Dietary Phytochemicals and Cancer Chemoprevention: A

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Perspective

Dietary phytochemicals and cancer chemoprevention: A perspective on oxidative stress, inflammation and epigenetics Wenji Li, Yue Guo, Chengyue Zhang, Renyi Wu, Anne Yu-Qing Yang, John M. Gaspar, and Ah-Ng Kong Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00413 • Publication Date (Web): 22 Nov 2016 Downloaded from http://pubs.acs.org on November 28, 2016

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Chemical Research in Toxicology

Dietary phytochemicals and cancer chemoprevention: A perspective on oxidative stress, inflammation and epigenetics Wenji Lia,b, Yue Guoa,b,c, Chengyue Zhanga,b,c, Renyi Wua,b, Anne Yuqing Yanga,b,c, John Gaspara,b, and Ah-Ng Tony Kong a,b,* a Center for Cancer Prevention Research, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA b Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA c Graduate Program in Pharmaceutical Sciences, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA

*Correspondence should be addressed to: Professor Ah-Ng Tony Kong Rutgers, The State University of New Jersey Ernest Mario School of Pharmacy, Room 228 160 Frelinghuysen Road, Piscataway, NJ 08854, USA Email: [email protected] Phone: 848-445-6369/8 Fax: 732-445-3134 Key Words: oxidative stress, inflammation, Nrf2, Epigenetics, Cancer chemoprevention, Next Generation Sequencing

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Table of Contents Graphic

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ABSTRACT Oxidative stress occurs when cellular reactive oxygen species (ROS) levels exceed the self-antioxidant capacity of the body. Oxidative stress induces many pathological changes, including inflammation and cancer. Chronic inflammation is believed to be strongly associated with the major stages of carcinogenesis. The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway plays a crucial role in regulating oxidative stress and inflammation by manipulating key antioxidant and detoxification enzyme genes via the antioxidant response element (ARE). Many dietary phytochemicals with cancer chemopreventive properties, such as polyphenols, isothiocyanates and triterpenoids, exert antioxidant and anti-inflammatory functions by activating the Nrf2 pathway. Furthermore, epigenetic changes, including DNA methylation, histone post-translational modifications (PTMs), and miRNA-mediated post-transcriptional alterations, also lead to various carcinogenesis processes by suppressing cancer repressor gene transcription. Using epigenetic research tools, including next-generation sequencing (NGS) technologies, many dietary phytochemicals are shown to modify and reverse aberrant epigenetic/epigenome changes potentially leading to cancer prevention/treatment. Thus, the beneficial effects of dietary phytochemicals on cancer development warrant further investigation to provide additional impetus for clinical translational studies.

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1 Introduction Oxidative stress occurs when cellular reactive oxygen species (ROS) levels exceed the self-antioxidant capacity of the body. Oxidative stress is believed to be the cause of many pathological changes, including inflammation and carcinogenesis. It is also thought to be a major cause of cellular malignancy and the initiation of cancer because it has roles in all the major stages of carcinogenesis. Chronic inflammation is believed to be strongly correlated to the initiation, promotion and progression of cancer. Dietary phytochemicals are beneficial for cancer chemoprevention due to their efficacy and relatively low toxicity. They have direct antioxidant and anti-inflammatory effects and indirectly exert those functions by activating the antioxidant and detoxification pathway mediated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which critically regulates antioxidative stress and anti-inflammatory responses. Epigenetic changes, including DNA methylation, histone post-translational

modifications

(PTMs),

and

miRNA-mediated

post-transcriptional

alterations, also lead to various cancers primarily by suppressing cancer repressor gene transcription. Abnormal epigenetic changes are more reversible than gene mutations and may be reversed by epigenetic modulators, including various dietary phytochemicals. We will explore the cancer-preventing effects of dietary phytochemicals from an epigenetic modification perspective. Next-generation sequencing (NGS) technologies have emerged as popular and powerful tools in cancer research because they deploy an “omics” approach to analyze alterations on a whole genome scale, from the sequencing of complete genomes and transcriptomes to the genome-scale analysis of DNA–protein interactions. The application of NGS to assess the roles of dietary phytochemicals in cancer prevention will also be discussed. 4


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Based on accumulating pre-clinical and clinical research findings, the consumption of dietary phytochemicals is a promising method to prevent carcinogenesis. In this review, we will discuss the progress of research on the ability of dietary phytochemicals to inhibit carcinogenesis (tumor initiation, promotion and progression) through antioxidant and anti-inflammatory effects, mainly by activating the Kelch-like ECH-associated protein 1 (Keap1)-Nrf2 signaling pathway and/or epigenetic modification mechanisms. Dietary phytochemicals, including polyphenols, isothiocyanates and triterpenoids, will be the focus of this review.

2 Oxidative stress, inflammation and cancer 2.1 Oxidative stress and its biomarkers The term ROS is typically used to refer to highly reactive oxygen-containing molecules such as free radicals.1 The internal origins of ROS include the mitochondria, cytochrome P450 metabolism, peroxisomes, and inflammatory cell activation.2 The external origins of ROS include non-genotoxic carcinogens, various xenobiotics, ultrasound, and microwave radiation.3, 4 Normal ROS levels are required for cell metabolism; however, excess ROS levels may induce cell damage. Oxidative stress is believed to occur when ROS production exceeds the cellular antioxidant capacity in the body. Oxidative stress will induce cellular alterations in proteins, nucleic acids, and lipids, which may further trigger inflammation or carcinogenesis.5-7 Levels of oxidative stress markers, including hydroxyl radical (OH-), superoxide ion radical (O2/H2O2), 8-hydroxy-2'-deoxyguanosine (8-OHdG), nitrotyrosine (ONOO), cyclooxygenase-2 (COX2), glutathione S-transferase-pi (GST-pi), inducible nitric 5



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oxide synthase (iNOS) and heme oxygenase 1 (HO-1), are highly associated with the severity of oxidative stress. Among these markers, OH- is believed to be the most powerful oxidative free radical in biological systems; it can react with most molecules in cells at a high rate.8 8-OHdG is an oxidized product in damaged DNA.9 Since 8-OHdG is excreted through the urine without being extensively metabolized, the urinary 8-OHdG level has become a popular, non-invasive biomarker of oxidative stress.10 HO-1 is often stimulated by oxidative stress and acts as a strong cellular defense barrier against excess ROS levels in various diseases.11

2.2 Oxidative stress and cancer Oxidative stress induces DNA damage and genomic instability, which is a major cause of cellular malignancy and the initiation of cancer.12-14 Excess ROS levels cause DNA mutations such as GC → TA transversion mutations,15 single strand breaks, and instability.16 In human tumors, a G to T transversion is the most frequent mutation in the p53 suppressor gene.17 Since excess ROS levels can generate 8-OHdG in DNA and cause GC → TA transversions,18 8-OHdG has been widely utilized as a biomarker of oxidative stress in studies of antioxidants and multiple ROS-related diseases.19 Excess ROS levels may also enhance carcinogenesis by inducing and maintaining the oncogenic phenotypes of cancer cells.14, 20, 21 In addition, cells that have adapted to oxidative stress inhibit apoptosis and undergo malignant transformation and metastasis, as well as develop resistance to anticancer drugs.22 The effects of oxidative stress on cancer development via epigenetic modifications have attracted attention. Oxidative stress induced by an H2O2 intervention inhibited the transcription of the tumor suppressor CDX1 in colorectal cancer by increasing the expression 6


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and activity of DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1).23 Upon chronic incubation with H2O2, MCF-7 human breast cancer cells transformed from estrogen-dependent, nonaggressive breast cancer cells into estrogen-independent, aggressive forms, potentially through epigenetic mechanisms such as histone H3 modifications and DNA methylation.24 Oxidative stress is associated with all three stages of carcinogenesis: initiation, promotion, and progression. As a carcinogenesis initiator, ROS contribute to the initiation of nuclear or mitochondrial DNA mutations, including point mutations, deletions, insertions, chromosomal translocations and others.25, 26 ROS may generate DNA damage by producing oxidative DNA modifications in cancer tissues.27 The initiation stage transforms normal cells into cancer cells. Following the initiation stage, the initiated cells are expanded into colonies in the promotion stage, accompanied by cell proliferation and/or inhibition of apoptosis.3 In this stage, ROS promote the expansion of malignant cells by regulating cell proliferation/apoptosis-related genes and transcription factors such as nuclear factor-kappaB (NF-κB), activator protein-1 (AP-1), Nrf2, and hypoxia-inducible factor (HIF).20, 28, 29 The progression stage is irreversible and exhibits more aggressive properties, including the secretion of metastasis-related proteases and invasion beyond the immediate primary tumor location.30

Excess

ROS

levels

may

prohibit

antiproteases,

upregulate

matrix-metalloproteinases (MMPs) and impair tissues.31, 32 In addition, ROS and NADPH oxidase complexes have been reported to be vital for triggering tumor angiogenesis by regulating vascular endothelial growth factor, angiotensin II, HIF, AP1, and inflammation.33

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2.3 Inflammation and cancer Excessive and chronic inflammations contribute to the development of many kinds of cancer. Various inflammatory mediators, which are mainly generated by immune cells, mesenchymal cells, and epithelial cells, are strongly correlated with the development of inflammation and cancer. Among these factors, interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 are the most well-studied major cytokines associated with inflammation and cancer development and are highly involved in cancer cell proliferation, migration, and angiogenesis.34,

35

These cytokines induce inflammation and cancer mainly by activating

NF-κB, inducing COX2 expression and increasing ROS production,36, 37 activating mitogen activated protein kinase (MAPK) cascades 38 and c-Jun N-terminal kinase (JNK).39 In the tumor initiation stage, the inflammatory microenvironment increases DNA mutation rates and the number of mutated cells,40 inactivates mismatch repair response genes,41 and induces the expression of growth factors and cytokines to promote cancer stem cell expansion mainly through the NF-κB and signal transducer and activator of transcription 3 (STAT3) pathways.42 Chronic inflammation also stimulates cancer promotion and progression. Tumor-promoting cytokines secreted by immune/inflammatory cells, such as IL-1β, TNF-α, and IL-6, are required for carcinogenesis.34, 35 Increase levels of inflammatory cytokines and macrophage infiltration promote tumor angiogenesis.43 Tumor growth and metastasis are highly attributed to chronic inflammation.44 Based on accumulating evidence, chronic inflammation is closely correlated to epigenetic alterations mediated by DNA and histone modifications, thus driving changes in the expression of many genes associated with inflammation, such as interleukin 1 receptor 8


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type 1 (IL1R1), IL-1β, toll-like receptor (TRL) 2, 15-lipoxygenase (15-LOX), COX2, chemokine (C-X-C motif) ligand 14 (CXCL14), chemokine (C-C motif) ligand 25 (CCL25), CXCL6, IL-13, IL-17C, and IL4R.45-52 Altered patterns of methylation and acetylation have been reported in inflammatory diseases and cancer and may provide biomarkers for early pre-malignant states. Abnormal DNA methylation is associated with many inflammatory disorders. DNA hypomethylation and upregulation of nucleotide-binding domain and leucine-rich repeat-containing (NLR) family caspase recruitment domain (CARD)-containing 4 (NLRC4), a critical component of obesity-related inflammation, have been reported in adipose tissue in mice fed a high-fat diet.53 In cystic fibrosis (CF), a chronic pulmonary inflammation disease, TLR2 has been shown to be upregulated in bronchial epithelial cell lines due to DNA hypomethylation in its promoter. 54 Histone acetyltransferases (HAT), transcriptional co-activators, exhibit aberrant expression in inflammatory diseases, suggesting that HDAC inhibitors exert anti-inflammatory effects by inducing cell death through the acetylation of transcription factors.55 Treatments with the HDAC inhibitors trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) significantly inhibit respiratory syncytial virus (RSV) replication and decrease RSV-induced airway inflammation.56 Furthermore, 50 nM TSA effectively decreases the levels of inflammatory cytokines in U-937 cell supernatants.57

2.4. Inflammation, oxidative stress and Nrf2 Inflammatory processes and oxidative stress, which is characterized by excess ROS levels, 9



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are reported to interact in many pathological conditions, including cancer.40, 58, 59 During inflammation, the cellular immune system generates ROS and pro-inflammatory cytokines and chemokine until the pathogens and other antigens are eliminated and the tissue repair process is complete.60, 61 However, a prolonged active inflammatory response may induce cell damage by generating excess ROS levels, which may cause permanent genomic mutations, such as point mutations, gene deletions, or gene rearrangements.62 Since cellular antioxidant systems are depleted during the chronic inflammatory response, the DNA damage induced by oxidative stress is more severe.63 Nrf2 has been shown to be vital in regulating the antioxidative stress response64-66 and is essential for the anti-inflammatory response, according to many clinical and pre-clinical studies.67, 68 Abundant dietary phytochemicals activate the Nrf2 pathway and exert inhibitory effects on pro-inflammatory pathways mediated by NF-κB69 and other inflammatory markers.70,

71

Importantly, the loss of Nrf2 attenuates the anti-inflammatory effects of

sulforaphane (SFN) and omega-3-fatty acids.72, 73 Mice lacking Nrf2 are more susceptible to azoxymethane (AOM)-dextran sulfate sodium (DSS)-induced colitis74 and colorectal carcinogenesis.75 Based on these results, oxidative stress and inflammation interact closely; a failure to stop these processes results in genetic/epigenetic changes that drive the initiation of carcinogenesis.76-78 A promising approach to examine the roles of dietary phytochemicals in preventing cancer is to activate the Nrf2 pathway to subsequently inhibit oxidative stress and the inflammatory response during carcinogenesis.

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3 The Keap1-Nrf2 pathway mediates antioxidant and anti-inflammatory responses to prevent cancer 3.1. The classical Keap1-Nrf2 signaling pathway Nrf2 is a basic leucine zipper transcription factor with a cap ‘n’ collar structure that controls a battery of phase ΙΙ detoxifying and antioxidant genes.79 Keap1 is a cysteine-rich protein that negatively regulates Nrf2 activity.80 The low expression of the Nrf2 protein in mammalian cells under basal conditions results from rapid proteasomal degradation, and the protein has a half-life of less than 1 h. Keap1 acts as a cytoplasmic adaptor between Nrf2 and the ubiquitination ligase Cullin-3 and promotes the ubiquitination of Nrf2.81-83 However, in the presence of various stimuli, such as electrophiles, reactive oxidants, and stress, Nrf2 degradation is suspended. Based on accumulating evidence, the sulfhydryl groups in the cysteine residues of Keap1 act as stress sensors,84, 85 leading to the “cysteine code” hypothesis, which proposes that the Keap1-Nrf2 complex differentially responds to various stressors.86, 87 The modification of thiols on Keap1 is thought to induce the dissociation of Nrf2 from the low-affinity binding site (DLG motif) as a result of conformational alterations in Keap1.88-90 Newly synthesized Nrf2 translocates into the nucleus, where it heterodimerizes with small musculoaponeurotic fibrosarcoma (Maf) proteins, binds to the antioxidant or electrophile response element (ARE/EpRE) located within the regulatory regions of a large number of genes involved in the response to cellular stress, and activates the transcription of target genes.91 The Nrf2 target genes that have been identified, including genes encoding the following: (1) proteins that regulate glutathione (GSH), such as glutathione S-transferase mu (GSTM); (2) antioxidant enzymes, such as glutathione peroxidase (GPX); (3) drug 11



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metabolism enzymes and transporters, such as NADPH:quinone oxidoreductase 1 (NQO1); and (4) other stress response proteins.92,

93

Thus, the Keap1-Nrf2 signaling pathway is

considered a pivotal cellular defense pathway that mediates the response to oxidative and electrophilic stresses by inducing the expression of cytoprotective genes.

3.2 Nrf2 and its antioxidant and anti-inflammatory effects on cancer prevention Moderate ROS levels are important intracellular signaling molecules for promoting cell proliferation and differentiation by activating cellular proliferation or survival signaling pathways.20 However, excessively high levels of ROS cause irreversible oxidative damage to biomacromolecules, apoptosis and cell death.14 Phase II antioxidant and detoxification enzymes (such as superoxide dismutase (SOD), HO-1, GST, NQO1, catalases, and glutathione peroxidase) are some of the vital defenses against oxidative stress. The transcription of these genes is mostly regulated by the ARE in the promoter.94 Nrf2 is well known for its protective effects against oxidative stress by regulating the transcription of phase II antioxidant and detoxification enzyme genes via binding to the ARE region in the promoters of its target genes.95, 96 Dietary phytochemical antioxidants (such as tocopherols, tocotrienols, carotenoids, and natural flavonoids) are important defensive factors that protects against oxidative stress.97, 98 Consumption of high levels of antioxidants was also correlated with a lower risk of cancer.99 Many dietary phytochemicals exert their antioxidant effects by increasing the expression of antioxidant and detoxification enzymes through the activation of the Keap1-Nrf2 signaling pathway.100,

101

Therefore, activating the Keap1-Nrf2 pathway

provides a rationale for analyzing the mechanism of action of dietary antioxidant 12


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phytochemicals. The cellular functions of the Keap1-Nrf2 signaling pathway are not limited to the maintenance of redox balance and xenobiotic metabolism by inducing the expression of antioxidant and phase ΙΙ detoxifying enzymes. Nrf2 may also regulate numerous genes involved in cell differentiation, proliferation, autophagy, apoptosis, and stem cell function.102, 103

Furthermore, functional interactions between Keap1-Nrf2 signaling and a wide range of

signaling pathways, including NF-κB, aryl hydrocarbon hydroxylase receptor (AhR), p53, and NOTCH, enable Nrf2 to modulate and influence more diverse cellular activities, such as inflammation, tissue regeneration, and metabolic reprogramming.64 Emerging evidence has revealed the interaction between the Nrf2 pathway and the anti-inflammatory response, although the underlying mechanism remains unclear. Nrf2 is postulated to assist in regulating the innate immune response and inhibiting the expression of pro-inflammatory genes.104 Nrf2 has been shown to play a crucial role in modulating the innate immune response in a lipopolysaccharide (LPS) - and TNF-α-induced sepsis model in Nrf2-deficient mice and embryonic fibroblasts derived from these mice.105 When exposed to hyperoxia to induce acute lung injury, Nrf2-deficient mice exhibited more severe inflammation, injury, and tissue damage compared with the wild type mice.106 Since the Nrf2 pathway protects cells from oxidative stress, modulation of the Nrf2 pathway may influence NF-κB, a transcription factor regulating redox-sensitive inflammation. The NF-κB pathway is a well-accepted pro-inflammatory signaling pathway.107 The Nrf2 pathway has been repressed by the pro-inflammatory NF-κB /p65 pathway by depriving Nrf2 of the CREB binding protein (CBP) and improving the recruitment of HDAC3, which is a 13



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corepressor of ARE.108 Nrf2 activators also inhibit NF-κB. A chalcone derivative inhibits TNF-α-induced NF-κB activation in HT-29 cells by activating Nrf2 and increasing HO-1 expression.109

Quercetin

significantly

inactivates

the

expression

of

NF-κB

and

pro-inflammatory cytokines in the livers of nickel-treated mice by activating the Nrf2 pathway and increasing HO-1 expression.110 Given the important effects of Nrf2 on regulating cellular activities and adaptive responses, the protective role of Nrf2 in preventing carcinogenesis in different organs has been extensively studied.66, 92, 111 Briefly, evidence supporting the cancer chemopreventive role of Nrf2 was presented in studies showing that Nrf2 knockout mice (Nrf2-/-) are more susceptible to carcinogens than wild type mice75, 112, 113 or by showing that activation of Nrf2 (by pharmacological or genetic approaches) suppresses carcinogenesis.114 Furthermore, the increased vulnerability of Nrf2-/- mice to drug- and oxidative stress-induced pathogenesis revealed that Nrf2 also protects against acute lung injury,115 chronic obstructive pulmonary diseases (COPD),116,

117

neurodegenerative diseases,

118, 119

diabetic nephropathy,120 and

cardiovascular diseases.121

3.3. Regulation of the Keap1-Nrf2 signaling pathway

The improved understanding of the regulatory mechanisms underlying the Keap1-Nrf2 pathway has shed light on the identification and development of Nrf2 activators and inhibitors. A wide range of structurally diverse phytochemicals (sulforaphane and curcumin), synthetic compounds (oleanane triterpenoids and dimethyl fumarate), therapeutic agents (oltipraz and auranofin), environmental toxins (paraquat and arsenic), and endogenous chemicals 14


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[(4-hydroxynonenal (4-HNE)] have been shown to activate Nrf2 and its target genes through distinct mechanisms.102 Some of the naturally occurring phytochemicals that activate Nrf2 signaling exhibit potential for the prevention of chronic diseases. Several small molecules, such as ascorbic acid, ochratoxin A, brusatol, and all-trans retinoic acid, suppress the Nrf2 pathway; this response could be developed to sensitize cancer cells to chemotherapeutic medications.122

Despite the extensive studies of the regulation of Nrf2 by Keap1, several other mechanisms have been studied and proposed to regulate Nrf2.123, 124 For example, covalent modification of Nrf2 by phosphorylation and acetylation may regulate its activity post-translationally. A number of upstream regulators of Nrf2 phosphorylation, such as MAPKs,125 phosphatidylinositol 3-kinase (PI3K),126 protein kinase C (PKC),127 glycogen synthase kinase-3 (GSK-3),128 and PKR-like endoplasmic reticulum kinase (PERK),129 phosphorylate various serine, threonine, and tyrosine residues in Nrf2 and thereby modulate the Keap1-Nrf2 pathway. At the translational level, an internal ribosomal entry site (IRES) has been shown to modulate the efficiency of Nrf2 mRNA translation under normal and stress conditions.130 Furthermore, as reported in several studies, Nrf2 activity is influenced by interactions with other proteins and signaling pathways, such as p21,131 p62,132 Ras GTPase-activating-like protein (IQGAP1),133and dipeptidyl peptidase 3 (DPP3).134 The transcriptional regulation of Nrf2 and Keap1 expression, which is less studied, has been reported in several cases. The transcription of the Nrf2 gene can be upregulated by Nrf2 itself through two ARE-like sequences in the proximal promoter region.135 Similarly, a functional ARE in the reverse strand of the proximal promoter of Keap1 upregulates Keap1 15



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expression.136 As shown in the study by Miao et al, the expression of the Nrf2 mRNA and its target genes is elevated by treatment with an aryl hydrocarbon receptor (AhR) inducer.137 In addition, Nrf2 transcription might be increased by the oncogenic activation of Kras, Braf, and Myc; this mechanism has been proposed to be an important pro-survival signal during carcinogenesis.138

Accumulating evidence has shown the Keap1-Nrf2 pathway is also regulated by epigenetic modifications such as CpG methylation, histone modification and interactions with miRNAs.139 In our previous study of a transgenic adenocarcinoma mouse prostate (TRAMP) mouse model of prostate cancer, the expression of Nrf2 was decreased by hypermethylation of the CpG promoter region.140 B. Wang et al. found that the classic HDAC inhibitor trichostatin A activates the Keap1-Nrf2 pathway and upregulates the Nrf2 downstream genes by inhibiting Nrf2 suppressor Keap1, enhancing the dissociation of Keap1/Nrf2, inducing Nrf2 nuclear translocation, and promoting Nrf2-ARE binding. In addition, the protective effect was absent in Nrf2 knockout mice.141 In breast cancer cells treated with the histone deacetylase inhibitor, miR-200a expression was increased and the Keap1-Nrf2 pathway was activated, with decreased Keap1 levels and increased Nrf2 and NQO1 expression.142

4. Roles of dietary phytochemicals in cancer chemoprevention through the Keap1-Nrf2 pathway Numerous dietary phytochemicals have been

shown

to

exert

their

cancer

chemopreventive effects by activating Nrf2 pathways.143, 144 Here, we will discuss the roles of 16


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major dietary phytochemicals, including polyphenols, isothiocyanates and triterpenoids in preventing cancer by activating the Nrf2 pathway. Table 1 summarizes the phytochemicals with cancer prevention properties that activate the Nrf2 pathway.

4.1 Dietary polyphenols (curcumin, Epigallocatechin gallate (EGCG) and genistein) Dietary polyphenols are natural compounds with multiple phenol structural units. Polyphenols have been shown to have potential effects on human health and disease. Many dietary polyphenols exert their antioxidant and anti-inflammatory functions by activating Nrf2 pathway. Curcumin (diferuloylmethane), commonly known as the main component of turmeric (also called curry powder), is a lipophilic polyphenol derived from the rhizome of

Curcuma Longa (an Indian plant). Commercially available curcumin also contains demethoxycurcumin (DMC, ~17%) and bisdemethoxycurcumin (BDMC, ~3%); however, curcumin has been shown to be more active than these two components.145 In addition to its common used as a spice in India for centuries, curcumin has also been regularly used in Ayurveda (Indian traditional medicine) and traditional Chinese medicine. Currently, curcumin is a popular dietary supplement and is generally considered pharmacologically safe.146 Epigallocatechin gallate (EGCG), the major active component in green tea, protects against many diseases, including cancer, via its antioxidant and anti-inflammatory activities.147-149 Genistein, an isoflavone found in many plants, including soybeans, has been identified as an angiogenesis inhibitor and inhibits the uncontrolled cell growth of cancer.150-152

4.1.1 Role of curcumin in preventing cancer through the Nrf2 pathway 17



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Accumulating evidence from preclinical and clinical studies suggested that curcumin is a promising therapeutic/preventive agent for different types of human pathologies, including cancers, neurocognitive disorders, kidney disorders, diabetes, cardiovascular diseases, and pulmonary diseases.153 As shown in mechanistic studies, curcumin regulates several critical molecular signaling pathways and targets involved in survival, oxidative stress, and inflammatory responses, among others.154, 155 Here, we discuss and review the mechanisms by which curcumin prevents cancer by regulating the Nrf2 pathway. Curcumin treatment exerted an anti-proliferative effect on MCF-7 human breast cancer cells, with an IC50 value of 35 µM. Curcumin simultaneously increased the expression of Nrf2 and decreased the expression of Flap endonuclease 1 (Fen1), a DNA repair-specific nuclease involved in breast cancer. In addition, in mechanistic investigations, curcumin treatment induced the translocation of Nrf2 from the cytoplasm to nucleus, decreased the recruitment of Nrf2 to the Fen 1 promoter, subsequently decreased Fen1 activity, and suppressed the proliferation of MCF-7 cells.156 The induction of Nrf2 by curcumin was also reported in an animal model after long-term use. Das et al investigated the effects of curcumin (intraperitoneal (i.p.) injections for 9 consecutive days) on the regulation of oxidative stress, inflammation, and tumor suppressor genes in the livers of mice induced with Dalton’s lymphoma.157 The Nrf2 signaling pathway and phase II antioxidant enzymes were suppressed during the progression of lymphoma in mice, but long-term administration of curcumin significantly activated Nrf2, restored the levels of phase II antioxidant genes (GST, glucocorticoid receptor (GR), and NQO1), upregulated p53 levels, and suppressed inflammatory signals. Therefore, the activation of Nrf2 signaling by curcumin might serve as 18


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the critical mechanism underlying the preventive effects of curcumin on lymphoma.157 In addition to its behavior as a chemopreventive agent, curcumin also showed promising potential as an adjuvant chemotherapeutic. The cytotoxic and apoptotic effects of cisplatin on the PE/CA-PJ15 human oral squamous carcinoma cell lines were enhanced when curcumin was administered in conjunction with cisplatin. Interestingly, although Nrf2 labeling (both cytoplasmic and nuclear) increased after curcumin monotherapy in cancer cells, the nuclear translocation of Nrf2 was decreased when curcumin was administered as an adjuvant to cisplatin. Therefore, adjuvant administration of curcumin may counteract the activation of Nrf2 and its nuclear translocation, thus favoring chemosensitization.158 In the same study, a protective effect of curcumin against the side effects of cisplatin was observed in the cochlea of Wistar rats.158 Furthermore, the activation of the Nrf2/HO-1 pathway by the adjuvant administration of curcumin potentiates the antioxidant defense mechanism against cisplatin-induced ototoxicity. By assessing the opposing activities of Nrf2 in cancer cells and normal cells, the author postulated that the administration of curcumin as an adjuvant to cisplatin provided protection against cisplatin-induced oxidative stress by enhancing Nrf2 signaling and the expression of its downstream antioxidant genes in normal cells undergoing cisplatin challenge, whereas adjuvant administration of curcumin with cisplatin sensitizes cisplatin-resistant cancer cells to oxidative stress by suppressing Nrf2 translocation.158

4.1.2 Roles of EGCG and genistein in preventing cancer through the Nrf2 pathway EGCG induces the expression of Nrf2-regulated genes in many cell lines and animal tissues. In the study by Han et al., EGCG was shown to protect endothelial cells from PCB 19



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126-induced inflammation by inducing the expression of Nrf2-related genes.147 The authors pretreated primary vascular endothelial cells with 50 µM EGCG, followed by exposure to coplanar PCB 126. PCB 126 increased cytochrome P450 1A1 expression and superoxide production. Interestingly, pretreatment with EGCG significantly attenuates the PCB 126-induced events. EGCG also reduces the binding of NF-κB to DNA and downstream expression of inflammatory markers, and increases expression of Nrf2-related antioxidant genes such as glutathione S transferase (GST) and NADPH quinone oxidoreductase 1 (NQO1).147 Kanaya et alstudied the protective effect of EGCG on oxalate-induced epithelial mesenchymal transition in tubular cells.148 The authors pretreated MDCK renal tubular cells with 25 µM EGCG for 1 h, followed by 0.5 mM sodium oxalate for 24 h. Vimentin and fibronectin expression were increased, whereas the levels of epithelial markers were decreased. EGCG pretreatment prevented these changes by inducing the expression of Nrf2-related antioxidant enzymes. Knockdown of Nrf2 with an siRNA abrogated the effects of EGCG. Zhou, P.et al also investigated the therapeutic potential of EGCG in a rat model of acute renal damage149 and found that EGCG exerts a protective effect on rats with obstructive nephropathy by activating the Nrf2 signaling pathway to suppress oxidative stress induced by free radicals. Rats were intraperitoneally injected with EGCG at dosages of 2.5, 5, and 10 mg/kg/day and EGCG exerted a significant preventive effect on damage induced by unilateral ureteral obstruction.149 Genistein has been shown to have antioxidant potential in many cell lines and animal tissues. Ma, W. et al. studied the neuroprotective effect of genistein in rat pheochromocytoma PC12 cells and found that this effect relies on the activation of Nrf2 and its target gene 20


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HO-1.150 PC12 cells were pretreated with 25 µM genistein for 2 h, followed by Αbeta25-35 for 24 h. Genistein reduced the ROS levels and increased the glutathione (GSH)/oxidized glutathione (GSSG) ratio and the expression of Nrf2 and HO-1.150 Zhang, T. et al studied the cytoprotective effect of genistein on the hydrogen peroxide-treated EA.hy926 human endothelial cell line.151 Pretreatment with 500 nM genistein induced the expression of Nrf2, HO-1, and peroxisome proliferator-activated receptor-γ (PPAR-γ) and then protected cells from hydrogen peroxide-induced damage.

151

Zhai et al studied the effects of genistein on

Caco-2 human colon cancer cells and showed that genistein induces the expression of Nrf2 and phase II detoxification genes.152 Caco-2 cells were pretreated with 10 µM genistein for 3 h, followed by 10 mM hydrogen peroxide (H2O2) for another 3 h. Genistein treatment significantly attenuated H2O2-induced cell death. Furthermore, genistein upregulates the expression of HO-1 and glutamate-cysteine ligase catalytic subunit (GCLC) through EKR1/2 and PKC/Nrf2 pathways in response to oxidative stress.152

4.2 Dietary isothiocyanates (allyl isothiocyanate (AITC), benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC) and SFN) Considerable epidemiological evidence suggests that the consumption of cruciferous vegetables is associated with a reduced cancer risk in humans.159, 160 These beneficial effects are mainly attributed to the isothiocyanate (ITC)-containing compounds. In the body, ITCs, which are characterized by the sulfur containing N=C=S functional group, are converted from glucosinolates, which are enriched in cruciferous vegetables, and exhibit a wide structural diversity. The effects of AITC from cabbage, mustard, and horseradish, BITC and PEITC 21



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from watercress and garden cress, and SFN from broccoli, cauliflower, and brassicas on a variety of human malignancies have been studied.161 Numerous studies using cell culture models have demonstrated that micromolar concentrations of ITCs exert a broad range of pharmacological activities against cancer.162, 163 In vivo studies provided evidence that these concentrations are achievable and well-tolerated in animals. For example, a peak plasma concentration of 20 µM SFN was observed 4 h after the oral administration of a 50-µmol dose in a pharmacokinetic study.164 In ApcMin/+ mouse models of colorectal cancer, SFN inhibited adenoma formation with a steady-state concentration of 3-13 nmol/g (roughly equivalent to 3-10 µM) in the gastrointestinal tract tissues.165 A pharmacokinetic evaluation of PEITC showed that the plasma concentrations could reach 9.2 and 42.1 µM after the oral administration of 10 and 100 µmol/kg doses, respectively, to rats.166

4.2.1 ITCs modulate phase II enzymes through Nrf2/ARE pathway The chemopreventive effects of ITCs are thought to be associated with their ability to induce

the

expression

of

phase

II

enzymes

(e.g.,

NQO-1;

GST;

and

UDP-glucuronosyltransferases, UGT; among others), which conjugate endogenous polar molecules onto the phase I metabolites. As a result, phase II metabolism facilitates xenobiotic (including carcinogens) elimination and excretion.167 SFN has been extensively documented to be a potent activator of phase II/antioxidant gene expression in both in vitro and in vivo studies.168 For example, a 40 µmol/kg/day oral dose of SFN increases GST and NQO1 activities in rat tissues, including the duodenum, forestomach, and bladder.169 SFN induces UGT1A1 and GSTA1 mRNA expression in human HepG2 cells and human hepatocytes, 22


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which

is

associated

with

decreased

formation

of

DNA

adducts

containing

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, commonly found in cooked meat and considered as a risk factor for cancer) in cells.170 Interestingly, PEITC decreases the number of PhIP-DNA adducts in rat tissues through a similar mechanism.171 Using a microarray approach, several GST isozymes were shown to be upregulated in the mouse liver 12 h after oral administration of PEITC.172 The promoter region of these phase II genes shares a common sequence named ARE/EpRE, which is the binding site for Nrf2. A number of studies on ITCs suggest that the induction of phase II/antioxidant enzymes relies on the function of Nrf2.72,

172, 173

Nrf2-deficient mice exhibit increased susceptibility in carcinogenesis models and reduced effectiveness of preventive treatments.75, 112 Therefore, Nrf2/ARE-mediated induction of the transcription of phase II enzymes is considered an important mechanism underlying the chemopreventive effects of ITCs. Nrf2 is a master regulator that maintains cellular homeostasis in response to environmental challenges. In unstimulated cells, Keap1 anchors Nrf2 in cytosol, and the Nrf2-Keap1 complex then undergoes Cul-3-dependent ubiquitination and proteosomal degradation.174 Under stress conditions, the disassociation of Nrf2 from Keap1 binding and the subsequent nuclear translocation activates the Nrf2 pathway. In the nucleus, Nrf2 forms heterodimers with Maf proteins, which bind to the ARE/EpRE sequences in the promoters and enhance the transcription of those phase II/antioxidant genes.175 In mechanistic studies, ITCs were shown to activate the Nrf2 pathway by modifying Nrf2-Keap1 interactions. Using a liquid-tandem mass spectrometry approach, Hong et al reported that SFN could directly react with the thiol groups of Keap1. The formation of SFN-Keap1 23



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thionoacyl adducts releases Nrf2 from the Nrf2-Keap1-Cul3 degradation complex; this stabilization of cellular Nrf2 subsequently results in Nrf2 nuclear translocation and activation.176 In contrast, PEITC does not directly interact with the Nrf2-Keap1 complex. A MAPK-dependent mechanism has been proposed, since PEITC-induced ARE activity is attenuated by JNK1 and extracellular signal-regulated kinase (ERK) inhibitors.173 In the same study, JNK1 and ERK2 were shown to directly phosphorylate the Nrf2 protein using in vitro kinase assays. Phosphorylation releases Nrf2 from the Keap1 complex, which in turn results in Nrf2 translocation and activation of the Nrf2/ARE pathway. In addition to inducing the expression of phase II/detoxifying enzymes, inactivation of the inflammatory pathway may be another important chemopreventive mechanism of ITCs. Interestingly, studies on the cross-talk between Nrf2 and NF-κB signaling have shown that Nrf2 downstream targets may negatively regulate the NF-κB pathway,177, 178 which lies on the molecular node of inflammation, cell survival, and cancer progression signaling.179 Accompanied by the reduced expression of inflammatory genes in colon tissues, increased expression of Nrf2-dependent genes was observed in the SFN-treated (25 mg/kg per day) group, which was associated with the mitigation of DSS-induced acute colitis in vivo.180 Similarly, SFN restored the number of sunburned cells back to the basal level in Nrf2 WT but not Nrf2 KO mice after UV irradiation. The levels of inflammatory markers were lower in Nrf2 WT tissues than in Nrf2 KO tissues after SFN treatment.181 In addition, LPS-stimulated peripheral macrophages from Nrf2 knockout mice showed an impaired anti-inflammatory response in response to PEITC treatment.182 Therefore, the activation of Nrf2 by ITCs may be a mechanism that partially contributes to the suppression of pro-inflammatory signaling 24


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pathways.

4.3 Dietary triterpenoids (ursolic acid, oleanolic acid and CDDOs (2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oic acids)) Many natural fruits and medicinal plants are enriched in triterpenoids.183 Triterpenoids are metabolites of isopentenyl pyrophosphate.184 Based on accumulating evidence, triterpenoids

have

various

pharmacological

activities,

including

anti-proliferative,

pro-apoptosis, antioxidative stress, anti-inflammatory, anti-allergic, anti-microbial, anti-viral, anti-pruritic, anti-angiogenic, anti-invasive, and anti-tumor properties.183, 185-187 Triterpenoids have been identified as a class of natural and synthetic molecules with pharmacological effects against ROS generation.188, 189 In this part of the review, we have summarized the effects of the types of triterpenoids and their roles in preventing cancer via the Nrf2 pathway.

4.3.1 Effects of ursolic acid (UA)/oleanolic acid (OA)/CDDO on cancer prevention through Nrf2 pathway 4.3.1.1 Ursolic acid Ursolic acid (UA, 3β-hydroxy-urs-12-ene-28-oic acid) is a pentacyclic terpenoid with various pharmaceutical effects, including reducing DNA damage and antioxidative stress and anti-inflammatory activities.190-192 UA is a secondary plant metabolite that is present in daily diets including fruits and herbs, such as apple peels, cranberry, bearberry, lavender, peppermint leaves, and holy basil.191 The health-promoting effects of UA have been known for centuries in folk medicine. Hence, UA is one of the most promising dietary 25



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chemopreventive compounds used to treat various cancers. Many studies have contributed to elucidating the mechanism underlying the effects of UA. UA prevented angiogenesis and metastasis and exerted its anti-cancer activity through the apoptotic pathway.193 In addition, UA protected the brain from ischemic injury by activating the Nrf2 pathway, contributing to its neuroprotective effects.194 In the liver, UA increased the translocation of Nrf2 from the cytosol to the nucleus and subsequently prevented hepatotoxicity and fibrosis by reducing oxidative stress, inflammation and apoptosis in a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis.195 Recently, UA has been reported to activate the Nrf2 pathway in mouse epidermal JB6 P+ cells by demethylating the Nrf2 promoter region.196 Thus, UA activates the Nrf2 pathway, exerts antioxidative stress and anti-inflammatory effects and contributes to chemoprevention.

4.3.1.2 Oleanolic acid Oleanolic acid (OA, 3β-hydroxyolean-12-en-28-oic acid) is a pentacyclic triterpenoid that can be obtained from approximately 1,600 different natural plants; it has various health promoting effects, including antioxidative, anti-inflammatory, and anti-cancer effects.197-199 OA significantly decreased MafK expression and MafK-mediated p65 acetylation, induced Nrf2 expression, and exhibited strong anti-inflammatory effects on mouse RAW 264.7 cells.200 The anti-inflammatory activities of triterpenoid derivatives of OA have been screened.188, 201 UA and OA exhibit anti-inflammatory activity in vitro; more potent synthetic derivatives of these compounds were developed to increase the potency and reduce the unexpected side effects.188 26


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4.3.1.3 CDDO and its analogues CDDO (2-cyano-3, 12-dioxooleana-1, 9(11)-dien-28-oic acid) and CDDO-Me (methyl2-cyno-3,12-dioxooleana1,9(11) dien-28-oate, or RTA 402) are synthetic triterpenoids derived from the OA backbone. CDDO was developed by chemically modifying three sites in OA: the C-28 carboxyl group, the C-12-C-13 double bond, and the C-3 hydroxy group. 202 Moreover, additional changes at C17 of CDDO have generated several types of derivatives, including a methyl ester (CDDO-Me) and imidazolides (CDDO-Im). 202, 203 CDDO and its derivatives are involved in various biological processes, including differentiation, proliferation, growth arrest, apoptosis, and inflammation.204 CDDO is reported to interact with specific cysteine residues on Keap1, allowing Nrf2 to translocate to the nucleus and resulting in the upregulation of downstream ARE-regulated genes. 201 CDDO-ME is reported to be among the most potent Nrf2 activators.187, 201 There are only a limited number of published preclinical studies of CDDO-Me because CDDO-Me is specifically metabolized into toxic metabolites in rodents. The emergence of CDDO-related analogues has opened up new opportunities by removing these limitations to reduce any untoward effects and to activate the Nrf2 pathway.

5 Epigenetic alterations in cancer Epigenetics is defined as a heritable change in gene expression which is not accompanied by changes in DNA sequence.205 Like genomics or proteomics, epigenomics refers to the research of the complete epigenetic modifications throughout the genome in a cell. The major 27



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epigenetic alterations include DNA methylation, histone PTMs, and miRNA-mediated post-transcriptional

alterations.206

Epigenetic modifications are vital in regulating

transcription, DNA repair, and replication. Hence, abnormal epigenetic changes may lead to the induction and maintenance of various cancers.207 In contrast to DNA mutations, epigenetic modifications are more reversible and more appropriate targets for cancer research.208 For this reason, phytochemicals that can reverse epigenetic changes would be a good focus of study for cancer prevention and treatment. The epithelial-mesenchymal transition (EMT) is popular example of an epigenetic change in cancer, which is marked by reduced expression of epithelial genes (e.g., E-cadherin) and increased expression of mesenchymal genes (e.g., vimentin, N-cadherin). The EMT is crucial in early cancer development, metastasis and recurrence209 and is highly associated with epigenetic changes, including histone modifications and microRNAs alterations.210 Phytochemicals inhibit the EMT by reversing the epigenetic alterations.211

5.1 DNA methylation DNA methylation refers to the methylation of 5' cytosine bases in CpG dinucleotides by DNA methyltransferases (DNMT1, DNMT3a and DNMT3b).212 Methylation of cytosine residues at CpG dinucleotides other than CpG islands is a common epigenetic change.213 In contrast, in cancer cells, the normally unmethylated CpG islands may become methylated.214 In normal cells, unmethylated CpG islands in promoter regions maintain the transcriptional activity of the related genes.215 However, in cancer cells, the hypermethylation of CpG islands would silence tumor repressor genes by preventing the recruitment of transcriptional proteins 28


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to the promoter regions of those genes.216 The hypermethylation of CpG islands in promoter regions and global genomic hypomethylation are now well-studied epigenetic changes in tumors. 215, 217 Therefore, chemicals with the capability of demethylating CpGs in promoter regions by inhibiting DNMT function are desirable. In recent studies, three ten-eleven translocation proteins

(TET

1–3)

5-hydroxymethylcytosine

were

shown

(5hmC),

which

to

revert provided

5-methylcytosine

(5mC)

another

for

strategy

to DNA

demethylation.218, 219 In our previous report, 3, 3'-diindolylmethane (DIM)220 and tocopherols

221

were shown

to demethylate and restore the expression of Nrf2 in prostate cancer models in vitro and in

vivo by inhibiting the activities of DNMTs. TETs have also been reported to influence the effects of natural dietary compounds on demethylation. In vitamin C-treated regulatory T cells, TETs mediated CNS2 demethylation, which is supported by in vivo findings in Tet2-/mice.219

5.2 Histone post-translational modifications (PTMs) The nucleosome is the basic functional subunit of chromatin and consists of a core histone octamer (with one H3/H4 tetramer and two H2A/H2B dimers) surrounded by 147 bp of wrapped superhelical DNA.222 With the help of linker histones (histone H1/H5), nucleosomes are further folded into 30 nm chromatin fibers, chromonema fibers and other higher-order chromatin structures.223 Each core histone contains an NH2-“tail” and COOH-“tail”, which protrude from the nucleosome and contain sites for PTMs.224 Histone 29



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PTMs are post-translational modifications that mainly occur along the NH2-terminal tails of the core histones and primarily refer to the addition of acetyl groups by HATs, methyl groups by histone methyltransferases (HMTs), phosphoryl groups by histone kinases, as well as the removal of acetyl groups by HDACs, methyl groups by histone demethylases (HDMs) and phosphoryl groups by histone phosphatases.225 The chromatin structure is highly related to gene transcription. Chromatin has an intrinsic condensed structure, which will repress gene transcription (condensed form).

226

In

contrast, a loosely packed chromatin structure allow the transcription sites to be more accessible and enhances gene transcription.227 The chromatin structure can be influenced by many factors, including histone PTMs. Generally, for histone acetylation, HATs act as transcriptional activators by neutralizing positive charges and disrupting electrostatic interactions within the histone N-terminal tails, whereas most HDACs act as transcriptional repressors by reversing lysine acetylation, restoring positive charges, and stabilizing the local chromatin structure.224 Dietary phytochemicals have a demonstrated ability to influence histone PTMs, therefore modulating gene expression to prevent cancer. EGCG suppresses human squamous carcinoma cell growth by promoting the ubiquitination of histone H2A, resulting in a less condensed chromatin structure and a subsequent increase in tumor suppressor gene expression.228 When used to treat HeLa human cervical cancer cells, SFN, a well-known plant derived HDAC inhibitor, exhibited anti-carcinogenesis effects epigenetically by reducing the expression of DNMT3B and HDAC1 and significantly reducing the activity of DNMTs and HDACs.229

30


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5.3 miRNAs MicroRNAs (miRNAs) are single stranded 18-25 nucleotide-long non-coding RNAs that participate in epigenetic regulation. miRNAs are associated with many disease processes, including COPD,230 ischemia,231 and cancer.232 miRNAs are prognostic and diagnostic markers of disease, and therefore research into miRNA is receiving more and more attention. According to the latest data from miRBase, the largest miRNA database that groups miRNA families according to sequence similarity, there are 1733 miRNA families with similar physiological functions.233 miRNAs are generally divided into two categories: the first category functions as cytoplasm mRNA inhibitors (e.g., miRNA-31, miRNA-29a, and miRNA-155) and the second category directly targets nuclear gene transcription (e.g., miR-211).234, 235 The expression of miRNAs is highly associated with DNA methylation or histone modifications. Approximately half of the miRNA genes are associated with DNA CpG methylation;236 miRNA expression is also regulated by histone modifications.237 Meanwhile, miRNAs are able to modulate the expression of DNA methyltransferases, HDACs, and polycomb group genes.238 miRNAs act as either oncogenes or tumor suppressor genes and correlate closely with DNA damage,239 the EMT240 and metastasis241 during cancer development. The ability of miRNAs to control EMT processes enable them to become novel biomarkers and therapeutic targets for cancer diagnosis and treatment.240 In addition, circulating miRNAs (cell-free miRNAs that exist in blood) have emerged as tumor-related biomarkers of all stages of cancer progression and drug sensitivities, hence providing a potential

non-invasive,

personized

regimen

for

cancer

therapy.242

Many

dietary

phytochemicals, such as curcumin,155 SFN,243 and OA,244 have been reported to inhibit 31



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carcinogenesis by inducing epigenetic modifications, including modulating the expression of miRNAs.

6 Next-generation sequencing (NGS) analysis of the roles of phytochemicals in preventing cancer 6.1 Introduction to NGS technology NGS technologies have revolutionized cancer research by deploying an “omics” approach to disclose the genomic, transcriptomic, and epigenomic landscapes of individual malignancies. 245 NGS technologies have emerged as a cost-effective way to analyze the function of phytochemicals on a whole genome scale, from the sequencing of complete genomes and transcriptomes to the genome-scale analysis of DNA-protein interactions.246 Modern biological research has been greatly aided by the development of NGS technologies, beginning with the emergence of Roche-454 over a decade ago. 247 This initial technology was followed by the introduction of Illumina 248 and IonTorrent,249 all of which utilize a sequencing-by-synthesis paradigm. Both Roche-454 and IonTorrent flow nucleotides sequentially and measure the signals generated from the incorporation of the bases complementary to the template molecules. In contrast, Illumina uses fluorophore-labeled bases to determine the sequence one base at a time. The flow cell also introduced the concept of paired-end sequencing, in which both ends of a template molecule are sequenced.

All of these technologies employ “massively parallel” sequencing, as they generate thousands (or, in the case of Illumina, hundreds of millions) of short DNA sequences, which 32


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are commonly termed “reads,” at a time. This high-throughput nature allows biological variations to be detected at an unprecedented level of sensitivity. On the other hand, the production of billions of base pairs of sequence leads to a significant amount of noise, even with an error rate of less than one percent. This noise is further magnified by mistakes that are introduced in the preparation of the DNA sample prior to the sequencing process (e.g., amplification by PCR). In some cases, errors are distributed in a nonrandom fashion; for example, both Roche-454 and IonTorrent are known to suffer from errors in and around long homopolymeric runs. 247 Regardless of the sources of errors, computational analyses of next-gen datasets must consider errors and utilize statistically rigorous tools to produce biologically meaningful results.

6.2 NGS analysis tools Quality assessment is a frequent initial step in the assessment of NGS data. FastQC 250 is a Java-based tool that provides convenient visualizations of quality scores and other issues that may indicate the need for additional processing of the reads prior to further analysis. For example, a DNA template that was shorter than the read length may lead to adapter contamination at the 3’ end of the read, which should be trimmed by a software tool such as cutadapt. 251

A critical component of most bioinformatics analyses is short-read alignment. In this step, each read is assessed to determine its location in the reference sequence (typically a genome) from which it was derived. Modern aligners, such as BWA 252 and Bowtie2,253 use compressed 33



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reference indexes to accelerate what would otherwise be a computationally burdensome process.

When aligning a read to a reference filled with repetitive elements, such as the human genome, a read will usually have multiple equivalent alignments. Therefore, these reads will be assigned a low mapping quality score by the alignment software. Depending on the bioinformatics application, researchers may choose to filter the alignments with a minimum mapping quality score, with the understanding that conclusions regarding certain genomic regions will not be able to be drawn. In general, longer reads and paired-end reads are resolved more precisely. In aligning sequenced transcripts (RNA-seq), tools such as TopHat, 254

which utilizes the algorithm of Bowtie2, will prefer an alignment that spans a splice

junction to an equivalent alignment that does not, since the latter may represent a pseudogene that does not produce transcripts.

Once the reads are aligned, the subsequent application determines what information in the alignments is the most useful. In whole genome or exome resequencing, assessing the specific differences between the reads and the reference is critical for detecting genetic variants. For other applications, such as RNA-seq, chromatin-immunoprecipitation sequencing (ChIP-seq), and methyl-DNA-IP sequencing (MeDIP-seq), the read-genome sequence comparison is of little consequence; instead, the determination of the relative numbers of reads that align to a given genomic location is more relevant.

34


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In mammalian genomes, the symmetric dinucleotide 5’-CG-3’ (commonly referred to as “CpG”), is typically methylated on both cytosines. However, certain regions of the genome contain high numbers of CpGs that are typically unmethylated. These CpG “islands” are often located in or near gene promoters and are associated with gene silencing when methylated.

The methylation status of cytosines can be determined using bisulfite sequencing (BS-seq; sometimes referred to as MethylC-seq). Bisulfite converts unmethylated cytosine to uracil, which will then be amplified and sequenced as thymine, but it has no effect on methylcytosine. This strategy allows for a straightforward determination of methylation at single-base resolution. Although whole genomes can be assayed using this method, the approach may be wasteful because genomic regions that are void of CpG dinucleotides or contain no functional methylation sites will be sequenced. More targeted approaches include hybridization-based enrichment and reduced representation bisulfite sequencing (RRBS), which involves treating the DNA with the restriction endonuclease MspI and selecting the resulting fragments based on size.

The alignment of reads derived from BS-seq is complicated because every unmethylated cytosine in the original DNA will appear as a C-T mismatch. One commonly used approach is to use a three-letter alignment, in which both the reference and the reads are converted with bisulfite in silico. The Bismark 255 bisulfite analysis package utilizes this technique and is followed by alignment with Bowtie2 and determination of the methylation status of each cytosine. 35



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With bisulfite sequencing, as well as other sequencing applications that involve comparing read-reference differences, one must be careful when analyzing datasets derived from paired-end sequencing. When these reads overlap, the overlapping sequence must not be counted twice, since the paired reads contain information derived from the same template molecule.

6.3 NGS analysis of the roles of phytochemicals in preventing cancer NGS analyses may help to provide a global view of whole genome changes and hence would identify novel genes and pathways and elucidate the cancer prevention mechanisms of phytochemicals.

By

analyzing

the

genome-wide

changes

in

transcription

in

curcumin/piperine-treated normal breast stem/progenitor cells using a combination of fluorescence-activated cell sorting and RNA-seq, curcumin was shown to target both stem cell populations by decreasing the expression of Aldehyde Dehydrogenase 1 Family Member A3 (ALDH1A3), CD49f, prominin-1 (PROM1), and tumor protein 63 (TP63).256 When comparing the changes in gene expression between malignant, normal and immortalized cell lines by genome-wide mRNA-Seq analysis, Shelley Z. et al. identified a novel molecular mechanism by which L-sulforaphane (LSF) regulates the anaphase promoting complex (APC) pathway in cell cycle progression by significantly decreasing the expression of cyclin-dependent kinase 1 (CDK1).257 ChIP-seq and RNA-seq were used to analyze endometrial cancer cells treated with bisphenol A (BPA), genistein (GEN), or 17β-estradiol. GEN and BPA generated thousands of ESR1 binding sites and over 50 changes in gene 36


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expression, which represented a subset of E2-induced changes in gene regulation.258

7 Roles of the epigenetic modifications induced by dietary phytochemicals in preventing cancer Recently, numerous experimental studies have supported the hypothesis that the initiation and progression of carcinogenesis involves aberrant epigenetic alterations. Given that epigenetics lies on the molecular interface between genetics and environmental factors, there is a growing interest in evaluating the potential of dietary phytochemicals that block or reverse the epigenetic abnormalities observed during cancer development. DNA methylation and histone PTMs are the major epigenetic events that together build the epigenome to store the epigenetic information regarding cell type-specific gene expression patterns.259 Here, we will mainly focus on the roles of phytochemicals in preventing cancer via DNA methylation and histone PTMs.

Many dietary phytochemicals have shown the potential to restore the expression of multiple tumor suppressor genes by regulating DNMTs and histone-modifying enzymes.260, 261 An in

vitro treatment with EGCG in human epidermoid carcinoma A431 cells restores the expression of the tumor suppressor genes p16/INK4a and Cip1/p21 by reducing the 5-methylcytosine levels, DNMT activity and expression, and histone deacetylase activity; increasing the acetylation of H3k9, H3k14, H4k5, H4k12 and H4K16; and decreasing the methylation of H3K9.262 SFN and 3,3'-diindolylmethane (DIM) have been reported to exert broad effects on inhibiting DNA methyltransferase expression in LNCaP and PC3 prostate 37



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cancer cells.263 In DIM-treated PC3 and LNCaP cells, HDAC activity was significantly inhibited by 66%, particularly HDAC2, which is correlated with an upregulation of the expression of the HDAC inhibitor p21.264 Here, we will discuss additional epigenetic changes induced by dietary phytochemicals as a cancer chemopreventive mechanism. Phytochemicals capable of inducing epigenetic modifications to prevent cancer are summarized in Table 2.

7.1 Dietary polyphenols (curcumin, EGCG, and genistein) 7.1.1 Effects of curcumin on DNA methylation and histone PTMs As reported in previous studies, curcumin acts as a hypomethylation agent to prevent multiple types of cancer. For example, studies conducted by our group on prostate cancer revealed that Nrf2 is silenced by DNA hypermethylation during the progression of prostate tumorigenesis in TRAMP mice,140 and curcumin treatment significantly decreases the methylation of the first 5 CpG sites in the promoter region of the Nrf2 gene in TRAMP C1 cells, an event associated with the re-expression of Nrf2 and its downstream targets.265 Given the preventive effect of curcumin on the development of prostate cancer in TRAMP mice,266 we postulated that the chemopreventive effect of curcumin on prostate cancer may involve the hypomethylation of the Nrf2 promoter and the subsequent activation of the Nrf2-mediated pathway.

We

further

tested

a

synthetic

curcumin

(3E,5E)-3,5-Bis(pyridin-2-methylene)-tetrahydrothiopyran-4-one

(FN1),

analogue, which

5

shows

stronger anticancer activity in prostate cancer cells.267 FN1 significantly inhibits the colony-forming ability of TRAMP-C1 cells, decreases the methylation of the Nrf2 promoter, 38


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and thus activates Nrf2 signaling. Furthermore, the inhibition of enzymes responsible for epigenetic modifications (DNMT1, DNMT3a, DNMT3b, and HDAC4) by FN1 may be associated with the ability of FN1 to demethylate Nrf2.268 In addition to Nrf2, curcumin also demethylates the promoter of the neurogenin 1 (Neurog1) gene and restores the expression of this cancer-related, CpG-methylated epigenetic marker gene in LNCaP cells.269 Methylation microarrays were conducted in three colon cancer cells (HCT116, HT29, and RKO) treated with curcumin or 5-aza-CdR. In contrast to the non-specific global DNA hypomethylation induced by 5-aza-CdR, curcumin induces selected changes in methylation at specific loci, with corresponding changes in gene expression.270 As shown in a recent study by our group, curcumin treatment reduces CpG methylation in the promoter region of the DLEC1 gene (deleted in lung and esophageal cancer 1), a tumor suppressor gene with promoter hypermethylation and reduced transcriptional activity in colon cancer, in HT29 cells. Furthermore, the curcumin-induced hypomethylation of the DLEC1 gene is associated with increased mRNA expression and reduced anchorage-independent growth.271 In MCF-7 breast cancer cells, curcumin reactivates Ras-association domain family protein 1A (RASSF1A) by reducing the methylation of its promoter. In addition, this demethylating event in the RASSF1A promoter may be correlated with suppressed DNMT1 activity due to the curcumin-induced disruption of the NF-kB/Sp1 complex at the promoter region of the DNMT1 gene.272 Curcumin treatment increases the expression of tumor suppressor gene retinoic acid receptor β (RARβ) by reducing the promoter methylation in A549 and H460 lung cancer cells and a xenograft nude mouse model.273 This specific demethylation of RARβ through the downregulation of the expression of the DNMT3b mRNA by curcumin might be 39



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one of the mechanisms underlying its protective effect on lung cancer growth.273 In acute myeloid leukemia (AML), curcumin suppresses p65 and Sp1 expression (the positive regulators of DNMT1) and their binding to the DNMT1 promoter. The curcumin-mediated decrease in DNMT1 expression is correlated with the reactivation of the tumor suppressor gene p15INK4B in multiple AML cells lines through the hypomethylation of its promoter.274 In addition to reducing the methylation of the promoters of specific epigenetically silenced tumor suppressor genes, curcumin also exerts its chemopreventive effect by inducing global DNA hypermethylation. A low concentration of curcumin (1 µM) is cytotoxic to HeLa cervical cancer cells, possibly by decreasing the expression of argyrophilic nucleolar protein (AgNOR protein) via global DNA hypermethylation. 275 Curcumin is also considered as a histone modifier that prevented multiple types of cancer by modulating the activities of several histone modifying enzymes, including HDACs, HATs, and enhancer of zeste homolog 2 (EZH2). In the myeloproliferative neoplastic cell lines K562 and HEL, curcumin restores the expression of suppressor of cytokine signaling 1 (SOCS1) and SOCS3 by inhibiting the expression and activity of class I HDACs (particularly HDAC8), thus increasing the levels of ac-H3 and ac-H4 in the promoter regions.276 Furthermore, the inhibitory effect of curcumin on HDACs has also been observed in DAOY medulloblastoma cells (mainly HDAC4)277 and in colorectal adenocarcinoma cells HT29 cells (mainly HDACs 4, 5, 6, and 8).271 Although the expression of HDAC1, 4, 5, and 8 is increased in human prostate LNCaP cells, curcumin treatment significantly decreases the expression of HDAC3 and the total HDAC activity, which is accompanied by decreased enrichment of H3K27me3 at the Neurog1 promoter region and increased transcription of Neurog1.269 In CEM leukemia 40


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cells, the induction of the expression of the epigenetically silenced genes p15 and CDH-1 (encodes E-cadherin) by DMC cannot be explained by DNA hypomethylation but is rather mediated by modulation of histone modifications. Specifically, the abundance of the H3K36me3 mark in the CpG islands of both the p15 and CDH-1 genes is significantly increased by treatment with DMC; however, this effect is not observed following treatment with curcumin.278

Although several studies have shown that curcumin exerted

histone-modifying effects by inhibiting HDACs, Kang et al showed that curcumin induces histone hypoacetylation by inhibiting HATs but not HDACs in human hepatoma Hep3B cells.279 In addition, the observed inhibition of HAT and histone hypoacetylation by curcumin is diminished by antioxidant enzymes, suggesting that ROS promote curcumin-mediated histone hypoacetylation.279 In MCF-7 human breast cancer cells, curcumin inhibits the activities of HAT and increases global levels of acetylated H3K18 and H4K16, potentially leading to the arrest of cell proliferation.280 Recently, curcumin was also shown to inhibit lung cancer cells by suppressing the expression of EZH2 in A549 cells, which further inhibited NOTCH1 signaling.281 EZH2, a key component of the human polycomb repressive complex 2 that is responsible for the trimethylation of histone H3K27,282 is overexpressed in multiple malignancies. However, the mechanism by which curcumin modulates the level of H3K27me3 by inhibiting EZH2 is unclear and requires further investigation.

7.1.2 Effects of EGCG/genistein on DNA methylation and Histone PTMs EGCG was reported to alter the DNA methylation patterns of genes in many cancer cell lines from different tissues, including breast, kidney, colon, lung, and lymphocytes.283-287 In 41



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the 786-0 human renal carcinoma cell line, EGCG inhibits DNA methyltransferase (DNMT) activity, and induces CpG demethylation and reactivation of methylation-silenced genes such as tissue factor pathway inhibitor-2 (TFPI-2). Overexpression of TFPI-2 may induce apoptosis in renal carcinoma cells. Interestingly, EGCG upregulates TFPI-2 expression by demethylating its promoter region.283 Morris et al studied the effects of EGCG on colon cancer and found that EGCG reduces DNA methylation in the RXRα promoter. The reduced methylation level in RXRα promoter restores its expression, providing a possible strategy for preventing and treating colon cancer.285 The chemoprevention potential of genistein in many diseases, including cancer, has been widely studied over the past decade. Based on several in vitro and in vivo studies, genistein may induce the expression of many tumor suppressor genes by demethylating their promoter regions. Genistein modulates DNA methylation and the expression of many tumor suppressor genes, including GSTP1, ATM, APC, and phosphatase and tensin homologue (PTEN).288, 289 In colon cancer cells, genistein was shown to alter the expression of genes involved in the Wnt pathway.290-292 In particular, SW1116 cells treated with genistein for 4 days exhibit demethylation of the Wnt5a promoter.290 Treatment with 75 µM genistein for 4 days increases the expression of sFRP2 and sFRP5 in DLD-1 human colon cancer cells.291 The elevated expression of these genes is due to decreased methylation in the promoter region.292 Many other studies also reported the chemoprotective effects of genistein on prostate cancer.293-297 Treatment with genistein significantly decreases the methylation of many genes, including estrogen receptor (ER), p21, p16, RAR-β, and O-6-methylguanine-DNA methyltransferase (MGMT).296 ER-β functions as a tumor suppressor and its expression is reduced during cancer 42


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progression.295 Treatment with 10 µM genistein reduces methylation at the ER-β promoter and leads to gene re-expression in LNCaP and LAPC-4 cells.295 Genistein was also reported to have chemopreventive effects on many other cancer cells, including leukemia, lung cancer and neuroblastoma cells.298-301 As reported by Liu et al, genistein mediates the radiosensitization of A549 non-small cell lung cancer cells by demethylating the Keap1 promoter region.300 Li et al studied the effects of genistein on SK-N-SH human neuroblastoma cells and found that treatment with genistein decreases the methylation of several tumor suppressor genes, such as p53 and chromodomain helicase DNA binding protein 5 (CHD5). Demethylation of these tumor suppressor genes leads to their reactivation.301 EGCG regulates gene expression by modifying histones. As reported by Lee et al EGCG suppresses prostate cancer cell growth by modulating histone acetylation of androgen receptor (AR), where EGCG acts as a HAT inhibitor and suppresses agonist-dependent AR activation and AR-regulated gene transcription.302 In another study by Nihal and colleagues, EGCG was suggested to improve the anti-melanoma effect of vorinostat. Vorinostat, also known as SAHA, is a histone deacetylase (HDAC) inhibitor that has been used to treat lymphoma. Vorinostat inhibits histone deacetylases, resulting in the accumulation of acetylated histones, which leads to the activation of many genes.303 Genistein regulates gene expression in many different cancer cell lines by modifying histones. Li et al studied the effect of genistein on breast cancer cells and found that genistein suppresses breast tumorigenesis by regulating multiple tumor-related genes.304,

305

The

expression of two tumor suppressor genes, p21/WAF1 and p16/INK4a, is increased by 43



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genistein. As shown in further studies, the elevated expression of these tumor suppressor genes is due to histone modification in the promoter region.305 In SW480 colon cancer cells, genistein induces dickkopf-related protein 1 (DKK1) expression by increasing histone H3 acetylation at its promoter region. Increased histone acetylation induces gene expression and then inhibits the WNT signaling pathway.306 Interestingly, Zhang et al found that genistein also decreases histone H3 acetylation at the promoters of several WNT pathway genes, including secreted frizzled-related protein 2 (Sfrp2), Sfrp5 and Wnt5a, thus suppressing expression of these genes.292 Kikuno et al studied the effects of genistein on LNCaP and PC-3 prostate cancer cells and found that genistein activates the expression of several aberrantly mutated tumor suppressor genes, including PTEN, ubiquitin carboxyl-terminal hydrolase (CYLD), p53 and forkhead box O3a (FOXO3a). The activation of these genes is involved in the demethylation and acetylation of H3 lysine 9 (H3K9) at their promoter regions.294 These findings increase our understanding of chemopreventive effects of genistein on prostate cancer.

7.2 Effects of dietary isothiocyanates (SFN and PEITC) on DNA methylation and histone PTMs DNA methylation refers to the addition of a methyl group at the 5’ position of the cytosine residues within CG dinucleotides by DNMTs. In a recent study, Wong et al. reported the effects of SFN on the promoter DNA methylation profile in prostate epithelial cells (PrEC) and androgen-dependent (LNCaP) and androgen-independent (PC-3) prostate cancer cells.263 The SFN treatment decreases the DNMT levels in all the tested cell lines. Although SFN has 44


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complex effects on the genome-wide DNA methylation patterns among normal prostate epithelial cells and prostate cancer cells, the genes with an altered methylation status are functionally similar within a single cell line (e.g., cell migration, cell adhesion, etc.). In various in vitro and in vivo studies, SFN or PEITC treatments appear to downregulate DNMT activity, thereby resulting in promoter demethylation and concomitant changes in the expression of a variety of genes.307, 308 The interactions between DNA methylation with histone modifications, transcription factors, transcriptional coactivators, and DNA binding proteins determines the status of gene transcription.309 The removal of acetyl groups from histone tails by HDACs is an important regulatory mechanism that results in a condensed chromatin structure and limited access to gene promoters. HDACs are often upregulated in cancers; therefore, HDAC inhibition is considered an important strategy in cancer prevention and therapy. As shown in molecular docking studies, metabolites of SFN and several structurally related ITCs directly interact with the HDAC catalytic core to inhibit enzyme activity.310 In a clinical study, a single dose of 68 g of broccoli sprouts (containing ~105 mg of SFN) significantly inhibited HDAC activity in peripheral blood mononuclear cells (PBMC) 3 and 6 h after consumption.311 Interestingly, DNA demethylation in promoter regions is often associated with a locally relaxed histone structure, although the precise mechanism remains to be elucidated. For example, in mouse epidermal JB6 (JB6 P+) cells, SFN demethylates the CpGs in the Nrf2 promoter region by inhibiting DNMT and HDAC activities. This change in the methylation pattern is associated with

increased

Nrf2

levels

and

a

phenotype

that

is

more

resistant

to

12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation.312 Epigenetic 45



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upregulation of the expression of the p21 gene by PEITC is associated with chromatin remodeling, which compromises dynamic changes in both histone acetylation and methylation.313 Notably, PEITC also exhibits the dual functions of CpG demethylation and HDAC inhibition in the epigenetic regulation of various genes. 314, 315

7.3 Dietary triterpenoids Triterpenoids have been reported to have various pharmacological activities, and many of them have also been shown to activate the Nrf2 pathway. Recently, few reports have shown that triterpenoids interacted with epigenetic regulators to exert their biological functions. In this review, we have summarized recent studies on the potential of triterpenoids to protect against human diseases by restoring abnormal epigenetic alterations related to DNA methylation, histone modifications and miRNAs.

7.3.1 Effects of UA on DNA methylation and histone PTMs Recently, UA has been reported to reduce the expression of enzymes regulating epigenetic modifications, including DNA methyltransferases (DNMT1 and DNMT3a) and HDACs (HDAC1, HDAC2, HDAC3, HDAC6, HDAC7 and HDAC8), and HDAC activity in mouse skin JB6 P+ cells, indicating its beneficial effects on the chemoprevention of skin cancer196. As shown in another study of HL-60 human acute myeloid leukemia cells, UA increases the acetylation of histone H3 and decreased HDAC activity, resulting in cell death.316 In addition, UA induces apoptosis by increasing expression of programmed cell death 4 (PCD4) and decreasing the expression of miR-21, a typical cancer-associated miRNA, 46


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in human glioblastoma cells.317, 318

7.3.2 Effects of OA on DNA methylation and histone PTMs OA suppresses the proliferation of lung cancer cells and lung cancer xenografts in mice by inducing the expression of miR-122, a potential target in cancer prevention that is hypermethylated in promoter regions; thus, OA exerts anti-tumor activity in liver cancer cells.244, 319, 320 In addition, Zhou et al reported the anti-diabetic activity of OA in a mouse model of type 2 diabetes is maintained even 4 weeks after the cessation of OA administration.321 In this study, OA administration increased the phosphorylation and acetylation of lysines 259, 262, and 271 in FOXO1, accompanied by an increase in the HAT1 levels and the inhibitory phosphorylation of HDAC4 and HDAC5 in mice with type 2 diabetes.321

7.3.3 Effects of CDDO and its derivatives on DNA methylation and histone PTMs CDDO and its derivatives are synthetic triterpenoids derived from OA. CDDO-Me, a CDDO derivative, has stronger anti-cancer potency than CDDO.322 CDDO enhances the pro-apoptotic and pro-differentiation effects of all trans-retinoic acid (ATRA) on ATRA-sensitive NB4 cells and ATRA-resistant derivative MR2 cells.323 Acetylation of histone H3-Lys9 is increased in the RARβ2 promoter and the expression of PPARγ is induced in these cells, resulting in increased apoptosis and differentiation.323 CDDO-Me inhibits proliferation and induces apoptosis in human pancreatic cancer cells.324 CDDO-Me reduces the expression of the telomerase reverse transcriptase (hTERT) mRNA and protein, as well as hTERT 47



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activity. Moreover, CDDO-Me inhibits the expression of the DNMT1 and DNMT3a proteins, resulting in the demethylation of the CpGs in the hTERT promoter region. The effects of CDDO-Me are associated with a reduction in acetylated histone H3-Lys9, acetylated histone H4, dimethyl-H3-Lys4, and trimethyl-H3-Lys9 at the hTERT promoter.324

8. Conclusions In conclusion, oxidative stress, chronic inflammation and epigenetic changes are all major causes of cancer; all three factors can also interact with each other. The activation of the Nrf2 pathway plays a pivotal role in inhibiting carcinogenesis by promoting antioxidant, detoxification, and anti-inflammatory reactions. Nrf2 function is also subjected to epigenetic alterations during carcinogenesis, similar to other cancer prevention genes/pathways. Dietary phytochemicals with cancer chemopreventive properties are able to prohibit, arrest, or reverse the initiation, progression and promotion of carcinogenesis. Their cancer prevention functions may arise from the activation of the Nrf2 antioxidant and detoxification pathways or the reversal of the epigenetic changes that occur during carcinogenesis. The findings on dietary phytochemicals may be valuable for clinical cancer prevention research.

AUTHOR INFORMATION Corresponding Author *Tel.: (848)-445-6368; Fax: 732-455-3134; E-mail: [email protected].

Funding Sources 48


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This work was supported in part by institutional funds and by R01-CA118947, R01-CA152826, R01-CA200129, from the National Cancer Institute (NCI), R01-AT009152 from the National Center for Complementary and Integrative Health (NCCIH) and R01-AT007065 from NCCIH and the Office of Dietary Supplements (ODS).

Notes The authors declare that there are no conflicts of interest.

Acknowledgments The authors express sincere gratitude to all of the members of Dr. Tony Kong's laboratory for their helpful discussions.

Abbreviations Nrf2, Nuclear factor erythroid-2 related factor 2; ROS, reactive oxygen species; PTMS, Post-translational modifications; NGS, Next-generation sequencing; HO-1, heme oxygenase-1; DNMT, DNA methyltransferase; HDAC, histone deacetylase; HAT, Histone acetyltransferases; NQO1, NAD[P] H:quinone oxidoreductase-1; GSTM, glutathione S-transferase mu; GPX, glutathione peroxidase;SOD, superoxide dismutase; GST, glutathione S-transferase; γ-GCL, γ-glutamyl cysteine ligase; SAHA, suberoylanilide hydroxamic acid; ARE, antioxidant response element; EpRE, electrophile response element; Keap-1, Kelch-like ECHassociated protein 1;TRL, toll-like receptor; COX-2, cyclooxygenase-2; DIM, 3,3'-diindolylmethane; 5-aza, 5-azadeoxycytidine; TSA, Trichostatin A ; TPA, 49



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tetradecanoylphorbol-13-acetate; SFN, sulforaphane; BGS, Bisulfite Genomic Sequencing; MeDIP, Methylated DNA Immunoprecipitation

50


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Author Biographies Dr. Wenji Li obtained his Ph.D. degree in Department of Pharmacy at the National University of Singapore, Singapore in 2011 with research focus on anti-cancer natural products evaluation and delivery. Afterward, Dr. wenji joined the faculty of pharmacy at MSU university in Malaysia and STCM college in Singapore as a lecturer successively. Since 2014, he joined Dr. Tony Kong’s laboratory in Rutgers University, USA, as a postdoc to perform research on the mechanisms of dietary phytochemicals in cancer prevention and anti-inflammation mainly focusing on epigenetics and next-generation sequencing study. Ms. Yue Guo is a Ph.D. candidate in the Graduate Program in Pharmaceutical Science at Ernest Mario School of Pharmacy, Rutgers University. She received her B.S in Pharmacy in 2011 at China Pharmaceutical University, Nanjing. Yue joined Dr. Kong’s laboratory in 2011. Her research is focused on the regulation of epigenetic mechanisms and Nrf2 signaling pathways in colon carcinogenesis and how dietary phytochemicals modulate these epigenetic mechanisms and Nrf2 activity in the prevention of colon cancer in vitro and in vivo. Mr. Chengyue Zhang obtained his BS and MS degree from Peking University, Beijing, China, majored in Pharmaceutical Sciences. In the fall of 2011, he joined Dr. Kong’s group in pursuit of a PhD degree. Given that many dietary compounds can prevent cancer initiation and development, how these chemo-preventive compounds can affect epigenetic events during carcinogenesis is his major research interest. He has published several works on DNA methylation associated Nrf2 transcription activities, elucidating the role of epigenetic regulation of Nrf2 in cancer prevention.

Dr. Renyi Wu obtained his B. Sc. in Biotechnology from Wuhan University in 2008 and Ph.D. degree in Cell Biology from Institute of Zoology, the Chinese Academy of Sciences in 2014. Since then, he has been working at Dr. Tony Kong's lab at School of Pharmacy, Rutgers University, as a postdoctoral associate. His research is mostly focused on studying the anti-cancer effect of phytochemicals on prostate and colon cancers in mouse model, and exploring the epigenetic changes during tumor progression by utilizing next generation sequencing and other technologies. Anne Yuqing Yang received her B.Sc. in Pharmacy from China Pharmaceutical University, Nanjing, China, in 2011. She is currently a Ph.D. candidate in Dr. Ah-Ng Tony Kong’s lab from the graduate program of Pharmaceutical Sciences at Rutgers University - New Brunswick. Her research focuses on exploring the effects of phytochemicals and their roles in skin cancer prevention related to Nrf2-mediated redox signaling and epigenetics/epigenomics. Dr. John Gaspar received his B.Sc. in Biology and Mathematics from Bowdoin College, USA in 1999, M.S. in Biology from Yeshiva University, USA in 2004 and Ph.D in Biochemistry from University of New Hampshire University, USA in 2014. Dr. Gaspar joined Computational Biology Institute in George Washington University in 2014 to carry out 76


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research on sensitive variant detection in targeted next next-gen resequencing in cancer. Since 2015, he joined Dr. Kong’s lab in investigating dietary phytochemicals in cancer prevention by next-generation sequencing.

Dr. Ah-Ng Tony Kong is a Distinguished Professor in Rutgers University. He obtained his B.Sc. in Pharmacy from University of Alberta, Canada and PhD in Pharmaceutics, Pharmacokinetics and pharmacodynamics under the mentorship of Professor William J. Jusko from SUNY at Buffalo. He has published more than 230 original research, review articles and book chapters in cancer prevention by dietary phytochemicals, epigenetics/epigenomics, Nrf2-mediated anti-oxidative stress and anti-inflammatory responses, pharmacokinetics, pharmacodynamics, PK-PD modeling, drug metabolism, pharmacogenomics, and cellular signaling. He has been continuously receiving funding from the NIH since 1993 and has continued to serve on numerous NIH panels since 1998.

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Table 1. Phytochemicals in cancer chemoprevention with Nrf2 activation properties Compounds Curcumin

Plant Curcuma Longa

EGCG

Green tea

Genistein

Genista tinctoria

SFN

Model 1. human breast cancer MCF-7 cells 2. livers from mice induced with Dalton’s Lymphoma 1. PCB 126-induced inflammation in endothelial cells

Molecular Targets References 156 ↑Nrf2, ↓Fen1 157 ↑Nrf2, GST, GR, NQO1, p53 ↓P450 1A1, ↓DNA binding of NF-kappaB, 147 ↑GST, NQO1 148 2. oxalate-induced epithelial mesenchymal transition in ↓vimentin, fibronectin, ↑Nrf2 MDCK renal tubular cells 149 3. rat acute renal damage model ↑Nrf2, γ-GCS 1. rat pheochromocytoma PC12 cells

↑gamma-GCS, Nrf2, HO-1.

150

2. human endothelial cell EA.hy926

↑Nrf2, HO-1, PPAR-γ.

151

3. human colon cancer caco-2 cells

↑HO-1,Nrf2,GCLC, ↑ERK1/2 and PKC pathway; ↑ GST, NQO1

152

broccoli, 1. rats duodenum, forestomach, and bladder. cauliflower, and 2. human HepG2 cells and human hepatocytes brassicas 3. mice liver 4. DSS-induced colitis mice model

UA

OA

cranberry, bearberry, holy basil,etc. olive oil, garlic, etc

1.mouse epidermal JB6 P+ cells

1.mouse RAW 264.7 cells

↑UGT1A1, GSTA1;↓ formation ↑GST, 2c55, 2 mu1

PhIP-DNA

169

adduct

↑NQO1, GCLC, PON-2, HSP70; ↓CXCL1, TNFα, IL1β, IL-6, IL10, IFNγ ↑Nrf2, HO-1, NQO1, UGT1A1; ↓DNMT1,DNMT3a,HDAC1,HDAC2,HDAC3, HDAC8, HDAC6,HDAC7 ↑Nrf2, ↓MafK, p65 acetylation

170

172 180

196

200

78


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CDDOs


synthetic derivatives

OA 1.Hepa1c1c7 murine hepatoma cells; mouse RAW 264.7 ↑Nrf2, NQO1;↓Cox-2, iNOS cells; Mouse embryonic fibroblasts (MEFs); human retinal pigment epithelial cells (ARPE-19)

201

79



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Table 2. Phytochemicals in cancer chemoprevention with epigenetics modification properties Compounds EGCG

SFN

PEITC DIM

Curcumin

Model Molecular Targets References 262 1.human epidermoid carcinoma A431 ↑ p16INK4a, Cip1/p21; cells ↓ 5-methylcytosine, DNMTs, HDACs; ↑ acetylation of H3k9, H3k14, H4k5, H4k12,H4K16;↓ methylation of H3K9 283 2.human renal carcinoma cell line ↓ CpG methylation in the promoter region of the TFPI-2 gene;↑TFPI-2 786-0 285 3. human colon cancer HCT116, ↓ CpG methylation in the promoter region of the RXRα gene;↑ RXRα HT29, SW48 cells 263 1. human prostate epithelial cells ↓DNMTs (PrEC), human prostate cancer LNCaP cells and PC-3 cells 311 2. human peripheral blood ↓HDAC activity mononuclear cells 3. mouse epidermal JB6 cells ↓DNMTs; ↓HDACs; ↓CpG methylation in the promoter region of the Nrf2 312 gene;↑Nrf2; 313 1. human prostate cancer LNCaP cells ↑p21WAF1 and p27; ↓HDACs;↑H3Ac and H3K4me, ↓H3k9me 263 1. human prostate epithelial cells ↓DNMTs (PrEC), human prostate cancer LNCaP cells and PC-3 cells 264 2.human prostate cancer cells lines ↓HDAC2, ↑p21 LNCaP and PC3 265 1.mouse prostate cancer TRAMP C1 ↑Nrf2, NQO-1,↓ CpG methylation in the promoter region of the Nrf2 gene cells 2. human prostate cancer LNCaP cells ↓ CpG methylation of the promoter region of the Neurog1 gene, H3K27me3, 269

80


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HDAC3, total HDAC activity, ↑ Neurog1 3. human Colorectal Adenocarcinoma ↓ CpG methylation in the promoter region of DLEC1, HDACs 4, 5, 6, HT29 cells 8;↑DLEC1 4. human breast cancer MCF-7 cells ↓ CpG methylation in the promoter region of RASSF1A, DNMT1;↑RASSF1A, 5. human lung cancer A549 cells and ↓ CpG methylation in the promoter region of RARβ, DNMT3b;↑ RARβ H460 cells and xenograft nude mice model 6. multiple acute myeloid leukemia ↓ CpG methylation in the promoter region of p15INK4B, DNMT1, p65,Sp1; (AML) cells lines ↑ p15INK4B 7. human cervical cancer HeLa cells ↓ AgNOR; ↑ global DNA hypermethylation 8.human myeloproliferative neoplasm ↑ SOCS1,SOCS3, ac-H3 and ac-H4 in the promoter regions;↓ HDAC8 K562 and HEL cell lines 9. human medulloblastoma DAOY ↓ HDAC4 cells 10. human breast cancer MCF-7 cells ↓ activities of HAT; ↑ acetylated H3K18 and H4K16 11. human lung cancer A549 cells ↓EZH2, NOTCH1 signaling FN1 (a synthetic 1. mouse prostate cancer TRAMP C1 ↓ CpG methylation in the promoter region of the Nrf2 gene, DNMT1, curcumin analogue) cells DNMT3a, DNMT3b, HDAC4,↑Nrf2, NQO1, HO-1, UGT1A1 Genistein 1. human colon cancer SW1116 cells ↓ CpG methylation in the promoter region of the Wnt5a gene;↑ Wnt5a 2. human colon cancer DLD-1 cells ↓ CpG methylation in the promoter region of the sFRP2 and sFRP5;↑ sFRP2, sFRP5; ↓ H3Ac at the promoter region of Sfrp2, Sfrp5 and Wnt5a; ↓H3K9Me3 and H3S10P at the promoter region of Sfrp2, Sfrp5 and Wnt5a 3. human prostate cancer LNCaP and ↓CpG methylation in the promoter region of the p21, p16, RAR-β, and MGMT; DuPro cells ↑ p21, p16, RAR-β, and MGMT; ↑ HAT, acetylated histones 3, 4, and H3/K4 at the p21 and p16 transcription start sites 4.human prostate cancer LNCaP and ↓ CpG methylation in the promoter region of the ER-β;↑ ER-β

271

272 273

274

275 276

277

280 281 268

290 291, 292

296

295

81



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LAPC-4 cells 5.human lung cancer A549 cells 6. human neuroblastoma SK-N-SH cells 7. human breast cancer MDA-MB-231 cells

UA

OA

CDDO CDDO-Me

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↓ CpG methylation in the promoter region of the keap1;↑keap1 ↓ CpG methylation in the promoter region of the p53, CHD5;↑p53, CHD5

300

↑p21, p16; ↑acetyl-H3 and H3K4me3 and ↓ H3K9me3 and H3K27me3 in the promoters of p16 and p21

305

8. human colon cancer SW480 cells ↑ histone H3 acetylation of DKK1promoter region; ↑ DKK1 9.human prostate cancer LNCaP and ↓H3-K9me and ↑H3k9Ac at the PTEN and CYLD promoter;↑PTEN, CYLD PC-3 cells 1. mouse skin JB6 P+ cells ↓DNMT1,DNMT3a,HDAC1,HDAC2,HDAC3,HDAC8, HDAC6,HDAC7;↑Nrf2, HO-1, NQO1, UGT1A1; 2.human acute myeloid leukemia ↑H3Ac; ↓HDACs; HL-60 cells 3. human glioblastoma U251 cells ↑PCD4; ↓miR-21 1. human lung cancer cell lines, A549, ↑miR-122; ↓CCNG1 and MEF2D NCI-H460, and NCI-H1299 2. high-fat diet induced type 2 diabetes ↑HAT1; ↓ phosphorylation of HDAC4 and HDAC5 mice model 1.human Acute Promyelocytic ↑H3K9Ac in RARβ2 promoter; ↑ PPARγ Leukemia NB4 cells and MR2 cells 1.human pancreatic cancer cells ↓hTERT;↓ DNMT1 and DNMT3a;↓H3K9Ac, H4Ac, H3K4me2, H3K9Me3,CpG methylation in the promoter region of the hTERT;

306

301

294

196

316

181 244

321

323

324

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Table of Contents Graphic 330x217mm (72 x 72 DPI)


Dietary Phytochemicals and Cancer Chemoprevention: A

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