Induction Of NQO1 Research Articles

A steroid like phytochemical Antcin M is an anti-aging reagent that eliminates hyperglycemia-accelerated premature senescence in dermal fibroblasts by direct activation of Nrf2 and SIRT-1.

The present study revealed the anti-aging properties of antcin M (ANM) and elucidated the molecular mechanism underlying the effects. We found that exposure of human normal dermal fibroblasts (HNDFs) to high-glucose (HG, 30 mM) for 3 days, accelerated G0/G1 phase arrest and senescence. Indeed, co-treatment with ANM (10 μM) significantly attenuated HG-induced growth arrest and promoted cell proliferation. Further molecular analysis revealed that ANM blocked the HG-induced reduction in G1-S transition regulatory proteins such as cyclin D, cyclin E, CDK4, CDK6, CDK2 and protein retinoblastoma (pRb). In addition, treatment with ANM eliminated HG-induced reactive oxygen species (ROS) through the induction of anti-oxidant genes, HO-1 and NQO-1 via transcriptional activation of Nrf2. Moreover, treatment with ANM abolished HG-induced SIPS as evidenced by reduced senescence-associated β-galactosidase (SA-β-gal) activity. This effect was further confirmed by reduction in senescence-associated marker proteins including, p21CIP1, p16INK4A, and p53/FoxO1 acetylation. Also, the HG-induced decline in aging-related marker protein SMP30 was rescued by ANM. Furthermore, treatment with ANM increased SIRT-1 expression, and prevented SIRT-1 depletion. This protection was consistent with inhibition of SIRT-1 phosphorylation at Ser47 followed by blocking its upstream kinases, p38 MAPK and JNK/SAPK. Further analysis revealed that ANM partially protected HG-induced senescence in SIRT-1 silenced cells. A similar effect was also observed in Nrf2 silenced cells. However, a complete loss of protection was observed in both Nrf2 and SIRT-1 knockdown cells suggesting that both induction of Nrf2-mediated anti-oxidant defense and SIRT-1-mediated deacetylation activity contribute to the anti-aging properties of ANM in vitro. Result of in vivo studies shows that ANM-treated C. elegens exhibits an increased survival rate during HG-induced oxidative stress insult. Furthermore, ANM significantly extended the life span of C. elegans. Taken together, our results suggest the potential application of ANM in age-related diseases or as a preventive reagent against aging process.

NAD(P)H:quinone oxidoreductase 1 inducer activity of some novel anilinoquinazoline derivatives.

The Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response elements pathway enables cells to survive oxidative stress conditions through regulating the expression of cytoprotective enzymes such as NAD(P)H:quinone oxidoreductase 1 (NQO1). This work presents the design and synthesis of novel anilinoquinazoline derivatives (2–16a) and evaluation of their NQO1 inducer activity in murine cells. Molecular docking of the new compounds was performed to assess their ability to inhibit Keap1–Nrf2 protein–protein interaction through occupying the Keap1–Nrf2-binding domain, which leads to Nrf2 accumulation and enhanced gene expression of NQO1. Docking results showed that all compounds can potentially interact with Keap1; however, 1,5-dimethyl-2-phenyl-4-(2-phenylquinazolin-4-ylamino)-1,2-dihydropyrazol-3-one (9), the most potent inducer, showed the largest number of interactions with key amino acids in the binding pocket (Arg483, Tyr525, and Phe478) compared to the native ligand or any other compound in this series.

Abstract 5093: Fibulin-5 supports pancreatic tumor growth through inhibition of integrin-induced ROS

Abstract Pancreatic ductal adenocarcinoma (PDA) is a highly recalcitrant disease. One factor contributing to chemoresistance is thought to be the desmoplastic response seen in these tumors. Fibronectin (FN) is a major constituent of the desmoplastic reaction of PDA. It has been shown that integrin activation by fibronectin can induce reactive oxygen species (ROS) production by stromal cells. Increased ROS has been observed in many cancers, including PDA. Therefore, we propose that oxidation therapy is an attractive approach to increase chemoresponse in PDA as this strategy takes advantage of the sensitivity of these tumors to further ROS insults. We discovered that the matricellular protein Fibulin5 (Fbln5) is upregulated in PDA and reduces FN-mediated integrin-induced ROS production by competing with FN for binding to α5β1 integrin and blocking integrin-induced ROS production. Mutation of the RGD-integrin binding domain in Fbln5 to RGE abolishes the binding of Fbln5 to the integrin and increases integrin-induced ROS production. Fbln5RGE/RGE (RGE) mice, display reduced tumor growth in implant and genetic models of PDA (KIC and KPC) as a direct consequence of elevated ROS levels in the tumor microenvironment. Furthermore, ROS induction correlates with reduced angiogenesis in tumors from RGE animals and had an additive therapeutic effect when combined with standard therapy. We have identified multiple oxidative stress-responsive genes that are elevated in stromal fibroblasts isolated from tumors from RGE animals. One of the most promising targets is NAD(P)H dehydrogenase (quinone 1) (Nqo1). In support of this observation we found that the level of Nqo1 is significantly higher in RGE fibroblasts compared with WT fibroblasts when plated on FN but not collagen or plastic. Furthermore we have demonstrated that FN-mediated induction of ROS and Nqo1 is dependent on β1 integrin and 5-lipooxygenase (5-LOX). Our studies reveal a unique integrin-based mechanism to enhance ROS production to a level that is specifically toxic to the tumor by exploiting the tumor-specific expression of Fbln5. Current studies are focused on understanding the mechanism by which Fbln5 is induced in PDA. Citation Format: Mary Topalovski, Miao Wang, Zachary Moore, David Boothman, Rolf Brekken. Fibulin-5 supports pancreatic tumor growth through inhibition of integrin-induced ROS. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5093.

Electron affinity of tricyclic, bicyclic, and monocyclic compounds containing cyanoenones correlates with their potency as inducers of a cytoprotective enzyme

Tricyclic, bicyclic, and monocyclic compounds containing cyanoenones induce various anti-inflammatory and cytoprotective enzymes through activation of the Keap1/Nrf2/ARE (antioxidant response element) pathway. The potency of these compounds as Nrf2 activators was determined using a prototypic cytoprotective enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) in Hepa1c1c7 murine hepatoma cells. The electron affinity (EA) of the compounds, expressed as the energy of their lowest unoccupied molecular orbital [E (LUMO)], was evaluated using two types of quantum mechanical calculations: the semiempirical (AM1) and the density functional theory (DFT) methods. We observed striking linear correlations [r=0.897 (AM1) and 0.936 (DFT)] between NQO1 inducer potency of these compounds and their E (LUMO) regardless of the molecule size. Importantly and interestingly, this finding demonstrates that the EA is the essentially important factor that determines the reactivity of the cyanoenones with Keap1.

American Indian Botanicals As Possible Alternatives To Hormone Therapy During Menopause

Although pharmaceutical hormone therapy (HT) remains the standard clinical treatment for managing menopausal symptoms, many women seek alternatives such as botanical dietary supplements because HT has been associated with increased risks of breast cancer, coronary heart disease and stroke. The leading botanicals that women take for menopause are black cohosh and red clover, which were also traditionally used by American Indians. While these two botanicals have been investigated extensively, there are still numerous American Indian plants that lack scientific studies on their safety and efficacy. In collaboration with the Chicago Botanic Garden, 15 native plants were evaluated for potential women's health-related benefits. Amorpha canescens Pursh. (Fabaceae) – Leadplant, Echinocystis lobata (Michx.) Torr. & A. Gray (Cucurbitaceae) – wild cucumber, and Silphium perfoliatum L. (Asteraceae) - cup plant inflorescent tissue were screened for estrogenicity, chemopreventive, and anti-inflammatory potential based on American Indian ethnobotany and published biological data. Leadplant MeOH extract was selected as the best candidate for bioassay-guided fractionation due to dose-dependent (40, 20, 10, 5, 2.5, 1.25 µg/mL) anti-estrogenic and anti-inflammatory activity in the Ishikawa and Griess assays (n = 9). However, leadplant extract (20 µg/mL) demonstrated low activity in the NQO1 induction assay for chemopreventive potential (n = 6). Currently, bioassay guided-fractionation is being performed on leadplant to ascertain the active constituents.

Bach1 differentially regulates distinct Nrf2-dependent genes in human venous and coronary artery endothelial cells adapted to physiological oxygen levels

The effects of physiological oxygen tension on Nuclear Factor-E2-Related Factor 2 (Nrf2)-regulated redox signaling remain poorly understood. We report the first study of Nrf2-regulated signaling in human primary endothelial cells (EC) adapted long-term to physiological O2 (5%). Adaptation of EC to 5% O2 had minimal effects on cell ultrastructure, viability, basal redox status or HIF1-α expression. Affymetrix array profiling and subsequent qPCR/protein validation revealed that induction of select Nrf2 target genes, HO-1 and NQO1, was significantly attenuated in cells adapted to 5% O2, despite nuclear accumulation and DNA binding of Nrf2. Diminished HO-1 induction under 5% O2 was stimulus independent and reversible upon re-adaptation to air or silencing of the Nrf2 repressor Bach1, notably elevated under 5% O2. Induction of GSH-related genes xCT and GCLM were oxygen and Bach1-insensitive during long-term culture under 5% O2, providing the first evidence that genes related to GSH synthesis mediate protection afforded by Nrf2–Keap1 defense pathway in cells adapted to physiological O2 levels encountered in vivo.

Activation of NQO1 in NQO1*2 polymorphic human leukemic HL-60 cells by diet-derived sulforaphane.

BackgroundThe NAD(P)H: quinone oxidoreductase (NQO1) confers protection against semiquinones and also elicits oxidative stress. The C609T polymorphism of the NQO1 gene, designated NQO1*2, significantly reduces its enzymatic activity due to rapid degradation of protein. Since down regulation of NQO1 mRNA expression correlates with increased susceptibility for developing different types of cancers, we investigated the link between leukemia and the NQO1*2 genotype by mining a web-based microarray dataset, ONCOMINE. Phytochemicals prevent DNA damage through activation of phase II detoxification enzymes including NQO1. Whether NQO1 expression/activity in leukemia cells that carry the labile NQO1*2 genotype can be induced by broccoli-derived phytochemical sulforaphane (SFN) is currently unknown.Methods and ResultsThe ONCOMINE query showed that: (1) acute lymphoblastic leukemia and chronic myelogenous leukemia are associated with reduced NQO1 levels, and (2) under-expressed NQO1 was found in human HL-60 leukemia cell line containing the heterozygous NQO1*2 polymorphism. We examined induction of NQO1 activity/expression by SFN in HL-60 cells. A dose-dependent increase in NQO1 level/activity is accompanied by upregulation of the transcription factor, Nrf2, following 1–10 μM SFN treatment. Treatment with 25 µM SFN drastically reduced NQO1 levels, inhibited cell proliferation, caused sub-G1 cell arrest, and induced apoptosis, and a decrease in the levels of the transcription factor, nuclear factor-κB (NFκB).ConclusionsUp to 10 μM of SFN increases NQO1 expression and suppresses HL-60 cell proliferation whereas ≥ 25 μM of SFN induces apoptosis in HL-60 cells. Further, SFN treatment restores NQO1 activity/levels in HL-60 cells expressing the NQO1*2 genotype.

Abstract B33: Regulation and function of Nrf2-associated long noncoding RNA

Abstract We hypothesized that the long non coding RNA (lncRNA), Loc344887, is induced by Nrf2 activation and acts as a transcriptional co-activator of the Nrf2 target gene NQO1. Nrf2 is a transcription factor that is important in oxidative stress responses. Upon oxidative stress, Nrf2 is released from its binding partner Keap1. Nrf2 translocates to the nucleus, partners with small Maf proteins, binds to antioxidant response elements (ARE) and induces detoxifying and metabolizing genes such as NQO1, HMOX1, and GSTs. We propose that Loc344887 is acting as a co-activator of Nrf2, either directly in the Nrf2-Maf complex, or indirectly by some other downstream mechanism, in order to regulate NQO1 expression. Loc344887 has significantly reduced expression in human colon tumors when compared to normal colon tissue (TCGA database). Human HCT116 colon cancer cells and CCD841 non-cancer colonic epithelial cells replicate this expression difference between tumor and non-transformed. We used these cell lines to study the regulation and function of Loc344887. HCT116 and CCD841 cells were treated with 15 μM sulforaphane (SFN), a dietary isothiocyanate known to have chemopreventative properties due to Nrf2 activation. Loc344887 increased 40-fold in HCT116 cells, but was not induced in CCD841 cells. Loc344887 was also induced by oltipraz and tert-butylhydroquinone, known Nrf2 activators. Keap1 knockdown upregulated Loc344887, and inducibility of Loc344887 by SFN was lost when Nrf2 was knocked down using RNAi. The gene promoter of Loc344887 contains putative AREs, and chromatin immunoprecipitation assays identified putative Nrf2 and MafK binding regions. Because Loc344887 is regulated by Nrf2, we hypothesized that Loc344887 may be involved in NRF2 gene activation. Knockdown of Loc344887 caused a loss of NQO1 induction by both SFN and Keap1 knockdown; however, this effect was not seen for HMOX1. Time-course assays showed that Loc344887 is induced as early as 1 h post-SFN treatment and peaks at ~8 h, similar to HMOX1. NQO1, however, was induced much slower, peaking after 24 h. This suggests that other factor(s), such as Loc344887, needs to be present for full induction to occur. This study shows that Loc344887 is an Nrf2 target gene and is required for complete NQO1 induction, but not for HMOX1 activation. Thus, there is a differential response for Nrf2 target genes that depends, in part, on lincRNAs as co-regulators. Further studies are required to access the mechanism of NQO1 regulation. We hypothesize that once Loc344887 is induced, it acts as a scaffold for MafK, Nrf2 and/or other transcription factors on the NQO1 promoter. Loc344887 localizes primarily to the nucleus, and preliminary RNA immunoprecipitation experiments show that Loc344887 binds directly to MafK. Further studies will also test whether Loc344887 is required for regulation of other Nrf2 target genes, or if other lncRNAs are involved in Nrf2 target gene induction. Studies supported in part by Chancellor's Research Initiative funding from Texas A&M University, and by NIH grants CA090890, CA122959, P30 ES00210, P30 ES023512. Citation Format: Gavin Johnson, Laura Beaver, David E. Williams, Emily Ho, Roderick H. Dashwood. Regulation and function of Nrf2-associated long noncoding RNA. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B33.

Protective Effects of Tetrahydrocurcumin and Curcumin against Doxorubicin and Cadmium-Induced Cytotoxicity in Chang Liver Cells

Purpose: To investigate the cytoprotective effect of tetrahydrocurcumin, (THC) and curcumin (CUR) on cytotoxicity induced by doxorubicin and cadmium in Chang liver cells.Methods: Cytotoxicity was determined by sulforhodamine B assay. The expression of nuclear factorerythroid- 2-related factor 2 (Nrf2) Nrf2 regulated cytoprotecetive enzymes, glutamylcysteine ligase catalytic subunit (GCLC) and NADP (H): quinone oxidoreductase1 (NQO1) was determined by Western blot analysis. Nuclear translocation of Nrf-2 was analyzed by immunofluorescence method. The level of superoxide formation was assayed by chemiluminescence method.Results: Treatment with THC or CUR significantly induced GCLC and NQO1 expression and the nuclear translocation of Nrf2. Exposure to doxorubicin (DOX) or Cd for 24 h induced cell death of about 50 %. However, pre-treatment with THC or CUR (1 or 6 μM) for 24 h significantly increased cell survival to 80 or 90 %, respectively (p < 0.05). Similar pre-treatment with THC or CUR significantly protected against Cd-induced cell death by a level of 80 and 85 %, respectively (p < 0.05). The cytoprotective effect of these compounds was associated with suppressed DOX- and Cd-induced superoxide formation and induction of GCLC and NQO1 expression.Conclusions: THC mediates its effects by activation of Nrf2 and its regulated enzymes, GCLC and NQO1. Induction of GCLC, NQO1 protein expression and suppression of superoxide are associated with the cytoprotective effect.Keywords: Chang hepatocyte, Curcumin, Tetrahydrocurcumin, Cytoprotection, Doxorubicin, Cadmium

The aryl hydrocarbon receptor suppresses cigarette-smoke-induced oxidative stress in association with dioxin response element (DRE)-independent regulation of sulfiredoxin 1

The aryl hydrocarbon receptor (AhR) is a ubiquitously expressed receptor/transcription factor that mediates toxicological responses of environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Emerging evidence indicates that the AhR suppresses apoptosis and proliferation independent of exogenous ligands, including suppression of apoptosis by cigarette smoke, a key risk factor for chronic obstructive pulmonary disease (COPD). As cigarette smoke is a potent inducer of oxidative stress, a feature that may contribute to the development of COPD, we hypothesized that the AhR prevents smoke-induced apoptosis by regulating oxidative stress. Utilizing primary lung fibroblasts derived from AhR+/+ and AhR−/− mice as well as A549 human lung adenocarcinoma cells deficient in AhR expression (A549-AhRko), we first show that AhR−/− fibroblasts and A549-AhRko epithelial cells have a significant increase in cigarette smoke extract (CSE)-induced oxidative stress compared to wild type. CSE induced a significant increase in the mRNA expression of key antioxidant genes, including Nqo1 and Srxn1, predominantly in AhR+/+ fibroblasts, with significantly less induction in AhR−/− cells. The induction of Srxn1, but not Nqo1, was independent of dioxin-response element (DRE) binding as AhRDBD/DBD lung fibroblasts, which express an AhR incapable of binding the DRE, increased Srxn1 to a degree similar to wild-type cells in response to CSE. There was no difference in Nrf2 expression or activation based on AhR expression. Lung fibroblasts derived from COPD subjects have significantly less AhR protein expression compared with both never-smokers (Normal) and smokers (At Risk). Consequently, COPD-derived fibroblasts were less robust in their induction of both Nqo1 and Srxn1 mRNA after exposure to CSE, which also failed to activate the AhR in the COPD fibroblasts. Taken together, these results support a new role for the AhR in regulating antioxidant defense in lung structural cells, such that low AhR expression may facilitate the development or progression of COPD.

Antioxidant activity of Moringa oleifera isothiocyanates

Moringa oleifera Lam. (Brassicales) is an edible tropical tree with medicinal properties. Here, we describe a fractionation process of moringa leaves by fast centrifugal partition chromatography (FCPC) to generate polyphenol and isothiocyanate (ITC) rich fractions (Fig. 1). The polyphenol-rich fraction showed direct antioxidant activity assayed by oxygen radical absorbance capacity (ORAC), while the ITC-rich fraction demonstrated indirect antioxidant properties assayed by NQO1 activity in Hepa1c1c7 cells. Also, purified 4-[(α-l-rhamnosyloxy) benzyl] isothiocyanate (ITC-1), and 4-[(4'-O-acetyl-α-l-rhamnosyloxy)benzyl] isothio-cyanate (ITC-4) were evaluated for their ORAC and NQO1 inducer potency in comparison with sulforaphane (SF) at 5µM (Fig. 2). Both ITCs were as potent as SF in inducing NQO1 activity. These findings suggest that edible M. oleifera leaves contain a potent mixture of antioxidants that could explain its health promoting effects.

Induction of NQO1 and Neuroprotection by a Novel Compound KMS04014 in Parkinson's Disease Models.

Parkinson's disease (PD) is a progressive neurodegenerative disorder associated with a selective loss of the neurons containing dopamine (DA) in the substantia nigra pars compacta. Lines of evidence suggest that oxidative stress is a major factor contributing to the vulnerability of DA cells and that the enzyme NAD(P)H quinone oxidoreductase (NQO1) provides protection in these cells. In the present study, we report the synthesis of a novel compound KMS04014 and show that it induces NQO1 gene expression and protects DAergic neuronal cells in both cell culture and animal models of PD. In vitro, KMS04014 increased both mRNA and protein levels of NQO1 and induced nuclear translocation of Nrf2 in the DAergic neuronal cell line CATH.a. It also protected the cells against oxidative stress generated by tetrahydrobiopterin, 1-methyl-4-phenylpyridinium (MPP(+)), and H2O2. In vivo, KMS04014 attenuated the loss of tyrosine hydroxylase-immunopositive DAergic neurons in the substantia nigra and reduced degeneration of the nigral neurons and striatal fibers in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, an animal model of PD. Taken together, KMS04014 may be utilized toward development of neuroprotective therapy for PD.

Direct and indirect antioxidant activity of polyphenol- and isothiocyanate-enriched fractions from Moringa oleifera.

Moringa oleifera Lam. is a fast-growing, tropical tree with various edible parts used as nutritious food and traditional medicine. This study describes an efficient preparatory strategy to extract and fractionate moringa leaves by fast centrifugal partition chromatography (FCPC) to produce polyphenol and isothiocyanate (ITC) rich fractions. Characterization and further purification of these fractions showed that moringa polyphenols were potent direct antioxidants assayed by oxygen radical absorbance capacity (ORAC), whereas moringa ITCs were effective indirect antioxidants assayed by induction of NAD(P)H quinone oxidoreductase 1 (NQO1) activity in Hepa1c1c7 cells. In addition, purified 4-[(α-l-rhamnosyloxy)benzyl]isothiocyanate and 4-[(4'-O-acetyl-α-l-rhamnosyloxy)benzyl]isothiocyanate were further evaluated for their ORAC and NQO1 inducer potency in comparison with sulforaphane (SF). Both ITCs were as potent as SF in inducing NQO1 activity. These findings suggest that moringa leaves contain a potent mixture of direct and indirect antioxidants that can explain its various health-promoting effects.

The dermato-protective effects of lucidone from Lindera erythrocarpa through the induction of Nrf2-mediated antioxidant genes in UVA-irradiated human skin keratinocytes

This study investigated the dermato-protective effects of lucidone, a cyclopentenedione from Lindera erythrocarpa dried fruits, against UVA-induced oxidative stress and apoptosis in human keratinocytes (HaCaT). Pre-treatment with lucidone (1–4 µM) significantly protects keratinocytes from UVA (15 J/cm2)-induced cell death, excessive ROS generation, LDH release, lipid peroxidation, and DNA damage. Notably, lucidone inhibited the UVA-induced apoptosis of HaCaT cells as measured by a reduction of DNA fragmentation, mitochondria dysfunction, and Bcl-2/Bax dysregulation. Furthermore, antioxidant potential of lucidone was directly correlated with the induction of antioxidant genes, including HO-1, NQO-1, and γ-GCLC by transcriptional activation of Nrf2. Treatment with hydrogen peroxide showed cellular byproducts of UVA irradiation-induced cell death, LDH release, and ROS generation, but these phenomena were inhibited by pre-treatment of lucidone. Besides, in Nrf2 knock-down cells, lucidone failed to protect UVA-induced oxidative stress or cell death. Our findings suggest that lucidone is capable of protecting skin cells from UVA-irradiated damage.

Kaposi's Sarcoma-Associated Herpesvirus Induces Nrf2 during De Novo Infection of Endothelial Cells to Create a Microenvironment Conducive to Infection

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS) and primary effusion B-cell lymphoma. KSHV induces reactive oxygen species (ROS) early during infection of human dermal microvascular endothelial (HMVEC-d) cells that are critical for virus entry. One of the downstream targets of ROS is nuclear factor E2-related factor 2 (Nrf2), a transcription factor with important anti-oxidative functions. Here, we show that KS skin lesions have high Nrf2 activity compared to healthy skin tissue. Within 30 minutes of de novo KSHV infection of HMVEC-d cells, we observed Nrf2 activation through ROS-mediated dissociation from its inhibitor Keap1, Ser-40 phosphorylation, and subsequent nuclear translocation. KSHV binding and consequent signaling through Src, PI3-K and PKC-ζ were also important for Nrf2 stability, phosphorylation and transcriptional activity. Although Nrf2 was dispensable for ROS homeostasis, it was essential for the induction of COX-2, VEGF-A, VEGF-D, Bcl-2, NQO1, GCS, HO1, TKT, TALDO and G6PD gene expression in KSHV-infected HMVEC-d cells. The COX-2 product PGE2 induced Nrf2 activity through paracrine and autocrine signaling, creating a feed-forward loop between COX-2 and Nrf2. vFLIP, a product of KSHV latent gene ORF71, induced Nrf2 and its target genes NQO1 and HO1. Activated Nrf2 colocalized with the KSHV genome as well as with the latency protein LANA-1. Nrf2 knockdown enhanced ORF73 expression while reducing ORF50 and other lytic gene expression without affecting KSHV entry or genome nuclear delivery. Collectively, these studies for the first time demonstrate that during de novo infection, KSHV induces Nrf2 through intricate mechanisms involving multiple signal molecules, which is important for its ability to manipulate host and viral genes, creating a microenvironment conducive to KSHV infection. Thus, Nrf2 is a potential attractive target to intervene in KSHV infection and the associated maladies.

Multitarget drug design strategy: quinone-tacrine hybrids designed to block amyloid-β aggregation and to exert anticholinesterase and antioxidant effects.

We report the identification of multitarget anti-Alzheimer compounds designed by combining a naphthoquinone function and a tacrine fragment. In vitro, 15 compounds displayed excellent acetylcholinesterase (AChE) inhibitory potencies and interesting capabilities to block amyloid-β (Aβ) aggregation. The X-ray analysis of one of those compounds in complex with AChE allowed rationalizing the outstanding activity data (IC50 = 0.72 nM). Two of the compounds showed negligible toxicity in immortalized mouse cortical neurons Neuro2A and primary rat cerebellar granule neurons. However, only one of them was less hepatotoxic than tacrine in HepG2 cells. In T67 cells, both compounds showed antioxidant activity, following NQO1 induction. Furthermore, in Neuro2A, they were able to completely revert the decrease in viability induced by Aβ. Importantly, they crossed the blood-brain barrier, as demonstrated in ex vivo experiments with rats. When ex vivo results were combined with in vitro studies, these two compounds emerged to be promising multitarget lead candidates worthy of further pursuit.

Camelina sativa Defatted Seed Meal Contains Both Alkyl Sulfinyl Glucosinolates and Quercetin That Synergize Bioactivity

Camelina sativa L. Crantz is under development as a novel oilseed crop, yet bioefficacy of camelina phytochemicals is unknown. Defatted camelina seed meal contains two major aliphatic glucosinolates (GSLs), glucoarabin (9-(methylsulfinyl)nonylglucosinolate; GSL 9) and glucocamelinin (10-(methylsulfinyl)decylglucosinolate; GSL 10), with traces of a third, 11(methylsulfinyl)undecylglucosinolate and several flavonoids, mostly quercetin glycosides. In Hepa1c1c7 cells, hydrolyzed GSLs (hGSLs) 9 and 10 upregulated the phase II detoxification enzyme quinone reductase (NQO1), with no effect on cytochrome P450 (CYP) 1A1 activity. Isobologram graphs revealed synergy of NQO1 induction for a combination of hGSL 9 and quercetin. These findings suggest that defatted camelina seed meal should be evaluated for anticancer activity, similar to broccoli and other Brassicaceae family members. Interestingly, synergy of NQO1 induction was also seen for physiologically relevant doses of sulforaphane (SF) and quercetin, two key bioactives present in broccoli. This suggests that SF within broccoli may be more potent than purified SF.

Sulforaphane attenuates homocysteine-induced endoplasmic reticulum stress through Nrf-2-driven enzymes in immortalized human hepatocytes.

quinone oxidoreductase 1 (NQO1), were involved in the protective effect of SFN against injury by HCY. The ER stress-specific proteins, such as glucose-regulated protein-78 (GRP78) and protein kinase RNA (PKR)-like ER kinase (PERK), were strikingly abolished by SFN. Furthermore, Nrf-2 translocation was enhanced by SFN, which lead to the induction of TrxR-1and NQO1.

Comparison of bioactivity between sulforaphane and neoglucobrassicin hydrolysis product in murine and human cell lines (372.5)

Although the biological activity of some glucosinolate (GSL) hydrolysis products from Brassica vegetables has been extensively studied, the activity of others, such as the hydrolysis product of the indole GSL neoglucobrassicin (hNeo), is relatively unknown. Here, we aim to determine how sulforaphane (SF) and hNeo act in human and murine cell lines, regarding induction of the phase II enzyme Quinone reductase (NQO1) and inhibition of nitrite (NO) generation. Haack and colleagues reported that hNeo inhibits the effect of SF on NQO1 induction in human HepG2 cells (Haack et al., 2010). Here, we confirm this and extend our knowledge to show that hNeo causes upregulation of NQO1 alone and does not inhibit SF induction of NQO1 in murine hepatoma cells. Furthermore, this species difference is reflected in phorbol 12‐myristate 13‐acetate (PMA) induced human monocytes (THP1) and in murine RAW 264.7 macrophages, where hNeo is inhibitory in human cells but additive in murine cells, both for NQO1 induction and inhibition of NO generation. These findings suggest that the mouse may not be a robust model for evaluating the human response to bioactive compounds from Brassica.Grant Funding Source: Supported by USDA/NIFA #AG 2010‐65200

Neuroprotective effects of cheonggukjang prepared with B. amyloliquefaciens MJ1‐4 and Bacillus sp EMD17 (629.12)

The health benefits of cheonggukjang are widely accepted. However, the sensory property of cheonggukjang such as unique odor is still a challenging issue which hampers extensive consumption.In this study, we investigated the neuroprotective activity of cheonggukjang prepared with B. amyloliquefaciens MJ1‐4 (MJ1‐4) and Bacillus sp. EMD17(EMD17) which have better odor. Cheonggukjang was made with MJ1‐4 alone or combination with MJ1‐4 and EMD17 as well as with different incubation times. Aqueous ethanol (80%, v/v) extracts of each cheonggukjang prepared with MJ1‐4 alone and combination with MJ1‐4 and EMD17 were subjected to determination of neuroprotective activities, using MTT cytotoxicity and 2´,7´‐dichlorofluorescein diacetate (DCF‐DA) assays. The results showed that cheonggukjang prepared with MJ1‐4 alone and combination with MJ1‐4 and EMD17 had relatively high protective effect against glutamate‐induced cytotoxicity, decreased ROS accumulation, and caused higher NQO1 induction, compared to normal cheonggukjang in mouse hippocampal HT22 cells.Grant Funding Source: Supported by NRF‐2010‐0027204 and 2011‐0009782 and IPET (High Value‐added Food TechnologyDevelopment Program, 2012, 112066‐3), S. Korea