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PDF file - 499K, Recombinant his-RXRa was unable to interact with GST-hNRF2 mutant fusion protein that lacked residues 209-316 between the Neh5 and Neh6 domains.

The transcription factor NRF2 (NFE2L2) is a pivotal activator of genes encoding cytoprotective and detoxifying enzymes that limit the action of cytotoxic therapies in cancer. NRF2 acts by binding antioxidant response elements (ARE) in its target genes, but there is relatively limited knowledge about how it is negatively controlled. Here, we report that retinoic X receptor alpha (RXRα) is a hitherto unrecognized repressor of NRF2. RNAi-mediated knockdown of RXRα increased basal ARE-driven gene expression and induction of ARE-driven genes by the NRF2 activator tert-butylhydroquinone (tBHQ). Conversely, overexpression of RXRα decreased ARE-driven gene expression. Biochemical investigations showed that RXRα interacts physically with NRF2 in cancer cells and in murine small intestine and liver tissues. Furthermore, RXRα bound to ARE sequences in the promoters of NRF2-regulated genes. RXRα loading onto AREs was concomitant with the presence of NRF2, supporting the hypothesis that a direct interaction between the two proteins on gene promoters accounts for the antagonism of ARE-driven gene expression. Mutation analyses revealed that interaction between the two transcription factors involves the DNA-binding domain of RXRα and a region comprising amino acids 209-316 in human NRF2 that had not been defined functionally, but that we now designate as the NRF2-ECH homology (Neh) 7 domain. In non–small cell lung cancer cells where NRF2 levels are elevated, RXRα expression downregulated NRF2 and sensitized cells to the cytotoxic effects of therapeutic drugs. In summary, our findings show that RXRα diminishes cytoprotection by NRF2 by binding directly to the newly defined Neh7 domain in NRF2. Cancer Res; 73(10); 3097–108. ©2013 AACR.

Supplementary Figure Legend from Generation of a Stable Antioxidant Response Element–Driven Reporter Gene Cell Line and Its Use to Show Redox-Dependent Activation of Nrf2 by Cancer Chemotherapeutic Agents

The NF-E2 p45-related factor 2 (Nrf2) regulates cytoprotective genes that contain an antioxidant response element (ARE) in their promoters. To investigate whether anticancer drugs can induce ARE-driven gene expression, we have developed a stable human mammary MCF7-derived reporter cell line called AREc32, which contains a luciferase gene construct controlled by eight copies of the cis-element. In these cells, luciferase activity was increased up to 50-fold following treatment with 50 μmol/L tert-butylhydroquinone (t-BHQ). Basal and inducible luciferase activities in AREc32 cells were increased by forced overexpression of Nrf2 and reduced by knockdown of endogenous Nrf2 expression with RNA interference. Depletion of cellular reduced glutathione (GSH) by treatment of AREc32 cells with l-buthionine-S,R-sulfoximine (BSO) did not influence basal levels of luciferase activity, but pretreatment with BSO augmented induction of luciferase activity by t-BHQ. Induction of reporter activity by t-BHQ in AREc32 cells was suppressed markedly by the antioxidants N-acetylcysteine and GSH but only modestly by vitamins C or E, suggesting that ARE-luciferase expression is induced primarily by thiol-active electrophiles rather than free radicals. The anticancer drugs cisplatin, etoposide, mitoxantrone, chlorambucil, melphalan, and carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)] weakly induced luciferase activity in AREc32 cells. Moreover, treatment of AREc32 cells with BSO immediately before exposure to anticancer drugs enhanced induction of ARE-driven luciferase activity by cisplatin, BCNU, chlorambucil, and melphalan and also induced endogenous AKR1C (AKR1C refers to AKR1C1 and AKR1C2), a target gene of Nrf2. Our findings show that Nrf2 can be activated by certain anticancer agents, and this will influence the effectiveness of chemotherapy. (Cancer Res 2006; 66(22): 10983-94)

PDF file - 572K, Cellular co-localization of ectopic GFP and RFP in HEK293T cells.

PDF file - 239K, The interaction of mNrf2 with RXRa in MCF7-RXRa cells.

PDF file - 288K, The binding of NRF2 to the ARE sites in MCF7 and MCF7-RXRo cells.

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PDF file - 97K, Includes plasmid construction data, GST pull-down assay, and immunoprecipitation data. Also includes supplementary references.

Supplementary Figure 1 from Generation of a Stable Antioxidant Response Element–Driven Reporter Gene Cell Line and Its Use to Show Redox-Dependent Activation of Nrf2 by Cancer Chemotherapeutic Agents

Four 2-phenylthiazole derivatives are synthesized, characterized, and evaluated as cholinesterase inhibitors. The structures of the 2-phenylthiazole derivatives are confirmed by 1H and 13C nuclear magnetic resonance spectroscopy, single-crystal X-ray diffraction studies, and Hirshfeld surfaces analysis. Hirshfeld surface analysis of the prepared compounds showed C–H···O intermolecular interactions. The cholinesterase inhibition activities of the synthesized compounds are tested by Ellman’s method. [2-(4-Benzyloxyphenyl)-thiazol-4-yl]-(3,5-dimethylpiperidin-1-yl)-methanone showed the best acetylcholinesterase inhibition activity with an IC50 value of 8.86 µM and the best butyrylcholinesterase inhibition activity with an IC50 value of 1.03 µM. A docking study demonstrates that the same compound interacts with the catalytic anionic site and peripheral anionic site of acetylcholinesterase and the catalytic anionic site of butyrylcholinesterase.

KEAP1 regulates the cytoprotection induced by NRF2 and has been reported to be a candidate tumor suppressor. Recent evidence has shown that mutations in several driver genes cause aberrant DNA methylation patterns, a hallmark of cancer. However, the correlation between KEAP1 mutations and DNA methylation in lung cancer has still not been investigated. In this study, we systematically carried out an integrated multi-omics analysis to explore the correlation between KEAP1 mutations and DNA methylation and its effect on gene expression in lung adenocarcinoma (LUAD). We found that most of the DNA aberrations associated with KEAP1 mutations in LAUD were hypomethylation. Surprisingly, we found several NRF2-regulated genes among the genes that showed differential DNA methylation. Moreover, we identified an 8-gene signature with altered DNA methylation pattern and elevated gene expression levels in LUAD patients with mutated KEAP1, and evaluated the prognostic value of this signature in various clinical datasets. These results establish that KEAP1 mutations are associated with DNA methylation changes capable of shaping regulatory network functions. Combining both epigenomic and transcriptomic changes along with KEAP1 mutations may provide a better understanding of the molecular mechanisms associated with the progression of lung cancer and may help to provide better therapeutic approaches.

Nuclear factor erythroid-derived-2-like 2(NRF2) regulates its downstream genes through binding with antioxidant responsive elements in their promoter regions. Hyperactivation of NRF2 results in oncogenesis and drug resistance in various cancers including non-small cell lung cancer (NSCLC). However, identification of the genes and pathways regulated by NRF2 in NSCLC warrants further investigation. We investigated the global NRF2 genomic binding sites using the high-throughput ChIP-Seq technique in KEAP1 (Kelch-like ECH-associated protein 1)-mutated A549 (NSCLC) cells. We next carried out an integrated analysis of the ChIP-Seq data with transcriptomic data from A549 cells with NRF2-knockdown and RNA-Seq data from TCGA patients with altered KEAP1 to identify downstream and clinically-correlated genes respectively. Furthermore, we applied transcription factor enrichment analysis, generated a protein-protein interaction network, and used kinase enrichment analysis. Moreover, functional annotation of NRF2 binding sites using DAVID v7 identified the genes involved in focal adhesion. Putative focal adhesion genes regulated by NRF2 were validated using qRT-PCR. Further, we selected one novel conserved focal adhesion gene regulated by NRF2-LAMC1 (laminin subunit gamma 1) and validated it using a reporter assay. Overall, the identification of NRF2 target genes paves the way for identifying the molecular mechanism of NRF2 signaling in NSCLC development and therapy. Moreover, our data highlight the complexity of the pathways regulated by NRF2 in lung tumorigenesis.

Nuclear factor erythroid 2-like factor 2 (NRF2) is a master regulator of the antioxidant enzymes and the detoxification proteins that play major roles in redox homeostasis. Although it plays a protective role against tumorigenesis, emerging evidence has shown that the NRF2 pathway is frequently altered in different types of cancer, including lung cancer. NRF2 activation influences many of the hallmarks of cancer and their signaling pathways, mainly apoptosis, proliferation, angiogenesis, metastasis, and metabolic reprogramming to establish cellular metabolic processes leading to "NRF2 addiction" in lung cancer cells. Intriguingly, constitutive activation of NRF2 promotes cancer development as well as resistance to chemotherapy and radiotherapy, and these malignant phenotypes lead to a poor prognosis in lung cancer patients. Therefore, targeted inhibition of the NRF2 together with traditional chemotherapy, radiotherapy, and immunotherapy, may be a promising approach to improving the survival rates of the NRF2-addicted lung cancer cases. Here we summarize the recent advances in NRF2-addicted lung cancer.

Alterations in KEAP1/ NF-E2 p45-related factor 2 (NFE2L2/Nrf2) signaling pathway have been reported in 23% lung adenocarcinoma patients, suggesting that deregulation of the pathway is a major cancer driver. Here we report that mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) drives tumor growth and drug resistance by up regulating transcription factor Nrf2. In non-small cell lung cancer (NSCLC) cells and xenografts, MKP-1 knockdown triggered the down-regulation of the metabolic enzymes and cytoprotective proteins, which are the target genes of Nrf2. Consequently, the cell growth was markedly inhibited with decrease of tumor metabolisms and GSH contents. Moreover, MKP-1 silencing sensitized NSCLC cells to cisplatin treatment. Mechanistically, MKP-1 inhibited the ubiquitylation of Nrf2 via a direct interaction with the transcription factor. Nrf2 was hence stabilized and its transcriptional program was activated. Notably, Nrf2 elevated MKP-1 expression at transcriptional level. In human lung adenoma tumor samples, high levels of expression of MKP-1, Nrf2, and its target gene heme oxygenase 1 were closely correlated. Thus, MKP-1 and Nrf2 form a forward feedback loop in lung cancer cells, which stabilizing and activating Nrf2 to promote anabolic metabolism and GSH biosynthesis. This study uncovers a novel role of MKP-1 in the malignant evolution of cancers.

Background The nuclear phosphatase mitogen-activate protein kinase phosphatase-1 (MKP-1) is a key negative regulator of the innate immune response through the regulation of the biosynthesis of proinflammatory cytokines. In colorectal cancer (CRC), which is induced mainly by chronic inflammation, Mkp-1 overexpression was found in addition to disturbances in Mkp-1 functions, which may play a role in cancer development in different types of tumors. However, the potential molecular mechanisms by which Mkp-1 influences CRC development is not clear. Here, we performed global gene expression profiling of Mkp-1 KO mice using RNA sequencing (RNA-seq) to explore the role of Mkp-1 in CRC progression using transcriptome analysis. Methods Azoxymethane/dextran sodium sulfate (AOM/DSS) mouse models were used to examine the most dramatic molecular and signaling changes that occur during different phases of CRC development in wild-type mice and Mkp-1 KO mice. Comprehensive bioinformatics analyses were used to elucidate the molecular processes regulated by Mkp-1. Differentially expressed genes (DEGs) were identified and functionally analyzed by Gene Ontology (GO), Kyoto Enrichment of Genes and Genomes (KEGG). Then, protein-protein interaction (PPI) network analysis was conducted using the STRING database and Cytoscape software. Results Persistent DEGs were different in adenoma and carcinoma stage (238 & 251, respectively) and in WT and MKp-1 KO mice (221& 196, respectively). Mkp-1 KO modulated key molecular processes typically activated in cancer, in particular, cell adhesion, ion transport, extracellular matrix organization, response to drug, response to hypoxia, and response to toxic substance. It was obvious that these pathways are closely associated with cancer development and metastasis. From the PPI network analyses, nine hub genes associated with CRC were identified. Conclusion These findings suggest that MKp-1 and its hub genes may play a critical role in cancer development, prognosis, and determining treatment outcomes. We provide clues to build a potential link between Mkp-1 and colitis-associated tumorigenesis and identify areas requiring further investigation. Graphical abstract

Many dietary compounds show promising protective activity against colon cancer by activating nuclear factor-erythroid 2 related factor 2 (Nrf2). Recently, we reported that mitogen-activated protein kinase phosphatase 1 (Mkp-1) exhibits crosstalk with the Nrf2 signaling pathway, protecting against intestinal inflammation. Here, we present evidence that Mkp-1 is required for the chemopreventive action of the Nrf2 activators butylated hydroxyanisole (BHA) and resveratrol (RSV). In an azoxymethane/dextran sulfate sodium model of colitis-associated tumorigenesis, Mkp-1−/− mice exhibited a phenotype similar to Nrf2−/− mice with significantly more tumors than WT mice. Tumors from Mkp-1−/− mice exhibited higher levels of macrophage infiltration than those from WT mice. This was accompanied by increased expression of nitrotyrosine and p53BP1, markers of oxidative stress and DNA damage, respectively. Moreover, dietary suppression of tumorigenesis using BHA (0.5%) or RSV (300 ppm) supplementation was achieved in WT but not in Mkp-1−/− mice. In adenomas from WT mice, the expression of Mkp-1 was markedly lower than in adjacent normal tissue, concomitant with the down-regulation of Nrf2 and its target genes. Our data revealed that Mkp-1 is required in the protective role of Nrf2 signaling against colitis-associated tumorigenesis.

Epidemiological studies have demonstrated an association between ambient particulate pollution and adverse health effects in humans. The antioxidant-responsive element (ARE) cytoprotective system mediated by the transcription factor NF-E2 p45-related factor 2 (Nrf2) serves as a primary defense against the oxidative stress triggered by particulate matter. In this study, using a cell-based ARE-reporter assay, the fine fractions of the fly ash collected from the municipal solid waste incinerators at four cities in China were examined for their ability to activate Nrf2/ARE signaling. We found that, at a non-lethal dose, all the fly ash samples were able to activate the ARE-reporter gene in a dose- and redox-dependent manner, and this was correlated with their cytotoxicity and their ability to induce DNA damage. Study of the kinetics revealed that fly ash particles elicited a prolonged activation of the ARE-reporter activity. Upon exposure to the particles, the ARE-luciferase activity significantly increased in 2 h, reached a peak at 24 h, and remained high level at 72 h. This was in contrast to the transient activation of the ARE-reporter gene triggered by the Nrf2 activators tert -butylhydroquinone and sulforaphane, while ARE-luciferase activity dropped to the basal level at 72 h from the peak at 24 h. These results demonstrate the robustness of using cell-based ARE-reporter assays to evaluate the oxidative potential of fly ash. Our novel findings suggest that the sustained activation of the Nrf2/ARE signaling pathway induced by fly ash particles perturbs cellular redox homeostasis, which in turn contributes to toxicity.

Mutations in KEAP1 and/or NRF2 genes have been identified across many cancers and the dysregulation of the NRF2 pathway due to these mutations leads to drug and radioresistance in several cancers. Identification of biomarkers associated with these mutations allows the researchers and clinicians to identify the personalized medicine and quicker diagnosis. In this current study, we carried out an integrated, multi-omics, multi-database analysis of exome, transcriptomics data's of KEAP1 mutated TCGA- Lung adenocarcinoma (LUAD) patients against non-mutated counterparts. Finally, we discovered the gene signature associated with KEAP1 mutations, prognostic genes which were highly correlated with the upregulation of the NRF2 pathway in the KEAP1 mutated LUAD patients. Our finding might be useful to identify the early diagnosis of KEAP1 mutated LUAD patients.

BackgroundKEAP1-NRF2 pathway alterations were identified in many cancers including, esophageal cancer (ESCA). Identifying biomarkers that are associated with mutations in this pathway will aid in defining this cancer subset; and hence in supporting precision and personalized medicine. MethodsIn this study, 182 tumor samples from the Cancer Genome Atlas (TCGA)-ESCA RNA-Seq V2 level 3 data were segregated into two groups KEAP1-NRF2-mutated (22) and wild-type (160).The two groups were subjected to differential gene expression analysis, and we performed Gene Set Enrichment Analysis (GSEA) to determine all significantly affected biological pathways. Then, the enriched gene set was integrated with the differentially expressed genes (DEGs) to identify a gene signature regulated by the KEAP1-NRF2 pathway in ESCA. Furthermore, we validated the gene signature using mRNA expression data of ESCA cell lines provided by the Cancer Cell Line Encyclopedia (CCLE). The identified signature was tested in 3 independent ESCA datasets to assess its prognostic value.ResultsWe identified 11 epithelial-mesenchymal transition (EMT) genes regulated by the KEAP1-NRF2 pathway in ESCA patients. Five of the 11 genes showed significant over-expression in KEAP1-NRF2-mutated ESCA cell lines. In addition, the over-expression of these five genes was significantly associated with poor survival in 3 independent ESCA datasets, including the TCGA-ESCA dataset.ConclusionAltogether, we identified a novel EMT 5-gene signature regulated by the KEAP1-NRF2 axis and this signature is strongly associated with metastasis and drug resistance in ESCA. These 5-genes are potential biomarkers and therapeutic targets for ESCA patients in whom the KEAP1-NRF2 pathway is altered.

BackgroundKEAP1-NRF2 pathway alterations were identified in many cancers including, esophageal cancer (ESCA). Identifying biomarkers that are associated with mutations in this pathway will aid in defining this cancer subset; and hence in supporting precision and personalized medicine. MethodsIn this study, 182 tumor samples from the Cancer Genome Atlas (TCGA)-ESCA RNA-Seq V2 level 3 data were segregated into two groups KEAP1-NRF2-mutated (22) and wild-type (160).The two groups were subjected to differential gene expression analysis and we performed Gene Set Enrichment Analysis (GSEA). Then, the enriched gene set was integrated with the differentially expressed genes (DEGs) to identify a gene signature regulated by the KEAP1-NRF2 pathway in ESCA. Furthermore, we validated the gene signature using mRNA expression data of ESCA cell lines provided by the Cancer Cell Line Encyclopedia (CCLE). The identified signature was tested in 3 independent ESCA datasets to assess its prognostic value.ResultsWe identified 11 epithelial-mesenchymal transition (EMT) genes regulated by the KEAP1-NRF2 pathway in ESCA patients. Five of the 11 genes showed significant over-expression in KEAP1-NRF2-mutated ESCA cell lines. In addition, over-expression of these five genes was significantly associated with poor survival in 3 independent ESCA datasets, including the TCGA-ESCA dataset.ConclusionAltogether, we identified a novel EMT 5-gene signature regulated by the KEAP1-NRF2 axis and this signature is strongly associated with metastasis and drug resistance in ESCA. These 5-genes are potential biomarkers and therapeutic targets for ESCA patients in whom the KEAP1-NRF2 pathway is altered.

Cancer is one of the leading causes of death worldwide. Chemotherapy and radiotherapy are the conventional primary treatments for cancer patients. However, most of cancer cells develop resistance to both chemotherapy and radiotherapy after a period of treatment, besides their lethal side-effects. This motivated investigators to seek more effective alternatives with fewer side-effects. In the last few years, resveratrol, a natural polyphenolic phytoalexin, has attracted much attention due to its wide biological effects. In this concise review, we highlight the role of resveratrol in the prevention and therapy of cancer with particular focus on colorectal and skin cancer. Also, we discuss the molecular mechanisms underlying its chemopreventive and therapeutic activity. Finally, we highlight the problems associated with the clinical application of resveratrol and how attempts have been made to overcome these drawbacks.

Inflammatory bowel disease (IBD) is associated with intense oxidative stress, contributes to colonic damage and tumorigenesis. Mitogen-activated protein kinase phosphatase 1 (Mkp-1) is an essential negative regulator of the innate immune response. However, its role in colitis, and its association with the nuclear factor-erythroid 2 related factor 2 (Nrf2), a master regulator of cytoprotection program against oxidative stress, in inflammatory response, is elusive. In this study, we found that increased expression of Mkp-1, Nrf2, and heme oxygenase 1 (Ho-1) was correlated in colonic tissues in patients with ulcerative colitis and Crohn's disease, as well as wild-type mice with colitis induced by dextran sodium sulfate (DSS). Mkp-1-/- mice were more susceptible to DSS-induced colitis with more severe crypt injury and inflammation. Mechanistically, directly interacting with the DIDLID motif of Nrf2, Mkp-1 increased Nrf2 stability and positively regulated the constitutive and lipopolysaccharide (LPS)-inducible Nrf2/Ho-1 expression. Conversely, upon exposure to LPS, Nrf2 activated Mkp-1 transcription through the antioxidant response elements in the promoter of Mkp-1. Our results revealed a novel link between Mkp-1 and Nrf2 signaling pathways in protecting against colonic inflammation. Mkp-1 might be a therapeutic target for IBD.

A series of novel bis-benzothiazole derivatives with head-to-head orientation were designed, synthesized, and evaluated for their anti-proliferative activities on U937, HL60, and HeLa cells. A significant part of the targets exhibited considerable in vitro anticancer activity, and some of them were more potent than the reference 5-FU. Molecular modeling, fluorescence, and viscosimetry studies revealed that these compounds could bind into the minor groove of DNA. Amongst them, the most active compound 7 with IC50 in a range of 6.76 to 8.67 μM against the tested three cell lines, which was 1.46–2.47 and 3.48–4.93 times more potent than 5-FU and Hoechst 33258, respectively, warrant further investigations.

Colorectal cancer (CRC) is the second most commonly diagnosed and one of the leading causes of mortality in cancer patients. CRC occurs due to the complications of chronic inflammatory disorders of the colon. Although the clinical features and molecular mechanisms associated with the CRC is not clear, but discovery of early diagnostic markers and the emergence of safe and effective chemopreventive agents plays a key role in preventing CRC. The purpose of this review is to elucidate and emphasize the different pathways associated with CRC and summarizes the compounds targeting the prevention of CRC. Researchers around the world have identified many potential phytochemical and synthetic compounds for the prevention and treatment of CRC. Based on previous and recent findings, we have reported series of natural and synthetic chemopreventive compounds which affects the specific pathways in CRC. These active chemicals can block CRC-associated signaling pathways, thereby reducing the risk of CRC. This review summarized the CRC pathogenesis, risk factors, difference between sporadic colorectal cancer (SCC) and colitis-associated colorectal cancer (CAC), CRC-related signaling pathways, and chemopreventive compounds against CRC in vitro and in vivo studies. Understanding the mechanistic effects of these compounds is critical for the prevention and therapeutic usage in patients with a high risk of CRC.

The NF-E2 p45-related factor 2 (NRF2)-Kelch-like ECH-associated protein 1 signaling pathway plays an important role in cytoprotection. In acute promyelocytic leukemia, fusion of the promyelocytic leukemia protein (PML) with retinoic acid receptor alpha (RARα) results in an oncogene, PML-RARα (PR). Although previous studies have shown that both RARα and PML inhibit NRF2 activity, how PR regulates NRF2 has not been reported. Here, we discovered that PR-S, the short isoform of PR, potentiates NRF2 activity in a tert-butylhydroquinone (tBHQ) concentration-dependent manner. Furthermore, PR-S colocalized with NRF2 in HeLa and HEK293T cells. The association of PR-S and NRF2 is mediated by the DNA-binding domains of RARα and the Neh7 domain of NRF2. Our results define a novel function of PR-S as a NRF2-transcriptional co-activator.

// Akhileshwar Namani 1, * , Qin Qin Cui 1, * , Yihe Wu 2 , Hongyan Wang 1, 3 , Xiu Jun Wang 3 and Xiuwen Tang 1 1 Department of Biochemistry, Zhejiang University, Hangzhou 310058, PR China 2 Department of Thoracic Surgery, First Affiliated Hospital, Zhejiang University, Hangzhou 310058, PR China 3 Department of Pharmacology, School of Medicine, Zhejiang University, Hangzhou 310058, PR China * These authors have contributed equally to this work Correspondence to: Xiuwen Tang, email: xiuwentang@zju.edu.cn Keywords: KEAP1, NRF2, NSCLC, biomarker, metabolism Received: February 02, 2017 Accepted: June 19, 2017 Published: July 18, 2017 ABSTRACT Mutations in Kelch-like ECH-associated protein 1 (KEAP1) cause the aberrant activation of nuclear factor erythroid-derived 2-like 2 (NRF2), which leads to oncogenesis and drug resistance in lung cancer cells. Our study was designed to identify the genes involved in lung cancer progression targeted by NRF2. A series of microarray experiments in normal and cancer cells, as well as in animal models, have revealed regulatory genes downstream of NRF2 that are involved in wide variety of pathways. Specifically, we carried out individual and combinatorial microarray analysis of KEAP1 overexpression and NRF2 siRNA-knockdown in a KEAP1 mutant-A549 non-small cell lung cancer (NSCLC) cell line. As a result, we identified a list of genes which were mainly involved in metabolic functions in NSCLC by using functional annotation analysis. In addition, we carried out in silico analysis to characterize the antioxidant responsive element sequences in the promoter regions of known and putative NRF2-regulated metabolic genes. We further identified an NRF2-regulated metabolic gene signature (NRMGS) by correlating the microarray data with lung adenocarcinoma RNA-Seq gene expression data from The Cancer Genome Atlas followed by qRT-PCR validation, and finally showed that higher expression of the signature conferred a poor prognosis in 8 independent NSCLC cohorts. Our findings provide novel prognostic biomarkers for NSCLC.

The adsorption capacities of a heterometallic metal–organic framework (CPM-200-In/Mg) to VOCs (HCHO, C2H4, CH4, C2H2, C3H8, C2H6, C2H3Cl, C2H2Cl2, CH2Cl2 and CHCl3) and some inorganic gas molecules (HCN, SO2, NO, CO2, CO, H2S and NH3), as well as its selectivity in ternary mixture systems of natural gas and post-combustion flue gas are theoretically explored at the grand canonical Monte Carlo (GCMC) and density functional theory (DFT) levels. It is shown that CPM-200-In/Mg is suitable for the adsorption of VOCs, particularly for HCHO (up to 0.39 g g−1 at 298 K and 1 bar), and the adsorption capacities of some inorganic gas molecules such as SO2, H2S and CO2 match well with the sequence of their polarizability (SO2 > H2S > CO2). The large adsorption capacities of HCN and HCHO in the framework result from the strong interaction between adsorbates and metal centers, based on analyzing the radial distribution functions (RDF). Comparing C2H4 and CH4 molecules interacting with CPM-200-In/Mg by VDW interaction, we speculate that the high adsorption capacities of their chlorine derivatives in the framework could be due to the existence of halogen bonding or strong electrostatic and VDW interactions. It is found that the basic groups, including –NH2, –N and –OH, can effectively improve both the adsorption capacities and selectivity of CPM-200-In/Mg for harmful gases. Note that the adsorption capacity of CPM-200-In/Mg–NH2 (site 2) (245 cm3 g−1) for CO2 exceeded that of MOF-74-Mg (228 cm3 g−1) at 273 K and 1 bar and that for HCHO can reach 0.41 g g−1, which is almost twice that of 438-MOF and nearly 45 times of that in active carbon. Moreover, for natural gas mixtures, the decarburization and desulfurization abilities of CPM-200-In/Mg–NH2 (site 2) have exceeded those of the MOF-74 series, while for post-combustion flue gas mixtures, the desulfurization ability of CPM-200-In/Mg–NH2 (site 2) is still comparable to those of the MOF-74 series at 303 K and 4 MPa. We hope that the current theoretical study could guide experimental research in the future.

Stearyl coenzyme A desaturase enzyme 1 (SCD1) is a key enzyme that catalyzes the conversion of saturated fatty acids (SFA) into monounsaturated fatty acids (MUFA) and plays a vital role in lipid metabolism of tumor cells. SCD1 is overexpressed in a variety of malignant tumors, and its related inhibitors showed significant anti-tumor activity in vitro and in vivo experiments, which is a new target for tumor therapy. The focus of this study is to identify novel SCD1 inhibitors from natural products through computer simulations. First, 176,602 compounds from natural product databases were virtually screened. By molecular dynamics (MD) simulations, the ligand-protein interactions of 5 compounds with high docking manifestation were analyzed accurately. Then, MM-GBSA and MM-PBMA methods were used to verify the results. Finally, ADMET prediction was performed for the 5 compounds. As a result, two natural products with potential inhibition towards SCD1 were identified, which had the excellent docking manifestation, binding mode within SCD1 pocket and stability during molecular dynamics simulation. This study provides a meaningful model for the development and optimization of new inhibitors and anti-tumor drugs targeting SCD1.

Exposure to outdoor concentrations of fine particulate matter (PM2.5) is a leading global health concern. Waste incineration emission has been recognized as a potential major contributor of ambient PM2.5. Respiratory inflammation is a central feature induced by PM2.5 exposure by inhalation. However, the molecular mechanisms are not fully understood. Dual-specificity phosphatase 1 (Dusp1) plays an instrumental role in the regulation of airway inflammation. In this study, fly ash particles (20 mg/kg BW) collected from a municipal waste incinerator in China were given to BALB/c wild-type (WT) and Dusp1−/− mice by intranasal administration daily for three consecutive days. While these particles induced mild inflammation in both genotypes, a significantly higher level of serum interleukin-6 (665 pg/ml) was measured in Dusp1−/− mice challenged with fly ash particles than in their WT counterparts. Genome-wide transcriptome profiling of pulmonary coding genes in response to the exposure were performed in both genotypes by RNA sequencing. We identified 487 differentially-expressed genes (DEGs) in fly ash-challenged Dusp1−/− mice versus their WT counterparts with a log2fold-change >1.5 and p

Exposure to combustion-derived nanoparticles is recognized as a major health hazard, but the molecular responses are still insufficiently described. The transcription factor erythroid 2-related factor 2 (Nrf2, also known as NFE2L2) is a master regulator of the pulmonary defense system against insults by particulate matter. However, its downstream molecular processes are not fully characterized. In the current study, BALB/c wild-type (WT) and Nrf2−/− mice were exposed by intranasal administration to fly ash particles (F3-S; 20 mg/kg BW), which were collected from a municipal waste incinerator in China, for three consecutive days. Using a comparative transcriptomics approach, the pulmonary global gene expression profiles to F3-S exposure were characterized for both genotypes. The preponderance of the differentially-expressed genes (DEGs) in WT mice induced by the fly ash particles, was related to inflammation. Functional enrichment and molecular pathway mapping of the DEGs specific to Nrf2−/− mice exposed to the particles revealed that all of the top 10 perturbed molecular pathways were associated with the inflammatory response. Our study identified a transcriptional signature related to the initial pulmonary injury in mouse upon fly ash exposure, and suggests an anti-inflammatory role of Nrf2 in protecting the lung against such exposure.

In this study, incorporation of gallic acid into typical phytosterols (β-sitosterol and stigmasterol) through Steglich esterification was optimized employing the protection and deprotection strategy. A novel mechanism leading to side esterification was discovered. Complication of the phenolic hydroxyl groups and side reactions were successfully reduced under the optimized conditions. The structural identity and purity of galloyl stigmasterol and galloyl β-sitosterol were confirmed by NMR, FT-IR, and HPLC-MS. Evaluation of galloyl β-sitosterol and galloyl stigmasterol revealed their excellent antioxidant and cholesterol-reducing activities. Significant enhancement of cholesterol-reducing activity by galloylation was unveiled especially for β-sitosterol. Galloyl β-sitosterol had slightly better antioxidant activity at ambient temperature and better cholesterol-reducing activity. Molecular modeling suggested that a subtle difference of galloyl β-sitosterol and galloyl stigmasterol in activities could be attributed to variation of molecular rigidity and conformation. The excellent properties of galloyl β-sitosterol and galloyl stigmasterol suggested their great potential application in the food industry.

Background and aims Acetaminophen (APAP) overdose induces severe liver injury and hepatic failure. While the activation of c-Jun NH2 -terminal kinase (JNK) has been implicated as a mechanism in APAP-induced liver injury, the hepatic defense system controlled by nuclear factor erythroid 2-related factor 2 (Nrf2) plays a central role in the mitigation of APAP toxicity. However, the link between the two signaling pathways in APAP-induced liver injury (AILI) remains unclear. Approach and results In this study, we demonstrated that the activation of JNK in mouse liver following exposure to APAP was correlated with the phosphorylation of Nrf2 and down-regulation of the antioxidant response element (ARE)-driven genes, NAD(P)H:quinone dehydrogenase 1, glutathione S-transferase α3, glutathione S-transferase M1, glutathione S-transferase M5, and aldo-keto reductase 1C. The JNK inhibitor, SP600125, or knockdown of JNK by infection of adenovirus expressing JNK small interfering RNA, ameliorated the APAP induced liver toxicity, and inhibited the phosphorylation of Nrf2 and down-regulation of detoxifying enzymes by stabilizing the transcription factor. Mechanistically, JNK antagonized Nrf2- and ARE-driven gene expression in a Kelch-like ECH-associated protein 1-independent manner. Biochemical analysis revealed that phosphorylated JNK (P-JNK) directly interacted with the Nrf2-ECH homology (Neh) 1 domain of Nrf2 and phosphorylated the serine-aspartate-serine motif 1 (SDS1) region in the Neh6 domain of Nrf2. Conclusions Mass spectrometric analysis identified serine 335 in the SDS1 region of mNrf2 as the major phosphorylation site for modulation of Nrf2 ubiquitylation by P-JNK. This study demonstrates that Nrf2 is a target of P-JNK in AILI. Our finding may provide a strategy for the treatment of AILI.

Resveratrol (RSV) is a naturally occurring polyphenol that exhibits pleiotropic health benefits, including anticolitis and colon cancer-protective activity. Recently, we identified the novel imine RSV analog (IRA), 2-methoxyl-3,6-dihydroxyl-IRA 3,4,5,4-tetramethoxystilbene (C33), as a putative activator of nuclear factor erythroid 2-related factor 2 (Nrf2). The present study was designed to evaluate the ability of C33 to activate the Nrf2 signaling pathway and its anticolitis effect in comparison to RSV. The anticolitis action of C33 was assessed in a mouse model of colitis induced by dextran sulfate sodium (DSS). The effect of C33 on the Nrf2 signaling pathway was examined in vitro and in vivo. Compared to RSV, C33 triggered a more dramatic increase in the expression of genes downstream of Nrf2 in LS174T cells as well as in the small intestine and colon of wild-type (WT) mice. Correlated with its superior ability to activate the cytoprotective Nrf2 pathway, C33 was significantly better in ameliorating DSS-induced colitis by improving the inflammation score, as well as downregulating the markers of inflammation in WT mice. Moreover, induction of the NOD-like receptors family pyrin domain containing 3 (NLRP3) inflammasome by colitis was also significantly inhibited by the IRA. Nrf2 knockout completely abolished the effects of C33, indicating that Nrf2 is the important mechanistic target of C33 in vivo. In conclusion, the novel IRA, C33, has stronger anticolitis effects than RSV. Further studies are warranted to evaluate C33 as a potential therapeutic agent for inflammatory bowel disease and cancer chemoprevention.

Twelve carbazole-coumarin hybrids were synthesized and biologically evaluated as dual binding site acetylcholinesterase inhibitors. The structures of the carbazole-coumarin hybrids were confirmed by IR, NMR, HRMS and single-crystal X-ray diffraction studies. The compound 3j had the crystal system of triclinic and the space group of P-1. The cholinesterase inhibitory activity of synthesized compounds was measured using colorimetric Ellman's method. Compound 3h exhibited good acetylcholinesterase (AChE) inhibitory activity (IC50 value of 6.72 μM) and a high selectivity over butyrylcholinesterase (BuChE). Compound 3k showed the best BuChE inhibitory activity with the IC50 of 0.50 μM. The SAR studies revealed that the linker length played a crucial role in determining AChE inhibitory activity and the structure of the coumarin moieties affected the BuChE-inhibition activities of the hybrids. Molecular docking study of compound 3h indicated that it interacts with the crucial amino acids present at the catalytic active site and peripheral anionic site of AChE. Compound 3h would be a promising drug candidate to treat AD as a selective and dual binding site inhibitor of AChE.

Butylated hydroxyanisole (BHA) is widely used as an antioxidant and preservative in food, food packaging and medicines. Its chemopreventive properties are attributing to its ability to activate the transcription factor NF-E2 p45-related factor 2 (Nrf2), which directs central genetic programs of detoxification and protection against oxidative stress. This study was to investigate the histological changes of Nrf2 and its regulated phase II enzymes Nqo1, AKR1B8, and Ho-1 in wild-type (WT) and Nrf2(-/-) mice induced by BHA. The mice were given a 200mg/kg oral dose of BHA daily for three days. Immunohistochemistry revealed that, in the liver from WT mice, BHA increased Nqo1 staining in hepatocytes, predominately in the pericentral region. In contrast, the induction of AKR1B8 appeared mostly in hepatocytes in the periportal region. The basal and inducible Ho-1 was located almost exclusively in Kupffer cells. In the small intestine from WT mice, the inducible expression patterns of Nqo1 and AKR1B8 were nearly identical to that of Nrf2, with more intense staining in the villus than that the crypt. Conversely, Keap1 was more highly expressed in the crypt, where the proliferative cells reside. Our study demonstrates that BHA elicited differential expression patterns of phase II-detoxifying enzymes in the liver and small intestine from WT but not Nrf2(-/-) mice, demonstrating a cell type specific response to BHA in vivo.

For a long time, the structural basis of TXA2 receptor is limited due to the lack of crystal structure information, till the release of the crystal structure of TXA2 receptor, which deepens our understanding about ligand recognition and selectivity mechanisms of this physiologically important receptor. In this research, we report the successful implementation in the discovery of an optimal pharmacophore model of human TXA2 receptor antagonists through virtual screening. Structure-based pharmacophore models were generated based on two crystal structures of human TXA2 receptor (PDB entry 6IIU and 6IIV). Docking simulation revealed interaction modes of the virtual screening hits against TXA2 receptor, which was validated through molecular dynamics simulation and binding free energy calculation. ADMET properties were also analyzed to evaluate the toxicity and physio-chemical characteristics of the hits. The research would provide valuable insight into the binding mechanisms of TXA2 receptor antagonists and thus be helpful for designing novel antagonists.

The adsorption capacities of a heterometallic metal-organic framework (CPM-200-In/Mg) to VOCs (HCHO, C