Nrf2 Promoter Region Research Articles

Epigenetics of Dietary Phytochemicals in Cancer Prevention: Fact or Fiction.

Cancer development takes 10 to 50 years, and epigenetics plays an important role. Recent evidence suggests that ~80% of human cancers are linked to environmental factors impinging upon genetics/epigenetics. Because advanced metastasized cancers are resistant to radiation/chemotherapeutic drugs, cancer prevention by relatively nontoxic "epigenetic modifiers" will be logical. Many dietary phytochemicals possess powerful antioxidant and anti-inflammatory properties that are hallmarks of cancer prevention. Dietary phytochemicals can regulate gene expression of the cellular genome via epigenetic mechanisms. In this review, we will summarize preclinical studies that demonstrate epigenetic mechanisms of dietary phytochemicals in skin, colorectal, and prostate cancer prevention. Key examples of the importance of epigenetic regulation in carcinogenesis include hypermethylation of the NRF2 promoter region in cancer cells, resulting in inhibition of NRF2-ARE signaling. Many dietary phytochemicals demethylate NRF2 promoter region and restore NRF2 signaling. Phytochemicals can also inhibit inflammatory responses via hypermethylation of inflammation-relevant genes to block gene expression. Altogether, dietary phytochemicals are excellent candidates for cancer prevention due to their low toxicity, potent antioxidant and anti-inflammatory properties, and powerful epigenetic effects in reversing procarcinogenic events.

Curcuminoid PBPD induces cuproptosis and endoplasmic reticulum stress in cervical cancer via the Notch1/RBP-J/NRF2/FDX1 pathway.

Curcumin has been shown to have antitumor properties, but its low potency and bioavailability has limited its clinical application. We designed a novel curcuminoid, [1-propyl-3,5-bis(2-bromobenzylidene)-4-piperidinone] (PBPD), which has higher antitumor strength and improves bioavailability. Cell counting kit-8 was used to detect cell activity. Transwell assay was used to detect cell invasion and migration ability. Western blot and quantitative polymerase chain reaction were used to detect protein levels and their messenger RNA expression. Immunofluorescence was used to detect the protein location. PBPD significantly inhibited the proliferation of cervical cancer cells, with an IC50 value of 4.16 μM for Hela cells and 3.78 μM for SiHa cells, leading to the induction of cuproptosis. Transcriptome sequencing analysis revealed that PBPD significantly inhibited the Notch1/Recombination Signal Binding Protein for Immunoglobulin kappa J Region (RBP-J) and nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathways while upregulating ferredoxin 1 (FDX1) expression. Knockdown of Notch1 or RBP-J significantly inhibited NRF2 expression and upregulated FDX1 expression, leading to the inhibition of nicotinamide adenine dinucleotide phosphate activity and the induction of oxidative stress, which in turn activated endoplasmic reticulum stress and induced cell death. The overexpression of Notch1 or RBP-J resulted in the enrichment of RBP-J within the NRF2 promoter region, thereby stimulating NRF2 transcription. NRF2 knockdown resulted in increase in FDX1 expression, leading to cuproptosis. In addition, PBPD inhibited the acidification of tumor niche and reduced cell metabolism to inhibit cervical cancer cell invasion and migration. In conclusion, PBPD significantly inhibits the proliferation, invasion, and migration of cervical cancer cells and may be a novel potential drug candidate for treatment of cervical cancer.

Fermented Syzygium samarangense leaves mitigate oxidative stress-triggered hepatotoxicity through Nrf2 activation and epigenetic regulation

The liver, which is a vital organ for metabolism and detoxification, is susceptible to damage caused by oxidative stress. Excessive consumption of acetaminophen (APAP), a commonly used analgesic ingredient, can cause liver injury due to oxidative stress. The aim of this study is to examine the protective effects and mechanisms of fermented Syzygium samarangense (wax apple) leaves (FWAL) against damage caused by APAP in mouse AML-12 hepatocytes. Ethanolic and water extracts of FWAL (FWAL-EE and FWAL-WE) were prepared, and both contained significant phenolic compounds such as gallic acid, EGCG, ellagic acid, myricitrin, and myricetin, with potent DPPH scavenging activity. FWAL-EE demonstrated superior effectiveness in lowering the death ratio, LDH leakage, and oxidative stress in APAP-treated AML-12 cells. The protective effects of FWAL-EE included the downregulation of inflammatory markers TNF-α and cox-2 mRNA, as well as the upregulation of Nrf2, HO-1, and NQO1 proteins in APAP-treated hepatocytes. Additionally, FWAL-EE reduced the expression of epigenetic enzymes HDACs 1, 2, 3, and 4, and DNMTs 1 and 3b while causing an increase in unmethylated DNA levels in the Nrf2 promoter region. Furthermore, EGCG, a prominent active compound in FWAL, protected AML-12 hepatocytes from APAP-induced damage by activating Nrf2 and NQO1, while also inhibiting epigenetic DNMTs and HDACs. These results suggested that FWAL-EE may effectively prevent APAP-induced liver damage by enhancing the activation of the Nrf2 antioxidant system through epigenetic regulation. FWAL-EE has potential as a nutraceutical for liver protection.

Histone deacetylase 3 inhibition alleviates 2,4-dinitrochlorobenzene-induced atopic dermatitis via epigenetically upregulating Nrf2/HO-1 signaling pathway

Atopic dermatitis (AD) is a frequent skin disorder that is associated with immune dysfunction and skin inflammation. Histone deacetylase 3 (HDAC3) possesses strong immune and inflammatory modulatory properties in multiple diseases. However, the role and mechanism of HDAC3 in AD remain unknown. Here, we reported that HDAC3 expression was aberrantly upregulated in 2,4-dinitrochlorobenzene (DNCB)-induced lesional AD skin in mice. Inhibition of HDAC3 by RGFP966 protected against DNCB-induced AD, indicated by improved histological damages, relieved inflammatory and immune dysfunction. Nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) signaling pathway activity in lesional AD skin was significantly decreased and RGFP966 attenuated the decrease. Inhibition of Nrf2/HO-1 signaling pathway via Nrf2 inhibitor ML385 blunted anti-AD effect of RGFP966 in DNCB-treated mice. Mechanistically, RGFP966 promoted Nrf2 expression and upregulated H3K27ac deposition on the promoter region of Nrf2. Collectively, HDAC3 inhibition protects against AD via epigenetically activating Nrf2 transcription to upregulate Nrf2/HO-1 signaling pathway activity. HDAC3 may act as a promising therapeutic target for the treatment of AD.

ETS1 Ameliorates Hyperoxia-Induced Bronchopulmonary Dysplasia in Mice by Activating Nrf2/HO-1 Mediated Ferroptosis.

Bronchopulmonary dysplasia (BPD) is associated with hyperoxia-induced oxidative stress-associated ferroptosis. This study examined the effect of E26 oncogene homolog 1 (ETS1) on oxidative stress-associated ferroptosis in BPD. Hyperoxia-induced A549 cells and neonatal mice were used to establish BPD models. The effects of ETS1 on hyperoxia-induced ferroptosis-like changes in A549 cells were investigated by overexpression of ETS1 plasmid transfection and erastin treatment. Glucose consumption, lactate production, and NADPH levels were assessed by the glucose, lactate, and NADP+/NADPH assay kits, respectively. The potential regulatory relationship between ETS1 and Nrf2/HO-1 was examined by treating hyperoxia-induced A549 cells with the Nrf2 inhibitor ML385. ETS1 effect on the Nrf2 promoter was explored by dual-luciferase reporter and chromatin immunoprecipitation assay. The effect of ETS1 on the symptoms of BPD mice was examined by injecting an adenovirus overexpressing ETS1. ETS1 overexpression increased hyperoxia-induced cell viability, glucose consumption, lactate production, and NADPH levels and reduced inflammation and apoptosis in A549 cells. In animal experiments, ETS1 overexpression prevented weight loss, airway enlargement, and reductions in radial alveolar counts in BPD mice, while reducing the mean linear intercept, mean alveolar diameter and inflammation. ETS1 overexpression suppressed PTGS2 and CHAC1 expression, reduced ROS, MDA and ferrous iron (Fe2+) production and increased GSH levels in hyperoxia-induced A549 cells and BPD mice. In addition, ETS1 can bind to the Nrf2 promoter region and thus promote Nrf2 transcription. ETS1 overexpression increased the mRNA and protein levels of Nrf2, HO-1, xCT, and GPX4 in hyperoxia-induced A549 cells and BPD mice. In hyperoxia-induced A549 cells, erastin and ML385 treatment abolished the effect of ETS1 overexpression. ETS1 is important in oxidative stress-related ferroptosis in a hyperoxia-induced BPD model, and the effect is partially mediated by the Nrf2/HO-1 axis.

Histone lysine methyltransferase SETDB2 suppresses NRF2 to restrict tumor progression and modulates chemotherapy sensitivity in lung adenocarcinoma.

Aberrant epigenetic remodeling represents a molecular hallmark in lung adenocarcinoma (LUAD). We aim to investigate the biological roles of SETDB2 and its underlying associations with oxidative stress, providing therapeutic targets for individualized treatment of LUAD. Differential analysis was conducted via Limma package, and Kaplan-Meier analysis was performed with survival package. CCK-8, cell proliferation assay, transwell assay, and in vivo assays were conducted to assess the function of SETDB2. Western blot assay, RT-qPCR, and immunohistochemistry (IHC) were conducted to assess the expression levels of SETDB2/NRF2. Chromatin immunoprecipitation (ChIP) assay and ChIP-qPCR were conducted to assess the epigenetic roles of SETDB2. We found that SETDB2 expression is decreased in tumor samples versus normal tissues in TCGA-LUAD cohort, LUAD-EAS cohort, GSE72094 dataset, and independent Soochow-LUAD dataset. Patients with low SETDB2 levels had a worse prognosis relative to those with high SETDB2. SETDB2 inhibition could significantly promote cell growth, migration ability, and stemness maintenance. Gene set enrichment analysis (GSEA) suggested that SETDB2 correlated with oxidative stress crosstalk and regulated NRF2 mRNA levels. ChIP assay suggested that SETDB2 mainly recruited the H3K9me3 enrichment at the NRF2 promoter region to suppress the mRNA levels of NRF2. Downregulated SETDB2 could activate NRF2 transcription and expression, thereby promoting its downstream targets, like NQO1, FTH1, and ME1. Functional experiments demonstrated that low SETDB2 allowed NRF2 to drive malignant processes of LUAD. SETDB2 overexpression attenuated the ability of NRF2 signaling to neutralize cellular reactive oxygen species (ROS) levels, leading to enhanced cell apoptosis. Overexpressed SETDB2 could inhibit tumor progression in vivo and further render LUAD cells sensitive to chemotherapy. In conclusion, these findings uncovered the suppressive role of SETDB2 in LUAD. SETDB2 negatively regulates NRF2 signaling to modulate tumor progression, which creates a therapeutic vulnerability in LUAD.

Sulforaphane inhibits angiotensin II-induced cardiomyocyte apoptosis by acetylation modification of Nrf2.

Oxidative stress is the central cause of angiotensin II (Ang II)-induced myocardial injury, and nuclear factor erythroid 2-related factor (Nrf2) is the core molecule of the anti-oxidant defense system. We have previously demonstrated that sulforaphane (SFN) can prevent Ang II-induced myocardial injury by activating Nrf2; however, the underlying molecular mechanism is still unclear. This study aimed to evaluate whether SFN prevents Ang II-induced cardiomyocyte apoptosis through acetylation modification of Nrf2. Wild-type and Nrf2 knockdown embryonic rat cardiomyocytes (H9C2) were exposed to Ang II to induce apoptosis, oxidative stress, and inflammatory responses. SFN treatment significantly reduced Ang II-induced cardiomyocyte apoptosis, inflammation and oxidative stress. Activation of Nrf2 played a critical role in preventing cardiomyocyte apoptosis. After Nrf2 was knockdown, the anti-inflammatory, antioxidant stress of SFN were eliminated. Furthermore, Nrf2 activation by SFN was closely related to the decreased activity of histone deacetylases (HDACs) and increased histone-3 (H3) acetylation levels in Nrf2 promoter region. These findings confirm that Nrf2 plays a key role in SFN preventing Ang II-induced cardiomyocyte apoptosis. SFN activates Nrf2 by inhibiting HDACs expression and activation.

Sulforaphane prevents angiotensin II-induced cardiomyopathy by activation of Nrf2 through epigenetic modification.

Nuclear factor erythroid 2‐related factor (Nrf2) is an important regulator of cellular antioxidant defence. We previously showed that SFN prevented Ang II‐induced cardiac damage via activation of Nrf2. However, the underlying mechanism of SFN’s persistent cardiac protection remains unclear. This study aimed to explore the potential of SFN in activating cardiac Nrf2 through epigenetic mechanisms. Wild‐type mice were injected subcutaneously with Ang II, with or without SFN. Administration of chronic Ang II‐induced cardiac inflammatory factor expression, oxidative damage, fibrosis and cardiac remodelling and dysfunction, all of which were effectively improved by SFN treatment, coupled with an up‐regulation of Nrf2 and downstream genes. Bisulfite genome sequencing and chromatin immunoprecipitation (ChIP) were performed to detect the methylation level of the first 15 CpGs and histone H3 acetylation (Ac‐H3) status in the Nrf2 promoter region, respectively. The results showed that SFN reduced Ang II‐induced CpG hypermethylation and promoted Ac‐H3 accumulation in the Nrf2 promoter region, accompanied by the inhibition of global DNMT and HDAC activity, and a decreased protein expression of key DNMT and HDAC enzymes. Taken together, SFN exerts its cardioprotective effect through epigenetic modification of Nrf2, which may partially contribute to long‐term activation of cardiac Nrf2.

Sulforaphane Prevents Angiotensin II-Induced Cardiomyopathy by Activation of Nrf2 Through Epigenetic Modification

Nuclear factor erythroid 2-related factor (Nrf2) is an important regulator of cellular antioxidant defense. We previously showed that SFN prevented Ang II-induced cardiac damage via activation of Nrf2. Furthermore, SFN treatment resulted in persistent activation of Nrf2 for at least another 3 months after withdrawing SFN. However, the underlying mechanism of SFN’s persistent cardiac protection remains unclear. This study aimed to explore the potential of SFN in activating cardiac Nrf2 through epigenetic mechanisms. Wild-type mice were injected subcutaneously with Ang II (0.5 mg/kg) every other day for 2 months, with or without SFN (0.5 mg/kg) treatment for 5 days each week for 3 months, and then kept for another 3 month. Administration of a subpressor dose of Ang II induced cardiac inflammatory factor expression, oxidative damage, fibrosis, and cardiac remodeling and dysfunction, all of which were effectively improved by SFN treatment, coupled with an upregulation of Nrf2 and downstream genes (CAT, HO-1). Bisulfite genome sequencing (BGS) and chromatin immunoprecipitation (ChIP) were then performed to detect the methylation level of the first 15 CpGs and histone H3 acetylation (Ac-H3) status in the Nrf2 promoter region, respectively. The results showed that SFN reduced Ang II-induced CpG hypermethylation and promoted Ac-H3 accumulation in the Nrf2 promoter region, accompanied by the inhibition of global DNMT and HDAC activity, and a decreased protein expression of key DNMT and HDAC enzymes: DNMT3a, DNMT3b and HDAC2, HDAC3, HDAC5. Taken together, SFN exerts its cardioprotective effect through epigenetic modification of Nrf2, which may partially contribute to long-term activation of cardiac Nrf2. Funding Statement: This work was supported by the National Natural Science Foundation of China (Grant number 395 81570344); National Key RD the Education Department Foundation of Jilin Province (Grant number JJKH20201036KJ); the Fundamental Research Funds for the Central Universities of Jilin University; the Health and Family Planning Commission of Jilin Province Foundations (Grant number 2016Q034 and 2017J11) and the Jilin Provincial Science and Technology Foundations (Grant number 20180414039GH and 20190201200JC). Declaration of Interests: The authors report no conflicts of interest in this work. Ethics Approval Statement: All animal procedures were approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences (Beijing, China).

Genetic Polymorphism of the Nrf2 Promoter Region (rs35652124) Is Associated with the Risk of Diabetic Foot Ulcers.

The genetic polymorphism in the nuclear factor erythroid 2-related factor 2 (Nrf2) gene has been reported as one of the prognosis markers for various diseases, including cancer. Nrf2 is a key transcription factor involved in wound healing by regulating angiogenesis. We investigated the genetic association of NRF2 single-nucleotide polymorphism rs35652124 with T2DM and DFU and assessed its functional impact. A total of 400 subjects were recruited for the study and categorized into three groups: infected DFU patients (DFU, n = 100), T2DM patients without complications (T2DM, n = 150), and healthy adults with normal glucose tolerance (NGT, n = 150). The subjects were genotyped by PCR-RFLP, and the polymorphism was identified by bidirectional Sanger sequencing. The expression of NRF2, IL-10, TNF-α, and IL-6 was studied by qPCR to evaluate the functional impact of rs35652124. The “TT” genotype of rs35652124 was associated with a significant risk for T2DM [OR = 2.2 (1.2-4.2), p = 0.01] and DFU [OR = 7.9 (4-14.9), p < 0.0001]. A significant decrease in transcriptional levels of NRF2 and IL-10 and a remarkable increase in TNF-α and IL-6 were observed in subjects with TT genotype. In conclusion, rs35652124 (TT) is a harmful genetic variant that predisposes to insulin resistance and impaired angiogenesis. Hence, it may serve as a diagnostic genetic marker for T2DM and DFU in combination with different inflammatory markers.

Demethylation of the NRF2 Promoter Protects Against Carcinogenesis Induced by Nano-SiO2

Nano silicon dioxide (Nano-SiO2) has been widely used in industries such as the field of biomedical engineering. Despite the existing evidence that Nano-SiO2 exposure could induce oxidative stress and inflammatory responses in multiple organ systems, the carcinogenicity of Nano-SiO2 exposure has rarely been investigated. Thus in this study, two types of human bronchial epithelial cell lines (16HBE and BEAS-2B) were selected as in vitro models to investigate the carcinogenicity of Nano-SiO2. Our results revealed that Nano-SiO2 induces a malignant cellular transformation in human bronchial epithelial cells according to the soft agar colony formation assay. The carcinogenesis induced by Nano-SiO2 was also confirmed in nude mice. By using immunofluorescence assay and high-performance capillary electrophoresis (HPCE), we observed a genome-wide DNA hypomethylation induced by Nano-SiO2. Besides the reduced enzyme activity of total DNMTs upon Nano-SiO2 treatment, altered expression of DNMTs and methyl-CpG binding proteins were observed. Besides, we found that the expression of NRF2 was activated by demethylation of CpG islands within the NRF2 promoter region and the overexpression of NRF2 could alleviate the carcinogenesis induced by Nano-SiO2. Taken together, our results suggested that Nano-SiO2 induces malignant cellular transformation with a global DNA hypomethylation, and the demethylation of NRF2 promoter activates the expression of NRF2, which plays an important role in protecting against the carcinogenesis induced by Nano-SiO2.

7-Hydroxycoumarin Attenuates Colistin-Induced Kidney Injury in Mice Through the Decreased Level of Histone Deacetylase 1 and the Activation of Nrf2 Signaling Pathway.

Colistin has been considered as the last line of defense against Gram-negative bacterial infections, however, the potential nephrotoxicity limited its clinical use. 7-Hydroxycoumarin (7-HC) possesses many beneficial pharmacological activities. This study aimed to investigate the nephroprotective effects of 7-HC against colistin-induced kidney injury. In vivo experiments showed that 7-HC alleviated kidney injury induced by colistin, as indicated by lower levels of serum neutrophil gelatinase-associated lipocalin, blood urea nitrogen and creatinine levels. Both in vivo and in vitro results demonstrated that 7-HC alleviated oxidative stress and apoptosis induced by colistin, as shown by decreased malondialdehyde levels, decreased caspase-3 and caspase-9 activities, and increased superoxide dismutase and catalase activities. We also found that colistin significantly induced histone deacetylase (HDAC) 1 expression that deacetylated histone 3 at Lys27 acetylation (H3K27AC) of Nrf2 promoter region and hence inhibiting Nrf2 signaling. 7-HC treatment restored histone acetylation at the Nrf2 promoter region and hence promoted Nrf2 expression. These results suggested that 7-HC alleviates colistin-induced renal injury and this effect was achieved by enhancement of renal antioxidant capacity with the decreased level of HDAC1 and the activation of Nrf2 signaling pathway.

Evaluation of the Epigenetic Demethylation of NRF2, a Master Transcription Factor for Antioxidant Enzymes, in Colorectal Cancer

Epigenetic changes in CpG islands of the promoter regions of homeostasis-related genes, including nuclear factor erythroid 2-related factor 2 (NRF2), have been shown to hold a significant role in the development of colorectal cancer. Therefore, we aimed to examine the DNA demethylation pattern of the NRF2 promoter region in cancerous lesions from patients with colorectal cancer and the association of methylation status with clinicopathological features in the Iranian population. In this cross-sectional study, 114 colorectal tissue samples were collected. These samples included: 34 tumour tissue samples, 60 precancerous polyps, and 20 normal tissue samples. The promoter methylation status of the NRF2 gene was examined using methylation-specific PCR. Additionally, the relationship between the methylation status and the clinicopathological features was investigated. The frequency of NRF2 demethylation in the tumour samples was significantly higher compared to the polyp tissues (p= 0.003) and normal tissue (p= 0.009), indicating that cancerous colorectal tissues exhibit increased demethylation of the NRF2 promoter. After examining the demethylation status of tissue samples, the clinicopathological features were compared to the demethylation results. No significant association was found between NRF2 promoter demethylation and the clinicopathological features of patient samples. Our findings suggest that the epigenetic modifications leading to NRF2 demethylation found in colorectal tumour samples may contribute to cancer progression from precancerous polyps to cancerous lesions.

Nrf2 gene mutation and single nucleotide polymorphism rs6721961 of the Nrf2 promoter region in renal cell cancer

BackgroundNuclear factor erythroid 2–related factor 2 (Nrf2) is involved in cell proliferation by promotion of metabolic activity. It is also the major regulator of antioxidants and has a pivotal role in tumor cell proliferation and resistance to chemotherapy. Accordingly, we investigated the role of Nrf2 in renal cell carcinoma (RCC).MethodsIn 50 patients who had metastatic RCC and received cytoreductive nephrectomy, we performed Nrf2 gene mutation analysis using targeted next-generation sequencing, as well as investigating a specific single nucleotide polymorphism (SNP; rs6721961) in the Nrf2 promoter region and Nrf2 protein expression.ResultsTargeted next-generation sequencing revealed that five tumors had SNPs of Nrf2 associated with amino acid sequence variation, while 11 tumors had SNPs of Kelch-like ECH-associated protein 1 gene, 35 had SNPs of von Hippel-Lindau gene, and none had SNPs of fumarate hydratase gene. The three genotypes of rs6721961 showed the following frequencies: 60% for C/C, 34% for C/A, and 6% for A/A. Nrf2 mutation and the C/A or A/A genotypes were significantly associated with increased Nrf2 protein expression (p = 0.0184 and p = 0.0005, respectively). When the primary tumor showed Nrf2 gene mutation, the C/A or A/A genotype, or elevated Nrf2 protein expression, the response of metastases to vascular endothelial growth factor-targeting therapy was significantly worse (p = 0.0142, p = 0.0018, and p < 0.0001, respectively), and overall survival was significantly reduced (p = 0.0343, p = 0.0421, and p < 0.0001, respectively). Elevated Nrf2 protein expression was also associated with shorter survival according to multivariate Cox proportional analysis.ConclusionThese findings suggest an associated between progression of RCC and Nrf2 signaling.

Sulforaphane-induced epigenetic regulation of Nrf2 expression by DNA methyltransferase in human Caco-2 cells.

The present study aimed to investigate the mechanism underlying sulforaphane-mediated epigenetic regulation of nuclear factor-erythroid derived 2-like 2 (Nrf2) expression in human colon cancer. Proteins were extracted from normal Caco-2 cells using sulforaphane and 5-aza-2'-deoxycytidine (5-Aza) combined with trichostatin A (TSA). The mRNA and protein expression levels and activity of DNA methyltransferase 1 (DNMT1) were determined. Methylation-specific polymerase chain reaction and bisulfite genomic sequencing were also used to measure the methylation levels of CpG sites in the Nrf2 promoter region. Nrf2 expression was measured using reverse transcription-quantitative PCR and western blot analysis. The results demonstrated that sulforaphane did not affect DNMT1 mRNA expression levels. DNMT1 protein expression was inhibited by sulforaphane and 5-Aza co-treatment with TSA. Nrf2 promoter methylation decreased significantly in the sulforaphane group compared with the control group. Nrf2 promoter methylation level in the 5-Aza+TSA group was the lowest among all groups. Nrf2 mRNA levels exhibited significant differences between the sulforaphane-treated and control groups, as well as between the 5-Aza+TSA and control groups, and the sulforaphane-treated and 5-Aza+TSA groups. Nrf2 protein expression was also inhibited by sulforaphane, as well as 5-Aza co-treatment with TSA. The results revealed that sulforaphane may promote demethylation of the Nrf2 promoter region to increase activation of Nrf2, which induces chemoprevention of colon cancer.

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.

Pelargonidin reduces the TPA induced transformation of mouse epidermal cells –potential involvement of Nrf2 promoter demethylation

Pelargonidin, a well-known natural anthocyanidin found in berries strawberries, blueberries, red radishes and other natural foods, has been found to possess health beneficial effects including anti-cancer effect. Herein, we investigated the effect of pelargonidin on cellular transformation in mouse skin epidermal JB6 (JB6 P+) cells induced by tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Pelargonidin treatment significantly decreased colony formation and suppressed cell viability of JB6 P+ cells. Pelargonidin also induced the anti-oxidant response element (ARE)-luciferase activation in HepG2-C8 cells overexpressing the ARE-luciferase reporter. Knockdown of nuclear factor E2-related factor 2 (Nrf2) in shNrf2 JB6 P+ cells enhanced TPA-induced colony formation and attenuated pelargonidin's blocking effect. Pelargonidin reduced the protein levels of genes encoding methyltransferases (DNMTs) and histone deacetylases (HDACs). Importantly, pelargonidin decreased the DNA methylation in the Nrf2 promoter region of JB6 P+ cells and increased Nrf2 downstream target genes expression, such as NAD(P)H/quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1), involved in cellular protection. In summary, our results showed that pelargonidin blocks TPA-induced cell transformation. The possible molecular mechanisms of its potential anti-cancer effects against neoplastic transformation may be attributed to its activation of Nrf2-ARE signaling pathway and its cytoprotective effect.

Luteolin promotes apoptotic cell death via upregulation of Nrf2 expression by DNA demethylase and the interaction of Nrf2 with p53 in human colon cancer cells

Luteolin, a dietary flavone, modulates various signaling pathways involved in carcinogenesis. In this study, we investigated the molecular mechanism that underlies the apoptotic effects of luteolin mediated by DNA demethylation of the nuclear factor erythroid 2-related factor 2 (Nrf2) promoter and the interaction of Nrf2 and p53, a tumor suppressor, in human colon cancer cells. Luteolin increased the expression of apoptosis-related proteins and antioxidant enzymes. In DNA methylation, luteolin inhibited the expression of DNA methyltransferases, a transcription repressor, and increased the expression and activity of ten-eleven translocation (TET) DNA demethylases, a transcription activator. Methyl-specific polymerase chain reaction and bisulfite genomic sequencing indicated that luteolin decreased the methylation of the Nrf2 promoter region, which corresponded to the increased mRNA expression of Nrf2. In addition, luteolin increased TET1 binding to the Nrf2 promoter, as determined using a chromatin immunoprecipitation (ChIP) assay. TET1 knockdown decreased the percentages of luteolin-treated cells in sub-G1 phase and cells with fragmented nuclei. Furthermore, complex formation between p53 and Nrf2 was involved in the apoptotic effects of luteolin. These results provide insight into the mechanism that underlies the anticancer effects of luteolin on colon cancer, which involve the upregulation of Nrf2 and its interaction with the tumor suppressor.

The dietary flavone luteolin epigenetically activates the Nrf2 pathway and blocks cell transformation in human colorectal cancer HCT116 cells.

Colorectal cancer remains a leading malignancy in humans. The importance of epigenetic modification in the development of this disease is now being recognized. The reversible and dynamic nature of epigenetic modifications provides a promising strategy in colorectal cancer chemoprevention and treatment. Luteolin (LUT), a flavone dietary phytochemical, can modulate various signaling pathways involved in carcinogenesis. Many studies have demonstrated that LUT inhibits colorectal carcinogenesis by activating the Nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant-responsive element (ARE) pathway. However, the potential epigenetic mechanism underlying Nrf2/ARE pathway activation remains unclear. In this study, we aimed to explore the anticancer potential of LUT in human colon cancer cells and the epigenetic regulation of the Nrf2/ARE pathway. Specifically, our data showed that LUT suppressed cell proliferation and cellular transformation of HCT116 and HT29 cells in a dose-dependent manner. Additionally, quantitative real-time polymerase chain reaction and Western blot analysis were performed to determine the mRNA and protein expression of Nrf2 and its downstream genes after LUT treatment. Bisulfite genomic sequencing revealed that methylation of the Nrf2 promoter region was decreased by LUT, corresponding with the increased mRNA expression of Nrf2. Decreased protein levels and enzyme activities of epigenetic modifying enzymes, such as DNA methyltransferases (DNMTs) and histone deacetylases (HDACs), were also observed in LUT-treated HCT116 cells. In summary, our findings suggest that LUT may exert its antitumor activity in part via epigenetic modifications of the Nrf2 gene with subsequent induction of its downstream antioxidative stress pathway.

Fucoxanthin Elicits Epigenetic Modifications, Nrf2 Activation and Blocking Transformation in Mouse Skin JB6 P+ Cells.

Nuclear factor erythroid-2-related factor-2 (Nrf2 or NFE2L2) is a master regulator of the anti-oxidative stress response, which is involved in the defense against many oxidative stress/inflammation-mediated diseases, including anticancer effects elicited by an increasing number of natural products. Our previous studies showed that the epigenetic modification of the Nrf2 gene plays a key role in restoring the expression of Nrf2. In this study, we aimed to investigate the epigenetic regulation of Nrf2 by astaxanthin (AST) and fucoxanthin (FX), carotenoids which are abundant in microalgae and seaweeds, in mouse skin epidermal JB6 P+ cells. FX induced the anti-oxidant response element (ARE)-luciferase and upregulated the mRNA and protein levels of Nrf2 and Nrf2 downstream genes in HepG2-C8 cells overexpressing the ARE-luciferase reporter. Both FX and AST decreased colony formation in 12-Otetradecanoylphorbol-13-acetate (TPA)-induced transformation of JB6 P+ cells. FX decreased the methylation of the Nrf2 promoter region in the JB6 P+ cells by the bisulfite conversion and pyrosequencing. Both FX and AST significantly reduced DNA methyltransferase (DNMT) activity but did not affect histone deacetylase (HDAC) activity in JB6 P+ cells. In summary, our results show that FX activates the Nrf2 signaling pathway, induces the epigenetic demethylation of CpG sites in Nrf2 and blocks the TPA-induced transformation of JB6 P+ cells, indicating the potential health-promoting effects of FX in skin cancer prevention.