Tien-Yuan Wu

Sulforaphane suppressed LPS-induced inflammation in mouse peritoneal macrophages through Nrf2 dependent pathway

Sulforaphane (SFN) is a natural isothiocyanate that is present in cruciferous vegetables such as broccoli and cabbage. Previous studies have shown that SFN is effective in preventing carcinogenesis induced by carcinogens in rodents, which is related in part to its potent anti-inflammation properties. In the present study, we compared the anti-inflammatory effect of SFN on LPS-stimulated inflammation in primary peritoneal macrophages derived from Nrf2 (+/+) and Nrf2 (-/-) mice. Pretreatment of SFN in Nrf2 (+/+) primary peritoneal macrophages potently inhibited LPS-stimulated mRNA expression, protein expression and production of TNF-alpha, IL-1beta, COX-2 and iNOS. HO-1 expression was significantly augmented in LPS-stimulated Nrf2 (+/+) primary peritoneal macrophages by SFN. Interestingly, the anti-inflammatory effect was attenuated in Nrf2 (-/-) primary peritoneal macrophages. We concluded that SFN exerts its anti-inflammatory activity mainly via activation of Nrf2 in mouse peritoneal macrophages.

Pharmacodynamics of curcumin as DNA hypomethylation agent in restoring the expression of Nrf2 via promoter CpGs demethylation

Prostate cancer (PCa) is one of the most deadly malignancies among men in the United States. Although localized prostate cancer can be effectively treated via surgery or radiation, metastatic disease is usually lethal. Recent evidence suggests that the development and progression of human prostate cancer involves complex interplay between epigenetic alterations and genetic defects. We have recently demonstrated that Nrf2, a master regulator of cellular antioxidant defense systems, was epigenetically silenced during the progression of prostate tumorigenesis in TRAMP mice. The aim of this study is to investigate the potential of curcumin (CUR), a dietary compound that we have reported to be able to prevent the development of prostate cancer in TRAMP mice, as a DNA hypomethylation agent. Using bisulfite genomic sequencing (BGS), treatment of TRAMP C1 cells we showed that CUR reversed the methylation status of the first 5 CpGs in the promoter region of the Nrf2 gene. Methylation DNA immunoprecipitation (MeDIP) analysis revealed that CUR significantly reduced the anti-mecyt antibody binding to the first 5 CpGs of the Nrf2 promoter, corroborated the BGS results. Demethylation of Nrf2 was found to be associated with the re-expression of Nrf2 and one of its downstream target gene, NQO-1, one of the major anti-oxidative stress enzymes, both at the mRNA and protein levels. Taken together, our current study suggests that CUR can elicit its prostate cancer chemopreventive effect, potentially at least in part, through epigenetic modification of the Nrf2 gene with its subsequent induction of the Nrf2-mediated anti-oxidative stress cellular defense pathway.

Epigenetic CpG Demethylation of the Promoter and Reactivation of the Expression of Neurog1 by Curcumin in Prostate LNCaP Cells

ABSTRACTCurcumin (CUR), a major bioactive polyphenolic component from turmeric curry, Curcuma longa, has been shown to be a potent anti-cancer phytochemical with well-established anti-inflammatory and anti-oxidative stress effects. Chromatin remodeling-related epigenetic regulation has emerged as an important mechanism of carcinogenesis, chemoprevention, and chemotherapy. CUR has been found to inhibit histone acetyltransferase activity, and it was also postulated to be a potential DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitor. In this study, we show that when human prostate LNCaP cells were treated with CUR, it led to demethylation of the first 14 CpG sites of the CpG island of the Neurog1 gene and restored the expression of this cancer-related CpG-methylation epigenome marker gene. At the protein level, CUR treatment had limited effects on the expression of epigenetic modifying proteins MBD2, MeCP2, DNMT1, and DNMT3a. Using ChIP assay, CUR decreased MeCP2 binding to the promoter of Neurog1 dramatically. CUR treatment showed different effects on the protein expression of HDACs, increasing the expression of HDAC1, 4, 5, and 8 but decreasing HDAC3. However, the total HDAC activity was decreased upon CUR treatment. Further analysis of the tri-methylation of histone 3 at lysine 27 (H3K27me3) showed that CUR decreased the enrichment of H3K27me3 at the Neurog1 promoter region as well as at the global level. Taken together, our present study provides evidence on the CpG demethylation ability of CUR on Neurog1 while activating its expression, suggesting a potential epigenetic modifying role for this phytochemical compound in human prostate cancer cells.

Anti-inflammatory/Anti-oxidative Stress Activities and Differential Regulation of Nrf2-Mediated Genes by Non-Polar Fractions of Tea Chrysanthemum zawadskii and Licorice Glycyrrhiza uralensis

Accumulating evidence from epidemiological studies indicates that chronic inflammation and oxidative stress play critical roles in neoplastic development. The aim of this study was to investigate the anti-inflammatory, anti-oxidative stress activities, and differential regulation of Nrf2-mediated genes by tea Chrysanthemum zawadskii (CZ) and licorice Glycyrrhiza uralensis (LE) extracts. The anti-inflammatory and anti-oxidative stress activities of hexane/ethanol extracts of CZ and LE were investigated using in vitro and in vivo approaches, including quantitative real-time PCR (qPCR) and microarray. Additionally, the role of the transcriptional factor Nrf2 (nuclear erythroid-related factor 2) signaling pathways was examined. Our results show that CZ and LE extracts exhibited potent anti-inflammatory activities by suppressing the mRNA and protein expression levels of pro-inflammatory biomarkers IL-1β, IL-6, COX-2 and iNOS in LPS-stimulated murine RAW 264.7 macrophage cells. CZ and LE also significantly suppressed the NO production of LPS-stimulated RAW 264.7 cells. Additionally, CZ and LE suppressed the NF-κB luciferase activity in human HT-29 colon cancer cells. Both extracts also showed strong Nrf2-mediated antioxidant/Phase II detoxifying enzymes induction. CZ and LE induced NQO1, Nrf2, and UGT and antioxidant response element (ARE)-luciferase activity in human hepatoma HepG2 C8 cells. Using Nrf2 knockout [Nrf2 (−/−)] and Nrf2 wild-type (+/+) mice, LE and CZ showed Nrf2-dependent transactivation of Nrf2-mediated antioxidant and phase II detoxifying genes. In summary, CZ and LE possess strong inhibitory effects against NF-κB-mediated inflammatory as well as strong activation of the Nrf2-ARE-anti-oxidative stress signaling pathways, which would contribute to their overall health promoting pharmacological effects against diseases including cancer.

Role of Nrf2 in Suppressing LPS-Induced Inflammation in Mouse Peritoneal Macrophages by Polyunsaturated Fatty Acids Docosahexaenoic Acid and Eicosapentaenoic Acid

This study is to investigate the role of Nrf2 in suppressing LPS-mediated inflammation in ex vivo macrophages by polyunsaturated fatty acids (PUFA) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Primary peritoneal macrophages from Nrf2 wild-type (+/+; WT) and Nrf2 knockout (-/-; KO) mice were treated with lipopolysaccharides (LPS) in the presence or absence of DHA or EPA. Quantitative real-time PCR (qPCR) analyses showed that LPS potently induced cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) in the macrophages collected from Nrf2 (+/+) wild-type mice. DHA and EPA inhibited LPS-induced COX-2, iNOS, IL-1β, IL-6, or TNF-α, but increased hemeoxygenase (HO-1) expression. DHA was found to be more potent than EPA in inhibiting COX-2, iNOS, IL-1β, IL-6, and TNF-α mRNA expression. DHA and EPA were also found to induce HO-1 and Nrf2 mRNA with a different dose-response. LPS induced COX-2, iNOS, IL-1β, IL-6, and TNF-α in the macrophages collected from Nrf2 (-/-) mice as well, however, DHA and EPA suppression of COX-2, iNOS, IL-1β, IL-6, and TNF-α was attenuated as compared to that in Nrf2 (+/+) macrophages. Taken together, using Western blotting, ELISA and qPCR approaches coupled with Nrf2 (-/-) mice, our study clearly shows for the first time that DHA/EPA would induce Nrf2 signaling pathway and that Nrf2 plays a role in DHA/EPA suppression of LPS-induced inflammation.

Pharmacodynamics of dietary phytochemical indoles I3C and DIM: Induction of Nrf2-mediated phase II drug metabolizing and antioxidant genes and synergism with isothiocyanates

The antioxidant response element (ARE) is a critical regulatory element for the expression of many phase II drug metabolizing enzymes (DME), phase III transporters and antioxidant enzymes, mediated by the transcription factor Nrf2. The aim of this study was to examine the potential activation and synergism of Nrf2‐ARE‐mediated transcriptional activity between four common phytochemicals present in cruciferous vegetables; the indoles: indole‐3‐carbinol (I3C), 3,3′‐diindolylmethane (DIM); and the isothiocyanates (ITCs): phenethyl isothiocyanate (PEITC) and sulforaphane (SFN). The cytotoxicity of the compounds was determined in a human liver hepatoma cell line (HepG2‐C8). The combination index was calculated to assess the synergistic effects on the induction of ARE‐mediated gene expressions. Quantitative real‐time polymerase chain reaction (qPCR) was employed to measure the mRNA expressions of Nrf2 and Nrf2‐mediated genes. I3C and DIM showed less cytotoxicity than SFN and PEITC. Compared with I3C, DIM was found to be a stronger inducer of ARE. Synergism was observed after combined treatments of 6.25 µm I3C + 1 µm SFN, 6.25 µm I3C + 1 µm PEITC and 6.25 µm DIM + 1 µm PEITC, while an additive effect was observed for 6.25 µm DIM + 1 µm SFN. Induction of endogenous Nrf2, phase II genes (GSTm2, UGT1A1 and NQO1) and antioxidant genes (HO‐1 and SOD1) was also observed. In summary, the indole I3C or DIM alone could induce or syngergistically induce in combination with the ITCs SFN or PEITC, Nrf2‐ARE‐mediated gene expression, which could potentially enhance cancer chemopreventive activity. Copyright

Requirement and Epigenetics Reprogramming of Nrf2 in Suppression of Tumor Promoter TPA-Induced Mouse Skin Cell Transformation by Sulforaphane

Nrf2 is a transcription factor that plays critical roles in regulating the expression of cellular defensive antioxidants and detoxification enzymes. However, the role of Nrf2 and Nrf2s epigenetics reprogramming in skin tumor transformation is unknown. In this study, we investigated the inhibitory role and epigenetics of Nrf2 on tumor transformation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse skin epidermal JB6 (JB6 P+) cells and the anticancer effect of sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables. After five days of treatment, SFN significantly inhibited TPA-induced JB6 cellular transformation and SFN enhanced the nuclear translocation of Nrf2 and increased the mRNA and protein levels of the Nrf2 target genes HO-1, NQO1 and UGT1A1. Knockdown of Nrf2 attenuated the induction of Nrf2, HO-1 and NQO1 by SFN, enhanced TPA-induced colony formation and dampened the inhibitory effect of SFN on TPA-induced JB6 transformation. Epigenetics investigation using bisulfite genomic sequencing showed that SFN decreased the methylation ratio of the first 15 CpGs of the Nrf2 gene promoter, which was corroborated by increased Nrf2 mRNA expression. Furthermore, SFN strongly reduced the protein expression of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b). SFN also inhibited the total histone deacetylase (HDAC) activity and decreased the protein expression of HDAC1, HDAC2, HDAC3 and HDAC4. Collectively, these results suggest that the anti-cancer effect of SFN against TPA-induced neoplastic transformation of mouse skin could involve the epigenetic reprogramming of anti-cancer genes such as Nrf2, leading to the epigenetic reactivation of Nrf2 and the subsequent induction of downstream target genes involved in cellular protection. Cancer Prev Res; 7(3); 319–29. ©2014 AACR.

Epigenetic Reactivation of Nrf2 in Murine Prostate Cancer TRAMP C1 Cells by Natural Phytochemicals Z-Ligustilide and Radix Angelica Sinensis via Promoter CpG Demethylation

Cancer development has been linked to epigenetic modifications of cancer oncogenes and tumor suppressor genes; in advanced metastatic cancers, severe epigenetic modifications are present. We previously demonstrated that the progression of prostate tumors in TRAMP mice is associated with methylation silencing of the Nrf2 promoter and a reduced level of transcription of Nrf2 and Nrf2 target genes. Radix Angelicae Sinensis (RAS; Danggui) is a medicinal herb and health food supplement that has been widely used in Asia for centuries. Z-Ligustilide (Lig) is one of the bioactive components of RAS. We investigated the potential of Lig and RAS to restore Nrf2 gene expression through epigenetic modification in TRAMP C1 cells. Lig and RAS induced the mRNA and protein expression of endogenous Nrf2 and Nrf2 downstream target genes, such as HO-1, NQO1, and UGT1A1. Bisulfite genomic sequencing revealed that Lig and RAS treatment decreased the level of methylation of the first five CpGs of the Nrf2 promoter. A methylation DNA immunoprecipitation assay demonstrated that Lig and RAS significantly decreased the relative amount of methylated DNA in the Nrf2 gene promoter region. Lig and RAS also inhibited DNA methyltransferase activity in vitro. Collectively, these results suggest that Lig and RAS are able to demethylate the Nrf2 promoter CpGs, resulting in the re-expression of Nrf2 and Nrf2 target genes. Epigenetic modifications of genes, including Nrf2, may therefore contribute to the overall health benefits of RAS, including the anticancer effect of RAS and its bioactive component, Lig.

Nrf2 knockout enhances intestinal tumorigenesis in Apcmin/+ mice due to attenuation of anti‐oxidative stress pathway while potentiates inflammation

Mutations in adenomatous polyposis coli (APC) gene are found in more than 80% of colorectal cancer (CRC) patients. The nuclear transcription factor Nrf2 plays a central role in the regulation of oxidative stress and inflammation. Previously, we have shown that chronic inflammation in Nrf2−/− (Nrf2 knockout; KO) mice resulted in higher expression of inflammatory markers and cytokines, coupled with higher inflammatory damage to the colonic crypt cells, as compared to the Nrf2+/+ (wild type; WT) mice. Induction of mutation in the colon by administration of carcinogen, AOM prior to DSS‐induced inflammation resulted in higher tumor incidence and numbers in Nrf2KO mice. These results indicate that Nrf2‐dependent inhibition of inflammation appears to be critical in inhibiting mutation‐initiated colorectal carcinogenesis. In this study, we aim to investigate if loss of Nrf2 would dose‐dependently promote intestinal tumorigenesis in Apcmin/+ mice. To demonstrate the in vivo mechanisms, we constructed both Apc mutated and Nrf2 deficient strain Apcmin/+ mice with C57BL/6 Nrf2KO mice to obtain F1, Apcmin/+;Nrf2+/− and F2, Apcmin/+;Nrf2−/−mice. Nrf2KO decreased the protein expression of antioxidant enzyme NQO1 in Apcmin/+. In contrast, Nrf2KO enhanced the expression of inflammatory markers such as COX‐2, cPLA, LTB4 in Apcmin/+. Finally, Nrf2KO resulted in higher level of PCNA and c‐Myc expression in intestinal tissue, indicating the deficiency of Nrf2 promotes proliferation of intestinal crypt cells in Apcmin/+. Taken together, our results suggest that Nrf2KO attenuates anti‐oxidative stress pathway, induces inflammation, and increases proliferative potential in the intestinal crypts leading to enhanced intestinal carcinogenesis and adenomas in Apcmin/+.

Epigenetic Modifications of Nrf2 by 3,3′-diindolylmethane In Vitro in TRAMP C1 Cell Line and In Vivo TRAMP Prostate Tumors

Abstract3,3′-diindolylmethane (DIM) is currently being investigated in many clinical trials including prostate, breast, and cervical cancers and has been shown to possess anticancer effects in several in vivo and in vitro models. Previously, DIM has been reported to possess cancer chemopreventive effects in prostate carcinogenesis in TRAMP mice; however, the in vivo mechanism is unclear. The present study aims to investigate the in vitro and in vivo epigenetics modulation of DIM in TRAMP-C1 cells and in TRAMP mouse model. In vitro study utilizing TRAMP-C1 cells showed that DIM suppressed DNMT expression and reversed CpG methylation status of Nrf2 resulting in enhanced expression of Nrf2 and Nrf2-target gene NQO1. In vivo study, TRAMP mice fed with DIM-supplemented diet showed much lower incidence of tumorigenesis and metastasis than the untreated control group similar to what was reported previously. DIM increased apoptosis, decreased cell proliferation and enhanced Nrf2 and Nrf2-target gene NQO1 expression in prostate tissues. Importantly, immunohistochemical analysis showed that DIM reduced the global CpG 5-methylcytosine methylation. Focusing on one of the early cancer chemopreventive target gene Nrf2, bisulfite genomic sequencing showed that DIM decreased the methylation status of the first five CpGs of the Nrf2 promoter region, corroborating with the results of in vitro TRAMP-C1 cells. In summary, our current study shows that DIM is a potent cancer chemopreventive agent for prostate cancer and epigenetic modifications of the CpG including Nrf2 could be a potential mechanism by which DIM exerts its chemopreventive effects.