Reactive metabolites from carcinogens and oxidative stress can drive genetic mutations, genomic instability, neoplastic transformation, and ultimately carcinogenesis. Numerous dietary phytochemicals in vegetables/fruits have been shown to possess cancer chemopreventive effects in both preclinical animal models and human epidemiological studies. These phytochemicals could prevent the initiation of carcinogenesis via either direct scavenging of reactive oxygen species/reactive nitrogen species (ROS/RNS) or, more importantly, the induction of cellular defense detoxifying/antioxidant enzymes. These defense enzymes mediated by Nrf2-antioxidative stress and anti-inflammatory signaling pathways can contribute to cellular protection against ROS/RNS and reactive metabolites of carcinogens. In addition, these compounds would kill initiated/transformed cancer cells in vitro and in in vivo xenografts via diverse anti-cancer mechanisms. These mechanisms include the activation of signaling kinases (e.g., JNK), caspases and the mitochondria damage/cytochrome c pathways. Phytochemicals may also have anti-cancer effects by inhibiting the IKK/NF-κB pathway, inhibiting STAT3, and causing cell cycle arrest. In addition, other mechanisms may include epigenetic alterations (e.g., inhibition of HDACs, miRNAs, and the modification of the CpG methylation of cancer-related genes). In this review, we will discuss: the current advances in the study of Nrf2 signaling; Nrf2-deficient tumor mouse models; the epigenetic control of Nrf2 in tumorigenesis and chemoprevention; Nrf2-mediated cancer chemoprevention by naturally occurring dietary phytochemicals; and the mutation or hyper-expression of the Nrf2–Keap1 signaling pathway in advanced tumor cells. The future development of dietary phytochemicals for chemoprevention must integrate in vitro signaling mechanisms, relevant biomarkers of human diseases, and combinations of different phytochemicals and/or non-toxic therapeutic drugs, including NSAIDs.
Dietary phytochemical; Nrf2; Antioxidant response; Inflammation; Epigenetics; Cancer stem cell
Tocopherol, a member of the vitamin E family, consists of four forms designated as α, β, γ, and δ. Several large cancer prevention studies with α-tocopherol have reported no beneficial results, but recent laboratory studies have suggested that δ- and γ-tocopherol may be more effective. In two different animal models of breast cancer, the chemopreventive activities of individual tocopherols were assessed using diets containing 0.3% of tocopherol (α-, δ- or γ-) or 0.3% of a γ-tocopherol rich mixture (γ-TmT). While administration of tocopherols did not prevent human epidermal growth factor receptor 2 (HER2/neu)-driven tumorigenesis, δ- and γ-tocopherols inhibited hormone-dependent mammary tumorigenesis in N-methyl-N-nitrosourea (NMU)-treated female Sprague Dawley rats. NMU-treated rats showed an average tumor burden of 10.6 ± 0.8 g in the control group at 11 weeks, whereas dietary administration of δ- and γ-tocopherols significantly decreased tumor burden to 7.2 ± 0.8 g (p<0.01) and 7.1 ± 0.7 g (p<0.01), respectively. Tumor multiplicity was also reduced in δ- and γ-tocopherol treatment groups by 42% (p<0.001) and 32% (p<0.01), respectively. In contrast, α-tocopherol did not decrease tumor burden or multiplicity. In mammary tumors, the protein levels of pro-apoptotic markers (BAX, cleaved-caspase 9, cleaved-caspase 3, cleaved-PARP) were increased, while anti-apoptotic markers (Bcl2, XIAP) were inhibited by δ-tocopherol, γ-tocopherol and γ-TmT. Furthermore, markers of cell proliferation (PCNA, PKC α), survival (PPARγ, PTEN, phospho-Akt) and cell cycle (p53, p21) were affected by δ- and γ-tocopherols. Both δ- and γ-tocopherols, but not α-tocopherol, appear to be promising agents for the prevention of hormone-dependent breast cancer.
Breast Cancer; Tocopherols; Apoptosis; Cell Cycle; PPARγ
This study assesses the pharmacokinetics (PK) and pharmacodynamics (PD) of Nrf2-mediated increased expression of Phase II drug metabolizing enzyme (DME) and antioxidant enzymes which represents an important component of cancer chemoprevention in rat lymphocytes following intravenous (i.v.) administration of an anti-cancer phytochemical sulforaphane (SFN)
SFN was administered intravenously to four groups of male Sprague-Dawley JVC rats each group comprising four animals. Blood samples were drawn at selected time points. Plasma were obtained from half of the blood samples and analyzed using a validated LC-MS/MS method. Lymphocytes were collected from the remaining blood samples using Ficoll-Paque™ Plus centrifuge medium. Lymphocyte RNAs were extracted, converted to cDNA, and quantitative real-time PCR analyses were performed and fold changes were calculated against those at time zero for the relative expression of Nrf2-target genes of phase II DME/antioxidant enzymes. PK-PD modeling was conducted based on Jusko’s indirect response model (IDR) using GastroPlus™ and Bootstrap Method.
SFN plasma concentration declined biexponentially and the pharmacokinetic parameters were generated. Rat lymphocyte mRNA expression levels showed no change for GSTM1, SOD, NF-κB, UGT1A1, or UGT1A6. Moderate increases (2-5 folds) over the time zero were seen for HO-1, Nrf2, and NQO1, and significant increase (> 5 folds) for GSTT1, GPx1, and Maf. PK-PD analyses using GastroPlus™ and Bootstrap method provided reasonable fitting for the PK and PD profiles and parameter estimates.
Our present study shows that SFN could induce Nrf2-mediated phase II DME/antioxidant mRNA expression for NQO1, GSTT1, Nrf2, GPx, Maf, and HO-1 in rat lymphocytes after i.v. administration, suggesting that Nrf2-mediated mRNA expression in lymphocytes may serve as surrogate biomarkers. The PK-PD IDR model simultaneously linking the plasma concentrations of SFN and the PD response of lymphocyte mRNA expression is valuable for quantitating Nrf2 mediated effects of SFN. This study may provide a conceptual framework for future clinical PK-PD studies of dietary cancer chemopreventive agents in human.
sulforaphane; pharmacokinetics; pharmacodynamics; lymphocyte; phase II genes; Nrf2
R-(-)-gossypol acetic acid (AT-101), a natural BH3 mimetic, is investigated in a Phase I/II clinical trial for the treatment of advanced solid tumor malignancies. Gossypol undergoes rapid degradation in solution phase, which causes major technical difficulty for its quantitation in plasma. We developed and validated a sensitive HPLC assay for pharmacokinetic evaluation of gossypol. Acetonitrile deproteinization method was chosen for sample preparation and Schiff's base derivative, R-(-)-gossypol-diamino-propanol (GDP), was used as internal standard. Chromatographic separation of gossypol in plasma was performed using a Zorbax Eclipse XDB column C18 at 30°C. The mobile phase consists of 10 mmol/L KH2PO4 (pH=3.0) and acetonitrile (20:80) at 1.0 mL/min flow rate. Linearity ranged over 56-3585 ng/mL (R2=0.9997±0.0003, n=4), and the limit of detection was 28 ng/mL. The intra- and inter-assay precision was less than 13.7% and the bias ranged from -7.4 to 7.0%. The method was successfully applied to characterize the pharmacokinetics of AT-101 in a Phase I clinical trial. The validated assay is accurate, and sensitive with minimum loss and rapid analysis time and suitable for quantification of gossypol for pharmacokinetics evaluation.
R-(-)-gossypol; HPLC-UV; pharmacokinetics
We evaluated the role of total dietary antioxidant capacity and of individual antioxidants on endometrial cancer risk in a population-based case-control study in New Jersey, including 417 cases and 395 controls. Dietary intake was ascertained using a food frequency questionnaire (FFQ), and total antioxidant capacity (TAC) intake was estimated using the USDA Oxygen Radical Absorbance Capacity (ORAC) Database and the University of Oslo’s Antioxidant Food Database (AFD) and FFQ-derived estimates of intake. Odds ratios and 95% confidence intervals were derived using multivariate logistic regression controlling for major endometrial cancer risk factors. Using the ORAC database, after adjusting for major covariates, we found decreased risks for the highest tertile of total phenolic intake compared to the lowest (OR: 0.62; 95% CI: 0.39–0.98). There was no association for TAC intake based on the AFD, which utilized the ferric reducing ability of plasma (FRAP) assay to assess antioxidant capacity. There was no strong evidence for an association with intake of any of the individual antioxidants. Our findings suggest that total phenolic consumption may decrease endometrial cancer risk.
Endometrial neoplasms; antioxidants; total antioxidant capacity; vitamin C; vitamin E; beta-carotene; selenium; lutein; lycopene; diet; phenolics
Activation of the antioxidant response element (ARE) up-regulates enzymes involved in detoxification of electrophiles and reactive oxygen species. The induction of ARE genes is regulated by the interaction between redox sensor protein, Keap1, and the transcription factor, Nrf2. Fluorescently labeled Nrf2 peptides containing the ETGE motif were synthesized and optimized as tracers in the development of a fluorescence polarization (FP) assay to identify small molecule inhibitors of Keap1-Nrf2 interaction. The tracers were optimized to increase the dynamic range of the assay and their binding affinities to the Keap1 Kelch domain. The binding affinities of Nrf2 peptide inhibitors obtained in our FP assay using FITC-9mer Nrf2 peptide amide as the probe were in good agreement with those obtained previously by a surface plasmon resonance (SPR) assay. The FP assay exhibits considerable tolerance towards DMSO and produced a Z'-factor greater than 0.6 in a 384-well format. Further optimization of the probe led to cyanine-labeled 9mer Nrf2 peptide amide, which can be used along with the FITC-9mer Nrf2 peptide amide in a high throughput screening (HTS) assay to discover small molecule inhibitors of Keap1-Nrf2 interaction.
Nrf2; Keap1; ARE; fluorescence polarization; high throughput screening; oxidative response
The Keap1-Nrf2 interaction plays important roles in regulation of Nrf2 activity and induction of chemopreventive enzymes. To better understand the interaction and to determine the minimal Nrf2 sequence required for Keap1 binding, we synthesized a series of Nrf2 peptides containing ETGE motif and determined their binding affinities to the Kelch domain of Keap1 in solution using a surface plasmon resonance (SPR)-based competition assay. The equilibrium dissociation constant for the interaction between 16mer Nrf2 peptide and Keap1 Kelch domain in solution (KDsolution) was found to be 23.9 nM, which is 10× lower than the surface binding constant (KDsurface) of 252 nM obtained for the direct binding of Keap1 Kelch domain to the immobilized 16mer Nrf2 peptide on a SPR sensor chip surface. The binding affinity of Nrf2 peptides to Keap1 Kelch domain was not lost until after deletion of 8 residues from the N-terminus of the 16mer Nrf2 peptide. The 9mer Nrf2 peptide has a moderate binding affinity with a KDsolution of 352 nM and the affinity was increased 15× upon removal of the positive charge at the peptide N-terminus by acetylation. These results suggest that the minimal Nrf2 peptide sequence required for Keap1 binding is the 9mer sequence of LDEETGEFL.
Keap1; Nrf2; protein-protein interaction; surface plasmon resonance; solution binding assay
NF-E2-related factor 2 (NRF2) regulates a battery of antioxidative and phase II drug metabolizing/detoxifying genes through binding to the antioxidant response elements (ARE). NRF2-ARE signaling plays a central role in protecting cells from a wide spectrum of reactive toxic species including reactive oxygen/nitrogen species (RONS). 4-hydroxylnonenal (4-HNE) is a major end product from lipid peroxidation of omega-6 polyunsaturated fatty acids (PUFA) induced by oxidative stress, and it is highly reactive to nucleophilic sites in DNA and proteins, causing cytotoxicity and genotoxicity. In this study, we examined the role of NRF2 in regulating the 4-HNE induced gene expression of antioxidant and detoxifying enzymes.
When HeLa cells were treated with 4-HNE, NRF2 rapidly transloated into the nucleus, as determined by the distribution of NRF2 tagged with the enhanced green fluorescent protein (EGFP) and increased NRF2 protein in the nuclear fraction. Transcriptional activity of ARE-luciferase was significantly induced by 0.01-10 μM of 4-HNE in a dose-dependent manner, and the induction could be blocked by pretreatment with glutathione (GSH). 4-HNE induced transcriptional expression of glutathione S-transferase (GST) A4, aldoketone reductase (AKR) 1C1 and heme oxygenase-1 (HO-1), and the induction was attenuated by knocking down NRF2 using small interfering RNA.
NRF2 is critical in mediating 4-HNE induced expression of antioxidant and detoxifying genes. This may account for one of the major cellular defense mechanisms against reactive metabolites of lipids peroxidation induced by oxidative stress and protect cells from cytotoxicity.
NRF2; 4-hydroxynonenal (4-HNE); Antioxidant response element (ARE); Oxidative stress
Prior studies from our laboratory have demonstrated the efficacy of a combined treatment of low doses of dietary agents curcumin and phenylethylisothiocyanate in effectively suppressing prostate cancer in vitro in human prostate cancer PC3 cells as well as in vivo in immunodeficient mice implanted with PC3 cells. Hence, this study was undertaken to examine the potential chemopreventive properties of the two agents against transgenic adenocarcinoma of the mouse prostate.
Materials and Methods
The efficacy of AIN-76A diet supplemented with 2% curcumin or 0.05% PEITC or a combination of 1% curcumin and 0.025% PEITC for periods of 10 and 16 weeks was tested against adenocarcinoma of the mouse prostate. Immunohistochemistry and Western blot analysis were used to examine the expression of proliferation and apoptotic biomarkers. All statistical tests were two-sided.
Supplementing AIN-76A diet with dietary phytochemicals curcumin or PEITC either alone or in combination, significantly decreased incidence of prostate tumor formation (P=0.0064). Immunohistochemistry revealed a significant inhibition of high-grade PIN (P=0.0006, 0.000069, 0.00029 for a treatment period of 10 weeks and P=0.02582, 0.022179, 0.0317 for a treatment period of 16 weeks) along with decreased proliferation and increased apoptotic index in the curcumin, PEITC or curcumin and PEITC treated animals, respectively. Furthermore, Western blot analysis revealed that downregulation of the Akt signaling pathway may in part play a role in decreasing cell proliferation ultimately retarding prostate tumor formation.
Our data lucidly evidence the chemopreventive merits of dietary phytochemicals curcumin and PEITC in suppressing prostate adenocarcinoma.
cancer chemoprevention; curcumin; PEITC; prostate cancer; TRAMP
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 anti-oxidant 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 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. 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 to I3C, DIM was found to be a stronger inducer of ARE. Synergism was observed after combined treatments of I3C 6.25 µM + SFN 1 µM, I3C 6.25 µM + PEITC 1 µM and DIM 6.25 µM + PEITC 1 µM, while additive effect was observed for DIM 6.25 µM + SFN 1 µM. 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.
Antioxidant response element (ARE); nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or Nrf2); indole-3-carbinol (I3C); 3,3’-diindolylmethane (DIM); isothiocyanates
PALB2/FANCN is mutated in breast and pancreatic cancers and Fanconi anemia (FA). It controls the intranuclear localization, stability, and DNA repair function of BRCA2 and links BRCA1 and BRCA2 in DNA homologous recombination repair and breast cancer suppression. Here, we show that PALB2 directly interacts with KEAP1, an oxidative stress sensor that binds and represses the master antioxidant transcription factor NRF2. PALB2 shares with NRF2 a highly conserved ETGE-type KEAP1 binding motif and can effectively compete with NRF2 for KEAP1 binding. PALB2 promotes NRF2 accumulation and function in the nucleus and lowers the cellular reactive oxygen species (ROS) level. In addition, PALB2 also regulates the rate of NRF2 export from the nucleus following induction. Our findings identify PALB2 as a regulator of cellular redox homeostasis and provide a new link between oxidative stress and the development of cancer and FA.
Curcumin (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.
curcumin; demethylation; hypermethylation; LNCaP; Neurog1
Cancer chemoprevention is a process of using either natural or synthetic compounds to reduce the risk of developing cancer. Observations that NF-E2-related factor 2 (Nrf2)-deficient mice lack response to some chemopreventive agents point to the important role of Nrf2 in chemoprevention. Nrf2 is a member of basic-leucine zipper transcription factor family and has been shown to regulate gene expression by binding to a response element, antioxidant responsive element. It is generally believed that activation of Nrf2 signaling is an adaptive response to the environmental and endogenous stresses. Under homeostatic conditions, Nrf2 is suppressed by association with Kelch-like ECH-associated protein 1 (Keap1), but is stimulated upon exposure to oxidative or electrophilic stress. Once activated, Nrf2 translocates into nuclei and upregulates a group of genes that act in concert to combat oxidative stress. Nrf2 is also shown to have protective function against inflammation, a pathological process that could contribute to carcinogenesis. In this review, we will discuss the current progress in the study of Nrf2 signaling, in particular, the mechanisms of Nrf2 activation by chemopreventive agents. We will also discuss some of the potential caveats of Nrf2 in cancer treatment and future opportunity and challenges on regulation of Nrf2-mediated antioxidant and antiinflammatory signaling in the context of cancer prevention. Antioxid. Redox Signal. 13, 1679–1698.
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.
anti-inflammatory; anti-oxidative stress; chrysanthemum; licorice; Nrf2; phase II drug metabolizing/detoxifying enzymes
This article reviews recent basic and clinical studies of ginseng, particularly the anti-cancer effects and the potential chemopreventive actions by activating the transcriptional factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2 or NFE2L2)-mediated anti-oxidative stress or anti-inflammatory pathways. Nrf2 is a novel target for cancer prevention as it regulates the antioxidant responsive element (ARE), a critical regulatory element in the promoter region of genes encoding cellular phase II detoxifying and anti-oxidative stress enzymes. The studies on the chemopreventive effects of ginseng or its components/products showed that Nrf2 could also be a target for ginseng's actions. A number of papers also demonstrated the anti-inflammatory effects of ginseng. Targeting Nrf2 pathway is a novel approach to the investigation of ginseng's cancer chemopreventive actions, including some oxidative stress and inflammatory conditions responsible for the initiation, promotion and progression of carcinogenesis.
The nuclear transcription factor E2-related factor 2 (Nrf2) has been shown to play pivotal roles in preventing xenobiotics-induced toxicity and carcinogen- related tumorigenesis. These protective effects are mainly attributed to the induction of Phase II drug metabolizing/detoxification and antioxidant enzymes through the Nrf2-antioxidant response element (ARE) pathways. In this review, we will summarize the current research status on the identification of Nrf2-regulated drug metabolism enzymes (DMEs), especially Phase II DMEs, and Phase III drug transporters. In addition, the molecular mechanisms underlying the coordinated regulation of Phase II DMEs and Pharse III transporters are also discussed based on finding published in the literatures.
Nrf2; Phase II drug metabolizing enzymes; Phase III transporters; cancer chemoprevention; dietary cancer chemopreventive agents
Phenethyl isothiocyanate (PEITC), an isothiocyanate abundantly found in cruciferous vegetables have been shown to induce apoptosis through MAPK pathway in prostate and colon cancer cells. In the present study, we investigate the effect of PEITC on cell cycle regulation of HT-29 colon cancer cells. Using flow cytometry and Western blot analyses, we found that PEITC significantly induced G1 cell cycle arrest in HT-29 cells. We showed that the cell cycle arrest was not related to beta-catenin translocation into the nucleus. Interestingly, inhibition of p38 attenuated the cell cycle arrest, suggesting that cell cycle arrest by PEITC was caused by the activation of MAPK pathway. Treatments of PEITC resulted in a dose-dependent down-regulation of cyclins A, D, E and pRb protein expression. The down-regulation can be attributed to the activation of the p38 pathway, since inhibition of its activities by specific inhibitor blocked PEITC’s ability to decrease the expression level of cyclins A and D and attenuate cell cycle arrest effect of PEITC. In conclusion, this study shows for the first time that PEITC can arrest HT-29 cells in G1 phase by down-regulation of cyclins through the activation of p38 MAPK signaling pathway.
cell cycle arrest; colon cancer; cyclin; PEITC; p38