * p ≤ 0.05, ** p ≤ 0.005, *** p ≤ 0.0005, **** p ≤ 0.0001; 2-way ANOVA Dunnett’s post-hoc test.
Figure 4
Figure 4
ROS induction (fluorescence microscopy using the ROS-dye CellROX orange) after 6 h, 12 h, and 24 h of incubation with SF5-SAHA and SAHA in DU145 (A) and Hep-G2 cells (B). Orange fluorescence indicates oxidation of the ROS-dye by formed ROS.
Figure 5
Figure 5
HDAC inhibitory effects of SF5-SAHA in cancer cells. (A) HDAC activity was measured by luminescence display of the acetylation state of histones. HeLa nuclear cell extracts, used as source for histones, where preincubated with substrate and inhibitor. Results are given as relatives to controls, not preincubated with inhibitor, as means ± SEM of n = 3 independent experiment. (B) Representative Western blot out of n = 3 experiments, showing changes in the expression of acetylated histone H3 after 24 h treatment with compounds in DU145 (left) and Hep-G2 (right) cells. (C) From the Western blots gray intensity, mean ± SEM, of acetylated histone H3 specific bands where quantified adjusted to protein loading and normalized to untreated control. * p ≤ 0.05, **** p ≤ 0.0001; 2-way ANOVA Dunnett’s post-hoc test.
Figure 6
Figure 6
HDAC inhibitory effects of SF5-SAHA in T-cell leukemia/lymphoma cells (Jurkat, Hut78, SMZ1, SupT11). Representative Western blots out of n = 3 experiments, showing changes in the expression of acetylated histone H3 and acetylated α-tubulin after 24 h treatment with SF5-SAHA.
Figure 7
Figure 7
Inhibition of HDAC1 (top), HDAC2 (middle) and HDAC6 (bottom) by SF5-SAHA and SAHA. HDAC activity was measured by luminescence display of the acetylation state of histones. Human recombinant HDAC1, HDAC2 and HDAC6 enzymes and adjacent fluorgenic HDAC substrates were used to determine subtype specific HDAC activity levels after preincubation with SF5-SAHA and SAHA. Results are given as relatives to controls, not preincubated with inhibitor, as means ± SEM of n = 3 independent experiments. ** p ≤ 0.005, *** p ≤ 0.0005, **** p ≤ 0.0001; 2-way ANOVA Dunnett’s post-hoc test.
Figure 8
Figure 8
Western blot determined expression of HDAC1, HDAC2, and HDAC6 in DU145 prostate cancer and Hep-G2 liver cancer cells.
Figure 9
Figure 9
Overlay images of docked SF5-SAHA (green) and SAHA (purple) with HDAC2 in cartoon view (top image) and surface view (bottom image). The interaction of inhibitors with active site amino acids (red) and zinc ion (sphere) is highlighted.
Figure 10
Figure 10
Inhibitory effects of SF5-SAHA on EGFR signaling pathway protein expression. (A) Representative Western blots of n = 3 independent experiments showing treatment induced changes in the expression of EGFR in DU145 cells after 24 h. β-actin was used as loading control. (B) From these Western blots gray intensity, mean ± SEM, of EGFRs specific bands were quantified and adjusted to protein loading and normalized to untreated control. ** p ≤ 0.005; 2-way ANOVA Dunnett’s post-hoc test.
Figure 11
Figure 11
Inhibitory effects of SF5-SAHA on DU145 tumor cell migration. Representative scratch assay images of n = 3 independent experiments showing a treatment induced retarded migration of DU145 cells after 24 h. Black lines indicate the initial scratch areas at the corresponding starting points (0 h). Control cells (ctrl) are untreated cells in medium.
Figure 12
Figure 12
Angiogenesis in fertilized chicken eggs (CAM assay). Representative images of n = 4 independent experiments showing antiangiogenic effects of SF5-SAHA (5 or 10 µM) after 72 h. Control (ctrl) image shows vessels of untreated CAM (PBS control). Red arrows indicate conspicuous branches and vessel diameters.
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