NQO1 Activity Research Articles

Berberine sensitizes immune checkpoint blockade therapy in melanoma by NQO1 inhibition and ROS activation

Unprecedented progress in immune checkpoint blockade (ICB) therapy has been made in cancer treatment. However, the response to ICB therapy is limited to a small subset of patients. The development of ICB sensitizers to improve cancer immunotherapy outcomes is urgently needed. Berberine (BBR), a well-known phytochemical compound isolated from many kinds of medicinal plants such as Berberis aristata, Coptis chinensis, and Phellondendron chinense Schneid, has shown the ability to inhibit the proliferation, invasion and metastasis of cancer cells. In this study, we investigated whether BBR can enhance the therapeutic benefit of ICB for melanoma, and explored the underlying mechanisms involved. The results showed that BBR could sensitize ICB to inhibit tumor growth and increased the survival rate of mice. Moreover, BBR stimulated intracellular ROS production partially by inhibiting NQO1 activity, which induced immunogenic cell death (ICD) in melanoma, elevated the levels of damage-associated molecular patterns (DAMPs), and subsequently activated DC cells and CD8 + T cells in vitro and in vivo. In conclusion, BBR is a novel ICD inducer. BBR could enhance the therapeutic benefit of ICB for melanoma. These effects were partially mediated through the inhibition of NQO1 and ROS activation.

Daphnetin induces ferroptosis in ovarian cancer by inhibiting NAD(P)H:Quinone oxidoreductase 1 (NQO1)

Ferroptosis, an emerging nonapoptotic, modulated cell death process characterized by iron accumulation and subsequent lipid peroxidation, has been intimately implicated in the development and progression of ovarian cancer (OC). Daphnetin (Daph), a natural product isolated from Daphne Korean Nakai, exhibits anticancer efficacy against various solid tumors. However, the specific role and potential mechanism underlying Daph-mediated modulation of ferroptosis in OC cells remain elusive. This study aims to analyze the proferroptotic impacts of Daph on OC cells and to further explore the underlying mechanisms involved. We used CCK-8, wound healing and Transwell assays to assess whether Daph can inhibit the proliferation and migration of OC cells. Additionally, transmission electron microscopy (TEM), iron measurement, reactive oxygen species (ROS) analysis, lipid peroxidation assays, qRT-PCR and western blotting were utilized to evaluate the impact of Daph on ferroptosis and elucidate the potential underlying mechanism. Furthermore, RNA sequencing analysis, molecular docking analysis, cellular thermal shift assays (CETSAs) and NQO1 activity assays were used to predict and validate the binding and mechanistic interactions between Daph and NQO1. Subcutaneous tumorigenesis models were utilized to examine the effectiveness of Daph (and/or cisplatin) in vivo. Daph exerted antitumor effects by inducing the death and suppressing the migration of A2780 and SKOV3 cells. Further, Daph induced ferroptosis in OC cells, as evidenced by the accumulation of intracellular ferrous iron (Fe2+), ROS and lipid peroxides, as well as the decreases in the glutathione/oxidized glutathione disulfide (GSH/GSSG) ratio and the expression of ferroptosis indicators (SLC7A11 and GPX4). RNA sequencing and molecular docking analyses revealed that the direct interaction between NQO1 and Daph reduced both the activity and expression of NQO1. Importantly, NQO1 overexpression effectively alleviated the effects of Daph on proliferation, migration, and ferroptosis in vitro and in vivo. Interestingly, we also found that combination treatment with Daph, a negative regulator of NQO1, and cisplatin synergistically induced cytotoxicity in OC cells. Our findings are the firstly demonstrated that Daph acts as a novel ferroptosis inducer in OC cells by specifically targeting NQO1 and is thus a promising candidate agent for OC treatment.

Antiviral, anti-inflammatory and antioxidant effects of curcumin and curcuminoids in SH-SY5Y cells infected by SARS-CoV-2

COVID-19, caused by SARS-CoV-2, affects neuronal cells, causing several symptoms such as memory loss, anosmia and brain inflammation. Curcuminoids (Me08 e Me23) and curcumin (CUR) are derived from Curcuma Longa extract (EXT). Many therapeutic actions have been linked to these compounds, including antiviral action. Given the severe implications of COVID-19, especially within the central nervous system, our study aims to shed light on the therapeutic potential of curcuminoids against SARS-CoV-2 infection, particularly in neuronal cells. Here, we investigated the effects of CUR, EXT, Me08 and Me23 in human neuroblastoma SH-SY5Y. We observed that Me23 significantly decreased the expression of plasma membrane-associated transmembrane protease serine 2 (TMPRSS2) and TMPRSS11D, consequently mitigating the elevated ROS levels induced by SARS-CoV-2. Furthermore, Me23 exhibited antioxidative properties by increasing NRF2 gene expression and restoring NQO1 activity following SARS-CoV-2 infection. Both Me08 and Me23 effectively reduced SARS-CoV-2 replication in SH-SY5Y cells overexpressing ACE2 (SH-ACE2). Additionally, all of these compounds demonstrated the ability to decrease proinflammatory cytokines such as IL-6, TNF-α, and IL-17, while Me08 specifically reduced INF-γ levels. Our findings suggest that curcuminoid Me23 could serve as a potential agent for mitigating the impact of COVID-19, particularly within the context of central nervous system involvement.

Identification of Pheophytin a and Hydroxy Pheophytin a from Rang Chuet (Thunbergia laurifolia Linn.) as Potent NQO-1 Inducers in Liver Cells.

Thunbergia laurifolia Linn. (Rang Chuet, RC), a Thai medicinal plant, possesses various bioactive compounds with potential health benefits. This study aimed to identify detoxifying compounds within RC crude extract. RC leaves were extracted using the Soxhlet method with chloroform. Total carotenoids, chlorophylls, extract yield, total phenolic contents (TPCs), and total flavonoid contents (TFCs) were measured. The extract's composition was analyzed. Cytotoxicity and effects on the detoxification enzyme NQO-1 were assessed in liver cell lines (AML12 and HepG2) using MTT and NQO-1 assays, respectively. Bioactive fractions were identified using fractionation techniques and mass spectrometry (LC-MS). RC extract displayed significant levels of carotenoids (0.375 mg/g), chlorophylls (2.682 mg/g), and favorable yield (15.3%). TPC and TFC were 363.776 mg/g and 112.22 mg/g of extract, respectively. Analysis revealed phenolic acids (gallic acid, caffeic acid), flavonoid (apigenin), chlorophylls (chlorophylls a, b, pheophytin a and b), and lutein. Among the fractions, Fraction 3 (F3) exhibited the highest NQO-1 enzyme activity. F3 contained pheophytin a and hydroxy pheophytin a, confirmed by LC-MS (m/z 871.59+ [M + H]+ and 887.59+ [M + H]+). F3 significantly induced NQO-1 activity in both HepG2 (3.908-fold) and AML12 (1.99-fold) cells. This study identified F3 from RC extract as a promising fraction containing pheophytin a and hydroxy pheophytin a, responsible for inducing the detoxification enzyme NQO-1 in liver cells. These findings suggest RC's potential for promoting detoxification.

A near-infrared fluorescent probe with two-photon excitation for in situ imaging of NQO1 in human colorectum cancer tissue

Colorectum cancer has become one of the most fatal cancer diseases, in which NAD(P)H: quinone oxidoreductase 1 (NQO1) plays a role in intracellular free radical reduction and detoxification and has been linked to colorectum cancer and chemotherapy resistance. Therefore, rational design of optical probe for NQO1 detection is urgent for the early diagnosis of colorectum cancer. Herein, we have developed a novel two-photon fluorescent probe, WHFD, which is capable of selectively detecting of intracellular NQO1 with two-photon (TP) absorption (800 nm) and near-infrared emission (620 nm). Combination with a substantial Stokes shift (175 nm) and biocompatibility, we have assessed its suitability for in vivo imaging of endogenous NQO1 activities from HepG2 tumor-bearing live animals with high tissue penetration up to 300 μm. Particularly, we for the first time used the probe to image NQO1 activities from human colorectum cancer samples by using TP microscopy, and proving our probe possesses reliable diagnostic performance to directly in situ imaging of cancer biomarker and can clearly distinguish the boundary between human colorectum cancer tissue and their surrounding normal tissue, which shows great potential for the intraoperative navigation.

Carbon Nanodots Inhibit Tumor Necrosis Factor-α-Induced Endothelial Inflammation through Scavenging Hydrogen Peroxide and Upregulating Antioxidant Gene Expression in EA.hy926 Endothelial Cells.

Carbon nanodots (CNDs) are a new type of nanomaterial with a size of less than 10 nanometers and excellent biocompatibility, widely used in fields such as biological imaging, transmission, diagnosis, and drug delivery. However, its potential and mechanism to mediate endothelial inflammation have yet to be explored. Here, we report that the uptake of CNDs by EA.hy926 endothelial cells is both time and dose dependent. The concentration of CNDs used in this experiment was found to not affect cell viability. TNF-α is a known biomarker of vascular inflammation. Cells treated with CNDs for 24 h significantly inhibited TNF-α (0.5 ng/mL)-induced expression of intracellular adhesion molecule 1 (ICAM-1) and interleukin 8 (IL-8). ICAM-1 and IL-8 are two key molecules responsible for the activation and the firm adhesion of monocytes to activated endothelial cells for the initiation of atherosclerosis. ROS, such as hydrogen peroxide, play an important role in TNF-α-induced inflammation. Interestingly, we found that CNDs effectively scavenged H2O2 in a dose-dependent manner. CNDs treatment also increased the activity of the antioxidant enzyme NQO1 in EA.hy926 endothelial cells indicating the antioxidant properties of CNDs. These results suggest that the anti-inflammatory effects of CNDs may be due to the direct H2O2 scavenging properties of CNDs and the indirect upregulation of antioxidant enzyme NQO1 activity in endothelial cells. In conclusion, CND can inhibit TNF-α-induced endothelial inflammation, possibly due to its direct scavenging of H2O2 and the indirect upregulation of antioxidant enzyme NQO1 activity in endothelial cells.

High-contrast NIR fluorescent probes for selective detection of NQO1 in breast cancer

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a potential biomarker for breast cancer (BC) diagnosis and prognosis. However, existing fluorescent probes for NQO1 detection have limitations such as short emission wavelength, weak fluorescence response, or large background interference. Here, we developed two novel near-infrared (NIR) fluorescent probes, DCl-Q and DCl2-Q, that selectively detect NQO1 activity in BC cells and tissues. They consist of a trimethyl-locked quinone as the recognition group and a donor-π-acceptor structure with halogen atoms as the reporter group. They exhibit strong fluorescence emission at around 660 nm upon binding to NQO1. We demonstrated that they can distinguish BC cells with different NQO1 expression levels and image endogenous NQO1 in tumor-bearing mice. Our probes provide a convenient and highly sensitive tool for BC diagnosis and prognosis based on NQO1 detection.

Application of a Specific and Sensitive NQO1 Turn-On Near-Infrared Fluorescence Probe for Live Cancer Cell and Xenografted Tumor Imaging in Nude Mice.

Sensitive activity stains for enzymes selectively expressed in human cancers offer valuable tools for imaging with wide applications in experimental, diagnostic, and therapeutic settings. The scant expression of the antioxidant enzyme NQO1 in normal tissues and its great abundance in malignant counterparts due to the increased redox stress and hypoxia is one such example. Previously, we described a potent nontoxic probe that remains nonfluorescent but releases an intense fluorogenic compound after intracellular cleavage by NQO1 catalysis. This infrared probe with a 644nm emission has excellent tissue penetrating ability and low background absorption. Described here are methods (fluorescence microscopy, flow cytometry, and in vivo animal imaging) to rapidly image NQO1 activity in hypoxic and non-hypoxic cancer cells and tumors developed in live mouse xenograft models. The specificity of the dye for NQO1 in all three procedures was verified, and the methods should be useful for both in vitro and in vivo studies.

Temporal dynamics of muscle mitochondrial uncoupling-induced integrated stress response and ferroptosis defense.

Mitochondria play multifaceted roles in cellular function, and impairments across domains of mitochondrial biology are known to promote cellular integrated stress response (ISR) pathways as well as systemic metabolic adaptations. However, the temporal dynamics of specific mitochondrial ISR related to physiological variations in tissue-specific energy demands remains unknown. Here, we conducted a comprehensive 24-hour muscle and plasma profiling of male and female mice with ectopic mitochondrial respiratory uncoupling in skeletal muscle (mUcp1-transgenic, TG). TG mice are characterized by increased muscle ISR, elevated oxidative stress defense, and increased secretion of FGF21 and GDF15 as ISR-induced myokines. We observed a temporal signature of both cell-autonomous and systemic ISR in the context of endocrine myokine signaling and cellular redox balance, but not of ferroptotic signature which was also increased in TG muscle. We show a progressive increase of muscle ISR on transcriptional level during the active phase (night time), with a subsequent peak in circulating FGF21 and GDF15 in the early resting phase. Moreover, we found highest levels of muscle oxidative defense (GPX and NQO1 activity) between the late active to early resting phase, which could aim to counteract excessive iron-dependent lipid peroxidation and ferroptosis in muscle of TG mice. These findings highlight the temporal dynamics of cell-autonomous and endocrine ISR signaling under skeletal muscle mitochondrial uncoupling, emphasizing the importance of considering such dissociation in translational strategies and sample collection for diagnostic biomarker analysis.

Gastrodin ameliorates postoperative cognitive dysfunction in aged mice by promoting nuclear translocation of Nrf2 through activation of AMPK

BackgroundPerioperative neurocognitive disorder (PND) remains a prevalent complication following anesthesia and surgery. Recent studies have revealed the therapeutic potential of gastrodin (GAS) in treating cognitive disturbances. This study delves deeper into the mechanisms through which GAS impacts PND. MethodsMale C57BL/6 mice (18 months old) underwent laparotomies and were administered GAS orally daily for three weeks preceding surgery and one week post-surgery. Thirty minutes before GAS administration, an intraperitoneal injection of Compound C was given. In vitro, H2O2-incubated SH-SY-5Y cells, with or without Nrf2-siRNA transfection, were set up and subjected to GAS or Compound C treatments. Cell viability was assessed via MTT assays, and apoptosis levels were assessed through flow cytometry. Cognitive function was evaluated using the Morris water maze, novel object recognition, and Y-maze tests. Oxidative stress markers, including MDA, SOD, GSH, GSH-px, and intracellular ROS (determined through immunofluorescence), were quantified. The expression of the genes Caspase3, Bax, Bcl-2, GST, and NQO1 was gauged using real-time RT-PCR. Brain, cortex and hippocampal pathologies were examined with hematoxylin-eosin (HE) and NeuN/TUNEL costaining. Finally, Nrf2 and p-AMPK were analyzed using Western blotting (WB) and immunofluorescence assays. ResultsGAS improved cognitive dysfunction in PND mice and reduced oxidative stress, neuro-apoptosis, and ROS levels both in vivo and in vitro experiment. In vivo, Immunofluorescence and Western blot outcomes indicated that postoperative p-AMPK and Nrf2 levels in the hippocampus were mitigated but were augmented by GAS. In vitro studies revealed GAS's protective effect against H2O2-induced oxidative stress and apoptosis and its upregulation of p-AMPK and Nrf2 in SH-SY-5Y cells. Notably, this protective effect was negated when Nrf2 siRNA was introduced. ELISA and PCR results highlighted the role of GAS in enhancing GST and NQO1 activity in both the mice hippocampus and SH-SY-5Y cells. Compound C, an AMPK inhibitor, both in vitro and in vivo, reversed the beneficial effects of GAS on Nuc-Nrf2/Cyt-Nrf2 expression and counteracted the positive influence of GAS on cognitive functions in PND mice. ConclusionGAS facilitates the nuclear translocation of Nrf2 via AMPK activation, offering a therapeutic avenue for alleviating postoperative cognitive impairments in mice, with a significant reduction in oxidative stress.

Structural dynamics at the active site of the cancer-associated flavoenzyme NQO1 probed by chemical modification with PMSF.

A large conformational heterogeneity of human NAD(P)H:quinone oxidoreductase 1 (NQO1), a flavoprotein associated with various human diseases, has been observed to occur in the catalytic site of the enzyme. Here, we report the X-ray structure of NQO1 with phenylmethylsulfonyl fluoride (PMSF) at 1.6 Å resolution. Activity assays confirmed that, despite being covalently bound to the Tyr128 residue at the catalytic site, PMSF did not abolish NQO1 activity. This may indicate that the PMSF molecule does not reduce the high flexibility of Tyr128, thus allowing NADH and DCPIP substrates to bind to the enzyme. Our results show that targeting Tyr128, a key residue in NQO1 function, with small covalently bound molecules could possibly not be a good drug discovery strategy to inhibit this enzyme.

Discovering the Protective Effects of Quercetin on Aflatoxin B1-Induced Toxicity in Bovine Foetal Hepatocyte-Derived Cells (BFH12).

Aflatoxin B1 (AFB1) induces lipid peroxidation and mortality in bovine foetal hepatocyte-derived cells (BFH12), with underlying transcriptional perturbations associated mainly with cancer, cellular damage, inflammation, bioactivation, and detoxification pathways. In this cell line, curcumin and resveratrol have proven to be effective in mitigating AFB1-induced toxicity. In this paper, we preliminarily assessed the potential anti-AFB1 activity of a natural polyphenol, quercetin (QUE), in BFH12 cells. To this end, we primarily measured QUE cytotoxicity using a WST-1 reagent. Then, we pre-treated the cells with QUE and exposed them to AFB1. The protective role of QUE was evaluated by measuring cytotoxicity, transcriptional changes (RNA-sequencing), lipid peroxidation (malondialdehyde production), and targeted post-transcriptional modifications (NQO1 and CYP3A enzymatic activity). The results demonstrated that QUE, like curcumin and resveratrol, reduced AFB1-induced cytotoxicity and lipid peroxidation and caused larger transcriptional variations than AFB1 alone. Most of the differentially expressed genes were involved in lipid homeostasis, inflammatory and immune processes, and carcinogenesis. As for enzymatic activities, QUE significantly reverted CYP3A variations induced by AFB1, but not those of NQO1. This study provides new knowledge about key molecular mechanisms involved in QUE-mediated protection against AFB1 toxicity and encourages in vivo studies to assess QUE's bioavailability and beneficial effects on aflatoxicosis.

Fighting drug-resistant lung cancer by induction of NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated ferroptosis.

Drug resistance is a major challenge in cancer treatment. The substrates of NAD(P)H:quinone oxidoreductase 1 (NQO1) show a promising anticancer effect in clinical trials. We previously identified a natural NQO1 substrate 2-methoxy-6-acetyl-7-methyljuglone (MAM) with a potent anticancer effect. The present study was designed to explore the efficacy of MAM in fighting against drug-resistant non-small cell lung cancer (NSCLC). The anticancer effect of MAM was evaluated in cisplatin-resistant A549 and AZD9291-resistant H1975 cells. The interaction of MAM with NQO1 was measured by cellular thermal shift assay and drug affinity responsive target stability assay. The NQO1 activity and expression were measured using NQO1 recombinant protein, Western blotting, and immunofluorescence staining assay. The roles of NQO1 were examined by NQO1 inhibitor, small interfering RNA (siRNA), and short hairpin RNA (shRNA). The roles of reactive oxygen species (ROS), labile iron pool (LIP), and lipid peroxidation were determined. MAM induced significant cell death in drug-resistant cells with similar potency to that of parental cells, which were completely abolished by NQO1 inhibitor, NQO1 siRNA, and iron chelators. MAM activates and binds to NQO1, which triggers ROS generation, LIP increase, and lipid peroxidation. MAM significantly suppressed tumor growth in the tumor xenograft zebrafish model. These results showed that MAM induced ferroptosis by targeting NQO1 in drug-resistant NSCLC cells. Our findings provided a novel therapeutic strategy for fighting against drug resistance by induction of NQO1-mediated ferroptosis.

Protective effect of hot-water and ethanol-aqueous extracts from Anneslea fragrans against acetaminophen-induced acute liver injury in mice.

Anneslea fragrans Wall. (AF) is an important medicinal and edible plant in China. The principal objectives of this study are to explore the hepatoprotective effect of ethanol-aqueous (AFE) and hot-water (AFW) extracts in vitro and in vivo. UPLC-ESI-MS/MS analysis showed that AFW and AFE are rich in dihydrochalcones. Both AFW and AFE significantly up-regulated the expressions of SOD, CAT and GSH, reduced the MDA content in acetaminophen (APAP)-induced HepG2 cells, and suppressed the expressions of NO, TNF-α, IL-1β, and IL-6 in LPS-induced RAW246.7cells. In APAP-induced mice, AFW and AFE administration significantly decreased the plasma levels of AST and ALT, and improved liver tissue damage, the collagen deposition and fibrosis formation. Moreover, AFW and AFE decreased the MDA and ROS accumulations via activating Nrf2 pathway to increase the hepatic GSH contents and activities of SOD, CAT, HO-1, and NQO-1, reduced the levels of NO, TNF-α, IL-1β, and IL-6 by suppressing the JNK/p38/ERK/NF-κB pathways, and alleviated apoptosis via regulating Bcl-2, Bax, caspase-3/9 protein expressions. This study provides a new sight that AFW and AFE may have a potential natural resource for the treatment of liver injury.

Trimethyl Lock Quinone-Based Organic Molecular Probes for NQO1 Sensing and Imaging

NAD(P)H: quinone oxidoreductase isozyme 1 (NQO1) is a flavoenzyme and involved in protection against oxidative stress and the regulation of metabolic functions, which is strongly implicated in neurodegenerative diseases and carcinogenic processes. Furthermore, NQO1 is also involved in the modes of action of redox-active drugs (e.g., antimalarials). Determining the activity and localization of NQO1 in living organisms is of great importance for early disease diagnosis and therapy. As a promising and convenient biosensing technique, trimethyl lock quinone-based organic molecular probes have been well established for the specific detection and imaging of NQO1 in living cells and in vivo. In this review, the recent progress of NQO1 probes based on organic small molecules is summarized from the perspectives of molecular design strategies, sensing mechanisms and bioimaging applications. We also elucidate the potential limitations and prospects of current NQO1 probes to further promote the development of versatile imaging tools for NQO1-related biomedical investigation.

Abstract 1427: NQO1/c-Fos/CKS1 signaling pathway regulates cell cycle progression at the G2/M phase

Abstract Overexpression of NAD(P)H:quinone oxidoreductase 1 (NQO1) is associated with tumor cell proliferation and growth in several human cancer types. However, the molecular mechanisms underlying the activity of NQO1 in cell cycle progression are currently unclear. Here, we report a novel function of NQO1 in modulation of cyclin-dependent kinase subunit (CKS1), a cell cycle regulator, at the G2/M phase, through effects on the stability of c-Fos. We analyzed the roles of NQO1/c-Fos/CKS signaling pathway in cell cycle progression in cancer cells using synchronization of cell cycle and flow cytometry. The mechanisms of NQO1/c-Fos/CKS1-mediated regulation of cell cycle progression in cancer cells were studied by siRNA, overexpression system, reporter assay, co-immunoprecipitation, pull-down assay, microarray analysis, and CDK1 kinase assay. In addition, publicly available data sets and immunohistochemistry were used to investigate the correlation between NQO1 expression levels and clinicopathological features in cancer patients. Our results suggest that NQO1 directly interacts with the unstructured DNA-binding domain of c-Fos implicated in proliferation, differentiation, development and survival of cancer and inhibits its proteasome-mediated degradation, thereby inducing CKS1 expression and regulating cell cycle progression at the G2/M phase. Notably, NQO1 deficiency in human cancer cell lines led to suppression of c-Fos-mediated CKS1 expression and cell cycle progression. Consistent with this finding, high NQO1 expression was correlated with increased CKS1 and poor prognosis in cancer patients. Our collective results support a novel regulatory role of NQO1 in the mechanism of cell cycle progression at the G2/M phase through effects on c-Fos/CKS1 signaling pathway in cancer. Citation Format: Eun-Taex Oh, Ha Gyeong Kim, Yunmi Cho, Heon Joo Park. NQO1/c-Fos/CKS1 signaling pathway regulates cell cycle progression at the G2/M phase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1427.

NQO1 regulates expression and alternative splicing of apoptotic genes associated with Alzheimer's disease in PC12 cells.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive memory loss and cognitive dysfunction. Quinone oxidoreductase 1 (NQO1) is an antioxidant enzyme that plays an important role in controlling cellular redox state, whose expression is altered in the brain tissues of AD patients. In addition to its traditional antioxidant effects, NQO1 also acts as a multifunctional RNA-binding protein involved in posttranscriptional regulation. Whether the RNA-binding activity of NQO1 influences AD pathology has not been investigated yet. The RNA-binding functions of NQO1 in rat pheochromocytoma (PC12) cells were investigated using siRNA knockdown followed by total RNA sequencing. Reverse transcription quantitative polymerase chain reaction was performed to explore the impact of NQO1 on the transcription and alternative splicing of apoptotic genes. NQO1 knockdown led to a significant increase in cellular apoptosis. Genes involved in certain apoptosis pathways, such as positive regulation of apoptotic processes and mitogen-activated protein kinase signaling, were under global transcriptional and alternative splicing regulation. NQO1 regulated the transcription of apoptotic genes Cryab, Lgmn, Ngf, Apoe, Brd7, and Stat3, as well as the alternative splicing of apoptotic genes BIN1, Picalm, and Fyn. Our findings suggest that NQO1 participates in the pathology of AD by regulating the expression and alternative splicing of the genes involved in apoptosis. These results extend our understanding of NQO1 in apoptotic pathways at the posttranscriptional level in AD.

Phenotypic Modulation of Cancer-Associated Antioxidant NQO1 Activity by Post-Translational Modifications and the Natural Diversity of the Human Genome

Human NAD(P)H:quinone oxidoreductase 1 (hNQO1) is a multifunctional and antioxidant stress protein whose expression is controlled by the Nrf2 signaling pathway. hNQO1 dysregulation is associated with cancer and neurological disorders. Recent works have shown that its activity is also modulated by different post-translational modifications (PTMs), such as phosphorylation, acetylation and ubiquitination, and these may synergize with naturally-occurring and inactivating polymorphisms and mutations. Herein, I describe recent advances in the study of the effect of PTMs and genetic variations on the structure and function of hNQO1 and their relationship with disease development in different genetic backgrounds, as well as the physiological roles of these modifications. I pay particular attention to the long-range allosteric effects exerted by PTMs and natural variation on the multiple functions of hNQO1.

Active Nrf2 signaling flexibly regulates HO-1 and NQO-1 in hypoxic Gansu Zokor (Eospalax cansus).

Gansu zokor (Eospalax cansus) is a typical subterranean rodent species with resistance to ambient hypoxia. The nuclear factor erythroid 2-related factor 2 (Nrf2) signaling plays a key role in regulating redox homeostasis. However, little is known about the regulation of Nrf2 signaling in Gansu zokor. We exposed Gansu zokors and SD rats to chronic hypoxia (44h at 10.5% O2) or acute hypoxia (6h at 6.5% O2) andmeasured the activities of heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO-1)，gene expression of HO-1, NQO-1, Nrf2, Kelch-like ECH-associated protein-1 (KEAP1), and β-transducin repeat-containing protein (β-TRCP) in the brain and liver. We found that Gansu zokor increased the NQO-1 protein content and activity, HO-1 protein content in the brain, and increased HO-1 activity and mRNA level, NQO-1 activity and protein content in the liver by up regulating Nrf2 gene expression under chronic hypoxia. Although acute hypoxia enhanced the expression of Nrf2 gene, only the level of HO-1 mRNA in the liver increased. Besides, the HO-1 and NQO-1 genes in the brain, HO-1 genes and NQO-1 mRNA in the Gansu zokor liver were significantly higher than those in SD rats under normoxia. Negative regulators of Nrf2 signaling were tissue specific: KEAP1 protein decreased in the brain, and β-TRCP decreased in the liver. The Nrf2 signaling and expression of downstream antioxidant enzymes were different under different oxygen concentrations, reflecting the flexible characteristics of Gansu zokor to deal with the hypoxic environment.

NQO1 regulates cell cycle progression at the G2/M phase.

Rationale: Overexpression of NAD(P)H:quinone oxidoreductase 1 (NQO1) is associated with tumor cell proliferation and growth in several human cancer types. However, the molecular mechanisms underlying the activity of NQO1 in cell cycle progression are currently unclear. Here, we report a novel function of NQO1 in modulation of the cell cycle regulator, cyclin-dependent kinase subunit-1 (CKS1), at the G2/M phase through effects on the stability of c‑Fos. Methods: The roles of the NQO1/c-Fos/CKS1 signaling pathway in cell cycle progression were analyzed in cancer cells using synchronization of the cell cycle and flow cytometry. The mechanisms underlying NQO1/c-Fos/CKS1-mediated regulation of cell cycle progression in cancer cells were studied using siRNA approaches, overexpression systems, reporter assays, co-immunoprecipitation, pull-down assays, microarray analysis, and CDK1 kinase assays. In addition, publicly available data sets and immunohistochemistry were used to investigate the correlation between NQO1 expression levels and clinicopathological features in cancer patients. Results: Our results suggest that NQO1 directly interacts with the unstructured DNA-binding domain of c-Fos, which has been implicated in cancer proliferation, differentiation, and development as well as patient survival, and inhibits its proteasome-mediated degradation, thereby inducing CKS1 expression and regulation of cell cycle progression at the G2/M phase. Notably, a NQO1 deficiency in human cancer cell lines led to suppression of c-Fos-mediated CKS1 expression and cell cycle progression. Consistent with this, high NQO1 expression was correlated with increased CKS1 and poor prognosis in cancer patients. Conclusions: Collectively, our results support a novel regulatory role of NQO1 in the mechanism of cell cycle progression at the G2/M phase in cancer through effects on c‑Fos/CKS1 signaling.