Nrf2 Binding Site Research Articles

(-)-Epicatechin attenuates hepatic sinusoidal obstruction syndrome by inhibiting liver oxidative and inflammatory injury

Hepatic sinusoidal obstruction syndrome (HSOS) is a rare liver disease with considerable morbidity and mortality. (-)-Epicatechin (EPI) is a natural flavonol. This study aims to investigate the protection of EPI against monocrotaline (MCT)-induced HSOS and its engaged mechanism. Results of serum alanine/aspartate aminotransferases (ALT/AST) activities, total bilirubin (TBil) and bile acids (TBA) amounts, liver histological evaluation, scanning electron microscope observation and hepatic metalloproteinase-9 (MMP-9) expression all demonstrated the protection by EPI against MCT-induced HSOS in rats. EPI attenuated liver oxidative injury induced by MCT. EPI enhanced the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and increased the expression of its downstream antioxidant genes in rats. Molecular docking results implied the potential interaction of EPI with the Nrf2 binding site in kelch-like ECH-associated protein-1 (Keap1). The EPI-provided protection against MCT-induced HSOS was diminished in Nrf2 knock-out mice when mice were treated with MCT for 24 h but not for 48 h. However, EPI reduced the increased liver myeloperoxidase (MPO) activity, hepatic infiltration of immune cells, pro-inflammatory cytokines expression and nuclear factor κB (NFκB) activation in both wild-type and Nrf2 knock-out mice when mice were treated with MCT for 48 h. EPI reduced the elevated serum heat shock protein 60 (HSP60) content, and reversed the decreased mitochondria expression of HSP60 and Lon in livers from MCT-treated rats. Furthermore, the MCT-induced HSOS was markedly alleviated in mice treated with anti-HSP60 antibody. Taken together, this study demonstrates that EPI attenuates MCT-induced HSOS by reducing liver oxidative injury via activating Nrf2 antioxidant pathway and inhibiting liver inflammatory injury through abrogating NFκB signaling pathway initiated by HSP60.

Effects of a novel biflavonoid of Lonicera japonica flower buds on modulating apoptosis under different oxidative conditions in hepatoma cells

Background: In our previous work, we purified a novel biflavonoid named Japoflavone D (JFD) from Lonicera japonica flower buds. Biflavonoids are chemical compounds characterized by their high levels of antioxidative activity. PurposeThe present study aimed to investigate the function and molecular mechanism of JFD under different oxidative conditions in hepatoma cells. MethodsMTT assay and apoptosis assay were used to evaluate the cytotoxic effect of JFD. The activities of SOD and CAT were detected to evaluate the oxidative level. Oxidative stress was induced by H2O2 stimulation. The molecular mechanism of JFD was investigated by analyzing relative signaling pathway. ResultsJFD inhibited cell viability in all hepatoma cell lines we examined. Under quiescent conditions, JFD treatment of SMMC-7721 cells resulted in upregulation of AKT/mTOR signal pathway and ERK activities and downregulation of KEAP1/NRF2/ARE signaling axis, together with apoptosis. However, under oxidative stress, JFD played a quite different role. Treatment of JFD suppressed the activation of ERK and mTOR and activated the KEAP1/NRF2/ARE signaling axis, which is a predominant regulator of cytoprotective responses to oxidative stress, thereby lessening the damage caused by excess reactive oxygen species (ROS). A molecular docking analysis suggested that JFD may interrupt the interaction between KEAP1 and NRF2 by competitively anchoring to the NRF2 binding site on KEAP1. ConclusionThe results indicate that JFD functions as a potent antioxidant and plays dual roles in modulating apoptosis under different oxidative conditions. JFD has the potential to be developed as a protective drug for diseases related with excess ROS.

Genome-wide identification and analysis of Nrf2 binding sites – Antioxidant response elements in zebrafish

In the post-genomic era, deciphering the Nrf2 binding sites - antioxidant response elements (AREs) is an essential task that underlies and governs the Keap1-Nrf2-ARE pathway – a cell survival response pathway to environmental stresses in the vertebrate model system. AREs regulate the transcription of a repertoire of phase II detoxifying and/or oxidative-stress responsive genes, offering protection against toxic chemicals, carcinogens, and xenobiotics. In order to identify and analyze AREs in zebrafish, a pattern search algorithm was developed to identify AREs and computational tools available online were utilized to analyze the identified AREs in zebrafish. This study identified the AREs within 30 kb upstream from the transcription start site of antioxidant genes and mitochondrial genes. We report for the first time the AREs of all the known protein coding genes in the zebrafish genome. Western blotting, RT2 profiler array PCR, and qRT-PCR were performed to test whether AREs influence the Nrf2 target genes expression in the zebrafish larvae using sulforaphane. This study reveals unique AREs that have not been previously reported in the cytoprotective genes. Nine TGAG/CNNNTC and six TGAG/CNNNGC AREs were observed significantly. Our findings suggest that AREs drive the dynamic transcriptional events of Nrf2 target genes in the zebrafish larvae on exposure to sulforaphane. The identified abundant putative AREs will define the Keap1-Nrf2-ARE network and elucidate the precise regulation of Nrf2-ARE pathway in not only diseases but also in embryonic development, inflammation, and aerobic respiration. Our results help to understand the dynamic complexity of the Nrf2-ARE system in zebrafish.

Abstract B38: The role of BQ323636.1, a novel splice variant of NCOR2, in modulation of oxidative stress in breast cancer

Abstract Breast cancer is the most common type of female cancer. Reactive oxygen species (ROS) plays an important role in the signaling pathways governing survival and proliferation of breast cancer cells. Several chemotherapeutic drugs, such as taxanes, platinum compounds, and anthracyclines, induce cell death by enhancing the levels of ROS. Nuclear factor erythroid 2-related factor 2 (NRF2) is the master transcription factor regulating antioxidative responses against ROS. Overexpression of NRF2 has been found in breast cancer and it modulates oxidative stress response, leading to drug resistance in cancers. Our previous splice array study identified a novel splice variant of NCOR2, BQ323636.1 (BQ), as predictor of tamoxifen resistance in breast cancer. In the current study, we show overexpression of BQ could promote cell proliferation and protect cells from oxidative stress. In addition, overexpression of BQ could reduce the levels of ROS in breast cancer cells. We therefore hypothesized that BQ could compromise oxidative stress via the NRF2 pathway. By qPCR assay, several downstream targets of NRF2 were found to be upregulated in BQ-overexpressing cells. This suggested that NRF2 transcriptional activity could be modulated by BQ. The results from luciferase reporter assay confirmed that NCOR2 could repress the transcriptional activity via antioxidant response element (ARE), the primary binding site of NRF2 in promoter region. BQ could reverse the repressive effect of NCOR2 on ARE, suggesting that BQ might modulate NRF2 activity via NCOR2. Co-immunoprecipitation indicated NCOR2 interacted with NRF2. BQ overexpression might inhibit such an interaction. Taken together, our results suggest that BQ regulates NRF2 signaling pathway via interfering with NCOR2 activity. Our findings reveal a novel role for BQ as a modulator of NRF2 and oxidative stress in breast cancer. Citation Format: Man Hong Leung, Ho Tsoi, Ellen Pui Sum Man, Eric Wing-Fai Lam, Ui Soon Khoo. The role of BQ323636.1, a novel splice variant of NCOR2, in modulation of oxidative stress in breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B38.

Glucose starvation induces LKB1-AMPK-mediated MMP-9 expression in cancer cells

Cancer cells utilise the glycolytic pathway to support their rapid growth and proliferation. Since cells in most solid tumours are subjected to severe microenvironmental stresses including low nutrient and oxygen availability, such cancer cells must develop mechanisms to overcome these unfavourable growth conditions by metabolic adaptation. Although the liver kinase B1 (LKB1)-adenosine monophosphate-activated kinase (AMPK) signalling pathway plays a pivotal role in maintaining energy homeostasis under conditions of metabolic stress, the role of LKB1-AMPK signalling in aiding cancer cell survival and in malignant tumours has not yet been fully elucidated. We show that glucose starvation promotes cancer cell invasiveness and migration through LKB1-AMPK-regulated MMP-9 expression. Most intriguingly, triggering the LKB1-AMPK signalling pathway by glucose starvation-induced oxidative stress facilitates selective autophagy, which in turn enhances Keap1 degradation and the subsequent activation of Nrf2. Following this, Nrf2 regulates the transactivation of MMP-9 via Nrf2 binding sites in the promoter region of the MMP-9 gene. These mechanisms also contribute to the suppression of excessive oxidative stress under glucose starvation, and protect against cell death. Our data clearly shows that LKB1-AMPK signalling not only maintains energy and oxidative stress homeostasis, but could also promote cancer progression during metabolic stress conditions by MMP-9 induction.

Peroxidasin is a novel target of the redox-sensitive transcription factor Nrf2

Peroxidasin (PXDN) facilitates peroxidative reactions via utilisation of hydrogen peroxide (H2O2) and has been shown to crosslink collagen IV through sulfilimine bond formation in the presence of hypohalous acids. Aberrant PXDN expression has been associated with kidney fibrosis, cancer, congenital eye defects and various cardiovascular disorders. Since PXDN expression is modified by H2O2, we hypothesized that a major antioxidant response transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), may regulate PXDN expression. PXDN expression in response to H2O2 and the Nrf2-specific inducers, tert-butylhydroquinone (tBHQ) and sulforaphane (SFN), was determined by western blotting and immunofluorescence microscopy, in HeLa and HEK293 cells. Chromatin immunoprecipitation and luciferase reporter assays were used to investigate the regulation of PXDN by Nrf2. We observed elevated Nrf2 nuclear translocation and increased PXDN protein expression in response to H2O2, tBHQ and SFN, in both cell lines. We found that Nrf2 binds to and increases luciferase reporter gene expression from the PXDN promoter via a putative Nrf2-binding site. In summary, we show that PXDN is a novel target of the redox sensitive transcription factor Nrf2. This finding further highlights the role of PXDN in redox-related processes and compliments the currently understood pathophysiological functions of PXDN.

Apigenin prevents metabolic syndrome in high-fructose diet-fed mice by Keap1-Nrf2 pathway.

Chronic dietary high fructose leads to various kinds of undesirable metabolic effects. Apigenin, a naturally occurring plant flavone, is plentiful in fruits and vegetables. The aim of this study was to identify the protective effects of apigenin on metabolic syndrome and elucidate potential underlying mechanisms. The animal model was established by 4-weeks high fructose feeding. Insulin resistance was estimated by oral glucose tolerance test and homeostasis model assessment-insulin resistance index. Liver function was evaluated by serum AST and ALT, hepatic histopathological alternation, and lipid accumulation in the liver. The alterations of lipid profile was evaluated by TG, TC, LDL-C and HDL-C levels in serum. Administration of apigenin exerted beneficial effects through improving insulin resistance, alleviating liver injury, and inhibiting the alterations of lipid profile in high fructose-fed mice. In addition, apigenin potently facilitated the accumulation of Nrf2 nuclear translocation and accompanied by increasing HO-1 and NQO1 protein expressions, which are responsible for attenuating oxidative stress. Molecular docking results demonstrated that potential interaction of apigenin with the Nrf2-binding site in the Keap1 protein. In summary, we demonstrated that apigenin prevented high fructose-induced metabolic syndrome probably by inhibiting binding of Keap1 to Nrf2, and thus Nrf2 nuclear translocation, subsequently resulting in increased the expressions of anti-oxidative genes including HO-1 and NQO1.

Liquiritigenin and liquiritin alleviated MCT-induced HSOS by activating Nrf2 antioxidative defense system

Hepatic sinusoidal obstruction syndrome (HSOS) is a serious and life-threatening liver disease. Liquiritigenin (LG) and liquiritin (LQ) are natural flavonoids distributed in Glycyrrhizae Radix et Rhizoma (Gan-cao). This study aims to investigate the protective effect and mechanism of LG and LQ against monocrotaline (MCT)-induced HSOS. Results of serum alanine/aspartate aminotransferases (ALT/AST) activities, liver histological evaluation and scanning electron microscope observation, and hepatic metalloproteinase-9 (MMP-9) expression demonstrated that LG and LQ both alleviated HSOS induced by MCT in rats. Results of hepatic reactive oxygen species (ROS), malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), oxidized glutathione (GSSG) and reduced glutathione (GSH) contents, glutathione reductase (GR) and superoxide dismutase (SOD) activities showed that LG and LQ attenuated MCT-induced liver oxidative stress injury. Furthermore, LG and LQ were found to promote Nrf2 nuclear translocation and lead to the increased expression of Nrf2 downstream antioxidative genes. Molecule docking analysis indicated the potential interaction of LG and LQ with Nrf2 binding site in the kelch-like ECH-associated protein-1 (Keap1) protein. Finally, Nrf2 knock-out mice were used. The results showed that LG and LQ both alleviated MCT-induced HSOS in wild-type mice, but such protection was totally diminished in Nrf2 knock-out mice. In conclusion, our study revealed that LG and LQ alleviated MCT-induced HSOS by inducing the activation of hepatic Nrf2 antioxidative defense system.

NFE2-Related Transcription Factor 2 Coordinates Antioxidant Defense with Thyroglobulin Production and Iodination in the Thyroid Gland.

The thyroid gland has a special relationship with oxidative stress. While generation of oxidative substances is part of normal iodide metabolism during thyroid hormone synthesis, the gland must also defend itself against excessive oxidation in order to maintain normal function. Antioxidant and detoxification enzymes aid thyroid cells to maintain homeostasis by ameliorating oxidative insults, including during exposure to excess iodide, but the factors that coordinate their expression with the cellular redox status are not known. The antioxidant response system comprising the ubiquitously expressed NFE2-related transcription factor 2 (Nrf2) and its redox-sensitive cytoplasmic inhibitor Kelch-like ECH-associated protein 1 (Keap1) defends tissues against oxidative stress, thereby protecting against pathologies that relate to DNA, protein, and/or lipid oxidative damage. Thus, it was hypothesized that Nrf2 should also have important roles in maintaining thyroid homeostasis. Ubiquitous and thyroid-specific male C57BL6J Nrf2 knockout (Nrf2-KO) mice were studied. Plasma and thyroids were harvested for evaluation of thyroid function tests by radioimmunoassays and of gene and protein expression by real-time polymerase chain reaction and immunoblotting, respectively. Nrf2-KO and Keap1-KO clones of the PCCL3 rat thyroid follicular cell line were generated using CRISPR/Cas9 technology and were used for gene and protein expression studies. Software-predicted Nrf2 binding sites on the thyroglobulin enhancer were validated by site-directed in vitro mutagenesis and chromatin immunoprecipitation. The study shows that Nrf2 mediates antioxidant transcriptional responses in thyroid cells and protects the thyroid from oxidation induced by iodide overload. Surprisingly, it was also found that Nrf2 has a dramatic impact on both the basal abundance and the thyrotropin-inducible intrathyroidal abundance of thyroglobulin (Tg), the precursor protein of thyroid hormones. This effect is mediated by cell-autonomous regulation of Tg gene expression by Nrf2 via its direct binding to two evolutionarily conserved antioxidant response elements in an upstream enhancer. Yet, despite upregulating Tg levels, Nrf2 limits Tg iodination both under basal conditions and in response to excess iodide. Nrf2 exerts pleiotropic roles in the thyroid gland to couple cell stress defense mechanisms to iodide metabolism and the thyroid hormone synthesis machinery, both under basal conditions and in response to excess iodide.

P-171 - Genome-scale dissection of regulatory network dysfunction in tumors with high NRF2 activity

NRF2 is a cytoprotective transcription factor, yet NRF2 hyperactivating mutations promote oncogenesis. We used an integrative genomics approach to get an unbiased view of gene dysregulation in tumors with oncogenic NRF2 mutations and identified a set of 32 direct NRF2 targets that are consistently upregulated in these tumors. This NRF2 cancer signature includes canonical redox-related NRF2 targets (e.g., NQO1) as well as several genes not previously linked to NRF2; upregulation of this gene set is independent of the organ where the tumor developed. A key distinguishing feature of the NRF2 cancer signature genes is that they are regulated by high affinity NRF2 binding sites (antioxidant response elements) that fall within DNA regions with a ubiquitously permissive chromatin signature. This implies that the NRF2 cancer target genes are broadly responsive to oncogenic NRF2 because they lack the epigenetic restraints that restrict expression of most NRF2 targets. We used this NRF2 cancer signature to infer NRF2 activity across 9000 tumors and found that high NRF2 activity is associated with decreased survival in many cancer types. Additionally, network-based screening for drivers of this universal NRF2 cancer signature has identified several novel genetic modifiers of NRF2 activity.

Neuroprotective Effects of Four Phenylethanoid Glycosides on H₂O₂-Induced Apoptosis on PC12 Cells via the Nrf2/ARE Pathway.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor against oxidative stress and neurodegenerative disorders. Phenylethanoid glycosides (PhGs; salidroside, acteoside, isoacteoside, and echinacoside) exhibit antioxidant and neuroprotective bioactivities. This study was performed to investigate the neuroprotective effect and molecular mechanism of PhGs. PhGs pretreatment significantly suppressed H2O2-induced cytotoxicity in PC12 cells by triggering the nuclear translocation of Nrf2 and reversing the downregulated protein expression of heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), glutamate cysteine ligase-catalytic subunit (GCLC), and glutamate-cysteine ligase modifier subunit (GCLM). Nrf2 siRNA or HO-1 inhibitor zinc protoporphyrin (ZnPP) reduced the neuroprotective effect. PhGs showed potential interaction with the Nrf2 binding site in Kelch-like ECH-association protein 1 (Keap1). This result may support the hypothesis that PhGs are activators of Nrf2. We demonstrated the potential binding between PhGs and the Keap1-activated Nrf2/ARE pathway, and that PhGs with more glycosides had enhanced effects.

The involvement of Nrf2 antioxidant signalling pathway in the protection of monocrotaline-induced hepatic sinusoidal obstruction syndrome in rats by (+)-catechin hydrate

Hepatic sinusoidal obstruction syndrome (HSOS) is a rare and life-threatening liver disease. (+)-Catechin is a natural dietary flavonol with high antioxidant capacity. This study aims to investigate the involvement of nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant signalling pathway in the protection of (+)-catechin hydrate (CAT) against monocrotaline (MCT)-induced HSOS. Results of serum alanine/aspartate aminotransferases (ALT/AST) activities, total bilirubin (TBil) and bile acids (TBA) amounts, liver histological observation, scanning electron microscope evaluation, and hepatic metalloproteinase-9 (MMP-9) expression all demonstrated the protection of CAT against MCT-induced HSOS in rats. CAT attenuated MCT-induced liver oxidative injury in rats and the formation of cellular reactive oxygen species (ROS) in human hepatic sinusoidal endothelial cells (HHSECs). CAT-enhanced Nrf2 nuclear translocation in livers from MCT-treated rats and in HHSECs treated with MCT, and further increased the expression of Nrf2-dependent genes including catalytic or modify subunit of glutamate-cysteine ligase (GCLC/GCLM), haem oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1). Moreover, GCL inhibitor L-buthionine-(S, R)-sulfoximine (BSO), NQO1 inhibitor diminutol (Dim), and HO-1 inhibitor zinc protoporphyrin (ZnPP) all abrogated CAT-provided the protection against MCT-induced cytotoxicity in HHSECs. The results of molecular docking analysis indicated the potential interaction of CAT with the Nrf2-binding site in kelch-like ECH-associated protein-1 (Keap1) protein. In summary, this study demonstrated the critical involvement of Nrf2 antioxidant signalling pathway in CAT-provided the protection against MCT-induced HSOS.

Nrf-2 transcriptionally activates P21Cip/WAF1 and promotes A549 cell survival against oxidative stress induced by H2O2.

Cancer cells possess elevated ROS coupled with increased levels of antioxidant enzymes which render them resistant against cytotoxic chemotherapies. Therefore, an understanding of the interaction between key molecules involved in stress adaptive mechanisms is important to innovate strategies against cancer cell chemoresistance. Here, the lung adenocarcinoma cell line A549 with constitutively expressed Nrf2 was found to be more tolerant to H2O2 (0.1, 0.2, 0.5 and 1 mM) than normal lung cell line L132 or p53 null lung cancer cell line H1299. Maximum cytoprotection was observed at 0.2 mM H2O2 accompanied by a significant increase in p21, Nrf2 and antioxidant enzymes in A549 cells. The increased p21 expression was independent of p53 but dependent on Nrf2 as evident from qPCR, Western blotting and dual luciferase assays after silencing Nrf-2 and p53 genes. Highly conserved Nrf-2 binding sites were identified in p21 promoter by bioinformatics and homology modeling which was further confirmed by ChIP and reporter assay.

Natural Polyphenol Chlorogenic Acid Protects Against Acetaminophen-Induced Hepatotoxicity by Activating ERK/Nrf2 Antioxidative Pathway.

Hepatotoxicity due to acetaminophen (APAP) overdose is a leading cause of drug-induced acute liver failure in clinic. Chlorogenic acid (CGA), a dietary polyphenol, was reported to prevent APAP-induced liver injury in our previous studies. This study aims to investigate the protection provided by CGA against APAP-induced hepatotoxicity via focusing on nuclear factor erythroid 2-related factor 2 (Nrf2) and extracellular regulated protein kinases (ERK)1/2. CGA prevented APAP-induced oxidative liver injury and enhanced Nrf2 activation in mice and in hepatocytes in vitro. CGA-provided the protection against APAP-induced hepatotoxicity was diminished after the application of Nrf2 siRNA in vitro and Nrf2 knockout mice in vivo. CGA enhanced the expression of heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase-1 (NQO1), and their inhibitors reduced the protection provided by CGA against APAP-induced cytotoxicity in hepatocytes. Molecular docking results indicated the potential interaction of CGA with Nrf2 binding site in Kelch-like ECH-associating protein-1 (Keap1). CGA decreased the expression of protein phosphatases including PP2A subunit A (PP2A-A) and PP5, and induced the sustained ERK1/2 phosphorylation. Moreover, ERK1/2 inhibitors (U0126 and PD98059) and ERK2 siRNA abrogated CGA-induced Nrf2 phosphorylation and its subsequent transcriptional activation, and also reduced the protection provided by CGA against APAP-induced cytotoxicity in hepatocytes. These results suggest that CGA protects against APAP-induced hepatotoxicity by activating Nrf2 antioxidative signaling pathway via blocking the binding of Nrf2 to its inhibitor protein Keap1, and ERK1/2 plays a critical role in regulating CGA-induced Nrf2 transcriptional activation. CGA is a promising therapeutic agent for the detoxification of APAP-induced hepatotoxicity.

Nrf2 induces cisplatin resistance via suppressing the iron export related gene SLC40A1 in ovarian cancer cells.

Induction of Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) has been demonstrated to be involved in cisplatin resistance in ovarian cancer. Solute carrier family 40 member 1 (SLC40A1) is an iron exporter, which possesses many putative Nrf2 binding sites. Here we hypothesize that it may be a possible downstream gene of Nrf2. Elevated level of Nrf2 and reduced level of SLC40A1 were found in cisplatin–resistant ovarian cancer cells as compared with cisplatin-sensitive ovarian cancer cells. Exogenous knockdown of Nrf2 leaded to increased expression of SLC40A1. While overexpression of Nrf2 resulted in decreased expression of SLC40A1. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assay revealed that Nrf2 inhibited the transcription of SLC40A1. Overexpression of SLC40A1 was able to reverse cisplatin resistance induced by Nrf2, while knockdown of SLC40A1 restored cisplatin resistance and increased iron concentration. Desferal, an iron chelator, was found to overcome cisplatin resistance through iron deprivation. Its function was boosted when combined with brusatol, an Nrf2 inhibitor. Taken together, this study first demonstrated that Nrf2 could transcriptionally suppress the expression of SLC40A1. Iron overload induced by SLC40A1 resulted in cisplatin resistance in ovarian cancer. Targeting iron metabolism may be a new therapeutic strategy to reverse drug resistance in ovarian cancer treatment.

Crosstalk between NRF2 and HIPK2 shapes cytoprotective responses

Homeodomain interacting protein kinase-2 (HIPK2) is a member of the HIPK family of stress-responsive kinases that modulates cell growth, apoptosis, proliferation and development. HIPK2 has several well-characterised tumour suppressor roles, but recent studies suggest it can also contribute to tumour progression, although the underlying mechanisms are unknown. Herein, we have identified novel crosstalk between HIPK2 and the cytoprotective transcription factor NRF2. We show that HIPK2 is a direct transcriptional target of NRF2, identifying a functional NRF2 binding site in the HIPK2 gene locus and demonstrating for the first time a transcriptional mode of regulation for this kinase. In addition, HIPK2 is required for robust NRF2 responsiveness in cells and in vivo. By using both gain-of-function and loss-of-function approaches, we demonstrate that HIPK2 can elicit a cytoprotective response in cancer cells via NRF2. Our results have uncovered a new downstream effector of HIPK2, NRF2, which is frequently activated in human tumours correlating with chemoresistance and poor prognosis. Furthermore, our results suggest that modulation of either HIPK2 levels or activity could be exploited to impair NRF2-mediated signalling in cancer cells, and thus sensitise them to chemotherapeutic drugs.

Abstract LB-143: Identification of STAT3-NRF2-hypoxia as a novel reinforcing mechanism for promoting cancer stemness

Abstract Signal transducer and activator of transcription 3 (STAT3) is a transcription factor constitutively activated in many cancer types, and has been identified as a major mediator of cancer stemness (Li et.al., PNAS 2015). The Nrf2 (nuclear factor erythroid 2 [NF-E2]-related factor 2) is the reactive oxygen species (ROS) sensor and a master regulator of antioxidant gene expression. NRF2 has recently been implicated in contributing to cancer stem cell phenotypes, chemoresistance, and is associated with poor clinical prognosis. Hypoxia is considered a major feature of the tumor microenvironment as it can promote tumor progression, increased stemness characteristic, and resistance to conventional therapeutic intervention. Hypoxia can regulate a series of stress-sensor transcription factors, notably, the reactive oxygen species (ROS) sensor and response modulator NRF2. We studied NRF2 transcript and protein levels in breast cancer cell lines MCF7 and AU565 cells under both hypoxic and normoxic conditions. Our data shows that in response to STAT3 pathway activators, NRF2 is upregulated at both mRNA and protein levels under hypoxia and this upregulation is STAT3 dependent. NRF2 ChIP-Seq experiments demonstrate that both hypoxia and STAT3 stimulation can modulate NRF2 binding in MCF-7 breast cancer cells. Surprisingly, under hypoxic conditions, STAT3 activation dramatically enhances (10 fold) NRF2 binding sites across the genome. Downstream analysis of NRF2 target genes revealed a significant change from stress response to activation of gene signaling pathways involved in stemness-high cancer cells and metastatic cells under both hypoxic and STAT3 stimulation. Finally, we investigated whether the stemness inhibitor BBI-608 (Napabucasin) could reduce chemotherapy-induced NRF2 upregulation. Our results indicate that STAT3 modulates NRF2 levels and transcriptional activity, and that stemness inhibitor BBI-608 can prevent this upregulation. Our data revealed STAT3-NRF2-Hypoxia as a novel reinforcing regulatory mechanism for promoting cancer stemness and chemoresistance. Citation Format: Luz Elisa Tavera, Karen Simon, Juying Li, Katherine Geromini, Zhuo Zhang, Sarah Keates, Harry A. Rogoff, Chiang J. Li. Identification of STAT3-NRF2-hypoxia as a novel reinforcing mechanism for promoting cancer stemness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-143. doi:10.1158/1538-7445.AM2017-LB-143

Wogonin, a plant derived small molecule, exerts potent anti-inflammatory and chondroprotective effects through the activation of ROS/ERK/Nrf2 signaling pathways in human Osteoarthritis chondrocytes

Osteoarthritis (OA), characterized by progressive destruction of articular cartilage, is the most common form of human arthritis. Here, we evaluated the potential chondroprotective and anti-inflammatory effects of Wogonin, a naturally occurring flavonoid, in IL-1β-stimulated human OA chondrocytes and cartilage explants. Wogonin completely suppressed the expression and production of inflammatory mediators including IL-6, COX-2, PGE2, iNOS and NO in IL-1β-stimulated OA chondrocytes. Further, Wogonin exhibits potent chondroprotective potential by switching the signaling axis of matrix degradation from catabolic towards anabolic ends and inhibited the expression, production and activities of matrix degrading proteases including MMP-13, MMP-3, MMP-9, and ADAMTS-4 in OA chondrocytes, and blocked the release of s-GAG and COL2A1 in IL-1β-stimulated OA cartilage explants. Wogonin also elevated the expression of cartilage anabolic factors COL2A1 and ACAN in chondrocytes and inhibited the IL-1β-mediated depletion of COL2A1 and proteoglycan content in the matrix of cartilage explants. The suppressive effect of Wogonin was not mediated through the inhibition of MAPKs or NF-κB activation. Instead, Wogonin induced mild oxidative stress through the generation of ROS and depletion of cellular GSH, thereby modulating the cellular redox leading to the induction of Nrf2/ARE pathways through activation of ROS/ERK/Nrf2/HO-1-SOD2-NQO1-GCLC signaling axis in OA chondrocytes. Molecular docking studies revealed that Wogonin can disrupt KEAP-1/Nrf-2 interaction by directly blocking the binding site of Nrf-2 in the KEAP-1 protein. Genetic ablation of Nrf2 using specific siRNA, significantly abrogated the anti-inflammatory and chondroprotective potential of Wogonin in IL-1β-stimulated OA chondrocytes. Our data indicates that Wogonin exerts chondroprotective effects through the suppression of molecular events involved in oxidative stress, inflammation and matrix degradation in OA chondrocytes and cartilage explants. The study provides novel insights into the development of Nrf2 as a promising candidate and Wogonin as a therapeutic agent for the management of OA.

Synthesis and biological evaluation of novel 2-phenylquinazoline-4-amine derivatives: identification of 6-phenyl-8H-benzo[g]quinazolino[4,3-b]quinazolin-8-one as a highly potent inducer of NAD(P)H quinone oxidoreductase 1

A novel series of quinazoline compounds (2–14) incorporating biologically active heterocyclic moieties were designed and synthesized. The structure of the newly synthesized compounds was recognized on the basis of elemental analyses, IR, 1H-NMR, 13C-NMR and mass spectral data. All compounds were evaluated for their ability to induce the cytoprotective enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) using a quantitative bioassay and a docking study was performed in the Kelch domain of Keap1 obtained from the Protein Data Bank (PDB ID: 4IQK) to explore the ability of the synthesized compounds to block the Nrf2-binding site of Keap1. All of the synthesized compounds showed concentration-dependent inducer activity with potencies in the low- or sub-micromolar range. Compound 12 was the most potent inducer in this new series, with a concentration that doubles the specific activity of NQO1 (CD value) of 70 nM. The identification of this compound offers a new chemical scaffold for future development of highly potent inducers.

Caffeic acid prevents acetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense system

Acute liver failure induced by acetaminophen (APAP) overdose is the main cause of drug-induced liver injury (DILI). Caffeic acid (CA) is a phenolic compound from many natural products. This study aims to investigate the protective mechanism of CA in APAP-induced liver injury. The results of serum alanine/aspartate aminotransferases (ALT/AST), liver myeloperoxidase (MPO) activity, liver glutathione (GSH) and reactive oxygen species (ROS) levels demonstrated the protection of CA against APAP-induced liver injury. Liver histological observation provided further evidences of CA-induced protection. CA was found to reverse the APAP-induced decreased cell viability in human normal liver L-02 cells and HepG2 cells. CA also reduced the increased cellular ROS level induced by APAP in hepatocytes. The results of luciferase assay and Western-blot analysis showed that CA increased the transcriptional activation of nuclear factor erythroid 2-related factor 2 (Nrf2) in the presence of APAP. Nrf2 siRNA reduced the protection of CA against APAP-induced hepatotoxicity. CA also reversed the APAP-induced decreased mRNA and protein expression of heme oxygenase 1 (HO-1) and NAD(P)H: quinone oxidoreductase 1(NQO1). In addition, HO-1 inhibitor zinc protoporphyrin (ZnPP) and NQO1 inhibitor diminutol (Dim) reduced the protection of CA against APAP-induced hepatotoxicity. CA also decreased the expression of kelch-like ECH-associated protein-1(Keap1). Molecular docking indicated the potential interacting of CA with Nrf2 binding site in the Keap1 protein. CA had little effect on the enzymatic activity of cytochrome P450 (CYP) 3A4 and CYP2E1 in vitro. In conclusion, we demonstrated that CA prevented APAP-induced hepatotoxicity by decreasing Keap1 expression, inhibiting binding of Keap1 to Nrf2, and thus activating Nrf2 and leading to increased expression of antioxidative signals including HO-1 and NQO1.