Anthocyanin Delphinidin Prevents Neoplastic Transformation of Mouse Skin JB6 P+ Cells: Epigenetic Re-activation of Nrf2-ARE Pathway.

Redox imbalance is a major contributor to the pathogenesis of melanoma and nonmelanoma skin cancer. Activation of the nuclear factor E2-related factor 2 (Nrf2) antioxidant responsive element (ARE) pathway is an intrinsic defense mechanism against oxidative stress. Flavonoids such as anthocyanidins, which are found abundantly in fruits and vegetables, have been shown to activate Nrf2. However, the epigenetic and genetic mechanisms by which anthocyanidins modulate the Nrf2-ARE pathway remain poorly understood in the context of skin cancer. In this study, delphinidin, one of the most potent and abundant anthocyanidins in berries, significantly inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic cell transformation in mouse epidermal JB6 P+ cells by 69.4 to 99.4%. The mechanism was elucidated based on observations of increased ARE-driven luciferase activity and elevated mRNA and protein expression of Nrf2 downstream genes, such as heme oxygenase-1 (Ho-1), in JB6 P+ cells. Activation of the Nrf2-ARE pathway was correlated with demethylation of 15 CpG sites in the mouse Nrf2 promoter region between nt - 1226 and - 863 from the transcription start site. The reduced CpG methylation ratio in the Nrf2 promoter region was consistent with observed decreases in the protein expression of DNA methyltransferases 1 (DNMT1), DNMT3a, and class I/II histone deacetylases (HDACs). Overall, our results suggest that delphinidin, an epigenetic demethylating agent of the Nrf2 promoter, can activate the Nrf2-ARE pathway, which can be applied as a potential skin cancer chemopreventive agent.

Sophora flavescens Containing-QYJD Formula Activates Nrf2 Anti-Oxidant Response, Blocks Cellular Transformation and Protects Against DSS-Induced Colitis in Mouse Model.

Qu-Yu-Jie-Du decoction (QYJD) is a commercially available traditional Chinese medicine (TCM). It is an aqueous extract of a Chinese herbal formula primarily consisting of eight TCM herbs: Taraxacum campylodes G.E. Haglund, Coix lacryma-jobi L., Smilax glabra Roxb., Sanguisorba officinalis L, Styphnolobium japonicum (L.) Schott, Prunus persica (L.) Batsch, Sophora flavescens Aiton, and Eupolyphaga sinensis Walker. Matrine and oxymatrine are two of the major phytochemical constituents of QYJD. Inflammation and oxidative stress are strongly associated with colon carcinogenesis. Colorectal cancer (CRC) is the third most common type of cancer. Therefore, cancer chemopreventive agents targeting CRC are urgently needed. This study was conducted to investigate the potential anticancer effects and the underlying mechanisms of QYJD and its active constituents, matrine and oxymatrine, in human colon cancer HT29 cells and in a dextran sulfate sodium (DSS)-induced colitis mouse model. QYJD and matrine effectively inhibited the proliferation and anchorage-independent growth of HT29 cells in a dose-dependent manner. QYJD and matrine also induced an Nrf2-mediated anti-oxidant response element-luciferase activity and upregulated the Nrf2-mediated anti-oxidative stress genes HO-1 and NQO1 at both the mRNA and protein levels. In the DSS-induced colitis mouse model, QYJD reduced the disease activity index (DAI) and alleviated colonic shortening. Elevated Nrf2 and HO-1 mRNA levels were also observed in QYJD-treated mice. These findings showed that QYJD could elicit anti-inflammatory and anti-oxidative stress response in vitro in a cell line and in vivo in a DSS-induced colitis mouse model. These responses may contribute to the overall anticolon cancer effect of QYJD.

Epigenetic alterations in TRAMP mice: epigenome DNA methylation profiling using MeDIP-seq.

PURPOSE: We investigated the genomic DNA methylation profile of prostate cancer in transgenic adenocarcinoma of the mouse prostate (TRAMP) cancer model and to analyze the crosstalk among targeted genes and the related functional pathways. METHODS: Prostate DNA samples from 24-week-old TRAMP and C57BL/6 male mice were isolated. The DNA methylation profiles were analyzed by methylated DNA immunoprecipitation (MeDIP) followed by next-generation sequencing (MeDIP-seq). Canonical pathways, diseases and function and network analyses of the different samples were then performed using the Ingenuity® Pathway Analysis (IPA) software. Some target genes with significant difference in methylation were selected for validation using methylation specific primers (MSP) and qPCR. RESULTS: TRAMP mice undergo extensive aberrant CpG hyper- and hypo-methylation. There were 2147 genes with a significant (log2-change ≥ 2) change in CpG methylation between the two groups, as mapped by the IPA software. Among these genes, the methylation of 1105 and 1042 genes was significantly decreased and increased, respectively, in TRAMP prostate tumors. The top associated disease identified by IPA was adenocarcinoma; however, the cAMP response element-binding protein (CREB)-, histone deacetylase 2 (HDAC2)-, glutathione S-transferase pi (GSTP1)- and polyubiquitin-C (UBC)-related pathways showed significantly altered methylation profiles based on the canonical pathway and network analyses. MSP and qPCR results of genes of interests corroborated with MeDIP-seq findings. CONCLUSIONS: This is the first MeDIP-seq with IPA analysis of the TRAMP model to provide novel insight into the genome-wide methylation profile of prostate cancer. Studies on epigenetics, such as DNA methylation, will potentially provide novel avenues and strategies for further development of biomarkers targeted for treatment and prevention approaches for prostate cancer.

Dietary Phytochemicals and Cancer Chemoprevention: A Perspective on Oxidative Stress, Inflammation, and Epigenetics.

Oxidative stress occurs when cellular reactive oxygen species 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. 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, 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 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.

Epigenetics Reactivation of Nrf2 in Prostate TRAMP C1 Cells by Curcumin Analogue FN1.

It has previously been shown that curcumin can effectively inhibit prostate cancer proliferation and progression in TRAMP mice, potentially acting through the hypomethylation of the Nrf2 gene promoter and hence activation of the Nrf2 pathway to enhance cell antioxidative defense. FN1 is a synthetic curcumin analogue that shows stronger anticancer activity than curcumin in other reports. We aimed to explore the epigenetic modification of FN1 that restores Nrf2 expression in TRAMP-C1 cells. Stably transfected HepG2-C8 cells were used to investigate the effect of FN1 on the Nrf2- antioxidant response element (ARE) pathway. Real-time quantitative PCR and Western blotting were applied to study the influence of FN1 on endogenous Nrf2 and its downstream genes. Bisulfite genomic sequencing (BGS) and methylated DNA immunoprecipitation (MeDIP) were then performed to examine the methylation profile of the Nrf2 promoter. An anchorage-independent colony-formation analysis was conducted to examine the tumor inhibition activity of FN1. Epigenetic modification enzymes, including DNMTs and HDACs, were investigated by Western blotting. The luciferase reporter assay indicated that FN1 was more potent than curcumin in activating the Nrf2-ARE pathway. FN1 increased the expression of Nrf2 and its downstream detoxifying enzymes. FN1 significantly inhibited the colony formation of TRAMP-C1 cells. BGS and MeDIP assays revealed that FN1 treatment (250 nM for 3 days) reduced the percentage of CpG methylation of the Nrf2 promoter. FN1 also downregulated epigenetic modification enzymes. In conclusion, our results suggest that FN1 is a novel anticancer agent for prostate cancer. In the TRAMP-C1 cell line, FN1 can increase the level of Nrf2 and downstream genes via activating the Nrf2-ARE pathway and inhibit the colony formation potentially through the decreased expression of keap1 coupled with CpG demethylation of the Nrf2 promoter. This CpG demethylation effect may come from decreased epigenetic modification enzymes, such as DNMT1, DNMT3a, DNMT3b, and HDAC4.

Natural compound-derived epigenetic regulators targeting epigenetic readers, writers and erasers.

Post-translational modifications can affect gene expression in a long-term manner without changes in the primary nucleotide sequence of the DNA. These epigenetic alterations involve dynamic processes that occur in histones, chromatin-associated proteins and DNA. In response to environmental stimuli, abnormal epigenetic alterations cause disorders in the cell cycle, apoptosis and other cellular processes and thus contribute to the incidence of diverse diseases, including cancers. In this review, we will summarize recent studies focusing on certain epigenetic readers, writers, and erasers associated with cancer development and how newly discovered natural compounds and their derivatives could interact with these targets. These advances provide insights into epigenetic alterations in cancers and the potential utility of these alterations as therapeutic targets for the future development of chemopreventive and chemotherapeutic drugs.

Association of aberrant DNA methylation in Apc(min/+) mice with the epithelial-mesenchymal transition and Wnt/ß-catenin pathways: genome-wide analysis using MeDIP-seq.

BACKGROUND: Aberrant DNA methylation at the 5-carbon on cytosine residues (5mC) in CpG dinucleotides is probably the most extensively characterized epigenetic modification in colon cancer. It has been suggested that the loss of adenomatous polyposis coli (APC) function initiates tumorigenesis and that additional genetic and epigenetic events are involved in colon cancer progression. We aimed to study the genome-wide DNA methylation profiles of intestinal tumorigenesis in Apc(min/+) mice. RESULTS: Methylated DNA immunoprecipitation (MeDIP) followed by next-generation sequencing was used to determine the global profile of DNA methylation changes in Apc(min/+) mice. DNA was extracted from adenomatous polyps from Apc(min/+) mice and from normal intestinal tissue from age-matched Apc(+/+) littermates, and the MeDIP-seq assay was performed. Ingenuity Pathway Analysis (IPA) software was used to analyze the data for gene interactions. A total of 17,265 differentially methylated regions (DMRs) displayed a ≥ 2-fold change (log2) in methylation in Apc(min/+) mice; among these DMRs, 9,078 (52.6 %) and 8,187 (47.4 %) exhibited increased and decreased methylation, respectively. Genes with altered methylation patterns were mainly mapped to networks and biological functions associated with cancer and gastrointestinal diseases. Among these networks, several canonical pathways, such as the epithelial-mesenchymal transition (EMT) and Wnt/ß-catenin pathways, were significantly associated with genome-wide methylation changes in polyps from Apc(min/+) mice. The identification of certain differentially methylated molecules in the EMT and Wnt/ß-catenin pathways, such as APC2 (adenomatosis polyposis coli 2), SFRP2 (secreted frizzled-related protein 2), and DKK3 (dickkopf-related protein 3), was consistent with previous publications. CONCLUSIONS: Our findings indicated that Apc(min/+) mice exhibited extensive aberrant DNA methylation that affected certain signaling pathways, such as the EMT and Wnt/ß-catenin pathways. The genome-wide DNA methylation profile of Apc(min/+) mice is informative for future studies investigating epigenetic gene regulation in colon tumorigenesis and the prevention of colon cancer.

Epigenetic DNA methylation of antioxidative stress regulator NRF2 in human prostate cancer.

Epigenetic control of NRF2, a master regulator of many critical antioxidative stress defense genes in human prostate cancer (CaP), is unknown. Our previous animal study found decreased Nrf2 expression through promoter CpG methylation/histone modifications during prostate cancer progression in TRAMP mice. In this study, we evaluated CpG methylation of human NRF2 promoter in 27 clinical prostate cancer samples and in LNCaP cells using MAQMA analysis and bisulfite genomic DNA sequencing. Prostate cancer tissue microarray (TMA) containing normal and prostate cancer tissues was studied by immunohistochemistry. Luciferase reporter assay using specific human NRF2 DNA promoter segments and chromatin immunoprecipitation (ChIP) assay against histone modifying proteins were performed in LNCaP cells. Three specific CpG sites in the NRF2 promoter were found to be hypermethylated in clinical prostate cancer samples (BPH

Nrf2 null enhances UVB-induced skin inflammation and extracellular matrix damages.

Nrf2 plays a critical role in defending against oxidative stress and inflammation. We previously reported that Nrf2 confers protection against ultraviolet-B (UVB)-induced inflammation, sunburn reaction, and is involved in sulforaphane-mediated photo-protective effects in the skin. In this study, we aimed to demonstrate the protective role of Nrf2 against inflammation-mediated extracellular matrix (ECM) damage induced by UVB irradiation. Ear biopsy weights were significantly increased in both Nrf2 wild-type (Nrf2 WT) and knockout (Nrf2 KO) mice one week after UVB irradiation. However, these weights increased more significantly in KO mice compared to WT mice, suggesting a greater inflammatory response in KO mice. In addition, we analyzed the protein expression of numerous markers, including macrophage inflammatory protein-2 (MIP-2), pro-matrix metalloproteinase-9 (MMP-9), and p53. p53, a regulator of DNA repair, was overexpressed in Nrf2 KO mice, indicating that the absence of Nrf2 led to more sustained DNA damage. There was also more substantial ECM degradation and increased inflammation in UVB-irradiated Nrf2 KO mice compared to UVB-irradiated WT mice. Furthermore, the protective effects of Nrf2 in response to UVB irradiation were mediated by increased HO-1 protein expression. Collectively, our results show that Nrf2 plays a key role in protecting against UVB irradiation and that the photo-protective effect of Nrf2 is closely related to the inhibition of ECM degradation and inflammation.

Genome-wide analysis of DNA methylation in UVB- and DMBA/TPA-induced mouse skin cancer models.

AIMS: Ultraviolet irradiation and carcinogens have been reported to induce epigenetic alterations, which potentially contribute to the development of skin cancer. We aimed to study the genome-wide DNA methylation profiles of skin cancers induced by ultraviolet B (UVB) irradiation and 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-1,3-acetate (TPA). MAIN METHODS: Methylated DNA immunoprecipitation (MeDIP) followed by next-generation sequencing was utilized to ascertain the DNA methylation profiles in the following common mouse skin cancer models: SKH-1 mice treated with UVB irradiation and CD-1 mice treated with DMBA/TPA. Ingenuity® Pathway Analysis (IPA) software was utilized to analyze the data and to identify gene interactions among the different pathways. KEY FINDINGS: 6003 genes in the UVB group and 5424 genes in the DMBA/TPA group exhibited a greater than 2-fold change in CpG methylation as mapped by the IPA software. The top canonical pathways identified by IPA after the two treatments were ranked were pathways related to cancer development, cAMP-mediated signaling, G protein-coupled receptor signaling and PTEN signaling associated with UVB treatment, whereas protein kinase A signaling and xenobiotic metabolism signaling were associated with DMBA/TPA treatment. In addition, the mapped IL-6-related inflammatory pathways displayed alterations in the methylation profiles of inflammation-related genes linked to UVB treatment. SIGNIFICANCE: Genes with altered methylation were ranked in the UVB and DMBA/TPA models, and the molecular interaction networks of those genes were identified by the IPA software. The genome-wide DNA methylation profiles of skin cancers induced by UV irradiation or by DMBA/TPA will be useful for future studies on epigenetic gene regulation in skin carcinogenesis.

The berry constituents quercetin, kaempferol, and pterostilbene synergistically attenuate reactive oxygen species: involvement of the Nrf2-ARE signaling pathway.

Quercetin, kaempferol, and pterostilbene are abundant in berries. The anti-oxidative properties of these constituents may contribute to cancer chemoprevention. However, their precise mechanisms of action and their combinatorial effects are not completely understood. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) regulates anti-oxidative stress enzymes and Phase II drug metabolizing/detoxifying enzymes by binding to antioxidant response element (ARE). This study aimed to investigate the anti-oxidative stress activities of quercetin, kaempferol, and pterostilbene individually and in combination, as well as the involvement of the Nrf2-ARE signaling pathway. Quercetin, kaempferol, and pterostilbene all exhibited strong free-radical scavenging activity in the DPPH assay. The MTS assay revealed that low concentration combinations we tested were relatively non-toxic to HepG2-C8 cells. The results of the DCFH-DA assay and combination index (CI) indicated that quercetin, kaempferol, and pterostilbene attenuated intracellular reactive oxygen species (ROS) levels when pretreated individually and had synergistic effects when used in combination. In addition, the combination treatment significantly induced ARE and increased the mRNA and protein expression of Nrf2-regulated genes. Collectively, our study demonstrated that the berry constituents quercetin, kaempferol, and pterostilbene activated the Nrf2-ARE signaling pathway and exhibited synergistic anti-oxidative stress activity at appropriate concentrations.

Astaxanthin and omega-3 fatty acids individually and in combination protect against oxidative stress via the Nrf2-ARE pathway.

Oxidative stress is a major driver of many diseases, including cancer. The induction of Nrf2-ARE-mediated antioxidant enzymes provides a cellular defense against oxidative stress. Astaxanthin (AST), a red dietary carotenoid, possesses potent antioxidant activity, and inhibits oxidative damages. Polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are important nutritional essentials and potent antioxidants found in fish oil. In the present study, we investigated whether AST in combination with low concentrations of DHA or EPA has a synergistic antioxidant effect in a HepG2-C8-ARE-luciferase cell line system. Using free radical scavenging DPPH assay, AST was more potent DPPH radical scavenger than DHA and EPA. MTS assay revealed that AST was non-toxic up to 100µM compared with more toxic DHA and EPA. The three compounds alone and in combination elevated cellular GSH levels, increased the total antioxidant activity, induced mRNA expression of Nrf2 and Nrf2 downstream target genes NQO1, HO-1, and GSTM2. Lower concentrations of AST show synergistic effects when combined with DHA or EPA. In summary, our study shows synergistic antioxidant effects of AST and PUFAs at low concentrations. The Nrf2/ARE pathway plays an important role in the antioxidative effects induced by AST, DHA, and EPA.

Pharmacodynamics of ginsenosides: antioxidant activities, activation of Nrf2, and potential synergistic effects of combinations.

Ginseng has long been used in Asian countries for more than 2000 years. Currently, in the "Western World or Western Medicines", many reports have indicated that they have used herbal medicines, and ginseng is one of the most popular herbs. Several recent reports have indicated that the antioxidant/antioxidative stress activities of ginseng play a role in the benefits of ginseng; however, the precise mechanism is lacking. The antioxidant response element (ARE) is a critical regulatory element for the expression of many antioxidant enzymes and phase II/III drug metabolizing/transporter genes, mediated by the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). The aim of this study was to examine the potential activation and synergism of Nrf2-ARE-mediated transcriptional activity between three common ginsenosides present in ginseng, ginsenoside Rb1 (Rb1), ginsenoside Rg1 (Rg1), and ginsenoside 20(S)-protopanaxatriol (20S). We tested whether these ginsenosides and their combinations could induce Nrf2-ARE activities in HepG2-C8 cells with stably transfected ARE luciferase reporter gene. Cell proliferation, antioxidant and ARE activities, Western blotting of Nrf2 protein, and qPCR of mRNA of Nrf2 were conducted for Rb1, Rg1, and 20S as well as the combinations of 20S with Rb1 or Rg1. To determine the combination effects, the combination index (CI) was calculated. Rb1 and Rg1 are relatively nontoxic to the cells, while 20S at 50 µM or above significantly inhibited the cell proliferation. Rb1, Rg1, or 20S induced total antioxidant activity and ARE activity in a concentration-dependent manner. Furthermore, combinations of 20S with either Rb1 or Rg1 induced total antioxidant and ARE activity synergistically. The induction of Nrf2 protein and mRNA was also found to be synergistic with the combination treatments. In summary, in this study, we show that ginsenosides Rb1, Rg1, and 20S possess antioxidant activity, transcriptionally activating ARE as well as the potential of synergistic activities. The Nrf2-ARE-mediated antioxidant pathway could play a role for the overall antioxidative stress activities, which could be important for ginseng's health beneficial effects such as cancer chemopreventive activities.