Chengyue Zhang

Sulforaphane enhances Nrf2 expression in prostate cancer TRAMP C1 cells through epigenetic regulation

Growing evidence suggests epigenetic alteration is involved during the development and progression of prostate cancer. Previously, we found Nrf2, a key regulator of cellular antioxidant defense systems, was silenced through epigenetic mechanism during tumorigenesis in vivo TRAMP mice and in vitro TRAMP C1 cells. Sulforaphane (SFN) in cruciferous vegetable has been demonstrated to be a potent cancer prevention agent for years. The aim of this study is to investigate the potential of SFN to restore Nrf2 expression in TRAMP C1 cells through epigenetic modifications. Bisulfite genomic sequencing results indicated that SFN treatment led to demethylation of the first 5 CpGs in the promoter region of the Nrf2 gene in TRAMP C1 cells. Using methylation DNA immunoprecipitation (MeDIP) assay, SFN significantly reduced the ratio of anti-mecyt antibody binding to the Nrf2 promoter containing the first 5 CpGs. SFN increased mRNA and protein expressions of Nrf2 and Nrf2 downstream target gene NQO-1. In addition, SFN decreased the protein levels of DNMT1 and DNMT3a. SFN treatment also attenuated the protein expression levels of HDACs 1, 4, 5, and 7 while increased the level of active chromatin marker acetyl-Histone 3 (Ac-H3). SFN treatments also increased chromatin-immunoprecipitated DNA of Nrf2 gene promoter using anti-Ac-H3 antibody. Taken together, our current study shows that SFN regulates Nrf2s CpGs demethylation and reactivation in TRAMP C1 cells, suggesting SFN may exert its chemopreventive effect in part via epigenetic modifications of Nrf2 gene with subsequent induction of its downstream anti-oxidative stress pathway.

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.

Curcumin inhibits anchorage-independent growth of HT29 human colon cancer cells by targeting epigenetic restoration of the tumor suppressor gene DLEC1

Colorectal cancer remains the most prevalent malignancy in humans. The impact of epigenetic alterations on the development of this complex disease is now being recognized. The dynamic and reversible nature of epigenetic modifications makes them a promising target in colorectal cancer chemoprevention and treatment. Curcumin (CUR), the major component in Curcuma longa, has been shown as a potent chemopreventive phytochemical that modulates various signaling pathways. Deleted in lung and esophageal cancer 1 (DLEC1) is a tumor suppressor gene with reduced transcriptional activity and promoter hypermethylation in various cancers, including colorectal cancer. In the present study, we aimed to investigate the inhibitory role of DLEC1 in anchorage-independent growth of the human colorectal adenocarcinoma HT29 cells and epigenetic regulation by CUR. Specifically, we found that CUR treatment inhibited colony formation of HT29 cells, whereas stable knockdown of DLEC1 using lentiviral short hairpin RNA vector increased cell proliferation and colony formation. Knockdown of DLEC1 in HT29 cells attenuated the ability of CUR to inhibit anchorage-independent growth. Methylation-specific polymerase chain reaction (MSP), bisulfite genomic sequencing, and methylated DNA immunoprecipitation revealed that CUR decreased CpG methylation of the DLEC1 promoter in HT29 cells after 5 days of treatment, corresponding to increased mRNA expression of DLEC1. Furthermore, CUR decreased the protein expression of DNA methyltransferases and subtypes of histone deacetylases (HDAC4, 5, 6, and 8). Taken together, our results suggest that the inhibitory effect of CUR on anchorage-independent growth of HT29 cells could, at least in part, involve the epigenetic demethylation and up-regulation of DLEC1.

A new salicylic acid-derivatized kojic acid vanadyl complex: synthesis, characterization and anti-diabetic therapeutic potential.

The molecular mechanisms of vanadium toxicity suggest that incorporation of antioxidant groups in the structure of vanadium complexes could be a preferable strategy in designing novel hypoglycemic vanadium complexes with proper efficacy and safety. By conjugating a pyrone skeleton to provide a coordination group and antioxidative motifs, we synthesized a novel complex of bis ((5-hydroxy-4-oxo-4H-pyran-2-yl) methyl 2-hydroxy- benzoatato) oxovanadium (IV) (BSOV). Evaluation of the anti-diabetic effects of BSOV using streptozotocin (STZ)-induced diabetic rats with bis (maltolato) oxovanadium (BMOV) as a positive control showed that BSOV effectively lowered blood glucose level, ameliorated damages of hepatic and renal function in diabetic rats and improved lipid metabolism. The signs of potential alteration of renal function caused by BSOV and BMOV were observed and are discussed. Overall, the experimental results suggest BSOV as a potent hypoglycemic agent and further studies using this strategy for anti-diabetic agents.

Nrf2 Knockout Attenuates the Anti-Inflammatory Effects of Phenethyl Isothiocyanate and Curcumin

The role of phytochemicals in preventive and therapeutic medicine is a major area of scientific research. Several studies have illustrated the mechanistic roles of phytochemicals in Nrf2 transcriptional activation. The present study aims to examine the importance of the transcription factor Nrf2 by treating peritoneal macrophages from Nrf2+/+ and Nrf2–/– mice ex vivo with phenethyl isothiocyanate (PEITC) and curcumin (CUR). The peritoneal macrophages were pretreated with the drugs and challenged with lipopolysaccharides (LPSs) alone and in combination with PEITC or CUR to assess their anti-inflammatory and antioxidative effects based on gene and protein expression in the treated cells. LPS treatment resulted in an increase in the expression of inflammatory markers such as cycloxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in both Nrf2+/+ and Nrf2–/– macrophages, detected by quantitative polymerase chain reaction (qPCR). Nrf2+/+ macrophages treated with PEITC and CUR exhibited a significant decrease in the expression of these anti-inflammatory genes along with an increase in the expression of hemeoxygenase-1 (HO-1), which is an antioxidative stress gene downstream of the Nrf2 transcription factor battery. Although there was no significant decrease in the expression of the anti-inflammatory genes or an increase in HO-1 expression in Nrf2–/– macrophages treated with either PEITC or CUR, there was a significant decrease in the protein expression of COX-2 and an increase in the expression of HO-1 in Nrf2+/+ macrophages treated with PEITC compared to that with CUR treatment. No significant changes were observed in the macrophages from knockout animals. Additionally, there was a significant decrease in LPS-induced IL-6 and TNF-α production following PEITC treatment compared with that following CUR in Nrf2+/+ macrophages, whereas no change was observed in the macrophages from knockout animals. The results from qPCR, western blot, and ELISA analyses in macrophages from Nrf2+/+ and Nrf2 –/– mice indicate that Nrf2 plays an important role in the anti-inflammatory and antioxidative effects of PEITC and CUR, as observed by their decreased activities in Nrf2–/– macrophages.

Phenethyl isothiocyanate (PEITC) suppresses prostate cancer cell invasion epigenetically through regulating microRNA‐194

SCOPE Tumor metastasis greatly contributes to the mortality of prostate cancer. The glucosinolate-derived phenethyl isothiocyanate (PEITC) has been widely documented to reduce the risk of prostate cancer by modulating multiple biologically relevant processes. Emerging evidence suggests that PEITC may exert its anti-cancer effects through epigenetic mechanisms including microRNAs. Altered levels of miRNA have been linked to tumor malignancy due to their capacity to regulate functional gene expression in carcinogenesis. Here, we assessed the effects of PEITC on miRNA expression which is related to PCa cell invasiveness. METHODS AND RESULTS Utilizing oligonucleotide microarray first identified the most affected miRNAs in LNCaP cells after PEITC treatment. Several top altered miRNAs were further validated using quantitative PCR. Interestingly, overexpression of miR-194 suppressed PC3 cell invasion in matrigel-coated Transwell chambers. Bone morphogenetic protein 1 (BMP1) was shown to be a direct target of miR-194. Downregulation of BMP1 by miR-194 or PEITC led to decreased expression of key oncogenic matrix metalloproteinases, MMP2 and MMP9. This in turn resulted in the suppression of tumor invasion. CONCLUSION Our results indicate that miR-194 downregulates the expression of oncogenic MMP2 and MMP9 by targeting BMP1, which suggests a potential new mechanistic target by which PEITC suppresses prostate cancer cell invasiveness.

Blocking of JB6 Cell Transformation by Tanshinone IIA: Epigenetic Reactivation of Nrf2 Antioxidative Stress Pathway

Increasing numbers of natural products have been found to possess anticancer effects. Nuclear factor erythroid-2-related factor-2 (Nrf2) is a master regulator of the antioxidative stress response, and our previous studies found that epigenetic modification of the Nrf2 gene appears to be a critical mechanism. Salvia miltiorrhiza, a Chinese herbal medicine widely used in Asian countries, has been shown to possess anticancer and antioxidant effects. Tanshinone IIA (TIIA), an active component in S. miltiorrhiza, has been reported to activate Nrf2 pathway. The objective of this study was to investigate the epigenetic regulation of Nrf2 by TIIA in mouse skin epidermal JB6 cells and the functional consequences for cell transformation. TIIA was found to induce antioxidant response element-luciferase and upregulate the mRNA and protein levels of Nrf2 and Nrf2 downstream target genes HO-1 and NQO-1. TIIA decreased the colony formation of JB6 cells by approximately 80%. TIIA decreased the protein levels of DNMT1, DNMT3a, DNMT3b, and HDAC3 and inhibited the enzymatic activity of HDACs. Bisulfite genomic sequencing indicated that TIIA demethylated the first five CpGs in the promoter region of the Nrf2 gene. Chromatin immunoprecipitation assays showed that TIIA treatment increased the recruitment of RNA polymerase II at Nrf2 transcription start site but had limited effects on enrichment of Ac-H3 in Nrf2 promoter. Taken together, our results show that TIIA activates the Nrf2 signaling pathway and induces epigenetic demethylation of the CpGs of Nrf2. The epigenetic reactivation of the Nrf2 signaling pathway by TIIA could potentially contribute to the attenuation of JB6 cellular transformation and anticancer effects.

MicroRNAs: new Players in Cancer Prevention Targeting Nrf2, Oxidative Stress and Inflammatory Pathways

AbstractmiRNAs are endogenous small non-coding RNAs of 20–22 nucleotides that repress gene expression at the posttranscriptional level. There is growing interest in the role of miRNAs in cancer chemoprevention, and several naturally occurring chemopreventive agents have been found to be modulators of miRNA expression both in vitro and in vivo. Moreover, these chemopreventive phytochemicals commonly possess anti-oxidative and/or anti-inflammatory properties, and Nrf2 has been extensively studied as a molecular target in cancer prevention. The crosstalk between miRNAs and the traditional cellular signaling pathways of chemoprevention remain to be fully elucidated. This review summarizes the data regarding the potential interactions between miRNAs and anti-oxidative and anti-inflammatory pathways. Cellular redox homeostasis can affect the biogenesis and processing of miRNAs, which in turn regulate the Nrf2 pathway of detoxifying/anti-oxidative genes. We also discuss the miRNA regulatory mechanisms in relation to inflammation-related cancer signaling pathways.

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.

The epigenetic effects of aspirin: the modification of histone H3 lysine 27 acetylation in the prevention of colon carcinogenesis in azoxymethane- and dextran sulfate sodium-treated CF-1 mice

Colorectal cancer (CRC) is the third most common cancer worldwide. Chronic inflammation appears to enhance the risk of CRC. Emerging evidence has suggested that epigenetic mechanisms play an important role in CRC. Aspirin [acetylsalicylic acid (ASA)] has been shown to prevent CRC; however, the epigenetic mechanisms of its action remain unknown. This study investigated the protective role of ASA in azoxymethane (AOM)-initiated and dextran sulfate sodium (DSS)-promoted colitis-associated colon cancer (CAC) and examined the epigenetic effects, particularly on histone 3 lysine 27 acetylation (H3K27ac), underlying the preventive effect of ASA. CF-1 mice were fed with AIN-93M diet with or without 0.02% ASA from 1 week prior to AOM initiation until the mice were killed 20 weeks after AOM injection. Our results showed that AOM/DSS + ASA significantly suppressed inflammatory colitis symptoms and tumor multiplicity. AOM/DSS + ASA reduced AOM/DSS-induced protein expression and the activity of histone deacetylases (HDACs) and globally restored H3K27ac. Furthermore, AOM/DSS + ASA inhibited AOM/DSS-induced enrichment of H3K27ac in the promoters of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) that corresponded to the dramatic suppression of the messenger RNA (mRNA) and protein levels. Surprisingly, no significant changes in the H3K27ac abundance in the prostaglandin-endoperoxide synthase 2 (Cox-2) promoters or in the Cox-2 mRNA and protein expression were observed. Collectively, our results suggest that a potential novel epigenetic mechanism underlies the chemopreventive effects of ASA, and this mechanism attenuates CAC in AOM/DSS-induced CF-1 mice via the inhibition of HDACs and the modification of H3K27ac marks that suppress iNOS, TNF-α and IL-6.