Molecular structures of Broxbam and vorinostat. Structures of the vascular disrupting agents combretastatin A-4 (CA-4), the histone deacetylase-inhibitor vorinostat (red) and the oxazole-bridged hybrid compound broxbam.

Real-time measurements of liver cancer and cholangiocellular carcinoma cell proliferation. Dose- and time-dependent effects of bb on (A) HepG2, (B) Huh7, (C) TFK1 and (D) EGI1 cell proliferation as assessed by the iCELLigence real-time monitoring system. Data show the cell viability index measured over 48 h after the application of broxbam. Representative graphs out of three independent experiments were shown for each cell line. Bb, broxbam.

LDH release by liver cancer cell lines HepG2 and Huh7 and the non-transformed hepatocyte cell line AML12 into the media after 24 h treatment with broxbam in different concentrations. Data are presented as the LDH release relative to those in untreated controls, where basal LDH release was set to 0%. Values are presented as the mean ± SEM. n=4. LDH, lactate dehydrogenase; ctrl, control.

Measurement of tumour size grown on the chorioallantoic membranes of fertilised chicken eggs. (A) Tumour mass of HepG2 tumours after treatment with different concentrations of bb and PBS as control for 72 h. Data are presented as the mean ± SEM from seven independent experiments. ^*P<0.05. (B) Representative images of control and bb-treated HepG2 tumours after excision. Scale bar 800 µm. bb, broxbam; Ctrl, control.

Detection of proapoptotic events in liver cancer cells. (A) Influence of bb and vs on the loss of mitochondrial membrane potential of liver cancer cell lines HepG2 and Huh7. Indicated values were measured by fluorometric measurement of JC-1-stained cells. The effect of IC₅₀ concentrations of bb and vs was monitored after 3, 6 and 18 h. Mitochondrial accumulation of JC-1 results in red fluorescence whereas loss of MMP results in monomeric JC-1, which emits green fluorescence. Data are presented as the ratio of green/red fluorescence of cells. Data are presented as the mean ± SEM from three experiments. One-way ANOVA with Tukey's post hoc test was used to test for significance. ^*P<0.05, ^***P<0.001 and ^****P<0.0001 vs. Ctrl. (B) Caspase-3 activity in HepG2and Huh7 cells upon treatment with bb for 24 h. Data are presented as the mean ± SEM percentage of untreated controls from four experiments. One-way ANOVA with Tukey's post hoc test was used to test for significance. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. ctrl. (C-H) HepG2 and Huh7 cells were treated either with solvent (Ctrl), bb at IC₅₀ (0.6 µM), bb at two-fold IC₅₀ (1.2 µM) or vs. at two-fold IC50 (4.0 µM). Western blotting results of (C) p53 expression and (D) phosphorylation levels in HepG2 cells. (E) p-p53/p53 ratio was also quantified in HepG2 cells. Western blotting results of (F) p53 expression and (G) phosphorylation levels in Huh7 cells. (H) p-p53/p53 ratio was also quantified in Huh7 cells. Corresponding representative western blotting images are shown in Fig. S2. n=3 per group. One-way ANOVA with Tukey's post hoc test was used to test for significance. Bb, broxbam; vs, vorinostat; Ctrl, control; Cas 3, caspase 3; p-, phosphorylated.

mRNA expression levels of HDACs 1-11 in untreated liver cancer cell lines HepG2 and Huh7. mRNA expression levels of HDACs 1-11 in liver cancer cell lines were measured using reverse transcription-quantitative PCR, which were then normalised to those of GAPDH, the housekeeping gene. Data are presented as the means ± SEM from three independent experiments. HDAC, histone deacetylase; Ctrl, control.

Quantification of the expression of selected HDAC proteins. HepG2 and Huh7 cells were treated for 24 h with either solvent (Ctrl), bb at IC₅₀ (0.6 µM), bb at two-fold IC₅₀ (1.2 µM) or vs. at two-fold IC₅₀ (4.0 µM). Western blotting was used to measure protein expression, using tubulin as loading control. (A) HDAC1 expression in Hep2G and Huh7 cells. (B) HDAC2 expression in Hep2G and Huh7 cells. (C) HDAC4 expression in Hep2G and Huh7 cells. ^**P<0.01 vs. ctrl. (D) HDAC6 expression in Hep2G and Huh7 cells. Corresponding representative western blotting images are provided in Fig. S2. n=3 per group. One-way ANOVA with Tukey's post hoc test was used to test for significance. HDAC, histone deacetylase; Ctrl,. control; bb, broxbam; vs, vorinostat.

Effects of inhibiting the selective HDACs of broxbam in Huh-7 cells. (A) Activities of HDAC1, -2, -4 and -6 after treatment with bb or vs at concen-trations corresponding to their respective IC₅₀ and two-fold IC₅₀. Data are presented as the means ± SEM percentage of untreated control, which was set to 100%, from three experiments. ^*P<0.05, ^**P<0.01 vs. Ctrl. One-way ANOVA with Tukey's post hoc test was used to test for significance (B) Dose-dependent inhibitory effect of bb on the activity of HDAC6 relative to control, represented as the means ± SEM from three independent experiments. ^****P<0.0001 vs. Ctrl. One-way ANOVA with Tukey's post hoc test was used to test for significance. (C) Western blot analysis and (D) quantification of siRNA-mediated knockdown of HDAC6 protein expression in Huh7 cells transfected with control (si-mock) or HDAC6-specific (si-HDAC6) siRNAs for 48 h. Each set of si-mock and si-HDAC6 lanes represents one transfection experiment. Tubulin was used as the loading control. Analysis of (E) Ki-67 and (F) E2F3 mRNA expression by reverse transcription-quantitative PCR. The mRNA expression levels were normalised to that of 18S rRNA. Data was presented as the mean ± SEM from six independent experiments. One-way ANOVA with Tukey's post hoc test was used to test for significance. HDAC, histone deacetylase; bb, broxbam; vs, vorinostat; si, small interfering.

Responses of the cytoskeleton in HepG2 cells to bb treatment. Confocal immunofluorescence images 24 h after incubation with bb (0.6 µM) or vs. (4 µM). α-Tubulin (red), f-actin (green) and DAPI-stained nuclei (blue) staining are shown. Scale bar, 30 µm. Ctrl, control; bb, broxbam; vs, vorinostat. Images are representative of n=3 independent experiments.

Effects of different concentrations of bb on the migration of HepG2 cells. HepG2 cell migration was measured using wound healing assays 24 h after treatment. (A) Representative images from five independent experiments, where the cells were treated with at IC₅₀ two-fold IC₅₀ of bb. (B) Cell migration was presented as the mean ± SEM percentage of control from three experiments. One-way ANOVA with Tukey's post hoc test was used to test for significance. ^*P<0.05 and ^**P<0.01 vs. ctrl. ctrl, control; bb, broxbam.

Morphology of the blood vessel system of transgenic Tg(fli1:EGFP)y1 mutant Casper zebrafish embryos 72 h post-fertilisation. Embryos were treated for 48 h with either a corresponding amount of DMSO, with bb concentrations of 1 µM, 750 or 500 nM. Brightfield and green fluorescent images, denoted as fli:GFP, are shown. The pericardial region was marked with asterisks whereas SIVs are marked with arrows. Fluorescent SIV areas are marked with yellow boxes, which were then magnified two-fold magnification. Individuals shown are representative for ≥20 embryos per concentration. bb, broxbam; GFP, green fluorescent protein; SIV, sub-intestinal veins.

Effects of bb on developing zebrafish embryos at 3 days post fertilisation. (A) Area of SIVs after bb treatment with concentrations of 1 µM, 750 and 500 nM for 48 h. Values were normalised to the negative control, DMSO. (B) Percentage pericardial diameter relative to the negative control, DMSO. Data are presented as the percentage means ± SEM of control from ≥20 experiments. Significance levels were determined using one-way ANOVA and Tukey's post hoc test. ^*P<0.05 and ^****P<0.0001 vs. ctrl. bb, broxbam; SIV, sub-intestinal veins.

Pleiotropic effects of broxbam. According to the present study, broxbam exerted anti-angiogenic functions, inhibited HDAC activity, disrupted cytoskeletal dynamics and induced apoptosis. HDAC, histone deacetylase.