Melinda C. Myzak

A Novel Mechanism of Chemoprotection by Sulforaphane Inhibition of Histone Deacetylase

Sulforaphane (SFN), a compound found at high levels in broccoli and broccoli sprouts, is a potent inducer of phase 2 detoxification enzymes and inhibits tumorigenesis in animal models. SFN also has a marked effect on cell cycle checkpoint controls and cell survival and/or apoptosis in various cancer cells, through mechanisms that are poorly understood. We tested the hypothesis that SFN acts as an inhibitor of histone deacetylase (HDAC). In human embryonic kidney 293 cells, SFN dose-dependently increased the activity of a β-catenin-responsive reporter (TOPflash), without altering β-catenin or HDAC protein levels. Cytoplasmic and nuclear extracts from these cells had diminished HDAC activity, and both global and localized histone acetylation was increased, compared with untreated controls. Studies with SFN and with media from SFN-treated cells indicated that the parent compound was not responsible for the inhibition of HDAC, and this was confirmed using an inhibitor of glutathione S-transferase, which blocked the first step in the metabolism of SFN, via the mercapturic acid pathway. Whereas SFN and its glutathione conjugate (SFN-GSH) had little or no effect, the two major metabolites SFN-cysteine and SFN-N-acetylcysteine were effective HDAC inhibitors in vitro. Finally, several of these findings were recapitulated in HCT116 human colorectal cancer cells: SFN dose-dependently increased TOPflash reporter activity and inhibited HDAC activity, there was an increase in acetylated histones and in p21Cip1/Waf1, and chromatin immunoprecipitation assays revealed an increase in acetylated histones bound to the P21 promoter. Collectively, these findings suggest that SFN may be effective as a tumor-suppressing agent and as a chemotherapeutic agent, alone or in combination with other HDAC inhibitors currently undergoing clinical trials.

Sulforaphane inhibits histone deacetylase in vivo and suppresses tumorigenesis in Apcmin mice

Sulforaphane (SFN) is an isothiocyanate from broccoli that induces phase 2 detoxification enzymes. We recently reported that SFN acts as a histone deacetylase (HDAC) inhibitor in human colon cancer cells in vitro, and the present study sought to extend these findings in vivo. In mice treated with a single oral dose of 10 ümol SFN, there was significant inhibition of HDAC activity in the colonic mucosa after 6 h, and immunoblots revealed a concomitant increase in acetylated histones H3 and H4, which returned to control levels by 48 h. Longer‐term treatment with SFN in the diet resulted in levels of acetylated histones and p21WAF1 in the ileum, colon, prostate, and peripheral blood mononuclear cells that were elevated compared with controls. Consistent with these findings, SFN suppressed tumor development in Apcmin mice, and there was an increase in acetylated histones in the polyps, including acetylated histones specifically associated with the promoter region of the P21 and bax genes. These results provide the first evidence for HDAC inhibition by SFN in vivo and imply that such a mechanism might contribute to the cancer chemoprotective and therapeutic effects of SFN, alone or in combination with other HDAC inhibitors currently undergoing clinical trials.

Sulforaphane Retards the Growth of Human PC-3 Xenografts and Inhibits HDAC Activity in Human Subjects

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables such as broccoli. This anticarcinogen was first identified as a potent inducer of Phase 2 enzymes, but evidence is mounting that SFN acts through other cancer chemopreventive mechanisms. We recently reported on a novel mechanism of chemoprotection by SFN in human colon cancer cells and prostate epithelial cells, namely the inhibition of histone deacetylase (HDAC). In the present investigation, we sought to test whether SFN also might inhibit HDAC activity in vivo. When consumed in the diet at an average daily dose of 7.5 μmol per animal for 21 days, SFN suppressed the growth of human PC-3 prostate cancer cells by 40% in male nude mice. There was a significant decrease in HDAC activity in the xenografts, as well as in the prostates and mononuclear blood cells (MBC), of mice treated with SFN, compared to controls. There also was a trend towards increased global histone acetylation in the xenografts, prostates, and MBC. In human subjects, a single dose of 68 g BroccoSprouts inhibited HDAC activity significantly in peripheral blood mononuclear cells (PBMC) 3 and 6 hrs following consumption. These findings provide evidence that one mechanism through which SFN acts as a cancer chemopreventive agent in vivo is through the inhibition of HDAC activity. Moreover, the data suggest that HDAC activity in PBMC may be used as a biomarker for assessing exposure to novel dietary HDAC inhibitors in human subjects.

Myeloperoxidase binds to low-density lipoprotein: potential implications for atherosclerosis

Myeloperoxidase (MPO), an abundant heme enzyme released by activated phagocytes, catalyzes the formation of a number of reactive species that can modify low‐density lipoprotein (LDL) to a form that converts macrophages into lipid‐laden or ‘foam’ cells, the hallmark of atherosclerotic lesions. Since MPO has been shown to bind to a number of different cell types, we investigated binding of MPO to LDL. Using the precipitation reagents phosphotungstate or isopropanol, MPO co‐precipitated with LDL, retaining its catalytic activity. The association of MPO with LDL was confirmed using native gel electrophoresis. MPO was also found to co‐precipitate with apolipoprotein B‐100‐containing lipoproteins in whole plasma. No precipitation of MPO was observed in lipoprotein‐deficient plasma, and there was a dose‐dependent increase in precipitation following addition of LDL to lipoprotein‐deficient plasma. Binding of MPO to LDL could potentially enhance site‐directed oxidation of the lipoprotein and limit scavenging of reactive oxygen species by antioxidants.

Dietary Agents as Histone Deacetylase Inhibitors

In cancer cells, an imbalance often exists between histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities, and various drug companies are actively seeking competitive HDAC inhibitors for chemotherapeutic intervention. Cancer cells appear to be more sensitive than nontransformed cells to HDAC inhibitors, which disrupt the cell cycle and induce apoptosis via derepression of genes such as P21 and BAX. However, in the search for potent HDAC inhibitors with cancer therapeutic potential, a tendency exists to overlook or dismiss weak ligands that could prove effective in cancer prevention. Butyrate, diallyl disulfide (DADS), and sulforaphane (SFN) are three dietary agents that exhibit HDAC inhibitory activity in vitro and/or in vivo, and other such dietary agents probably will be discovered that affect HDAC activity. We make the distinction between ‘pharmacologic’ agents that potently derepress gene expression, during therapeutic intervention, and dietary HDAC inhibitors that, as weak ligands, might subtly regulate the expression of genes involved in cell growth and apoptosis. An important issue for future study is to determine the extent to which dietary HDAC inhibitors, by modulating genes such as p21 and Bax, enable normal, nontransformed cells to respond most effectively to external stimuli and toxic insults.

Phosphorylation and ubiquitination of oncogenic mutants of β-catenin containing substitutions at Asp32

β-Catenin, a member of the Wnt signaling pathway, is downregulated by glycogen synthase kinase-3β (GSK-3β)-dependent phosphorylation of Ser/Thr residues in the N-terminus of the protein, followed by ubiquitination and proteosomal degradation. In human and rodent cancers, mutations that substitute one of the critical Ser/Thr residues in the GSK-3β region of β-catenin stabilize the protein and activate β-catenin/TCF/LEF target genes. This study examined three oncogenic β-catenin mutants from rat colon tumors containing substitutions adjacent to amino-acid residue Ser33, a key target for phosphorylation by GSK-3β. Compared with wild-type β-catenin (WT), the β-catenin mutants D32G, D32N, and D32Y strongly activated TCF-4-dependent transcription in HEK293 cells, and there was accumulation of β-catenin in the cell lysates. Immunoblotting with phosphospecific antibodies indicated that there was little if any effect on the phosphorylation of Ser37, Thr41 or Ser45; however, the phosphorylation of Ser33 appeared to be affected in the β-catenin mutants. Specifically, antiphospho-β-catenin 33/37/41 antibody identified high, intermediate and low expression levels of phosphorylated β-catenin in cells transfected with D32G, D32N and D32Y, respectively. Experiments with the proteosome inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) revealed ubiquitinated bands on all three mutant β-catenins, as well as on WT β-catenin. The relative order of ubiquitination was WT>D32G>D32N>D32Y, in parallel with findings from the phosphorylation studies. These results are discussed in the context of previous studies, which indicated that amino-acid residue D32 lies within the ubiquitination recognition motif of β-catenin.

Myeloperoxidase-dependent caspase-3 activation and apoptosis in HL-60 cells: protection by the antioxidants ascorbate and (dihydro)lipoic acid

Abstract The heme enzyme myeloperoxidase (MPO) has recently been implicated in hydrogen peroxide H2O2-induced apoptosis of HL-60 human leukemia cells. The purpose of this study was to investigate the molecular mechanism(s) of MPO-mediated apoptosis, in particular caspase-3 activation, and to determine the effects of the antioxidants ascorbate and (dihydro)lipoic acid. Incubation of HL-60 cells (1 x 106 cells/ml media) with H2O2 (0-200 μM) resulted in dose-dependent stimulation of caspase-3 activity, DNA fragmentation, and morphological changes associated with apoptosis. Caspase-3 activity, DNA fragmentation and apoptosis were maximal at approximately 50 μM H2O2. Pre-incubation of the cells with the MPO-specific inhibitor 4-aminobenzoic acid hydrazide (ABAH) and the heme enzyme inhibitor 3-aminotriazole (100 μM each) resulted in complete and partial inhibition, respectively, of intracellular MPO, caspase-3 activity, and apoptosis following addition of 50 μM H2O2. Enhancement of cellular antioxidant status by pre-incubation of the cells with dehydro-ascorbic acid and lipoic acid, which are reduced intracellularly to ascorbate and dihydrolipoic acid, respectively, afforded protection against caspase-3 activation and apoptosis following addition of H2O2. Addition of high concentrations of H2O2 (200 μM) to cells pre-incubated with lipoic acid, however, resulted in cytotoxicity. Overall, our data indicate that MPO-derived oxidants, rather than H2O2 itself, are involved in caspase-3 activation and apoptosis in HL-60 cells, and the antioxidants ascorbate and (dihydro)lipoic acid inhibit caspase-3 activation and apoptosis in these cells, likely via scavenging the MPO-derived oxidants.