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.
It has previously been demonstrated that curcumin is effective against prostate cancer growth and progression in TRAMP mice, potentially acting through the epigenetic modification of the Nrf2 gene and the subsequent induction of the Nrf2-mediated anti-oxidative stress cellular defense pathway. FN1 is a synthetic curcumin analog that shows stronger anti-cancer activity than curcumin. The purpose of this study was to investigate a potential epigenetic effect of FN1 that restores Nrf2 gene expression in TRAMP-C1 cells. Stably transfected HepG2-C8 cells were used to investigate the effect of FN1 on the Nrf2-ARE pathway. Real-time quantitative PCR and western blotting were used 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 assay was conducted to test the tumor inhibitory effect of FN1. Epigenetic modification enzymes, including DNMTs and HDACs, were investigated by western blotting. Luciferase reporter assay indicated FN1 was more potent than curcumin in activating the Nrf2-ARE pathway. FN1 increased the mRNA and protein expression of Nrf2 and downstream genes, such as HO-1, NQO1, and UGT1A1. FN1 significantly inhibited the colony formation of TRAMP-C1 cells. BGS and MeDIP assays revealed that FN1 treatment (250 nM for 3 days) decreased the level of CpG methylation of the Nrf2 promoter. FN1 also downregulated epigenetic modification enzymes. In conclusion, our results suggest that FN1 is a novel anti-cancer agent for prostate cancer. FN1 can activate the Nrf2-ARE pathway, inhibit the colony formation of TRAMP-C1 cells and increase the expression of Nrf2 and downstream genes 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.
Astaxanthin (AST), a red dietary carotenoid, has synergistic antioxidant effects with polyunsaturated fatty acids at low concentrations via Nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or Nrf2)/antioxidant response element (ARE) signaling. In addition, chromatin remodeling and DNA methylation-based gene silencing represent a common mechanism in prostate carcinogenesis and tumor progression from normal cells to pre-initiated cells and ultimately to invasive carcinoma. Therefore, the control of epigenetic modification and the transcriptional/translational control of the activation of Nrf2 and Nrf2-target genes, including glutathione S-transferases (GSTs), appear to be an important mechanism that protects cells against injuries from oxidative stress and cancer development. In this study, we aim to investigate the role of AST in reactivating the expression of Nrf2 and GSTP1 through epigenetic modification in human prostate LNCaP cells. Treatment with AST in human LNCaP cells reduced the methylation of 21 CpG sites of the GSTP1 CpG island but did not affect the three CpG sites of the Nrf2 promoter region. AST induced the mRNA expression and protein expression of both Nrf2 and GSTP1. It also increased the mRNA expression of NQO1 in sh-mock LNCaP cells but not in sh-SETD7 LNCaP cells. Furthermore, AST reduced the protein expression of DNMT3b and significantly inhibited DNMT and HDAC activities in vitro. Taken together, these results suggest that AST decreased the methylation status of the GSTP1, and these epigenetic modifying effects may originate from the decreasing activities of epigenetic modification enzymes, contributing to the overall beneficial health effects of AST.
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