Dietary HDAC inhibitors: time to rethink weak ligands in cancer chemoprevention?

Roderick H.Dashwood 0 2 Melinda C.Myzak 0 1 Emily Ho 0 1 0 Linus Pauling Institute, Oregon State University , Corvallis, OR 97331-6512 , USA 1 Department of Nutrition & Exercise Sciences 2 Department of Environmental & Molecular Toxicology To whom correspondence should be addressed. Email: There is growing interest in the various mechanisms that regulate chromatin remodeling, including modulation of histone deacetylase (HDAC) activities. Competitive HDAC inhibitors disrupt the cell cycle and/or induce apoptosis via de-repression of genes such as P21 and BAX, and cancer cells appear to be more sensitive than non-transformed cells to trichostatin A and related HDAC inhibitory compounds. This apparent selectivity of action in cancer cells makes HDAC inhibitors an attractive avenue for drug development. However, in the search for potent HDAC inhibitors with cancer therapeutic potential there has been a tendency to overlook or dismiss weak ligands that could prove effective in cancer prevention, including agents present in the human diet. Recent reports have described butyrate, diallyl disulfide and sulforaphane as HDAC inhibitors, and many other dietary agents will be probably discovered to attenuate HDAC activity. Here we discuss 'pharmacologic' agents that potently de-repress gene expression (e.g. during therapeutic intervention) versus dietary HDAC inhibitors that, as weak ligands, might subtly regulate the expression of genes involved in cell growth and apoptosis. An important question is the extent to which dietary HDAC inhibitors, and other dietary agents that affect gene expression via chromatin remodeling, modulate the expression of genes such as P21 and BAX so that cells can respond most effectively to external stimuli and toxic insults. Introduction Chromatin remodelingdirect versus indirect HDAC inhibition Considerable attention has focused on the silencing and unsilencing of genes through changes in DNA methylation (1), but such epigenetic modifications in DNA often require prior alterations at the level of the histones. The histone code refers to an ever increasing complexity of histone modifications, including acetylation, methylation, phosphorylation, ubiquitination and biotinylation (2). There is growing interest Abbreviations: APL, acute promyelocytic leukemia; CLA, conjugated linoleic acid; HDAC, histone deacetylase; RAR, retinoic acid receptors; RARE, retinoic acid response element; SAHA, suberoylanilide hydroxamic acid; SFN, sulforaphane; SFNCys, SFNcysteine; SFNGSH, SFN glutathione; SFNNAC, SFNN-acetylcysteine. in these post-translational changes and their implications for cancer development. Global loss of monoacetylation and trimethylation of histone H4 is a common hallmark of human tumor cells (3). A recent commentary (4) also discussed novel protective functions of p53 associated with chromatin remodeling on a global scale, including transcriptional mechanisms that recruit or displace histone deacetylase (HDAC). Direct HDAC inhibitors also can affect changes in gene expression and impact on key regulators of apoptosis and the cell cycle (510), such as p21Cip1/Waf1, cyclins (A, E, B1, D1 and D3), apoptosis mediators (CD95, Bax and Bcl-2), transcription factors (GATA-2, c-Myc) and retinoic acid receptors (RAR). RARs are targets of retinoids, which have been reviewed extensively in terms of their promise and pitfalls for cancer prevention (11,12). One area of particular interest has been that of acute promyelocytic leukemia (APL), because APL patients respond to pharmacologic doses of retinoic acid with disease remission. Molecular studies have focused on the PML/RAR oncogenic transcription factor, and models have been developed to explain the therapeutic mechanisms of retinoids in APL (Figure 1) (http://www.nature.com/nrc/journal/v1/ n3/animation/nrc1201-181a_swf_MEDIA1.html, http://www. broad.mit.edu/chemobio/lab_schreiber/anims/animations/ trich_retin.html). In brief, RAR/PML binding to the retinoic acid response element (RARE) recruits the CoR/SIN3/HDAC complex and represses transcription (Figure 1A), but agonists such as retinoic acid interact with the RAR and displace CoR/ SIN3/HDAC (13), thereby activating gene expression in association with CoA/HAT complexes (Figure 1B). Resistance to retinoic acid treatment can occur in APL, due to the presence of a RARPLZF fusion protein and the inability to effectively displace HDAC (14,15). However, therapeutic efficacy in such cases can be improved when retinoids are combined with agents such as trichostatin A or suberoylanilide hydroxamic acid (SAHA), which interact directly with HDAC as competitive inhibitors (1619) (Figure 1C). The latter compounds have provided valuable insights into the role of specific residues in the HDAC catalytic mechanism and the geometry of the substrate-binding pocket (6). Important structural features for HDAC inhibition appear to be a functional group that interacts with the buried zinc atom, a spacer or linker arm that fits within the HDAC pocket, and in many (but not all) inhibitors a cap group that sits just outside the active site (610). HDAC inhibitors have been reported to disrupt the cell cycle in G2, allowing cells to prematurely enter the M phase, as well as interfering directly with the mitotic spindle checkpoint (20). Cell cycle arrest and/or apoptosis is mediated through the de-repression of genes such as P21 and BAX, and cancer cells appear to be more sensitive than non-transformed cells to the actions of HDAC inhibitors. The mechanistic basis for this apparent selectivity of action against cancer cells is far from clear, although recent studies have implicated thioredoxin and intracellular thiol status, the accumulation of reactive oxygen species, and induction of TRAIL (Apo2L, TNFSF10), DR4 and DR5 (21,22). For the reasons alluded to above, HDAC inhibitors provide an attractive avenue for drug development, and considerable attention has focused on potent, high-affinity agents related to trichostatin A and SAHA (710). However, in the search for HDAC inhibitors with cancer therapeutic potential, we believe that there has been a tendency to overlook or dismiss weak ligands that could prove effective in cancer prevention, including agents present in the human diet. Dietary HDAC inhibitorsweak ligands in cancer prevention A recent review discussed the cancer chemopreventive properties of three reported dietary HDAC inhibitors (23), namely butyrate, diallyl disulfide (DADS) and sulforaphane (SFN). In general, these dietary agents are weak ligands and inhibit HDAC activity at higher concentrations than trichostatin A or SAHA, which are effective in the nanomolar to low micromolar range. A pertinent question, then, concerns the concentrations needed for inhibition of HDAC activity by dietary compounds, and the likelihood that these levels might be achieved under normal physiological conditions. Butyrate is the smallest known HDAC inhibitor and contains a sim (...truncated)