Dysfunction of Nrf2-regulated cellular defence system and JNK activation induced by high dose of fly Ash particles are associated with pulmonary injury in mouse lungs.

The mechanisms of the exposure to fine particulate matter (PM) as a risk factor for pulmonary injury are not fully understood. The transcription factor, NF-E2-related factor 2 (Nrf2), plays a key role in protection lung against PM insult and cancer chemoprevention. In this study, F3-S fly ash particles from a municipal waste incinerator were evaluated as a PM model. We found that F3-S triggered hierarchical oxidative stress responses involving the prolonged activation of the cytoprotective Nrf2 transcriptional program via Keap1 Cys151 modification, and c-Jun NH2-terminal kinase (JNK) phosphorylation at higher doses. In mouse lungs exposed to fly ash particles at a low dose (10-20 mg/kg), Nrf2 signalling was upregulated, while in those exposed to a high fly ash particle dose (40 mg/kg), there was significant activation of JNK, and this correlated with Nrf2 phosphorylation and the downregulation of antioxidant response element (ARE)-driven genes. The JNK inhibitor, SP600125, reversed Nrf2 phosphorylation, and downregulation of detoxifying enzymes. Silencing JNK expression in mouse lungs using adenoviral shRNA inhibited JNK activation and Nrf2 phosphorylation, promoted ARE-driven gene expression, and reduced pulmonary injury. Furthermore, we found that the 452-515 amino acid region within the Neh1 domain of Nrf2 was required for its interaction with P-JNK. We demonstrated that Nrf2 was an important P-JNK target in fly ash-induced pulmonary toxicity. JNK phosphorylated Nrf2, leading to a dysfunction of the Nrf2-mediated defence system.

NRF2 has a splicing regulatory function involving the survival of motor neuron (SMN) in non-small cell lung cancer.

The nuclear factor erythroid 2-like 2 (NFE2L2; NRF2) signaling pathway is frequently deregulated in human cancers. The critical functions of NRF2, other than its transcriptional activation, in cancers remain largely unknown. Here, we uncovered a previously unrecognized role of NRF2 in the regulation of RNA splicing. Global splicing analysis revealed that NRF2 knockdown in non-small cell lung cancer (NSCLC) A549 cells altered 839 alternative splicing (AS) events in 485 genes. Mechanistic studies demonstrated that NRF2 transcriptionally regulated SMN mRNA expression by binding to two antioxidant response elements in the SMN1 promoter. Post-transcriptionally, NRF2 was physically associated with the SMN protein. The Neh2 domain of NRF2, as well as the YG box and the region encoded by exon 7 of SMN, were required for their interaction. NRF2 formed a complex with SMN and Gemin2 in nuclear gems and Cajal bodies. Furthermore, the NRF2-SMN interaction regulated RNA splicing by expressing SMN in NRF2-knockout HeLa cells, reverting some of the altered RNA splicing. Moreover, SMN overexpression was significantly associated with alterations in the NRF2 pathway in patients with lung squamous cell carcinoma from The Cancer Genome Atlas. Taken together, our findings suggest a novel therapeutic strategy for cancers involving an aberrant NRF2 pathway.

Identification of potential biomarkers with colorectal cancer based on bioinformatics analysis and machine learning.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Biomarker discovery is critical to improve CRC diagnosis, however, machine learning offers a new platform to study the etiology of CRC for this purpose. Therefore, the current study aimed to perform an integrated bioinformatics and machine learning analyses to explore novel biomarkers for CRC prognosis. In this study, we acquired gene expression microarray data from Gene Expression Omnibus (GEO) database. The microarray expressions GSE103512 dataset was downloaded and integrated. Subsequently, differentially expressed genes (DEGs) were identified and functionally analyzed via Gene Ontology (GO) and Kyoto Enrichment of Genes and Genomes (KEGG). Furthermore, protein protein interaction (PPI) network analysis was conducted using the STRING database and Cytoscape software to identify hub genes; however, the hub genes were subjected to Support Vector Machine (SVM), Receiver operating characteristic curve (ROC) and survival analyses to explore their diagnostic values. Meanwhile, TCGA transcriptomics data in Gene Expression Profiling Interactive Analysis (GEPIA) database and the pathology data presented by in the human protein atlas (HPA) database were used to verify our transcriptomic analyses. A total of 105 DEGs were identified in this study. Functional enrichment analysis showed that these genes were significantly enriched in biological processes related to cancer progression. Thereafter, PPI network explored a total of 10 significant hub genes. The ROC curve was used to predict the potential application of biomarkers in CRC diagnosis, with an area under ROC curve (AUC) of these genes exceeding 0.92 suggesting that this risk classifier can discriminate between CRC patients and normal controls. Moreover, the prognostic values of these hub genes were confirmed by survival analyses using different CRC patient cohorts. Our results demonstrated that these 10 differentially expressed hub genes could be used as potential biomarkers for CRC diagnosis.

Quinone oxidoreductase 1 is overexpressed in gastric cancer and associated with outcome of adjuvant chemotherapy and survival.

BACKGROUND: Quinine oxidoreductase 1 (NQO1) plays a vital role in protecting normal cells against oxidative damage and electrophilic attack. It is highly expressed in many solid tumors, suggesting a role in cancer development and progression. However, the role of NQO1 in gastric cancer and its effect on cancer development and prognosis have not been fully investigated. AIM: To investigate the clinical relevance of NQO1 protein expression in gastric cancer and to explore the potential of NQO1 to serve as a prognostic biomarker and therapeutic target. METHODS: In this retrospective study, gastric cancer specimens of 175 patients who were treated between 1995 and 2011 were subjected to immunohistochemistry analyses for NQO1. The correlation of NQO1 expression with gastric cancer prognosis and clinical and pathological parameters was investigated. RESULTS: NQO1 protein was overexpressed in 59.43% (104/175) of the analyzed samples. Overexpression of NQO1 was associated with a significantly inferior prognosis. In addition, multivariate analysis suggested that NQO1 overexpression, along with tumor stage and patient age, are prominent prognostic biomarkers for gastric cancer. Moreover, NQO1 overexpression was correlated to a better response to 5-fluorouracil (5-FU)-based adjuvant chemotherapy. CONCLUSION: NQO1 overexpression is associated with a significantly poor prognosis and better response to 5-FU in patients with gastric cancer. These findings are relevant for improving therapeutic approaches for gastric cancer patients.

Transcriptome analysis of potential candidate genes and molecular pathways in colitis-associated colorectal cancer of Mkp-1-deficient mice.

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.

High-performance state feedback controller for permanent magnet synchronous motor.

Due to the outstanding characteristics of permanent magnet synchronous motor (PMSM), such as fast response speed, high torque and power density, it has been widely used in the automation industry. However, it remains a challenge to obtain high control performance caused by its dynamic complexity. In order to achieve favorable control performance, a novel state feedback control algorithm and parameter tuning method for permanent magnet synchronous motor is proposed in this paper. The development of the presented control method starts with the analyses of current state-space equation in rotor reference frame and then provides the design procedure of state feedback controller from the first-order to third-order system. An enhanced Proportional-Integral (PI) plus state feedback controller is designed, which includes the information of current, the error of current and the integral of the current error. The stability and convergence of the proposed control approach, as the extension of the conventional PI regulator, is mathematically justified in state feedback theory. The simulation and experimental results compared with the classical state feedback control method illustrate that the proposed PI plus state feedback control scheme can obtain better control performance in the presence of parameter changes and disturbance.

Integrated data analysis reveals significant associations of KEAP1 mutations with DNA methylation alterations in lung adenocarcinomas.

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.

c-Jun NH2 -Terminal Protein Kinase Phosphorylates the Nrf2-ECH Homology 6 Domain of Nuclear Factor Erythroid 2-Related Factor 2 and Downregulates Cytoprotective Genes in Acetaminophen-Induced Liver Injury in Mice.

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.

Transcriptomic profiling identifies a critical role of Nrf2 in regulating the inflammatory response to fly ash particles in mouse lung.

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.

Comparative transcriptome analysis reveals Dusp1 as a critical regulator of inflammatory response to fly ash particle exposure in mouse.

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 < 0.05. Functional enrichment and molecular pathway mapping of the DEGs specific to Dusp1-/- mice exposed to the particles revealed that the top 10 perturbed molecular pathways were associated with the immune response. Our study demonstrates the anti-inflammatory role of Dusp1 in protecting the lung against insults by fly ash particles, suggesting that Dusp1 might be a therapeutic target for the treatment of PM2.5-induced respiratory diseases.

Metformin reverses chemoresistance in non-small cell lung cancer via accelerating ubiquitination-mediated degradation of Nrf2.

BACKGROUND: The therapeutic efficacy of cisplatin-based chemotherapy for non-small cell lung cancer (NSCLC) is limited by drug resistance. In NSCLC, hyperactivation of nuclear factor erythroid 2-related factor 2 (Nrf2) counteracts oxidative stress to promote chemoresistance. Metformin-mediated downregulation of Nrf2 plays a pivotal role in overcoming drug resistance in NSCLC cells. Therefore, a deeper understanding of the molecular mechanisms of combination therapy and the role of Nrf2 in chemotherapeutic response is critical to clinical translation. METHODS: The effects of combination therapy with metformin and cisplatin on cell proliferation and apoptosis, intracellular reactive oxygen species (ROS) levels, and xenograft tumor formation were analyzed in NSCLC cells. Co-immunoprecipitation (co-IP) and Phos-tag assays were used to explore the mechanism of metformin-mediated Nrf2 suppression. Immunohistochemical (IHC) staining was performed to detect Nrf2 expression in matched tumor samples before and after neoadjuvant chemotherapy. RESULTS: Metformin was observed to synergistically augment cisplatin-induced cytotoxicity by strongly inhibiting the level of Nrf2, thereby weakening the antioxidant system and detoxification ability of Nrf2 and enhancing ROS-mediated apoptosis in NSCLC. The synergistic antitumor effect of combination therapy is blocked by treatment with the ROS scavenger N-acetyl cysteine (NAC) as well as overexpression of Nrf2 and its downstream antioxidant protein. Mechanistically, metformin extensively dephosphorylates Nrf2 by attenuating the interaction between Nrf2 and extracellular signal-regulated kinases 1/2 (ERK1/2), which then restores its polyubiquitination and accelerates its proteasomal degradation. Moreover, for the first time, an association of non-decreased Nrf2 expression in patients after neoadjuvant chemotherapy with poor survival and chemoresistance in NSCLC was revealed. CONCLUSIONS: Our findings illustrate the mechanism of metformin-mediated Nrf2 degradation through posttranslational modifications (PTMs), which weakens the ROS defense system in NSCLC. Fluctuations in Nrf2 expression have a strong predictive ability for chemotherapeutic response in neoadjuvant NSCLC patients. Targeting of the Nrf2 pathway could be a therapeutic strategy for overcoming chemoresistance, with metformin as the first choice for this strategy.

Interplay of MKP-1 and Nrf2 drives tumor growth and drug resistance in non-small cell 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.

Systematic Identification of Multi Omics-based Biomarkers in KEAP1 Mutated TCGA Lung Adenocarcinoma.

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.

Genome-wide global identification of NRF2 binding sites in A549 non-small cell lung cancer cells by ChIP-Seq reveals NRF2 regulation of genes involved in focal adhesion pathways.

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.

Synthetic Imine Resveratrol Analog 2-Methoxyl-3,6-Dihydroxyl-IRA Ameliorates Colitis by Activating Protective Nrf2 Pathway and Inhibiting NLRP3 Expression.

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.

"NRF2 addiction" in lung cancer cells and its impact on cancer therapy.

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.

Identification of novel Nrf2 target genes as prognostic biomarkers in colitis-associated colorectal cancer in Nrf2-deficient mice.

AIMS: Colorectal cancer (CRC) is the third most common cancer worldwide. Nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulator of many cytoprotective genes, plays a protective role in carcinogenesis. Recent studies have identified a specific gene-expression signature regulated by the Nrf2 pathway in lung adenocarcinoma and head-and-neck squamous cell cancer. However, the roles of Nrf2 in the development of colitis-associated colorectal cancer (CACC) have not been well characterized. Nrf2 target genes as prognostic biomarkers in CACC remain to be explored. Thus, this work aimed to identify the molecular changes that occur during mouse CACC progression to facilitate the development of diagnostic and prognostic biomarkers. MAIN METHODS: The CACC model was established using azoxymethane (AOM) with dextran sulfate sodium salt (DSS) in BALB/c mice for 3 weeks to induce colitis-associated adenoma (CAA, early stage) and for 9 weeks to induce colitis-associated carcinoma (CAC, late stage). Using RNA-sequencing and bioinformatics analyses we examined the mRNA expression profiles of 6 groups: wild-type control (WT-C), WT-CAA, WT-CAC, Nrf2 knockout control (Nrf2KO-C), Nrf2KO-CAA, and Nrf2KO-CAC. KEY FINDINGS: In the AOM/DSS model of colitis-associated tumorigenesis, Nrf2-/- mice showed a phenotype similar to WT mice, but with significantly more tumors and a much higher percentage of adenocarcinomas. We identified 47 novel Nrf2 genes via gene expression profiling of tumor samples. Survival analysis showed that 23 of these genes were biomarkers of a poor prognosis in colon cancer patients. SIGNIFICANCE: Nrf2 target genes deserve exploration as prognostic and therapeutic targets for CRC.

Enhancement of Galloylation Efficacy of Stigmasterol and ß-Sitosterol Followed by Evaluation of Cholesterol-Reducing Activity.

In this study, incorporation of gallic acid into typical phytosterols (ß-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 ß-sitosterol were confirmed by NMR, FT-IR, and HPLC-MS. Evaluation of galloyl ß-sitosterol and galloyl stigmasterol revealed their excellent antioxidant and cholesterol-reducing activities. Significant enhancement of cholesterol-reducing activity by galloylation was unveiled especially for ß-sitosterol. Galloyl ß-sitosterol had slightly better antioxidant activity at ambient temperature and better cholesterol-reducing activity. Molecular modeling suggested that a subtle difference of galloyl ß-sitosterol and galloyl stigmasterol in activities could be attributed to variation of molecular rigidity and conformation. The excellent properties of galloyl ß-sitosterol and galloyl stigmasterol suggested their great potential application in the food industry.

Mkp-1 is required for chemopreventive activity of butylated hydroxyanisole and resveratrol against colitis-associated colon tumorigenesis.

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.