NQO1 Inhibitor Research Articles

SP600125, a selective JNK inhibitor, is a potent inhibitor of NAD(P)H: quinone oxidoreductase 1 (NQO1).

The c-Jun N-terminal kinases (JNKs) has been identified as a critical modulator in multiple cellular processes, including stress stimulus, inflammation, cell proliferation, apoptosis, etc. SP600125 is a widely used ATP-competitive reversible JNKs inhibitor. NAD(P)H: quinone oxidoreductase 1 (NQO1) is a flavoprotein mediated two or four electron-reduction of quinones. Here, we showed that SP600125 bind to the active pocket of NQO1 and inhibit NQO1 activity. SP600125 exhibits comparable inhibitory effects on NQO1-mediated quinone bioactivation, H2O2 generation, and cell death, as the specific NQO1 inhibitor dicoumarol (DIC). Importantly, the inhibitory effects of SP600125 on NQO1 are independent of JNKs inhibition. These results suggested that SP600125 is a novel NQO1 inhibitor, which provides new insights into the mechanism of action of SP600125. Furthermore, SP600125 should be used more cautiously as a JNKs inhibitor, especially when NQO1 is highly expressed. SP600125 competed with β-Lap (NQO1-bioactivated drugs) for binding to NQO1, and inhibited NQO1-dependent cell death.

Discovery of Potent Benzothiazole Inhibitors of Oxidoreductase NQO2, a Target for Inflammation and Cancer.

Inhibitors of NQO2 (NRH: quinone oxidoreductase) have potential application in several areas of medicine and pharmacology, including cancer, neurodegeneration (PD and AD), stroke, and diabetes. Here, resveratrol, a known inhibitor of NQO2, was used as the lead by replacing the double bond in resveratrol with a benzothiazole scaffold. Fifty-five benzothiazoles were designed as NQO2 inhibitors and synthesized, comprising five benzothiazole series with 3,5-dimethoxy, 2,4-dimethoxy, 2,5-dimethoxy, 3,4-dimethoxy, and 3,4,5-trimethoxy substituents, the key synthetic step being a Jacobson cyclisation with the appropriate thiobenzamide. All compounds were evaluated in an NQO2 enzyme inhibition assay, with four compounds having IC50 values of <100 nM. The most active (IC50 25 nM) was 6-hydroxy-2-(3',5'-dihydroxyphenyl)benzo[d]thiazole (15), a good mimetic of resveratrol. Three of the 3',4',5'-trimethoxybenzothiazole analogues, with 6-methoxy (40, IC50 51 nM), 6-amino (48, IC50 79 nM), and 6-acetamide (49, IC50 31 nM) substituents, were also potent inhibitors of NQO2. Computational modelling indicated the most active compounds exhibited good shape complementarity and polar interactions with the NQO2 active site. Through the inhibition of NQO2, benzothiazole-based compounds may have the potential to enhance the efficiency of cancer therapies or minimise oxidative damage in neuroinflammation.

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.

DPP9 regulates NQO1 and ROS to promote resistance to chemotherapy in liver cancer cells

Chemotherapy has been the standard treatment for liver cancer. However, intrinsic or acquired drug resistance remains a major barrier to successful treatment. At present, the underlying molecular mechanisms of chemoresistance in liver cancer have not been elucidated. Dipeptidyl peptidase 9 (DPP9) is a member of the dipeptidyl peptidase IV family that has been found to be highly expressed in a variety of tumors, including liver cancer. It is unclear whether DPP9 affects chemoresistance in liver cancer. In this study, we find that DPP9 weakens the responses of liver cancer cells to chemotherapy drugs by up-regulating NQO1 and inhibiting intracellular ROS levels. In terms of mechanism, DPP9 inhibits ubiquitin-mediated degradation of NRF2 protein by binding to KEAP1, up-regulates NRF2 protein levels, promotes mRNA transcription of NQO1, and inhibits intracellular ROS levels. In addition, the NQO1 inhibitor dicoumarol can enhance the efficacy of chemotherapy drugs in liver cancer cells. Collectively, our findings suggest that inhibiting DPP9/NQO1 signaling can serve as a potential therapeutic strategy for liver cancer.

DIPG-88. TARGETING THE ANTIOXIDANT RESPONSE ELEMENT AXIS SENSITIZES DIFFUSE MIDLINE GLIOMA CELLS TO ONC201

Abstract BACKGROUND Diffuse midline glioma (DMG) is a universally fatal CNS tumor, with a median overall survival of &amp;lt;12-months. Recent studies, including our own, have identified overexpression of the mitochondrial protease ClpP in DMG patient samples. ClpP plays a pivotal role in regulating energy production. Brain-penetrant ClpP agonists, including ONC201, ONC206, and TR107, induce non-specific degradation of the mitochondrial electron transport chain. However, there is considerable variability in the response of DMG cell lines to these therapies, with only about half reaching IC50. Notably, TP53-mutant cell lines exhibit reduced sensitivity. METHODS/RESULTS Further analysis using quantitative proteomics of TP53-mutant DMG cell lines exhibited hallmark activation of ClpP, increased mitochondrial stress and dysfunction. Importantly, ClpP agonism increased the activity of the transcription factor NRF2, driving expression of NQO1 (p=0.0017). NQO1 is a cytoprotective enzyme, promoting redox balance and cell survival. This highlights a potential adaptive response to ClpP agonist therapy in DMG cells. Analysis of DMG patient samples through publicly available RNA sequencing data identified ubiquitously high NRF2 expression compared to other pediatric CNS tumors. This led us to hypothesize that a clinically relevant, selective NQO1 inhibitor could elicit a strong response to ClpP agonism. Therefore, we examined ARQ761, a CNS-active therapy that disrupts NQO1 leading to the formation of an unstable hydroquinone, causing oxidative DNA damage and apoptosis. When ARQ761 was used concurrently with a ClpP agonist, the combination showed additive effects, average Bliss Score of 9.91 (n=4). Interestingly, administering ARQ761 24 h after ClpP agonist exposure combined synergistically, average Bliss Score of 15.87 (n=4). This synergy was further potentiated using ARQ761 48 h after ClpP agonism, when NQO1 expression peaked, resulting in an average Bliss Score of 20.30 (n=4). CONCLUSIONS This study has unveiled an intrinsic antioxidant defense mechanism in DMG, which facilitates evasion from treatments inducing oxidative stress. This defense mechanism appears particularly robust in cell lines harboring TP53-mutations, which are also known to exhibit resistance to radiotherapy.

NQO1 Mediates Lenvatinib Resistance by Regulating ROS-induced Apoptosis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-associated death worldwide. As a first-line drug for advanced HCC treatment, lenvatinib faces a significant hurdle due to the development of both intrinsic and acquired resistance among patients, and the underlying mechanism remains largely unknown. The present study aims to identify the pivotal gene responsible for lenvatinib resistance in HCC, explore the potential molecular mechanism, and propose combinatorial therapeutic targets for HCC management. Cell viability and colony formation assays were conducted to evaluate the sensitivity of cells to lenvatinib and dicoumarol. RNA-Seq was used to determine the differences in transcriptome between parental cells and lenvatinib-resistant (LR) cells. The upregulated genes were analyzed by GO and KEGG analyses. Then, qPCR and Western blotting were employed to determine the relative gene expression levels. Afterwards, the intracellular reactive oxygen species (ROS) and apoptosis were detected by flow cytometry. PLC-LR and Hep3B-LR were established. There was a total of 116 significantly upregulated genes common to both LR cell lines. The GO and KEGG analyses indicated that these genes were involved in oxidoreductase and dehydrogenase activities, and reactive oxygen species pathways. Notably, NAD(P)H:quinone oxidoreductase 1 (NQO1) was highly expressed in LR cells, and was involved in the lenvatinib resistance. The high expression of NQO1 decreased the production of ROS induced by lenvatinib, and subsequently suppressed the apoptosis. The combination of lenvatinib and NQO1 inhibitor, dicoumarol, reversed the resistance of LR cells. The high NQO1 expression in HCC cells impedes the lenvatinib-induced apoptosis by regulating the ROS levels, thereby promoting lenvatinib resistance in HCC cells.

1,8-Cineole alleviates OGD/R-induced oxidative damage and restores mitochondrial function by promoting the Nrf2 pathway.

This study examined the effects of 1,8-cineole on reducing oxidative stress injury and restoring mitochondrial function in oxygen-glucose deprivation and reoxygenation (OGD/R) HT22 cells via the nuclear factor erythrocyte 2 related factor 2 (Nrf2) pathway. The optimal concentration of 1,8-cineole to reduce OGD/R injury was screened via cell morphology, cell survival rate, and lactate dehydrogenase (LDH) leakage rate. Oxidative damage was observed by measuring superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) activities, and reactive oxygen species (ROS), glutathione (GSH), protein carbonyl, malondialdehyde (MDA), lipid peroxidation (LPO) content, and 8-hydroxy-2 deoxyguanosine (8-OHDG) expression. Mitochondrial function was observed by mitochondrial membrane potential (MMP) and ATPase activity. Nrf2 pathways were observed by the expression levels of total Nrf2, nucleus Nrf2, NAD(P)H: quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1), the mRNA levels of HO-1 and NQO1. Among different concentrations of 1,8-cineole for promoting HT22 cell proliferation and attenuated OGD/R injury, 10 μmol/L 1,8-cineole was the best. After 1,8-cineole treatment, SOD, GSH-PX, and CAT activities and GSH content increased, while ROS, MDA, LPO, protein carbonyl, and 8-OHDG levels decreased. 1,8-Cineole could restore MMP and increase mitochondrial enzyme activity. It could also increase the total Nrf2, nucleus Nrf2, NQO1, and HO-1, and Nrf2 inhibitor brusatol reduced the effect of 1,8-cineole. Immunofluorescence assay showed that 1,8-cineole could facilitate the transfer of Nrf2 into the nucleus. 1,8-cineole increased the mRNA levels of NQO1 and HO-1. The above results showed that 1,8-cineole could alleviate OGD/R-induced oxidative damage and restores mitochondrial function by activating the Nrf2 signal pathway.

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.

Nicotinamide mononucleotide inhibits oxidative stress-induced damage in a SIRT1/NQO-1-dependent manner

Oxidative stress causes endothelial dysfunction, which is associated with vascular cellular aging and is causally related to cardiovascular disease pathogenesis. Preclinical studies indicate that a nicotinamide adenine dinucleotide (NAD+) precursor, nicotinamide mononucleotide (NMN), alleviates oxidative stress in aged vessels, granting vasoprotective effects. However, the associated cellular mechanism remains largely unclear. In this study, we used human umbilical vein endothelial cells (HUVECs) to demonstrate that NMN inhibits oxidative stress-induced damage by activating the sirtuin 1 (SIRT1)/NAD(P)H: quinone oxidoreductase 1 (NQO-1) axis. We found that NMN inhibited H2O2-induced cytotoxicity and senescence-associated protein expression, such as p16 and p21. Furthermore, NMN prevented H2O2-induced actin cytoskeletal disorganization via inhibiting reactive oxygen species (ROS) production. NMN increased NQO-1 mRNA and protein expression that in turn was abrogated by SIRT1 inhibition, suggesting that NMN-inducible NQO-1 was associated with SIRT1 activity. SIRT1 and NQO-1 inhibition attenuated the inhibitory effect of NMN on H2O2-inducible cytotoxicity, senescence-related protein upregulation, and actin cytoskeletal disorganization. Our findings provide new insights into the mechanism by which NMN exerts protective effects against vascular oxidative stress.

Abstract 2169: NAD(P)H: quinone oxidoreductase 1 (NQO1)- A viable biomarker in colorectal cancer

Abstract Background/Objective: Colorectal cancer (CRC) is aggressive and widespread (3rd among malignancies) in the United States. NAD(P)H: Quinone Oxidoreductase 1 (NQO1) is an enzyme that contributes to chemoprotection for CRC. The aim of this meta-analysis is to test the connection between NQO1 C609T polymorphisms and CRC risk. Furthermore, the study explores the binding capacity and efficacy of BBI 608 against NQO1 in CRC through molecular docking. The inhibitor will then be tested in vitro. Methods: PubMed, Web of Science, and Google Scholar were used in bibliographic searches. For the NQO1 C609T analysis, a total of 18 studies were used. Data was gathered and subsequently calculated using the pooled odds ratio (OR) (95% confidence interval, CI). Molecular docking, Western Blot and QRT-PCR were used to determine the molecular function of BBI 608 and NQO1. Results: The relation between NQO1 polymorphism and CRC risk (TT + CT vs. CC: OR=1.19, 95% CI =1.06-1.34, p&amp;lt;0.001) was significant. Furthermore, stratified investigation based on ethnicity indicated significant association between NQO1 polymorphism and CRC risk (TT + CT vs. CC: OR=1.17, 95% CI =1.08-1.27, p&amp;lt;0.001). This study indicates that the C609T polymorphism of NQO1 is linked with CRC risk in Asians and Caucasians. To observe the molecular properties of BBI 608 (NQO1 inhibitor) and to analyze the exact mechanism of NQO1 on CRC, computational approaches were applied. This investigation provides a detailed understanding of the interaction between NQO1 and BBI 608 and its implication in CRC therapy. To support this computational analysis, in vitro studies were performed. BBI 608 creates higher cytotoxicity in a dose dependent manner in CRC cell lines. BBI 608 treatment significantly (p&amp;lt;0.001) reduced the NQO1 expression at protein and messenger levels in both CRC (HCT 116 and RKO) cell lines. Conclusion: Based on meta-analysis and computational approach, NQO1 is a viable biomarker and targeted molecule in CRC. In vitro results showed that inhibiting NQO1 with the drug BBI 608 decreased cell proliferation in both CRC cell lines. Knockout or overexpression or site directed mutagenesis is essential for a better understanding about BBI 608 targeted NQO1 and its amino acids. Citation Format: Chaithanya Ganji, Bhaskar VK Lakkakula, Santosh Kumar Behera, Bassel F. El-Rayes. NAD(P)H: quinone oxidoreductase 1 (NQO1)- A viable biomarker in colorectal cancer [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 2169.

β-lapachone-mediated WST1 Reduction as Indicator for the Cytosolic Redox Metabolism of Cultured Primary Astrocytes

Electron cycler-mediated extracellular reduction of the water-soluble tetrazolium salt 1 (WST1) is frequently used as tool for the determination of cell viability. We have adapted this method to monitor by determining the extracellular WST1 formazan accumulation the cellular redox metabolism of cultured primary astrocytes via the NAD(P)H-dependent reduction of the electron cycler β-lapachone by cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1). Cultured astrocytes that had been exposed to β-lapachone in concentrations of up to 3 µM remained viable and showed an almost linear extracellular accumulation of WST1 formazan for the first 60 min, while higher concentrations of β-lapachone caused oxidative stress and impaired cell metabolism. β-lapachone-mediated WST1 reduction was inhibited by the NQO1 inhibitors ES936 and dicoumarol in a concentration-dependent manner, with half-maximal inhibition observed at inhibitor concentrations of about 0.3 µM. β-lapachone-mediated WST1 reduction depended strongly on glucose availability, while mitochondrial substrates such as lactate, pyruvate or ketone bodies allowed only residual β-lapachone-mediated WST1 reduction. Accordingly, the mitochondrial respiratory chain inhibitors antimycin A and rotenone hardly affected astrocytic WST1 reduction. Both NADH and NADPH are known to supply electrons for reactions catalysed by cytosolic NQO1. Around 60% of the glucose-dependent β-lapachone-mediated WST1 reduction was prevented by the presence of the glucose-6-phosphate dehydrogenase inhibitor G6PDi-1, while the glyceraldehyde-3-phosphate dehydrogenase inhibitor iodoacetate had only little inhibitory potential. These data suggest that pentose phosphate pathway-generated NADPH, and not glycolysis-derived NADH, is the preferred electron source for cytosolic NQO1-catalysed reductions in cultured astrocytes.

Skullcapflavone II, a novel NQO1 inhibitor, alleviates aristolochic acid I-induced liver and kidney injury in mice.

Aristolochic acid I (AAI) is a well established nephrotoxin and human carcinogen. Cytosolic NAD(P)H quinone oxidoreductase 1 (NQO1) plays an important role in the nitro reduction of aristolochic acids, leading to production of aristoloactam and AA-DNA adduct. Application of a potent NQO1 inhibitor dicoumarol is limited by its life-threatening side effect as an anticoagulant and the subsequent hemorrhagic complications. As traditional medicines containing AAI remain available in the market, novel NQO1 inhibitors are urgently needed to attenuate the toxicity of AAI exposure. In this study, we employed comprehensive 2D NQO1 biochromatography to screen candidate compounds that could bind with NQO1 protein. Four compounds, i.e., skullcapflavone II (SFII), oroxylin A, wogonin and tectochrysin were screened out from Scutellaria baicalensis. Among them, SFII was the most promising NQO1 inhibitor with a binding affinity (KD = 4.198 μmol/L) and inhibitory activity (IC50 = 2.87 μmol/L). In human normal liver cell line (L02) and human renal proximal tubular epithelial cell line (HK-2), SFII significantly alleviated AAI-induced DNA damage and apoptosis. In adult mice, oral administration of SFII dose-dependently ameliorated AAI-induced renal fibrosis and dysfunction. In infant mice, oral administration of SFII suppressed AAI-induced hepatocellular carcinoma initiation. Moreover, administration of SFII did not affect the coagulation function in short term in adult mice. In conclusion, SFII has been identified as a novel NQO1 inhibitor that might impede the risk of AAI to kidney and liver without obvious side effect.

Role of Human Aldo-Keto Reductases and Nuclear Factor Erythroid 2-Related Factor 2 in the Metabolic Activation of 1-Nitropyrene via Nitroreduction in Human Lung Cells.

1-Nitropyrene (1-NP) is a constituent of diesel exhaust and classified as a group 2A probable human carcinogen. The metabolic activation of 1-NP by nitroreduction generates electrophiles that can covalently bind DNA to form mutations to contribute to cancer causation. NADPH-dependent P450 oxidoreductase (POR), xanthine oxidase (XO), aldehyde oxidase (AOX), and NAD(P)H/quinone oxidoreductase 1 (NQO1) may catalyze 1-NP nitroreduction. We recently found that human recombinant aldo-keto reductases (AKRs) 1C1-1C3 catalyze 1-NP nitroreduction. NQO1 and AKR1C1-1C3 are genes induced by nuclear factor erythroid 2-related factor 2 (NRF2). Despite this knowledge, the relative importance of these enzymes and NRF2 to 1-NP nitroreduction is unknown. We used a combination of pharmacological and genetic approaches to assess the relative importance of these enzymes and NRF2 in the aerobic nitroreduction of 1-NP in human bronchial epithelial cells, A549 and HBEC3-KT. 1-NP nitroreduction was assessed by the measurement of 1-aminopyrene (1-AP), the six-electron reduced metabolite of 1-NP, based on its intrinsic fluorescence properties (λex and λem). We found that co-treatment of 1-NP with salicylic acid, an AKR1C1 inhibitor, or ursodeoxycholate, an AKR1C2 inhibitor, for 48 h decreased 1-AP production relative to 1-NP treatment alone (control) in both cell lines. R-Sulforaphane or 1-(2-cyano-3,12,28-trioxooleana-1,9(11)-dien-28-yl)-1H-imidazole (CDDO-Im), two NRF2 activators, each increased 1-AP production relative to control only in HBEC3-KT cells, which have inducible NRF2. Inhibitors of POR, NQO1, and XO failed to modify 1-AP production relative to control in both cell lines. Importantly, A549 wild-type cells with constitutively active NRF2 produced more 1-AP than A549 cells with heterozygous expression of NFE2L2/NRF2, which were able to produce more 1-AP than A549 cells with homozygous knockout of NFE2L2/NRF2. Together, these data show dependence of 1-NP metabolic activation on AKR1Cs and NRF2 in human lung cells. This is the second example whereby NFE2L2/NRF2 is implicated in the carcinogenicity of diesel exhaust constituents.

Riboflavin compounds show NAD(P)H dependent quinone oxidoreductase-like quinone reducing activity.

NAD(P)H-dependent quinone oxidoreductase (NQO) is an essential enzyme in living organisms and cells protecting them from oxidative stress. NQO reduces coenzyme Q (CoQ) using NAD(P)H as an electron donor. In the present study, we searched for coenzyme Q10 reducing activity from fractions of gel filtration-fractionated rat liver homogenate. In addition to the large-molecular-weight fraction containing NQO, CoQ10 reducing activity was also detected in a low-molecular-weight fraction. Furthermore, dicumarol, a conventional inhibitor of NQO1 (DT diaphorase), did not inhibit the reduction but quercetin did, suggesting that the activity was not due to NQO1. After further purification, the NADH-dependent CoQ10-reducing compound was identified as riboflavin. Riboflavin is an active substituent of other flavin compounds such as FAD and FMN. These flavin compounds also reduced not only CoQ homologues but also vitaminK homologues in the presence of NADH. The mechanism was speculated to work as follows: NADH reduces flavin compounds to the corresponding reduced forms, and subsequently, the reduced flavin compounds immediately reduce bio-quinones. Furthermore, the flavin-NADH system reduces CoQ10 bound with saposin B, which is believed to function as a CoQ transfer protein in vivo. This flavin-dependent CoQ10 reduction, therefore, may function in aqueous phases such as the cell cytosol and bodily fluids.

FAD-deficient P187S mutation of NAD(P)H:quinone oxidoreductase 1 (NQO1*2) binds and accelerates β-amyloid aggregation.

Alzheimer's disease (AD) is one of the most prominent neurodegenerative diseases. Results from animal and cellular models suggest that FAD-deficient forms of NAD(P)H quinone oxidoreductase 1 (NQO1) may accelerate the aggregation of Alzheimer's amyloid-β peptide (Aβ1-42). Here, we examined in vitro whether NQO1 and its FAD-deficient P187S mutation (NQO1*2) directly interact with Aβ1-42 and modify its rate of aggregation. When monitored using the fluorescence of either noncovalent thioflavin T (ThT) or HiLyte Fluor 647 (HF647) dye covalently attached to the Aβ1-42 peptide, the aggregation kinetics of Aβ1-42 were markedly more rapid in the presence of NQO1*2 than the wild-type (WT) NQO1. Experiments using apo-NQO1 indicate that this increase is linked to the inability of NQO1*2 to bind to FAD. Furthermore, dicoumarol, an NQO1 inhibitor that binds near the FAD-binding site and stabilizes NQO1*2, markedly decreased the aggregation kinetics of Aβ1-42. Imaging flow cytometry confirmed in-vitro coaggregation of NQO1 isoforms and Aβ1-42. Aβ1-42 alone forms rod-shaped fibril structures while in the presence of NQO1 isoforms, Aβ1-42 is incorporated in the middle of larger globular protein aggregates surrounded by NQO1 molecules. Isothermal titration calorimetry (ITC) analysis indicates that Aβ1-42 interacts with NQO1 isoforms with a specific stoichiometry through a hydrophobic interaction with positive enthalpy and entropy changes. These data define the kinetics, mechanism, and shape of coaggregates of Aβ1-42 and NQO1 isoforms and the potential relevance of FAD-deficient forms of NQO1 for amyloid aggregation diseases.

β-caryophyllene ameliorates hepatic ischemia reperfusion-induced injury: the involvement of Keap1/Nrf2/HO 1/NQO 1 and TLR4/NF-κB/NLRP3 signaling pathways.

Hepatic ischemia-reperfusion (H I/R) injury is a frequent clinical event during which the leading contributing players are inflammation and oxidative stress responses. β-caryophyllene (BCP), a natural bicyclicsesquiterpene, is an essential oil component of different plant species and edibles. This study aims to identify whether BCP pretreatment could avert H I/R injury with inspections of the underlying mechanisms. Animals were devised into five groups; Sham and BCP + Sham; the animals were administered saline or BCP (200 mg/kg, orally) respectively; H I/R group, the animals were administered saline orally for 14 days before induction of H I/R; BCP100 and BCP200, the animals were administered BCP (100 and 200 mg/kg, respectively) for 14 days, followed by induction of H I/R. H I/R showed markedly increased ALT, AST, MDA, and lowered antioxidant enzyme activities, while the Nrf2/HO1/NQO1 pathway components were significantly augmented. The TLR4/NF-κB/NLRP3 elements were deterred, and subsequently, escalations in the inflammatory mediators (IL-1β, IL-6, and TNF-α), adhesion molecule ICAM-1, neutrophils infiltration (MPO), and apoptotic markers were observed. Pretreatment with BCP amplified the antioxidant enzyme activities and Keap1/Nrf2/HO1/NQO1 pathway components. BCP pretreatment lowered TLR4/NF-κB/NLRP3 pathway elements, which mitigated inflammatory mediators, ICAM-1, MPO, and apoptotic markers. The protective effect of BCP against hepatic I/R induced injury might be accomplished via mitigation of oxidative stress by regulating the Keap1/Nrf2/HO1/NQO1 pathway and inhibition of the inflammatory process via manipulating the TLR4/ NF-κB/ NLRP3, reflected by inflammatory markers, neutrophils recruitment, and adhesion molecules reduction. BCP might be a potential therapeutic agent for alleviating hepatic I/R-induced injury.

Role and mechanism of benzo[a]pyrene in the transformation of chronic obstructive pulmonary disease into lung adenocarcinoma.

This experiment is explores the genes that play a key role, their expression changes and the biological processes in the transformation of chronic obstructive pulmonary disease (COPD) into lung adenocarcinoma (LAC). Meanwhile, identify the effects of Benzo[a]pyrene (BaP) in the conversion of COPD into LAC. 1. Differential expression genes of COPD and LAC were screened and analyzed by high-throughput microarray data between the two diseases and their respective control groups. 2. The screened genes were used for routine bioinformatics analysis such as functional analysis, expression verification, protein interaction analysis and functional enrichment. 3. Cigarette smoke extract (CSE) combined with lipopolysaccharide (LPS) was used to establish an in vitro COPD model. 4. MTT assay was used to detect the influence of B(a)P in effect on A549 cell proliferation. CCK-8, Transwell invasion test and scratch test were used to detect the cell proliferation, invasion and migration ability, while qPCR and Western Blot tests were used to observe the cell proliferation, apoptosis and changes in related indicators such as EMT. 5. Experimental method of separately adding agonists (tBHQ) and inhibitors (DIC) of NQO1 was used to confirm the effect of NQO1 on A549 cell proliferation, apoptosis, migration and invasion. 6. To further clarify whether BaP exerted effect on cell proliferation, apoptosis, migration and invasion through NQO1, we knocked down NQO1 gene and then infecting cells with BaP. 1. We screened genes of COPD and LAC using datasets from GSE151052, GSE118370, and GSE140797. After screening, the genes upregulated in COPD and downregulated in LAC were RTKN2, SLC6A4, and HBB, the gene downregulated in COPD and upregulated in LAC was NQO1, the genes downregulated in both COPD and LAC were FPR1, LYVE1 and PKHD1L1. 2. The main signaling pathways in which the target genes were enriched are cell cycle, EMT, PI3K/AKT, and apoptosis. In the data included GEPIA, PKHD1L1, FPR1, LYVE1, RTKN2, HBB, and SLC6A4 were significantly downregulated and NQO1 was upregulated in LAC relative to controls. In addition, there were 46 interaction proteins in the target genes, and the functions they enriched included hydrogen peroxide catabolism, etc. 3. When A549 cell was stimulated with 100ng/mL LPS+ 10% CSE, the COX-2 expression indicated that COPD model in vitro was successfully established. 4. The optimal dose and action time were screened which were 1μM and 24h. Compared to the control group, COPD and BaP group increased cell proliferation and invasion capabilities. On the basis of COPD, adding BaP could further increase the proliferation and migration capabilities. Interestingly, the levels of NQO1 decreased in COPD models, while increased by BaP. 5. tBHQ can increase the proliferation and migration capacity of A549 cells, which is inhibited by the addition of DIC. 6. The enhanced proliferation, migration and invasion of A549 cells by BaP were attenuated after knockdown of NQO1. Our study reveals that PKHD1L1, FPR1, LYVE1, RTKN2, HBB, SLC6A4 and NQO1 may play an important role in the conversion of COPD to LAC. High NQO1 expression may increase the proliferation and migration ability of A549 cells, and BaP may promote the EMT state by increasing the expression of NQO1, thereby making the COPD model in vitro expose the tumor characteristics.

Hypoxia-responsive nanocarriers for chemotherapy sensitization via dual-mode inhibition of hypoxia-inducible factor-1 alpha

The overexpression of hypoxia-inducible factor-1 alpha (HIF-1α) in solid tumor compromises the potency of chemotherapy under hypoxia. The high level of HIF-1α arises from the stabilization effect of reduced nicotinamideadeninedinucleotide(phosphate) NAD(P)H: quinone oxidoreductase 1 (NQO1). It was postulated that the inhibition of NQO1 could degrade HIF-1α and sensitize hypoxic cancer cells to antineoplastic agents. In the current work, we report hypoxia-responsive polymer micelles, i.e. methoxyl poly(ethylene glycol)-co-poly(aspartate-nitroimidazole) orchestrate with a NQO1 inhibitor (dicoumarol) to sensitize the ovarian cancer cell line (SKOV3) to a model anticancer agent (sorafenib) at low oxygen conditions. Both cargos were physically encapsulated in the nanoscale micelles. The placebo micelles transiently induced the depletion of reduced nicotinamideadeninedinucleotidephosphate (NADPH) as well as glutathione and thioredoxin under hypoxia, which further inactivated NQO1 because NADPH was the cofactor of NQO1. As a consequence, the expression of HIF-1α was repressed due to the dual action of dicoumarol and polymer. The degradation of HIF-1α significantly increased the vulnerability of SKOV3 cells to sorafenib-induced apoptosis, as indicated by the enhancement of cytotoxicity, and increase of caspase 3 and cytochrome C. The current work opens new avenues of addressing hypoxia-induced drug resistance in chemotherapy.

Neferine Exerts Ferroptosis-Inducing Effect and Antitumor Effect on Thyroid Cancer through Nrf2/HO-1/NQO1 Inhibition.

Thyroid cancer is the most prevalent endocrine malignancy with an increasing incidence in the past few decades. Neferine possesses various pharmacological activities, which have been applied in diverse disease models, including various tumors. However, the detailed effect and mechanism of neferine on thyroid cancer are still unclear. In the current study, the viability of IHH-4 and CAL-62 cells was examined by the CCK-8 assay. The effect of neferine on the proliferation, apoptosis, invasion, vascular endothelial growth factor (VEGF), epithelial-mesenchymal transition (EMT), and ferroptosis was evaluated by CCK-8, flow cytometry, western blot, and spectrophotometry assays. Mechanically, the expressions levels of Nrf2/HO-1/NQO1 signaling were first determined by a western blot, which was then verified by Nrf2 overexpression. In vivo validation was also conducted on BALB/c nude mice with an inoculation dose of 2 × 106 IHH-4 cells. The results showed that neferine repressed the viability of both IHH-4 and CAL-62 cells both in a dose-dependent way and in a time-dependent fashion, in which the IC50 value of neferine on IHH-4 and CAL-62 cells was 9.47 and 8.72 μM, respectively. Besides, neferine enhanced apoptosis but suppressed invasion, angiogenesis, and EMT of IHH-4 and CAL-62 cells. Moreover, neferine induced the activation of ferroptosis in thyroid cancer cells. Notably, it was revealed that the Nrf2/HO-1/NQO1 pathway was strongly associated with the effect of neferine on the modulation of thyroid cancer. Furthermore, these outcomes were validated in xenografted mice. Therefore, neferine exerted an antitumor effect and ferroptosis-inducing effect on thyroid cancer via inhibiting the Nrf2/HO-1/NQO1 pathway.

Synthesis of Analogues of Natural Product ‘Antheminone A’ and Evaluation of Their Anti-Cancer Activity

Synthesis of two novel compounds (1a and 1b) derived from analogue of antheminone A (1c) and their evaluation of anti-cancer activities are hereby described. This synthesis involved a multi-step reaction sequence involving the use of natural product (-)-quinic acid (1d) and unsymmetrical dicoumarol (1e) as precursors (Figure 1). Unsymmetrical dicoumarol (1e), a potent inhibitor of NQO1 was synthesized by coupling 4-hydroxyl coumarin and an appropriate benzaldehyde. Thus, in order to facilitate drug penetration through the barriers of cell membrane to NQO1 location, compound (1e) was re-modified by coupling with an analogue of antheminone A (1c). Spectral analyses of the products were carried out in order to confirm the identity of the compounds. Interestingly, the compounds which were obtained in good to moderate yield (51-68%) exhibited toxicity against the non-small cancer cell line, A549.