Cancer chemoprevention by dietary polyphenols: Promising role for epigenetics

Abstract

Epigenetics refers to heritable changes that are not encoded in the DNA sequence itself, but play an important role in the control of gene expression. In mammals, epigenetic mechanisms include changes in DNA methylation, histone modifications and non-coding RNAs. Although epigenetic changes are heritable in somatic cells, these modifications are also potentially reversible, which makes them attractive and promising avenues for tailoring cancer preventive and therapeutic strategies. Burgeoning evidence in the last decade has provided unprecedented clues that diet and environmental factors directly influence epigenetic mechanisms in humans. Dietary polyphenols from green tea, turmeric, soybeans, broccoli and others have shown to possess multiple cell-regulatory activities within cancer cells. More recently, we have begun to understand that some of the dietary polyphenols may exert their chemopreventive effects in part by modulating various components of the epigenetic machinery in humans. In this article, we first discuss the contribution of diet and environmental factors on epigenetic alterations; subsequently, we provide a comprehensive review of literature on the role of various dietary polyphenols. In particular, we summarize the current knowledge on a large number of dietary agents and their effects on DNA methylation, histone modifications and regulation of expression of the non-coding miRNAs in various in vitro and in vivo models. We emphasize how increased understanding of the chemopreventive effects of dietary polyphenols on specific epigenetic alterations may provide unique and yet unexplored novel and highly effective chemopreventive strategies for reducing the health burden of cancer and other diseases in humans.

Introduction

Cancer is widely perceived as a heterogeneous group of disorders, which is caused by a series of clonally selected ‘genetic’ changes in key tumor suppressor genes and oncogenes. However, accumulating evidence in the recent years indicate that tumor cell heterogeneity is in part due to significant contribution of ‘epigenetic’ alterations in cancer cells. Consequently, it is now becoming apparent that epigenetic plasticity together with genetic lesions drives tumor progression, and that cancer is the manifestation of both genetic and epigenetic modifications [1], [2], [3], [4]. Although a small proportion of tumors can be inherited, it is believed that majority of cancers result from changes that accumulate throughout the life because of exposure to various endogenous factors such as nutrients, infections, physical activity, social behavior and other environmental factors. Even when cancer initiation and progression is driven by acquired genetic alterations, epigenetic disruption of gene expression plays an equally important role in the development of disease [5], and arguably diet and environment-mediated epigenetic perturbations play a crucial role in cancer progression in humans [6], [7], [8].

The term ‘epigenetics’, which was first coined by the developmental biologist Conrad H. Waddington in 1942, is defined as reversible heritable changes in gene expression that occur without alteration in DNA sequence, but changes that are sufficiently powerful to regulate the dynamics of gene expression [9]. Three distinct and intertwined mechanisms are known to be part of the “epigenome”, which includes DNA methylation, histone modifications, and post transcriptional gene regulation by non-coding microRNAs (miRNAs) [2]. These processes affect transcript stability, DNA folding, nucleosome positioning, chromatin compaction, and complete nuclear organization of the genetic material (Figure 1). Synergistically and cooperatively they determine whether a gene is silenced or expressed, as well as the timing and tissue-specificity of the expression of these genes. Disruption of the epigenome certainly underlies disease development. Therefore, disease susceptibility is clearly a result of complex interplay between one's genetic endowment and epigenetic marks imprinted on one's genome by endogenous and exogenous factors [10].

From a clinical point of view, epigenetics offers a very promising and attractive avenue. This is because, unlike genetic changes (mutations, gene deletions, etc.), epigenetic alterations are potentially reversible. What this means is that unlike mutations, which exist for the lifetime, epigenetically modified genes can be restored; methylation silenced genes can be demethylated, and histone complexes can be rendered transcriptionally active by modification of acetylation and methylation of various histones via nutrients, drugs and other dietary interventions. This is really fascinating, as this provides a perfect opportunity for designing optimal chemopreventive and therapeutic strategies. The mechanism of interaction between various epigenetic factors and regulation of chromatin structure, dynamics, and ultimately gene expression is an active area of research, and recent understanding of these epigenetic mechanisms is highlighted in the sections below.

DNA methylation of cytosines at CpG dinucleotides is perhaps the most extensively studied epigenetic modification in mammals. DNA methylation, in association with histone modifications is an essential component of the epigenetic machinery, which regulates gene expression and chromatin architecture [11]. In mammalian cells, DNA methylation occurs at the 5′ position of the cytosine residues within CpG dinucleotides through addition of a methyl group to form 5-methylcytosine [12]. CpG dinucleotides are not uniformly distributed throughout the human genome, but are often enriched in the promoter regions of genes, as well as regions of large repetitive sequences (e.g. centromeric repeats, LINE and ALU retrotransposon elements) [13]. Short CpG-rich regions are also called as “CpG islands”, and these are present in more than 50% of human gene promoters [14]. Whilst most of the CpG dinucleotides in the genome are methylated, the majority of CpG islands usually remain unmethylated during development and in undifferentiated normal cells [15]. Hypermethylation of CpG islands within gene promoters can result in gene silencing, while promoters of transcriptionally active genes typically remain hypomethylated [15]. DNA methylation can lead to gene silencing by either preventing or promoting the recruitment of regulatory proteins to DNA. For example, it can inhibit transcriptional activation by blocking transcription factors from accessing target-binding sites, e.g. c-myc [16]. In other instances, it can provide binding sites for methyl-binding (sequestering) domain proteins, which can orchestrate gene repression through interaction with histone modifying enzymes [17]. Thus DNA methylation uses a variety of mechanisms to silence genes, and a direct association between DNA methylation and the phenotype of a cell can be postulated.

The modification at 5-methylcytosine is catalyzed by various DNA methyltransferases (DNMTs). There are three main DNMTs; DNMT1, which is the major maintenance enzyme that preserves existing methylation patterns following DNA replication by adding methyl groups to the hemi-methylated (partially-methylated) CpG sites [18], [19]; DNMT3a and DNMT3b on the other hand serve as de novo methyltransferases, which act independent of replication and show equal preference for both unmethylated and hemi-methylated DNA [20], [21]. The role of DNA methylation-induced transcriptional silencing of genes is now well-established in multiple human malignancies [22]. In fact, analogous to mutations and deletions, DNA methylation of genes in most human cancers is now believed to be a most frequent mechanism for the transcriptional silencing of tumor suppressor genes [23]. Several detailed and informative reviews on the association between DNA methylation and cancer are available, but these are beyond the scope of this review [11], [19], [24], [25], [26], [27].

In addition to direct methylation of DNA, chromatin structure is frequently influenced by diverse histone modifications, which also play an important role in gene regulation and tumorigenesis [3], [4]. Chromatin proteins serve as building blocks to package eukaryotic DNA into higher order chromatin fibers. Each nucleosome encompasses ∼146 bp of DNA wrapped around an octamer of histone proteins. These octamers consist of double subunits of H2A, H2B, H3 and H4 core histone proteins [28]. The histone proteins coordinate the changes between tightly packed DNA (or heterochromatin), which is inaccessible to transcription, and lightly packed DNA (or euchromatin), which is available for active transcription through binding of transcription factors [29]. These changes typically occur in the ‘histone tails’, which extend from the core octamer. The histone tails comprise of a globular C-terminal domain and an unstructured N-terminal tail [30]. The N-terminal histone tails are the major sites for post-translational modifications including methylation, acetylation, phosphorylation, ribosylation, ubiquitination, sumoylation and biotinylation [31]. The majority of these modifications take place at lysine, arginine and serine residues within the histone tails and regulate key cellular processes such as transcription, replication and repair [31]. Unlike DNA methylation, histone modifications can lead to either activation or repression depending upon which residues are involved, and the type of modification present. For instance, lysine acetylation associates with transcriptional activation, while its methylation leads to transcriptional activation or repression depending upon which specific lysine is modified. For instance, tri-methylation of lysine 4 on histone H3 (H3K4me3) is enriched at transcriptionally active gene promoters [32], whereas tri-methylation of H3K9 (H3K9me3) and H3K27 (H3K27me3) is present at transcriptionally repressed promoters [31]. H3K9me3 and H3K27me3 histone modifications together constitute the two main silencing mechanisms in mammalian cells.

Similar to DNA methylation changes, various histone modifications are potentially reversible, and are dynamically regulated by groups of enzymes that add or remove covalent modifications to histone proteins [3], [33]. Histone acetyltransferases (HATs) and histone methyltransferases (HMTs) add acetyl and methyl groups, respectively, whereas histone deacetylases (HDACs) and histone demethylases (HDMs) remove acetyl and methyl groups, respectively, from histone proteins [34], [35], [36]. A number of histone-modifying enzymes including various HATs, HMTs, HDACs, and HDMs have been identified in the recent years, including a large number of dietary polyphenols enumerated in the later sections of this review.

Besides DNA methylation and histone modifications, miRNAs are emerging as key mediators of epigenetic gene regulation in mammals. Non-coding RNAs, including miRNAs, were initially noted to perform catalytic functions in facilitating RNA splicing. In recent years it has been recognized that they participate in the post-transcriptional gene regulation [37], [38]. miRNAs are small single-stranded RNAs, ∼19–24 nucleotides in length, that regulate gene expression through post-transcriptional silencing of the target genes. Sequence specific base pairing of miRNAs with 3′ untranslated regions of the target messenger RNA (mRNA) results in degradation or translational inhibition [39]. miRNAs are expressed in a tissue-specific manner and control a wide spectrum of biological processes including cell proliferation, apoptosis and differentiation. Although miRNA are vital to normal cell physiology, aberrant expression of these small non-coding RNAs has been linked to carcinogenesis. In fact, miRNA profiles are now being used to classify human cancers [40], [41], [42]. One of the interesting features of miRNAs is that similar to regular genes, their own expression can be regulated by other epigenetic mechanisms, such as DNA methylation [43]. The influence of miRNA on the epigenetic machinery and the reciprocal epigenetic regulation of miRNA expression suggest that its deregulation during carcinogenesis has an important implication for global regulation of epigenetics and cancer. Additionally, interaction among various components of the epigenetic machinery re-emphasizes the integrated nature of epigenetic mechanisms involved in the maintenance of global gene expression patterns in mammals. Several detailed and informative reviews of the association between miRNA and cancer have been published previously [37], [41], [44], [45], [46].