The impact of cruciferous vegetable isothiocyanates on histone acetylation and histone phosphorylation in bladder cancer

Abstract

Cruciferous vegetable intake is associated with reduced risk of bladder cancer, yet mechanisms remain unclear. Cruciferous vegetable isothiocyanates (ITCs), namely sulforaphane (SFN) and erucin (ECN), significantly inhibit histone deacetylase (HDAC) activity in human bladder cancer cells representing superficial to invasive biology (59-83% inhibition with 20μM, 48h treatment), and in bladder cancer xenografts (59±3% ECN inhibition). Individual HDACs inhibited by SFN and ECN include HDACs 1, 2, 4 and 6. Interestingly, global acetylation status of histones H3 or H4 remain unaltered. The interplay between HDAC inhibition and modest modulation of AcH3 and AcH4 status is partially explained by decreased histone acetyl transferase activity (48.8±5.3%). In contrast, a significant decrease in phosphorylation status of all isoforms of histone H1 was observed, concomitant with increased phosphatase PP1β and PP2A activity. Together, these findings suggest that ITCs modulate histone status via HDAC inhibition and phosphatase enhancement. This allows for reduced levels of histone H1 phosphorylation, a marker correlated with human bladder cancer progression. Therefore, ITC-mediated inhibition of histone H1 phosphorylation presents a novel direction of research in elucidating epidemiological relationships and supports future food-based prevention strategies.

Significance: Collectively, our findings suggest that the cruciferous vegetable isothiocyanates: sulforaphane (SFN) and erucin (ECN), impact histones status in bladder cancer cells by modulating specific HDACs and HATs, and enhancing phosphatase activity, resulting in reduction of histone H1 phosphorylation. These findings are significant due to the fact that our previous work positively correlated histone H1 phosphorylation with bladder cancer carcinogenesis and progression. Therefore, we propose that SFN and ECN may inhibit bladder carcinogenesis via epigenetic modulation of gene expression associated with histone H1 phosphorylation. These efforts may elucidate biomarkers useful in epidemiologic studies related to cruciferous vegetable intake and cancer risk or provide intermediate biomarkers for food-based clinical intervention studies in high-risk cohorts.

Keywords: Bladder cancer; Broccoli; Cruciferous vegetables; Erucin; Histone acetylation; Histone phosphorylation; Isothiocyanates; Sulforaphane.

Figure 1. Sulforaphane and erucin inhibits HDACs in bladder cancer in vitro and in vivo.

A. Human bladder cancer cells ranging from superficial (RT4) to invasive (J82 and UMUC3) were treated with DMSO vehicle control or 5, 10 and 20 μM sulforaphane (SFN) and erucin (ECN) for 48h. Protein lysates were obtained under non-denaturing conditions followed by concentration determination and normalization via the BCA Assay. HDAC activity was assessed by Fluor de Lys fluorometric HDAC activity assay. The data was collected, normalized to the DMSO “0” control and is expressed as the mean Relative HDAC Activity ± standard deviation (SD) and represents three independent experiments. Statistical significance was set at *, p<0.05 relative to DMSO controls. B. HDAC activity was also assessed using tissue from a mouse xenograft study where athymic nude mice were subcutaneously injected with 0.05 × 10⁶ UMUC3 cells and gavaged daily with either vehicle control (soybean oil), 295 μmol/kg body weight SFN or 295 μmol/kg body weight ECN (n = 12 mice/group) for 2 weeks until the tumors reached approximately 1.2 cm in diameter and were sacrificed. Protein extraction was performed from normal bladder tissue (Bladder) and UMUC3 tumor xenografts (Tumor) from each treatment group; the amount of protein was quantified/normalized and subjected to the HDAC Activity Assay. HDAC activity assay data was collected and normalized to DMSO treated mice and is expressed as the mean Relative HDAC Activity ± SD and represents data from four bladder tissue samples (normal bladder tissue, labeled “Bladder”) and four UMUC3 tumor samples (UMUC3 tumor xenograft, labeled “Tumor”) from each treatment group. Statistical significance is set at *, p<0.05 relative to the DMSO Control.

Figure 2. Sulforaphane and erucin inhibit HDAC1, 2, 4 and 6 with no effect on other HDACs or Sirtuins

UMUC3 (invasive human bladder cancer cells) were treated with either DMSO vehicle control, 20 μM sulforaphane (SFN) or erucin (ECN) for 48h and protein lysates were obtained under non-denaturing conditions. Input protein concentration was determined and normalized via the BCA Assay. Subsequently, each HDAC/Sirtuin was immunoprecipitated (IP) sequentially and protein beads were utilized to assess HDAC activity of each individual IP HDAC/SIRT. The data was collected and normalized to the DMSO vehicle condition and is expressed as the mean Relative HDAC or SIRT Activity ± SD and represents three independent experiments. Statistical significance was set at *, p<0.05 relative to the DMSO (Control).

Figure 3. Histone acetylation status is not robustly modulated by sulforaphane and erucin treatment

A. RT4 and UMUC3 cells were treated with 5, 10 and 20 μM SFN and ECN for 48h, protein lysates were obtained and analyzed by western blot analysis utilizing a site-specific (K9 and K13) acetylated histone H3 (AcH3) antibody. The antibody was raised against amino acids 1–20 of histone H3 (ARTKQTAR[K*]STGG[K*]APRKQLC, where K* is acetylated). P21 and thymidylate synthase (TS) protein expression was also analyzed and protein expression was quantified by densitometry relative to GAPDH. The data was normalized to the DMSO control “C” and is expressed as the mean (AcH3, p21, and Thymidylate Synthase Protein) Relative Densitometry (ratio to control) ± SD and represents three independent experiments. Statistical significance was set at *, p<0.05 relative to the “C” control. B. UMUC3 cells were treated with 20 μM SFN or ECN for 48h, protein lysates were obtained and analyzed by western blot analysis utilizing acetylated tubulin antibody and quantified by densitometry relative to GAPDH. The data was normalized to the DMSO treated control and is expressed as the mean Ac-Tubulin Protein (Fold Change) ± standard error of the mean (SEM) and represents 3 independent experiments. Statistical significance was set as *, p<0.05 relative to the DMSO control. Representative gels in (A–B) are shown from three independent experiments.

Figure 4. HAT activity is also inhibited by broccoli ITCs

UMUC3 cells were treated with DMSO vehicle control or 5, 10, 20 μM SFN or ECN for 48h, nuclear extract was obtained under non-denaturing conditions and HAT activity was assessed. The data was normalized to the DMSO vehicle control and is expressed as the mean Relative HAT Activity ± SD and represents three independent experiments. Statistical significance was set at *, p<0.05 relative to the DMSO control.

Figure 5. Modulation of histone H1 phosphorylation occurs with SFN and ECN treatment

RT4 and UMUC3 cells were treated with DMSO vehicle control or 20 μM SFN or ECN for 3h, cells were flash frozen, histones extracted and analyzed by LC-MS. The deconvoluted mass spectra of all four variants of histone H1 (H1.2, H1.3, H1.4 and H1.5) of RT4 and UMUC3 is shown. An 80 Da shift to the right indicates an increase in phosphorylation of histone H1. A representative spectra is shown from three independent experiments.

Figure 6. Modulation of histone H1 phosphorylation occurs earlier and appears to be more robust than histone acetylation changes with SFN and ECN treatment

Data from the treatment of RT4 and UMUC3 cells with DMSO, SFN or ECN treatment (20 μM, 3h) is shown. A. The ratio of the summed peak heights from the LC-MS data for the phosphorylated isoforms to the unphosphorylated isoform for all four variants of histone H1 (H1.2, H1.3, H1.4 and H1.5) is shown. Data represents four independent experiments, error bars represent ± standard error and statistically significant differences are indicated (*, p<0.05; **, p<0.01; ***, p<0.001). B. Histone H1 phosphorylation changes were also analyzed by western blot analysis where representative gels are shown from at least two independent experiments. C. Densitometry from the western blot analysis from B. is shown. The data was normalized to the DMSO control and is expressed as the mean Relative Densitometry (ratio to control) ± SEM and represents two independent experiments. Statistical significance was set at *, p<0.05 and **, p<0.01 relative to the DMSO control.

Figure 7. An increase in PP1β and PP2A activity occurs with SFN and ECN treatment

UMUC3 cells were treated with DMSO vehicle control or 20 μM SFN or ECN for 3h, cells were lysed in non-denaturing cell lysis buffer, PP1β and PP2A were immunoprecipitated and phosphatase activity was assayed. The data was collected in triplicate and is expressed as the mean Phosphatase Activity (O.D.) ± SD and represents four independent experiments. A significant increase in PP1β and PP2A activity resulted for all of the treatments (SFN and ECN) relative to the control where statistically significant differences are indicated (*, p<0.05, ***, p<0.001).