Dietary histone deacetylase inhibitors: from cells to mice to man

Abstract

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables, such as broccoli and broccoli sprouts. This anticarcinogen was first identified as a potent inducer of Phase 2 detoxification enzymes, but evidence is mounting that SFN also acts through epigenetic mechanisms. SFN has been shown to inhibit histone deacetylase (HDAC) activity in human colon and prostate cancer lines, with an increase in global and local histone acetylation status, such as on the promoter regions of P21 and bax genes. SFN also inhibited the growth of prostate cancer xenografts and spontaneous intestinal polyps in mouse models, with evidence for altered histone acetylation and HDAC activities in vivo. In human subjects, a single ingestion of 68 g broccoli sprouts inhibited HDAC activity in circulating peripheral blood mononuclear cells 3-6 h after consumption, with concomitant induction of histone H3 and H4 acetylation. These findings provide evidence that one mechanism of cancer chemoprevention by SFN is via epigenetic changes associated with inhibition of HDAC activity. Other dietary agents such as butyrate, biotin, lipoic acid, garlic organosulfur compounds, and metabolites of vitamin E have structural features compatible with HDAC inhibition. The ability of dietary compounds to de-repress epigenetically silenced genes in cancer cells, and to activate these genes in normal cells, has important implications for cancer prevention and therapy. In a broader context, there is growing interest in dietary HDAC inhibitors and their impact on epigenetic mechanisms affecting other chronic conditions, such as cardiovascular disease, neurodegeneration and aging.

Fig. 1

Working hypothesis for the role of dietary histone deacetylase (HDAC) inhibitors. HDAC/co-repressor complexes maintain a tightly restricted chromatin configuration, which limits access of transcription factors to DNA, and represses genes required for cell cycle checkpoint control and apoptosis. HDAC inhibition by SFN and other dietary agents enables histone acetyltransferase/co-activator (HAT/CoA) complexes to add acetyl groups to histone tails, loosening DNA/chromatin interactions, and allowing access of transcription factors to the promoters of genes such as P21 and bax. Re-expression of these genes facilitates cell cycle arrest and apoptosis in the context of cancer chemoprevention or therapy.

Fig. 2

HDAC inhibition by SFN in human colon and prostate cells, and suppression of xenograft growth in mice. Human colon or prostate cell lines were seeded at ∼1 × 10⁶ and 24 h later treated with SFN. Whole cell lysates or nuclear extracts were obtained after 48 h and examined for protein expression by immunoblotting, or HDAC activity using a commercial kit (BioMol). In chromatin immunoprecipitation (ChIP) assays, antibody to acetylated histone H4 was followed by primers to the promoter region of the P21 gene. In the xenograft studies, 5-week-old male athymic nude BALB/c (nu/nu) mice were randomized to 10 animals per group and fed control AIN93G diet or AIN93G diet containing 443 mg SFN/kg. Human prostate cancer PC-3 cells were mixed in a 1:1 ratio of complete media (RPMI 1640 + 10% FBS) and High Concentration Growth Factors Matrigel Matrix (Becton Dickinson). A suspension of 10⁶ cells (50 μl) was injected subcutaneously into the right flank of each mouse. Tumor volume was calculated using the following formula for the volume of an ellipsoid: length × width² × 0.5236 (π/6). After 21 days xenografts and host tissues were examined for HDAC activity using the BioMol kit. Error bars indicate mean±S.E.; *P < 0.05, **P < 0.01, ***P < 0.001. Full details of the studies in colon and prostate cells can be found in Refs. [11,17], respectively, and xenograft experiments were reported in Ref. [18]. PBMC, peripheral blood mononuclear cells.

Fig. 3

HDAC inhibition by SFN in mouse colon, and suppression of intestinal polyps. In a short-term pilot study, 3 mice/group were treated by single oral gavage with 10 μmol SFN, 10 μmol of SFN-N-acetylcysteine (SFN-NAC), or DMSO vehicle alone. Colonic mucosa was scraped 6 h later and examined in the BioMol HDAC activity assay. In a longer-term experiment, Apc^min mice ingested on average ∼6 μmol SFN/day for 70 days in AIN93 diet, whereas controls received AIN93 diet alone. Polyps were enumerated at the end of the study, and the intestinal mucosa was immunoblotted for acetylated histones and β-actin. In addition, intestinal mucosa was subjected to ChIP, as described in Fig. 2 legend. Error bars indicate mean±S.E.; **P < 0.01, ***P < 0.001. For further details, see Ref. [19].

Fig. 4

HDAC inhibition by SFN-rich broccoli sprouts in human volunteers. Three healthy human volunteers refrained from cruciferous vegetable intake for 48 h. Each subject consumed 68 g broccoli sprouts (∼105 mg SFN) with a bagel and cream cheese, and blood was collected at times indicated. PBMCs were immunoblotted for acetylated histones H3 and H4 (acH3, acH4), or the corresponding total histone (H3, H4), and HDAC activities were determined using the BioMol kit. Error bars indicate mean±S.E.; *P < 0.05, n=3. For further details, see Ref. [18].

Fig. 5

Dietary HDAC inhibitors. HDAC inhibition has been reported in vitro and/or in vivo for butyrate, garlic organosulfur compounds, and metabolites of SFN, such as SFN-NAC and SFN-Cys, whereas other compounds shown are hypothetical HDAC inhibitors (see text). c9, t11-CLA, cis-9, trans-11-conjugated linoleic acid. Inset: SFN-Cys was manually docked into the active site of human HDAC8, according to the following constraints: (i) carboxylate binding in a bidentate fashion to the active site zinc, (ii) minimal steric conflict between substrate and enzyme, based on a fixed protein, (iii) favorable torsion angles, (iv) hydrogen-bond partners for buried polar atoms, and (v) following the favored position of a bound hydroxamic acid inhibitor [31].