Studies during the last two decades have revealed the involvement of epigenetic modifications in the development of human cancer. It is now recognized that the interplay of DNA methylation, posttranslational histone modification, and non-coding RNAs can interact with genetic defects to drive tumorigenesis.
The early onset, reversibility, and dynamic nature of such epigenetic modifications enable them to be developed as promising cancer biomarkers and preventive/therapeutic targets. In addition to the recent approval of several epigenetic therapies in the treatment of human cancer, emerging studies have indicated that dietary phytochemicals might exert cancer chemopreventive effects by targeting epigenetic mechanisms. In this review, we will present the current understanding of the epigenetic alterations in carcinogenesis and highlight the potential of targeting these mechanisms to treat/prevent cancer. The latest findings, published in the past 3 years regarding the effects of dietary phytochemicals in modulating epigenetic mechanisms, will also be discussed.
Dietary Phytochemicals Modulate Epigenetic Modifications
Environmental and dietary factors can influence the pathological progression of diseases, including cancer. Some naturally occurring phytochemicals that are common secondary metabolites in fruits and vegetables have been demonstrated to be beneficial for human health through various actions, including ameliorating oxidative stress, inducing detoxification enzymes, inhibiting nitrosamine formation, binding/diluting carcinogens in the digestive tract, altering hormone metabolism, and modulating carcinogenic cellular and signaling events . Recently, accumulating research has demonstrated that dietary phytochemicals can alter the epigenome and may help to prevent and treat human cancer. Here, we review the most recent studies regarding the epigenetic role of dietary phytochemicals, including polyphenols [quercetin, apigenin, (−)-epigallocatechin-3-gallate (EGCG), genistein, resveratrol, and curcumin], organosulfur compounds [sulforaphane (SFN), phenethyl isothiocyanate (PEITC), diallyl disulfide (DADS)], and indoles [diindolylmethane (DIM)] in cancer chemoprevention and therapy. We also discussed the latest progress in the identification of chemopreventive phytochemicals from Chinese herbal medicine in modulating epigenetic mechanisms. The epigenetic modifications regulated by phytochemicals are summarized in Table 1.Table 1
Epigenetic modifications by phytochemicals
Quercetin, a flavonol with yellow color, is widely found in fruits, vegetables, and grains. Quercetin was shown to inhibit the recombinant prokaryotic SssI DNMT- and human DNMT1-mediated DNA methylation . Quercetin suppresses the growth of the human colon cancer cell line RKO via demethylation of the p16INK4a gene promoter . Quercetin has been found to block the binding of the transactivators cAMP-response element-binding protein 2 (CREB2), c-Jun, CCAAT-enhancer-binding protein beta (C/EBPβ), and nuclear factor kappa B (NF-κB) to the COX-2 promoter. In addition, quercetin suppresses COX-2 expression in breast cancer cells by attenuating p300/HAT-mediated signaling . Moreover, quercetin induces Fas ligand-related apoptosis through the activation of the c-Jun/AP-1 signaling pathway, the induction of HAT, and the inhibition of HADC in HL-60 cells . Quercetin was also found to induce senescence in glioma cells via the inhibition of HDACs . A quercetin-rich diet has been reported to influence miRNA expression in human lung cancer tissues, including the tumor suppressor let-7 family and carcinogenesis-related miR-146, miR-26, and miR-17 . Quercetin also up-regulates miR-142-3p, a negative regulator of heat shock protein 70, which is related to the inhibition of the cell proliferation of pancreatic ductal adenocarcinoma cells (MIA PaCa-2, Capan-1, and S2-013) .
Apigenin is a yellow flavone compound in fruits and vegetables, especially in parsley, celery, and chamomile tea. In our recent study, we found that apigenin effectively demethylated the Nrf2 promoter, resulting in an increase in the mRNA and protein expression of Nrf2 and the Nrf2 downstream target gene NAD[P]H:quinine oxidoreductase-1 (NQO1) in skin epidermal JB6 P+ cells. This effect was associated with the reduced expression of epigenetic proteins, including DNMT1, DNMT3a, DNMT3b, and some HDACs . Apigenin also induces growth arrest and apoptosis in human prostate cancer cells through the up-regulation of global histone H3 and H4 acetylation and hyperacetylation of histone H3 on the p21/waf1 promoter in prostate cancer PC-3 and 22Rv1 cells. These effects may be caused by the inhibitive effect of apigenin on HDAC enzyme activity and the expression of HDAC1 and HDAC3 . The tumor suppressor miR-138 is correlated with telomerase activity in many human cancers, and apigenin-induced overexpression of miR-138 has been demonstrated to powerfully induce apoptosis of human malignant neuroblastoma in cell culture and animal models .
(−)-Epigallocatechin-3-gallate (EGCG) is one of the most abundant catechins in tea leaves and has been identified as a non-nucleoside DNMT inhibitor. The restoration of Wnt inhibitory factor 1 (WIF-1) expression by EGCG treatment, occurring via the demethylation of the WIF-1 promoter, has been found in lung cancer H460 and A549 cell lines . A recent study reported that EGCG treatment inhibits DNMT transcript levels and the protein expression of DNMT1, HDAC1, and MeCP2, effectively reactivating genes silenced by promoter methylation, such as estrogen receptor α (ERα), progesterone receptor B (PRB), target of methylation-induced silencing-1 (TMS1), Cyclin D2 (G1/S-specific cyclin-D2), and MGMT in MCF-7 cells . EGCG treatment was found to reactivate the tumor suppressor gene p16INK4a and Cip1/p21 by reducing DNA methylation and increasing histone acetylation in human epidermoid carcinoma A431 cells . EGCG may delay breast cancer progression and invasion via the induction of matrix metalloproteinase-3 (TIMP-3) expression. The proposed mechanism for this effect is that EGCG decreases EZH2 localization and H3K27 trimethylation enrichment at the TIMP-3 promoter, with a concomitant increase in histone H3K9/18 acetylation, in breast cancer cells . EGCG also induces the expression of miR-210, a major miRNA regulated by hypoxia-induced factor (HIF)-1α, in lung cancer cells, resulting in a reduced cell proliferation rate and anchorage-independent growth . EGCG can suppress the expression of p53-targeting miRNAs, including miR-25, miR-92, miR-141, and miR-200a, which are induced by the environmental carcinogen benzo[a]pyrene (BaP) in multiple myeloma, a common and deadly cancer of blood plasma cells .
Genistein, an isoflavone, is a major phytoestrogen compound in soy beans (Glycine max). Genistein has been demonstrated to suppress global DNA methylation, DNMT activity, and DNMT1 expression. These effects lead to promoter hypomethylation and increased mRNA expression of multiple tumor suppressor genes, including ataxia telangiectasia mutated (ATM), adenomatous polyposis coli (APC), phosphatase and tensin homolog (PTEN), and mammary serpin peptidase inhibitor (SERPINB5), in human breast cancer MCF-7 and MDA-MB-231 cells . Genistein induces the expression of two tumor suppressor genes, secreted frizzled-related protein 1 (sFRP1), and Smad4 (mothers against decapentaplegic homolog 4), via the demethylation of their promoter regions and histone modifications, such as H3K9-me2, H3K9-me3, and H3K27-me3, in prostate cancer cells . Genistein also down-regulates onco-miRNA-1260b in prostate cancer cells, resulting in the up-regulation of sFRP1 and Smad4 and the inhibition of cell proliferation and invasion . miR-27a down-regulation by genistein leads to enhanced apoptosis and reduced cell growth and invasion in pancreatic cancer cells .
Resveratrol is a stilbenoid, a type of natural polyphenol, and is found in blueberries, cranberries, and grapes. DNA methylation of the tumor suppressor gene RASSF1A (Ras association domain-containing protein 1) was reported to be reduced by resveratrol intake (twice daily for 12 weeks) in the breasts of women at high breast cancer risk . Resveratrol suppressed the increase in DNMT3b expression in estradiol-induced mammary tumor tissue in female ACI rats, an effect that may increase the expression of miRNA-129, miRNA-204, and miRNA-489 . The role of resveratrol as a HDAC inhibitor has also been demonstrated in glioma cells and human-derived hepatoblastoma cells . Recent studies suggested that resveratrol inhibits prostate cancer growth and metastasis and promotes the apoptosis of pancreatic cancer cells by inhibiting a miRNA-21-mediated pathway [84, 85].
Curcumin, a curcuminoid, is the primary component in the most popular Indian spice, turmeric (Curcuma longa). Growing evidence shows that curcumin harbors DNA demethylation potential in various cancer cell lines and might be a DNMT inhibitor [86, 87, 88, 89]. For example, studies conducted in our laboratory suggested that curcumin restored the expression of Nrf2 and Neurogenin-1 (Neurog1) in murine prostate cancer Tramp C1 cells and human prostate cancer LnCap cells, respectively, by suppressing DNA methylation in the promoter region [86, 87]. The hypomethylation effect of some novel synthetic curcumin analogs, such as EF31 and UBS109, has also been described to activate silenced genes, including p16, secreted protein acidic and rich in cysteine (SPARC), and epithelial cadherin (E-cadherin), in pancreatic cancer MiaPaCa-2 and PANC-1 cells . Curcumin has also been reported to modulate the activities of HDAC and HAT. Curcumin restored the expression of SOCS1 and SOCS3, suppressors of cytokine signaling, via the inhibition of HDAC activity (especially HDAC8), resulting in increased histone acetylation in the SOCS1 and SOCS3 promoter regions of the myeloproliferative neoplasm cell lines K562 and HEL . In breast tumor MCF-7 cells, the inhibitory effects of curcumin in the activities of HAT have also been demonstrated, with increased global levels of acetylated H3K18 and H4K16, potentially leading to the arrest of cell proliferation . Curcumin may also induce apoptosis of ovarian cancer SKOV3 cells through inducing the miRNA-9-mediated Akt/forkhead box protein O1 (FOXO1) pathway . The up-regulation of miRNA-181b by curcumin was found to suppress the expression of the pro-inflammatory cytokines chemokine (C–X–C motif) ligand 1 (CXCL1) and CXCL2, leading to the diminished proliferation and invasion of breast cancer cells .
Sulforaphane (SFN) is a bioactive isothiocyanate, a group of organosulfur compounds, which are abundant in cruciferous vegetables, such as broccoli, cabbage, and brussels sprouts. According to our recent studies, SFN suppresses DNA methylation of the Nrf2 promoter in mouse skin JB6 and prostate Tramp C1 cells by down-regulating DNMTs and HDACs. These effects may contribute to its preventive potentials against TPA-induced skin transformation and prostate carcinogenesis, respectively [95•, 30•]. SFN has also been demonstrated to exhibit antiproliferative effects on LnCaP prostate cancer cells by epigenetically restoring the expression of cyclin D2 . The restoration of miR-140 by SFN, accompanied by the reduced expression of SOX9 and aldehyde dehydrogenases 1 (ALDH1), has been reported to result in decreased breast tumor growth in vivo . SFN also inhibits the epithelial-to-mesenchymal transition (EMT) process in human bladder cancer T24 cells, and the up-regulation of miRNA-200c by SFN may be one of the mechanisms underlying this effect .
Phenethyl isothiocyanate (PEITC), another isothiocyanate, exists in some cruciferous vegetables. PEITC has been reported to be able to demethylate and reactivate pi-class glutathione S-transferase (GSTP1). This protein is a frequently silenced detoxifying enzyme that is highly associated with prostate carcinogenesis through its regulation of the cross-talk between DNA and chromatin in LNCaP cells . PEITC was also observed to modify the acetylation and methylation of histone 3 in human colon cancer SW480 cells, leading to the down-regulation of some inflammation-related genes, such as chemokine ligand 2 (CCL2), CD40, C–X–C motif chemokine 10 (CXCL10), colony stimulating factor 2 (CSF2), interleukin 8 (IL-8), NF-κB, and tumor necrosis factor, alpha-induced protein 3 (TNFaip3) . PEITC treatment significantly increased the expression of miRNA-17 and decreased the expression of p300/CBP-associated factor (PCAF) in dihydrotestosterone-stimulated LNCaP cells, which might contribute to the inhibitory effect of PEITC against androgen receptor (AR) transcriptional activity and cell growth in prostate cancer 
Diallyl disulfide (DADS) is one of the principal sulfur compounds in Allium vegetables, such as garlic (Allium sativum). DADS has been found to exhibit an inhibitory effect on HDAC, resulting in hyperacetylation of histone 4 in the breast cancer MCF-7 cell line . In addition, DADS treatment has been demonstrated to impair proliferation and enhance apoptosis in both human gastric cell lines and xenograft models. This effect occurred through the Wnt-1 signaling pathway and was mediated by the up-regulation of miRNA-200b and miRNA-22 .
3,3′-Diindolylmethane (DIM), an indole compound, is derived from glucosinolate indole-3-carbinol (I3C) in cruciferous vegetables, including broccoli, cabbage, cauliflower, and brussels sprouts. In addition to SFN, DIM can alter the DNA methylation status of many cancer-associated gene promoters in normal PrECs as well as in the prostate cancer cell lines LnCap and PC3 . Similarly, DIM exerts its chemopreventive effects in prostate tumorigenesis by epigenetically demethylating the Nrf2 promoter and up-regulating the expression of Nrf2 and its downstream gene NQO1 . The proteasome-mediated degradation of class I HDACs (HDAC1, HDAC2, HDAC3, and HDAC8) induced by DIM triggers cell cycle arrest and apoptosis in human colon cancer HT-29 cells and in tumor xenografts . DIM also inhibits cell proliferation in human breast cancer MCF-7 (estrogen-dependent) and MDA-MB-468 (estrogen receptor-negative, p53 mutant) cells via miRNA-21-mediated Cdc25A (cell division cycle 25 homolog A) degradation . A phase II clinical study in patients prior to radical prostatectomy suggested that formulated DIM intervention could attenuate prostate cancer aggressiveness via the up-regulation of miRNA let-7 and down-regulation of EZH2 expression in tissue specimens .
Phytochemicals from Traditional Chinese Herbal Medicine
During the last few decades, great progress had been made in the identification of chemopreventive agents and anticancer drugs in traditional Chinese herbal medicine. Recently, the potential of the components from Chinese herbs to influence epigenetic mechanisms in cancer prevention have been recognized. Ginseng is one of the most commonly used herbs in East Asia. Compound K [20-O-β-(d-glucopyranosyl)-20(S)-protopanaxadiol], the main metabolite of ginseng saponin, was found to inhibit the proliferation of human HT29 human colon cancer cells by demethylating and reactivating runt-related transcription factor 3 (RUNX 3), which is associated with reduced DNMT1 activity . Ginsenoside Rh2 is another biologically active triterpene saponin extracted from ginseng. The chemopreventive effect of Rh2 in inhibiting the proliferation of human glioma cells had been demonstrated to involve epigenetic modifications, such as the regulation of miRNAs. Specifically, the up-regulation of miR-128 by the treatment with Rh2 had been shown to trigger apoptosis-related signaling . Similarly, using miRNA microarray analysis, An et al.  identified a network of miRNAs regulated by treatment with Rh2 in nonsmall cell lung cancer A549 cells, which may contribute to the antiproliferative effect of Rh2. A research study from our group demonstrated that the Chinese herb Radix angelicae sinensis (RAS; Danggui) and its bioactive component Z-Ligustilide (Lig) are able to hypomethylate the Nrf2 promoter, resulting in the restoration of Nrf2 and downstream targets such as NQO1, heme oxygenase 1 (HO-1), and UDP-glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) in murine prostate cancer TRAMP C1 cells . Another Chinese herb with great promise in altering epigenetic mechanisms is Salvia miltiorrhiza, also known as Danshen. We found that tanshinone IIA, one of the main active components from Danshen, blocks TPA (12-O-tetradecanoylphorbol-13-acetate)-mediated JB6 transformation through epigenetic regulation of the Nrf2 signaling pathway . Treatment with tanshinone IIA reduced the methylation of the Nrf2 promoter, elevated the expression of Nrf2 and downstream targets, suppressed the protein levels of DNMT1, DNMT3a, DNMT3b, and HDAC3, and inhibited HDAC activity . Another study showed that tanshinone IIA decreases inflammatory responses in LPS-induced macrophages and inhibits the proliferation of inflammation-stimulated colon cancer cells by inhibiting the overexpressed miR-155 in macrophages . Tanshinone I, another main component derived from Danshen, has been shown to trigger cell cycle arrest in several breast cancer cells by down-regulating Aurora A gene expression via the reduction of H3 acetylation levels in the Aurora A promoter . In addition to tanshinones, the primary components, minor components, including tanshindiols, are currently under investigation for their potential antitumor ability by targeting epigenetic modifications. Using molecular docking and an enzyme kinetics approach, Woo et al.  proposed that tanshindiols B and C are potential EZH2 inhibitors, resulting in the inhibition of the growth of several cancer cell lines.
Other Dietary Phytochemicals
In addition to above-mentioned dietary phytochemicals, various other natural compounds are currently under investigation regarding their cancer chemopreventive potential through epigenetic modifications. Boswellic acids, a pentacyclic terpenoid derived from the plant Boswellia serrata, have long been used as anti-inflammatory and cancer chemopreventive agents. Recently, Shen et al. demonstrated that boswellic acids inhibit DNMT activity and induce genome-wide demethylation, permitting the restoration of tumor suppressor genes, such as sterile α motif domain containing 14 (SAMD14) and sphingomyelin phosphodiesterase 3 (SMPD3) in colorectal cancer cells . In addition to modulating DNA methylation, boswellic acids were found to significantly up-regulate tumor-suppressive miRNAs, such as let-7 and miR-200, and to modulate the expression of downstream targets in several colon cancer cells and tumor xenografts in nude mice . Experimental evidence demonstrated that ursolic acid, another naturally occurring pentacyclic triterpene, suppresses proliferation and induces apoptosis in the human glioma cell line U251 by mediating the miR-21 pathway . A recent study proposed that the antitumor activity of rosemary extracts with high contents of phenolic diterpene carnosic acid and carnosol might involve the up-regulation of glycosyltransferase 3 (GCNT3) and down-regulation of miR-15b in colon and pancreatic cancer cells .
Conclusions and Perspectives
Great accomplishments have been made in recent years in advancing our understanding of epigenetic alterations in the development of cancer. These epigenetic abnormalities are now believed to exist in all cancer types and drive tumor progression along with genetic defects. The reversible and dynamic nature of epigenetic modifications strongly encouraged clinicians and pharmaceutical industries to develop epigenetic biomarkers and therapeutic targets in cancer diagnosis and treatment. However, the complexity of epigenetic pathways, including the interplay of the different epigenetic mechanisms in regulating gene transcription and the genetic mutations in epigenetic regulators, need to be addressed before we can fully apply our current understanding to the clinical field. For example, histone modification enzymes such as HDACs might be abnormally regulated by genetic or DNA methylation changes in cancer cells. Thus, further systematic studies may facilitate the development of epigenetic research in preventing and treating cancer.
The approval of several DNMT and HDAC inhibitors for clinical use has opened up a new avenue in cancer therapy. However, it could be reasonably argued that epigenetic interventions may be more effective in hematopoietic malignancies than solid malignancies. Factors such as the microenvironment, epigenetic landscape, drug exposure, and drug metabolism appear to be largely different in solid tumors than in hematopoietic malignances. However, more intensive studies regarding these cellular or epigenetic differences are urgently needed to successfully apply the concept of epigenetic therapy across a broader spectrum. Furthermore, adverse effects and a lack of selectivity have hindered the road towards effective epigenetic therapies. Investigations should be conducted regarding whether a selective subset or large numbers of genes will be influenced by the drugs or phytochemicals that target epigenetic modifications. Additionally, based on the crosstalk between genetic and epigenetic mechanisms, combining conventional antitumor drugs with epigenetic therapies or dietary phytochemicals that target epigenetic mechanisms might be a promising strategy for reducing toxicity and resistance.
Accumulating evidence indicates that some dietary phytochemicals can modulate epigenetic mechanisms. Here, we summarized and discussed the latest findings in the past 3 years. Together with numerous reports published more than 3 years ago, it is now clear that these natural compounds hold great promise in cancer prevention via acting on a variety of epigenetic targets. However, we should also notice that the success of epigenetic interventions elicited by phytochemicals was mostly limited in preclinical models. Thus, future studies should be carefully designed on the translation of these natural agents’ effects to prevent human malignancies in clinical settings. Moreover, most phytochemicals have been reported to influence a wide range of epigenetic regulators. Therefore, understanding the global patterns of epigenetic modifications that are induced by phytochemicals will help to optimize strategies to prevent and treat cancer.
In summary, aberrant epigenetic modifications, such as DNA methylation, histone modifications, and miRNA, add another layer of complexity to the development of human cancer. The identification of dietary phytochemicals that modulate epigenetic modifications offers promising benefits in the management of human cancer.
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