Epigenetic mechanisms in anti-cancer actions of bioactive food components – the implications in cancer prevention

DNA methylation

DNA methylation is a covalent modification of DNA that in mammalian cells takes place mainly at the fifth position of the cytosine pyrimidine ring located predominantly within CpG sequences (Hotchkiss, 1948; Wyatt, 1950; Gruenbaum et al., 1981). CG-rich regions, called CpG islands, are mostly unmethylated in normal cells and located in regulatory regions of housekeeping genes, tissue-specific genes and tumour suppressors (Hermann et al., 2004). CpG islands are also present in promoters of some oncogenes, where their methylated state is involved in the formation of an inactive chromatin structure leading to transcriptional silencing (Szyf et al., 2004). Recent data indicate that methylation can also occur in cytosines within dinucleotide sequences other than CpGs, although the role of non-CpG methylation remains to be elucidated (Lister et al., 2009).

DNA methylation in normal cells is implicated in oncogene repression, the control of expression of genes crucial for cell proliferation, differentiation and normal development as well as in parental imprinting, X chromosome inactivation, and preservation of chromosomal integrity by the silencing of transposons and repetitive elements (Hermann et al., 2004; Szyf et al., 2004). An inverse correlation between the extent of DNA methylation and gene expression was reported in many studies (Rauch et al., 2009; Stefanska et al., 2011a) and several mechanisms were proposed for DNA methylation-mediated transcriptional silencing (Stein et al., 1982). Firstly, some transcription factors, such as CREB (cAMP response element binding protein), E2F (elongation 2 factor), NF-κB and AP-2 (activator protein-2), are unable to recognize specific sequences when they are methylated or methylation occurs in their proximity (Comb and Goodman, 1990; Inamdar et al., 1991; Paluszczak and Baer-Dubowska, 2005). Secondly, the binding of transcription factors to regulatory elements within promoters and enhancers can be hindered by methyl-CpG-binding domain proteins (MBDs) that bind with high affinity to methylated DNA and cover recognition elements (Nan et al., 1997; Reik and Dean, 2001). The third mechanism is associated with the MBD-mediated recruitment of HDACs and histone methyltransferases that set up a compacted inactive chromatin state around the gene (Nan et al., 1997; Eden et al., 1998; Das and Singal, 2004). Numerous data show that the hallmark of cancer is global hypomethylation and hypermethylation of specific regions, mainly within promoters of tumour suppressor genes. The increase in promoter DNA methylation was reported as a common mechanism of tumour suppressor gene silencing observed in many types of cancer (Baylin et al., 2001; Szyf et al., 2004; Hatziapostolou and Iliopoulos, 2011), and the reversal of the aberrant DNA hypermethylation reactivated gene transcription (Szyf, 2005; Stefanska et al., 2010; 2011b). Recent whole-genome approaches demonstrate that loss of DNA methylation in gene promoters can have a detrimental effect in cancer as hypomethylation was linked to the activation of genes implicated in metastasis, invasion and other biological functions typical for cellular transformation (Stefanska et al., 2011a).

DNA methylation is carried out by DNMTs, which catalyze the transfer of a methyl group from the ubiquitous methyl donor, S-adenosyl-L-methionine (SAM), to the fifth position of a cytosine pyrimidine ring (Gruenbaum et al., 1981). The main enzyme responsible for the maintenance of DNA methylation patterns is DNMT1 (Chen et al., 2007a), whereas de novo methylation activity is attributed to DNMT3A and DNMT3B (Okano et al., 1999; Robertson et al., 1999). Both DNMT3s play a role in genomic imprinting and silencing of transposons and repetitive elements during embryonic development (Okano et al., 1998; 1999) and their substrate recognition is regulated by another member of DNMTs, DNMT3L (Bourc'his et al., 2001; Bourc'his and Bestor, 2004).

DNA methylation has been considered as a non-reversible reaction for decades. It was assumed that demethylation can only occur in a passive way by blocking the activity of DNMTs in dividing cells. However, evidence has been obtained showing that active demethylation does take place, such as demethylation of paternal genome, during embryogenesis or demethylation in post-mitotic neurons (Weaver et al., 2004; Mastronardi et al., 2007; Feng et al., 2010). Szyf et al. (Bhattacharya et al., 1999; Ramchandani et al., 1999; Detich et al., 2002) showed that one of the MBDs, MBD2, has demethylase activity and demethylates both fully methylated and hemimethylated DNA in vitro. One of the mechanisms of the active enzymatic removal of a methyl group from DNA can involve oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, followed by the release of a methyl group in formaldehyde (Hamm et al., 2008). This concept becomes more probable in the light of recent findings demonstrating the presence of 5-hydroxymethylcytosine in mammalian DNA (Kriaucionis and Heintz, 2009; Ndlovu et al., 2011). Although the biological function of this modification still remains poorly understood, recent reports providing insights into 5-hydroxymethylcytosine genomic distribution suggest it is potentially highly relevant in gene regulation (Ndlovu et al., 2011). The ten-eleven translocation (TET) family of enzymes has been demonstrated to catalyze the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. In addition, TET1 was required for the maintainance of demethylation of nanog in embryonic stem cells (Ito et al., 2010). This raises the possibility that this reaction might be an intermediate form of active DNA demethylation (Ndlovu et al., 2011). An alternative mechanism suggested for DNA demethylation is based on the DNA repair system, where 5-methylcytosine is replaced with non-methylated cytosine (Razin et al., 1986; Barreto et al., 2007; Rai et al., 2008). Deamination of 5-methylcytosine to thymine carried out by the activation-induced (cytosine) deaminase (Rai et al., 2008) creates a G : T mismatch that is recognized by a specific thymine glycosylase, Mbd4, and the repair is mediated by the growth arrest and DNA-damage inducible (Gadd45) protein (Barreto et al., 2007).

Histone modifications and non-coding RNAs

Chromatin consists of DNA, histones and non-histone proteins, and plays a role in compacting DNA, preventing DNA damage, controlling gene expression and DNA replication (Kouzarides, 2007; Cedar and Bergman, 2009). DNA is wrapped around a histone octamer that comprises two subunits of each of the core histones, H2A, H2B, H3 and H4 (Kouzarides, 2007; Kanwal and Gupta, 2011), containing amino acid residues with an amino group (Kouzarides, 2007; Bannister and Kouzarides, 2011; Fullgrabe et al., 2011). The positively charged amino group interacts with the negatively charged phosphate groups of DNA, which results in the compaction of chromatin and the formation of transcriptionally inactive heterochromatin. It is well established that post-translational modifications of the histone tails regulate gene expression by determining chromatin structure; these modifications include methylation, acetylation, ubiquitination, phosphorylation, sumoylation, ADP ribosylation, biotinylation and proline isomerization (Jenuwein and Allis, 2001; Kouzarides, 2007; Bannister and Kouzarides, 2011). These modifications are dynamic and controlled by a variety of enzymes that add and remove specific groups. The most studied are histone acetylation mediated by histone acetyltransferases (HATs) and HDACs, and histone methylation controlled by methyltransferases (HMTs) and demethylases (HDMs) (Kouzarides, 2007; Yang and Seto, 2007; Bannister and Kouzarides, 2011). Actively transcribed genes are enriched with active histone marks such as acetylation of H3K9, H3K14 and H3K4, trimethylation of H3K4, dimethylation of H3K79 and monomethylation of H3K4, H3K9, H3K27, H3K79 and H2BK5, whereas regions transcriptionally inactive are characterized by dimethylation of H3K9 and H3K27, and trimethylation of H3K9, H3K27 and H2BK5 (Barski et al., 2007; Koch et al., 2007; Li et al., 2007; Steger et al., 2008; Rosenfeld et al., 2009; Karlic et al., 2010b). Recent studies with genome-wide approaches revealed that histone modifications have different functional outcomes on gene transcription and chromatin structure, depending on their positional context as well as the interplay among various histone modifications (Li et al., 2007; Batta et al., 2011).

Although still controversial, non-coding RNAs have been included as a component of the epigenome (Choudhuri, 2011). Non-coding RNAs are encoded by a major part of our genome and regulate gene expression by controlling translation of mRNA into proteins. Without changing the DNA sequence, non-coding RNAs and more specifically micro-RNAs (miRNAs) are involved in heritable changes in gene expression. More interestingly, they are implicated in reciprocal interconnections between all the components of the epigenome. Aberrant miRNA regulation, especially that associated with cancer, can be influenced by DNA methylation and histone covalent modifications (Lu et al., 2005; Iorio et al., 2010). On the other hand, miRNA affects the epigenome by targeting enzymes of the epigenetic machinery (Iorio et al., 2010; Zhou et al., 2010). DNMT1 and DNMT3B are targets of miR-148a in cholangiocarcinoma and cervical cancer, respectively. HDAC1 is targeted by miR-449a and b in prostate cancer, and HDAC4 by miR-1 in HCC. miRNA genes are transcribed in the nucleus and the miRNA transcript is processed in the cytoplasm where it is cut by the endoribonuclease Dicer to approximately 22 nucleotide fragments. This short, double-stranded RNA is part of the RNA-induced silencing complex where one strand of miRNA is eliminated and the other strand is orientated by Argonaute protein, Ago2, for interaction with a target mRNA. Complementary binding to mRNA is followed by its cleavage, degradation and inhibition of translation (Fire et al., 1998; Zhou et al., 2010; Choudhuri, 2011).

Aberrations in both histone modifications and miRNA regulation have been shown to be associated with several diseases, including cancer (Kanwal and Gupta, 2010; Lovat et al., 2011). Deregulation of miRNAs is linked to tumour initiation and progression in many human cancers (Lovat et al., 2011).

Folate and other nutrients of one-carbon metabolism

A group of nutrients involved in one-carbon metabolism such as folate, cobalamin, riboflavin, pirydoxine and methionine have a direct effect on the level of a methyl donor, SAM (Figure 1). Folate is a water-soluble form of vitamin B9, which is important for DNA synthesis, repair and methylation. After dietary intake, folate is converted to tetrahydrofolate, which is involved in the remethylation of homocysteine to methionine (Scott and Weir, 1998; Selhub, 2002). Methionine is a precursor of SAM, which is the primary methyl group donor for most methylation reactions (Waterland, 2006). After transferring a methyl group, SAM is converted to S-adenosyl-L-homocysteine (SAH), an inhibitor of methylation reactions (Figure 1). Numerous studies have shown a causal role of a folate deficiency in cancer development (Duthie et al., 2004), including amongst others colon (Kim, 2004; Duthie, 2011), prostate (Bistulfi et al., 2010) and liver cancer (James et al., 2003), as well as head and neck squamous cell carcinoma (Kraunz et al., 2006). Malignant transformation was accompanied by SAM depletion, global DNA hypomethylation, gene-specific hypomethylation and activation of oncogenes, and paradoxically in hypermethylation and silencing of tumour suppressor genes (James et al., 2003; Davis and Uthus, 2004; Duthie et al., 2004). Folate deficiency has been shown to alter components of the DNA methylation machinery with the increase in DNMT1, DNMT3B, MBD2 and MBD4 mRNA and protein levels during the early stages of hepatocarcinogenesis (Ghoshal et al., 2006). The increase in DNMTs may explain the hypermethylation observed, whereas the stimulation of MBD2 and MBD4 may be responsible for hypomethylation of oncogenes and prometastatic genes, as both proteins have been implicated in active DNA demethylation (Bhattacharya et al., 1999; Ramchandani et al., 1999; Detich et al., 2002; Rai et al., 2008; Popp et al., 2010). Hence, folic acid supplementation may have a chemopreventive effect; this has been demonstrated in a few cancer models, including hepatocarcinogenesis. In rats fed a folic acid-enriched diet followed by the chemical initiation of liver cancer, cell growth and the number of preneoplastic lesions as well as c-myc oncogene expression were diminished compared with a control group (Chagas et al., 2011). Although no changes in global DNA methylation were detected in this study, the SAM/SAH ratio significantly increased, which has the potential to induce gene-specific alterations. Furthermore, folic acid supplementation has been found to reverse cervical dysplasia in patients using oral contraceptives and prevent cervical cancer (Butterworth et al., 1982; Whitehead et al., 1989). Recent prospective and epidemiological studies of 718 Chinese breast cancer cases, Shanghai Women's Health Study (1997–2008), indicated that high folate intake reduces breast cancer risk in premenopausal women and that low intakes of methionine, pyridoxine, cobalamin, niacin and riboflavin are associated with breast carcinogenesis (Shrubsole et al., 2011). A protective role of folate in breast cancer, particularly in oestrogen receptor (ER)-negative cancer, was also shown in another prospective study (Maruti et al., 2009).

Other B vitamins implicated in one-carbon metabolism include riboflavin (B2), pyridoxine (B6) and cobalamin (B12) (Figure 1). Riboflavin is a component of FAD (flavin adenine dinucleotide) that acts as a cofactor for methylenetetrahydrofolate (MTHF) reductase catalyzing the conversion of 5,10-MTHF to 5-MTHF, a substrate for homocysteine remethylation to methionine (Slattery et al., 1999). As shown in Figure 1, blocking this enzyme activity will diminish methionine synthesis and DNA methylation reactions. It was reported that a reduced riboflavin intake can lead to changes in the methyl supply, followed by alterations in the DNA methylation state (Singh et al., 2003). Pyridoxine acts not only as a cofactor for serine hydroxymethyltransferase in the synthesis of 5,10-MTHF from THF (Selhub, 2002) but also participates in the trans-sulfuration reactions, where glutathione is formed from homocysteine (Figure 1) (Maruti et al., 2009). A low pyridoxine plasma concentration has been linked to an increased risk of colorectal cancer in a prospective nested case–control study of female participants (The Nurses' Health Study; Wei et al., 2005). As a consequence of pyridoxine depletion, the SAH level increases, resulting in DNA hypomethylation through inhibition of methyltranferases (Maruti et al., 2009). Cobalamin is another vitamin B that, by affecting one-carbon metabolism, is involved in the regulation of DNA methylation. As a cofactor for methionine synthase, cobalamin is important for the methylation of homocysteine to methionine (Figure 1) (Selhub, 2002). Indeed, in many studies, a diet deficient in major dietary sources of the methyl group, including folate and cobalamin, has been linked to the development of HCC (Newberne and Rogers, 1986; Kanduc et al., 1988; Asada et al., 2006; Calvisi et al., 2007). Promoter hypomethylation and increased expression of oncogenes were detected in livers of rats fed methyl-deficient diets (Zapisek et al., 1992). More interestingly, the effects of folate, pyridoxine and cobalamin supplementation on DNA methylation and cancer risk were dependent on MTHFR gene polymorphisms. Colon cancer risk was reduced by 30–40% in individuals with variant TT genotype of the MTHFR gene who consumed adequate amounts of the nutrients compared to those with CC genotype whose diet was low in nutrients (Slattery et al., 1999; Davis and Uthus, 2004).

Retinoic acid and vitamin D3

Contrary to nutrients and vitamins such as folic acid or cobalamin, which act directly on the synthesis of a methyl group donor, the epigenetic activity of retinoic acid (RA) and vitamin D3 (Vit.D3) is mainly mediated by an interaction with their receptors. RA, which is generated via oxidative conversion from β-carotene absorbed from fruits and vegetables, binds to nuclear retinoic acid receptors (RARs) α, β and γ that heterodimerize with retinoid X receptors (RXRs). This complex further modulates transcription through cognate response elements in the promoters of target genes including p21 and AP-1 (Liu et al., 1996; Wang et al., 2001; Benkoussa et al., 2002; Yu et al., 2008). RA activated p21 transcription in cervical squamous carcinoma (Wang et al., 2001; Arany et al., 2003), and inhibited AP-1 transcriptional activity in gastric cancer cells (Wu et al., 2002). As p21 competes with DNMT1 for the same binding site on PCNA that is crucial for DNMT1 activity (Chuang et al., 1997; Milutinovic et al., 2000; Iida et al., 2002), RA-mediated p21 induction decreases DNA methylation efficiency (Figure 2B). On the other hand, AP-1 stimulates DNMT1 transcription through binding sites located within the DNMT1 regulatory region (Bigey et al., 2000). Therefore, inhibiton of AP-1 activity by RA will down-regulate AP-1 responsive genes, including DNMT1 (Wu et al., 2002) and decrease DNA methylation (Figure 2C). As changes in DNA methylation affect gene function and signalling pathway networks (Maradeo and Cairns, 2011), a modulation of DNA methylation patterns can contribute to the anti-proliferative, differentiating and pro-apoptotic actions of RA, which have been observed in a variety of cancers, such as leukaemia (Degos et al., 1995), breast (Fujii et al., 2008), ovarian (Zhang et al., 2000), and head and neck cancer (Youssef et al., 2004). Treatment of non-invasive MCF-7 cells with RA caused a reduction in promoter methylation and an increase in the expression of RARβ2 and PTEN tumour suppressor genes, which was associated with inhibition of breast cancer cell growth (Stefanska et al., 2010; 2011b). Although a decrease in DNMT1 mRNA level was not observed in these studies, genome-wide analysis of all-trans RA on gene methylation and expression in neuroblastoma revealed the down-regulation of methyltransferases DNMT1 and DNMT3B, along with the up-regulation of endogenous miRNAs targeting these DNMTs (Das et al., 2010). These changes in DNMT expression were accompanied by the demethylation and overexpression of 82 genes, including NOS1 that is required for neural cell differentiation. MiRNA-mediated attenuation of DNMTs after RA treatment constitutes another mechanism for the demethylating activity of this compound. Furthermore, a decrease in DNA promoter methylation has been linked to the ability of RA to up-regulate the activity of glycine N-methyltransferase (GNMT) that catalyzes the conversion of SAM to SAH (Rowling et al., 2002). Activation of GNMT leads to the loss of methyl groups crucial for SAM-dependent methylation reactions (Figure 1).

Vit.D3, synthesized upon sun exposure and from diet, acts in a similar manner to RA by binding to vitamin D receptors (VDR) that can homodimerize or heterodimerize with RXR or RAR and affect gene transcription through VDR responsive elements (VDRE) in target genes including p21 (Saramaki et al., 2009) and PTEN (Pan et al., 2010). Epigenetic effects of Vit.D3 that can be partially linked to its activity as a chemopreventive and anti-cancer agent (Dace et al., 1997; Gurlek et al., 2002; Lin et al., 2002; Palmer et al., 2003) were primarily observed as histone modifications, which will be discussed in the next chapter. However, demethylating effects were also reported, for instance in osteocalcin promoter in MG63 osteosarcoma cells (Haslberger et al., 2006) and promoters of tumour suppressor genes in breast cancer cells (Stefanska et al., 2010; 2011b). Exposure to Vit.D3 caused hypomethylation and induction of PTEN and RARβ2 tumour suppressor genes in breast cancer cells (Stefanska et al., 2010; 2011b). Vit.D3-mediated promoter demethylation was also observed in CDH1 gene encoding for E-cadherin, which resulted in the activation of this gene and differentiation of breast cancer cells (Lopes et al., 2012). Similar to RA, changes in DNA methylation patterns induced by Vit.D3 treatment may be attributed to the negative regulation of DNMT1 activity via stimulation of p21 expression and inhibiton of AP-1 activity (Figure 2). In breast cancer, Vit.D3 increases p21 expression (Wang et al., 2001; Stefanska et al., 2011b), whereas in breast cancer and leukaemic cells, Vit.D3 and its derivatives induce PTEN transcription (Hisatake et al., 2001; Stefanska et al., 2011b). Activation of PTEN that is another negative regulator of AP-1 (Chung et al., 2006) may down-regulate AP-1 responsive genes, including DNMT1 (Figure 2C) (Bigey et al., 2000). More interestingly, the effects of Vit.D3 as well as RA seem to depend on cell invasiveness and ER status. Previous studies in human breast cancer cells indicated that activation of ERs stimulates the activity of c-Ha-ras, which leads to induction of the Ras/Raf/MAPK/AP-1 signalling pathway (Pethe and Shekhar, 1999). As the AP-1 transcription complex activates the DNMT1 promoter (Bigey et al., 2000), inactivation of ERs, by causing inhibition of AP-1, can consequently reduce DNMT1 expression. Vit.D3 as well as RA were shown to down-regulate ER abundance and function in MCF-7 cells (Pratt et al., 1996; Swami et al., 2000), which can explain their stronger epigenetic effects in this cell line compared with ER-negative MDA-MB-231 cells (Stefanska et al., 2010; 2011b).

Phytoestrogens

Several polyphenols called phytoestrogens such as resveratrol (RES) and genistein interact with ERs and regulate oestrogen-responsive genes such as p21 (Bowers et al., 2000; Mandal and Davie, 2010). They can act as oestrogen agonists or antagonists depending on their concentrations, tissue type and genes tested on, as well as the presence of oestradiol (Pozo-Guisado et al., 2002; 2004). RES (3,4′,5-trihydroxystilbene) is a dietary antioxidant found in a wide variety of plant species, including mulberries, peanuts, grapes, apricots and pineapples (Le Corre et al., 2004). This polyphenol was shown to modulate the risk of cardiovascular disease (atherosclerosis) and breast cancer, inhibit chemical carcinogenesis in rodents and growth promotion of preneoplastic lesions by effects on multiple signalling pathways, inhibit cell proliferation, block cell cycle progression and induce apoptosis in numerous types of human cancer cell lines (Bowers et al., 2000; Cornwell et al., 2004; Whitsett and Lamartiniere, 2006). Genistein (4′,5,7-trihydroxyisoflavone) is a major isoflavone from soybean. This polyphenol has been shown to prevent carcinogenesis in animal models (Dixon and Ferreira, 2002), reduce mammary carcinogenesis in rats exposed to carcinogens (Cabanes et al., 2004), inhibit growth of transplanted human prostate cancer cells in mice (Zhou et al., 1999) and inhibit carcinogenesis and metastasis in various types of cancer (Arai et al., 2000; Fang et al., 2005). Moreover, it has been reported that genistein inhibits cancer cell proliferation and angiogenesis as well as induces apoptosis and cell cycle arrest (Li et al., 1999; Fang et al., 2005). Dietary intake of genistein decreases mammary tumour incidence and increases tumour latency in rats exposed to chemical carcinogens. These breast cancer protective effects were found to be associated with the down-regulation of the oncogenic Wnt-signalling pathway and the up-regulation of PTEN tumour suppressor gene in the mammary gland, which was coincident with increased apoptosis and enhanced differentiation (Dave et al., 2005; Su et al., 2007). Changes in the epigenome upon treatment with these phytoestrogens have been reported in numerous studies. In non-invasive MCF-7 breast cancer cells, RES exposure led to hypomethylation and reactivation of RARβ2 and PTEN tumour suppressor genes with a concomitant decrease in DNMT1 expression and up-regulation of p21 (Stefanska et al., 2011b). RES prevented BRCA1 promoter hypermethylation and silencing induced by an aromatic hydrocarbon receptor ligand in MCF-7 cells (Papoutsis et al., 2011). Treatment of KYSE510 oesophageal squamous cell carcinoma cells with genistein resulted in a partial reversal of p16, RARβ and MGMT (O⁶-methylguanine methyltransferase) promoter hypermethylation and in reactivation of these genes, which was associated with the inhibition of DNMT activity (Fang et al., 2005). Similarly, methylation-silenced RARβ was hypomethylated and reactivated in LNCaP and PC3 prostate cancer cells exposed to genistein (Fang et al., 2007). Genistein significantly decreased promoter methylation and reactivated BTG3 tumour suppressor gene in A498, ACHN and HEK-293 renal cell carcinoma cell lines, as well as LNCaP and PC3 prostate carcinoma cell lines (Majid et al., 2009). Its effect was comparable with that of 5-aza-cytydine, which is a potent demethylating agent. Changes in DNA methylation were accompanied by increased histone acetylation and RNA polymerase II binding at the BTG3 promoter, suggesting active chromatin formation (Majid et al., 2009). Daidzein is another phytoestrogen found in soy (Barnes et al., 1996). Multiple studies have indicated that daidzein may act as a growth modulator in various types of cancer, such as colon (Bielecki et al., 2011), prostate (Vardi et al., 2010), breast (Choi and Kim, 2008) and pancreatic cancer (Guo et al., 2004). Treatment of human prostate cancer cells with daidzein led to demethylation of glutathione S-transferase P1 (GSTP1) and ephrin B2 (EPHB2) promoters and to an increase in their protein levels (Vardi et al., 2010). Demethylation and activation of tumour suppressor gene p16 was also detected in RKO colon cancer cells exposed to nordihydroguaiaretic acid found in the creosote bush (Cui et al., 2008) and to quercetin, which is another flavonoid found in a wide variety of vegetables, fruits, leaves and grains (Tan et al., 2009).

The epigenetic activity of phytoestrogens can be attributed to their ability to stimulate p21 via ERE elements in its promoter or other indirect mechanisms (Le Corre et al., 2004; Matsumura et al., 2008; Mandal and Davie, 2010) as well as to suppress AP-1 transcriptional activity via antagonism of ERs (Pethe and Shekhar, 1999) or inducing PTEN expression (Chung et al., 2006). As mentioned earlier, both p21 and AP-1 are involved in the regulation of DNMT1 expression/activity (Figure 2) (Chuang et al., 1997; Bigey et al., 2000; Iida et al., 2002). RES-mediated p21 up-regulation has been reported in many types of cancer, including prostate, bladder, colorectal and breast cancer (Tessitore et al., 2000; Bai et al., 2010; Stefanska et al., 2010; 2011b; Hsieh et al., 2011). Genistein treatment has been shown to induce an up-regulation of p21 in mouse B16-F1 melanoma cells, mouse fibroblasts (Kuzumaki et al., 1998) and breast cancer cells (Privat et al., 2010). Also, exposure to both RES and genistein is associated with an up-regulation of PTEN tumour suppressor gene in breast and prostate cancer (Waite et al., 2005; Kikuno et al., 2008; Rahal and Simmen, 2010; Stefanska et al., 2011b).

Polyphenols with catechol group

Certain polyphenols such as bioflavonoids (quercetin, fisetin and myricetin), tea catechins [catechin, epicatechin, (−)-epigallocatechin-3-gallate (EGCG) ] and coffee polyphenols (caffeic acid, chlorogenic acid) exert a profound inhibitory effect on DNA methylation and DNMT activities (Yang et al., 2008). Treatment of MCF-7 and MDA-MB-231 breast cancer cells with caffeic acid or chlorogenic acid decreased RARβ promoter methylation (Lee and Zhu, 2006). Moreover, consumers of high doses of chlorogenic acid had an increased plasma concentration of total homocysteine, which is an inhibitor of methylation reactions (Olthof et al., 2001). EGCG, a major polyphenol from green tea, inhibited UVB-induced global hypomethylation and protected mice from photocarcinogenesis and transformation of papillomas to carcinomas (Mittal et al., 2003). Exposure of human oesophageal cancer KYSE510 cells to EGCG resulted in demethylation and reactivation of p16, RARβ, MGMT and hMLH1 tumour suppressor genes (Lu et al., 2006). Promoter methylation-mediated silencing of p16 and RARβ was also found to be reversed by EGCG in colon cancer HT-29, prostate cancer PC3, breast cancer MCF-7 and MDA-MB-231 cell lines (Lee et al., 2005; Fang et al., 2007).

The tea catechins and bioflavonoids have also been shown to inhibit the prokaryotic SssI methyltransferase-mediated DNA methylation as well as the human DNMT1-mediated DNA methylation reaction in a concentration-dependent manner with diverse potencies (Lee et al., 2005). The presence of a catechol group in the structure of all these compounds can play a key role in inhibiting methyltransferase activity as compounds with a catechol group are excellent substrates for the methylation mediated by catechol-O-methyltransferase (COMT) (Figure 2D). COMT-mediated methylation results in the depletion of the methyl donor SAM and the formation of SAH, which is a potent feedback inhibitor of DNA methylation (Lee and Zhu, 2006). EGCG can exert a dual inhibitory effect on DNMT-mediated DNA methylation, which explains its high efficacy at inhibiting DNA methylation catalyzed by SssI methyltransferase and human DNMT1 compared with other compounds tested (Lee et al., 2005). Like other catechol-containing polyphenols, EGCG inhibits methyltransferase activity by increase the level of SAH in the reaction catalyzed by COMT. In addition, this tea polyphenol was shown to interact directly with the catalytic site of human DNMT1. Molecular modelling studies indicate that EGCG is well accommodated in a hydrophilic pocket of DNMT1 and effectively tethered within the DNMT1 binding site by at least four hydrogen bonds (Fang et al., 2007) and stabilized by magnesium ions (Figure 2B) (Lee et al., 2005). EGCG analogues without the gallic acid moiety were found to be poor inhibitors of DNMTs, therefore, the galloyl moiety is assumed to be crucial for EGCG binding to DNMTs. Furthermore, myricetin, which has a pyrogallic acid moiety similar to the galloyl group of EGCG, is a more potent, direct inhibitor of DNMT activity than other bioflavonoids, such as quercetin or fisetin, in the presence of magnesium ions (Lee et al., 2005).

Parthenolide and curcumin

Parthenolide was initially shown to have anti-cancer properties by inhibiting NF-κB activation through alkylation of Cys38 of p65 (Wiedhopf et al., 1973; Woynarowski and Konopa, 1981; Garcia-Pineres et al., 2004; Guzman et al., 2005). This compound, with a sesquiterpene lactone structure, is naturally present in the plant feverfew. Recent findings indicate that parthenolide inhibits DNMT1 activity and decreases DNMT1 expression; in K562, Kasumi-1, MV4-11 leukaemia cell lines and MCF-7 breast cancer cells, it decreased the global methylation and led to hypomethylation and activation of the HIN-1 tumour suppressor gene (Liu et al., 2009a). This inhibition of DNMT1 is probably linked to alkylation of the proximal thiolate of Cys1226 of the DNMT1 catalytic domain by parthenolide's γ-methylene lactone, as well as to interference with Sp1 binding to DNMT1 promoter and down-regulation of DNMT1 owing to the induction of subG1 cell cycle arrest (Liu et al., 2009a). Another natural compound directly regulating activity of DNMTs is curcumin, which is present in the spice turmeric. This agent was found to induce global DNA methylation in MV4-11 leukaemia cells (Liu et al., 2009b) and to hypomethylate and reactivate RARβ2 and WIF-1 tumour suppressor genes in human cervical and non-small cell lung cancer cells, respectively (Jha et al., 2010; Liu et al., 2011). Furthermore, exposure to curcumin led to promoter demethylation and re-expression of NEUROG1 and NRF1 in human prostate cancer cells (Khor et al., 2011; Shu et al., 2011). Curcumin can directly interact with SssI methyltransferase, a homologue of DNMT1, and covalently bind to the catalytic thiol group of Cys1226 present in DNMT1, which causes the inhibition of DNMT1 catalytic activity (Figure 2B) (Liu et al., 2009b).

Compounds targeting UHRF1

Down-regulation of UHRF1 (ubiquitin-like containing PHD ring finger 1) may be one of the mechanisms by which natural compounds exert their effect on DNA methylation. Thymoquinone from black cumin oil (Alhosin et al., 2011) and polyphenols from red wine such as RES, anthocyanins and procyanidins (Sharif et al., 2010) have been shown to suppress UHRF. UHRF1 is a putative oncogene that was found to be overexpressed in multiple cancer types such as prostate, lung, cervical, pancreatic, breast, bladder and kidney (Bronner et al., 2007; Unoki et al., 2009). Through its SRA (set and ring associated) domain, UHRF1 recognizes hemimethylated DNA and interacts with DNMT1 in order to form a complex responsible for copying the DNA methylation pattern from a maternal to a daughter strand (Bostick et al., 2007; Sharif et al., 2007). The same domain plays an important role in the cooperation of UHRF1 with HDAC1 (Unoki et al., 2004). UHRF1 up-regulation in cancer is associated with hypermethylation and inhibition of tumour suppressor genes (Daskalos et al., 2011). DNA demethylation caused by natural compounds may be a consequence of UHRF1 inhibition and subsequent attenuation of DNMT activity. UHRF1 suppression may also result from up-regulation of negative regulators such as p53 and p73 that are activated by many natural compounds, leading to cell cycle arrest and apoptosis (Arima et al., 2004; Alhosin et al., 2011).