NRH:quinone Oxidoreductase 2 Research Articles

Autophagy and neuroprotection in astrocytes exposed to 6-hydroxydopamine is negatively regulated by NQO2: relevance to Parkinson’s disease

Dopaminergic degeneration is a central feature of Parkinson’s disease (PD), but glial dysfunction may accelerate or trigger neuronal death. In fact, astrocytes play a key role in the maintenance of the blood–brain barrier and detoxification. 6-hydroxydopamine (6OHDA) is used to induce PD in rodent models due to its specific toxicity to dopaminergic neurons, but its effect on astrocytes has been poorly investigated. Here, we show that 6OHDA dose-dependently impairs autophagy in human U373 cells and primary murine astrocytes in the absence of cell death. LC3II downregulation was observed 6 to 48 h after treatment. Interestingly, 6OHDA enhanced NRH:quinone oxidoreductase 2 (NQO2) expression and activity in U373 cells, even if 6OHDA turned out not to be its substrate. Autophagic flux was restored by inhibition of NQO2 with S29434, which correlated with a partial reduction in oxidative stress in response to 6OHDA in human and murine astrocytes. NQO2 inhibition also increased the neuroprotective capability of U373 cells, since S29434 protected dopaminergic SHSY5Y cells from 6OHDA-induced cell death when cocultured with astrocytes. The toxic effects of 6OHDA on autophagy were attenuated by silencing NQO2 in human cells and primary astrocytes from NQO2−/− mice. Finally, the analysis of Gene Expression Omnibus datasets showed elevated NQO2 gene expression in the blood cells of early-stage PD patients. These data support a toxifying function of NQO2 in dopaminergic degeneration via negative regulation of autophagy and neuroprotection in astrocytes, suggesting a potential pharmacological target in PD.

Highly efficient and simultaneous production of thirteen taxanes from Taxus × media and mining of their new bioactivity

Taxus serves as a primary natural source of paclitaxel, a valuable anticancer drug. Developing an efficient and synchronous separation and purification process for the abundant taxanes in Taxus, as well as mining their potential bioactive, can not only improve the production efficiency of paclitaxel but also further reduce the production cost. In this study, the abundant taxanes in the leaves and branches of Taxus × media, as well as the intermediates obtained during the purification process, were analyzed by high-performance liquid chromatography (HPLC) in combination with high-resolution accurate-mass spectrometry (LC-HR-MS/MS). Subsequently, thirteen taxanes were successfully separated and purified to a purity of over 98% with a recovery rate of 61% in a pilot-scale production. Notably, the purity and recovery of paclitaxel were increased to over 99.2% and 78.2%, respectively. Interestingly, taxuspine M was identified for the first time to exhibit superior binding properties with poly ADP-ribose polymerase 1 (PARP1) and NRH:quinone oxidoreductase 2 (NQO2) proteins, which were highly associated with breast cancer, through virtual screening across the human disease therapeutic protein in the therapeutic target database (TTD). Moreover, taxuspine M demonstrated significant anti-proliferative ability against the Michigan Cancer Foundation-7 (MCF-7) cell line with an IC50 value of 195.5 nM, indicating its potential as an anticancer agent. This study provides an improved scalable process for the highly efficient and comprehensive utilization of plant-source pharmaceutical resources.

3-Arylidene-2-oxindoles as Potent NRH:Quinone Oxidoreductase 2 Inhibitors.

The enzyme NRH:quinone oxidoreductase 2 (NQO2) plays an important role in the pathogenesis of various diseases such as neurodegenerative disorders, malaria, glaucoma, COVID-19 and cancer. NQO2 expression is known to be increased in some cancer cell lines. Since 3-arylidene-2-oxindoles are widely used in the design of new anticancer drugs, such as kinase inhibitors, it was interesting to study whether such structures have additional activity towards NQO2. Herein, we report the synthesis and study of 3-arylidene-2-oxindoles as novel NRH:quinone oxidoreductase inhibitors. It was demonstrated that oxindoles with 6-membered aryls in the arylidene moiety were obtained predominantly as E-isomers while for some 5-membered aryls, the Z-isomers prevailed. The most active compounds inhibited NQO2 with an IC50 of 0.368 µM. The presence of a double bond in the oxindoles was crucial for NQO2 inhibition activity. There was no correlation between NQO2 inhibition activity of the synthesized compounds and their cytotoxic effect on the A549 cell line.

The influence of NQO2 on the dysfunctional autophagy and oxidative stress induced in the hippocampus of rats and in SH-SY5Y cells by fluoride.

For investigating the mechanism of brain injury caused by chronic fluorosis, this study was designed to determine whether NRH:quinone oxidoreductase 2 (NQO2) can influence autophagic disruption and oxidative stress induced in the central nervous system exposed to a high level of fluoride. Sprague-Dawley rats drank tap water containing different concentrations of fluoride for 3 or 6months. SH-SY5Y cells were either transfected with NQO2 RNA interference or treated with NQO2 inhibitor or activator and at the same time exposed to fluoride. The enrichment of gene signaling pathways related to autophagy was evaluated by Gene Set Enrichment Analysis; expressions of NQO2 and autophagy-related protein 5 (ATG5), LC3-II and p62, and mammalian target of rapamycin (mTOR) were quantified by Western-blotting or fluorescent staining; and the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) assayed biochemically and reactive oxygen species (ROS) detected by flow cytometry. In the hippocampal CA3 region of rats exposed to high fluoride, the morphological characteristics of neurons were altered; the numbers of autophagosomes in the cytoplasm and the levels of NQO2 increased; the level of p-mTOR was decreased, and the levels of ATG5, LC3-II and p62 were elevated; and genes related to autophagy enriched. In vitro, in addition to similar changes in NQO2, p-mTOR, ATG5, LC3 II, and p62, exposure of SH-SY5Y cells to fluoride enhanced MDA and ROS contents and reduced SOD activity. Inhibition of NQO2 with RNAi or an inhibitor attenuated the disturbance of the autophagic flux and enhanced oxidative stress in these cells exposed to high fluoride. Our findings indicate that NQO2 may be involved in regulating autophagy and oxidative stress and thereby exerts an impact on brain injury caused by chronic fluorosis.

Apigenin and Luteolin Regulate Autophagy by Targeting NRH-Quinone Oxidoreductase 2 in Liver Cells.

Dietary flavonoids stimulate autophagy and prevent liver dysfunction, but the upstream signaling pathways triggered by these compounds are not well understood. Certain polyphenols bind directly to NRH-quinone oxidoreductase 2 (NQO2) and inhibit its activity. NQO2 is highly expressed in the liver, where it participates in quinone metabolism, but recent evidence indicates that it may also play a role in the regulation of oxidative stress and autophagy. Here, we addressed a potential role of NQO2 in autophagy induction by flavonoids. The pro-autophagic activity of seven flavonoid aglycons correlated perfectly with their ability to inhibit NQO2 activity, and flavones such as apigenin and luteolin showed the strongest activity in all assays. The silencing of NQO2 strongly reduced flavone-induced autophagic flux, although it increased basal LC3-II levels in HepG2 cells. Both flavones induced AMP kinase (AMPK) activation, while its reduction by AMPK beta (PRKAB1) silencing inhibited flavone-induced autophagy. Interestingly, the depletion of NQO2 levels by siRNA increased the basal AMPK phosphorylation but abrogated its further increase by apigenin. Thus, NQO2 contributes to the negative regulation of AMPK activity and autophagy, while its targeting by flavones releases pro-autophagic signals. These findings imply that NQO2 works as a flavone receptor mediating autophagy and may contribute to other hepatic effects of flavonoids.

Association Between a Tri-allelic Polymorphism in the Estrogen Metabolism Oxidoreductase NRH:Quinone Oxidoreductase 2 Gene and Risk of Breast Cancer by Molecular Subtype

Background: We hypothesized that NRH:quinone oxidoreductase 2 (NQO2) is a candidate susceptibility gene for breast cancer because of its known enzymatic activity on estrogen-derived quinones. A tri-allelic polymorphism in the NQO2 gene might be associated with the risk of luminal-like breast cancer. Methods: In this case-control study, 2,865 women were recruited, including 1,164 patients with pathologically confirmed breast cancer and 1,701 cancer-free controls. The tri-allelic genetic polymorphism (I-29, I-16, and D alleles) was genotyped by a polymerase chain reaction and restriction fragment length polymorphism (RFLP)-based assay. Because the I-16 allele frequency is rare (approximately 1.0%), individuals carrying the I-16 allele were excluded from the analysis. Breast cancer subtypes were classified according to ER, PR, HER2, and grade. Results: In the association analysis of allele, an increased risk of breast cancer is associated with I-29 allele [82.5% in case group and 79.0% in the control group; odds ratio (OR), 1.25; 95% CI, 1.09–1.43, compared with D allele, p = 0.0015]. In the association analysis of genotype, the I-29-containing genotype was significantly correlated with breast cancer under a dominant model (adjusted OR, 1.31, 95% CI, 1.12–1.54, p = 0.001). Moreover, in the subtype analysis, there was a significant association of the I-29/D polymorphism with luminal-like breast cancer (adjusted OR, 1.54, 95% CI, 1.22–1.94, p = 0.001 for luminal-A disease; adjusted OR, 1.37, 95% CI, 1.06–1.76, p = 0.014 for luminal-B disease) but not with HER2-enriched or triple-negative subtypes. Conclusion: The tri-allelic polymorphism in the NQO2 gene is associated with breast cancer risk, especially for the luminal-like subtype. Our findings provide a new piece of molecular epidemical evidence supporting the hypothesis that estrogen and its metabolites are carcinogens of luminal-like breast cancer. Further external validation studies are needed.

Curcumol Overcomes TRAIL Resistance of Non-Small Cell Lung Cancer by Targeting NRH:Quinone Oxidoreductase 2 (NQO2).

Resistance to tumor‐necrosis‐factor‐related apoptosis‐inducing ligand (TRAIL) of cancer cell remains a key obstacle for clinical cancer therapies. To overcome TRAIL resistance, this study identifies curcumol as a novel safe sensitizer from a food‐source compound library, which exhibits synergistic lethal effects in combination with TRAIL on non‐small cell lung cancer (NSCLC). SILAC‐based cellular thermal shift profiling identifies NRH:quinone oxidoreductase 2 (NQO2) as the key target of curcumol. Mechanistically, curcumol directly targets NQO2 to cause reactive oxygen species (ROS) generation, which triggers endoplasmic reticulum (ER) stress‐C/EBP homologous protein (CHOP) death receptor (DR5) signaling, sensitizing NSCLC cell to TRAIL‐induced apoptosis. Molecular docking analysis and surface plasmon resonance assay demonstrate that Phe178 in NQO2 is a critical site for curcumol binding. Mutation of Phe178 completely abolishes the function of NQO2 and augments the TRAIL sensitization. This study characterizes the functional role of NQO2 in TRAIL resistance and the sensitizing function of curcumol by directly targeting NQO2, highlighting the potential of using curcumol as an NQO2 inhibitor for clinical treatment of TRAIL‐resistant cancers.

Role of Nrf2-antioxidant in radioprotection by root extract of Inula racemosa

Purpose: Inula racemosa, a Trans-Himalayan plant is an important medicinal herb. In this study, the radio-modulatory efficacy of aqueous root extract of I. racemosa was investigated.Materials and methods: Normal Kidney Epithelium cells were treated with extract (50–200 μg/ml) and exposed to 3 Gy of γ-radiation, while C57BL/6 mice were administered with extract (300–600 mg/kg BW) intraperitoneally prior to exposure to 7.5 Gy of γ-radiation to assess radiation modulatory efficacy.Results: The administration of extract (30 min and 1 h) prior to radiation exposure improved the survival of NKE cells (as measured by proliferation), restored MMP and ROS levels as compared to radiation-exposed alone cells. These cells showed up-regulated Nrf2 protein levels at 7 h and increased expression of HO-1 and NOQ1 protein at 24 h In mice, the 30 days whole body survival study demonstrated that extract pre-treatment increases survival or delays the onset of radiation-induced mortality as against 70% mortality of 7.5 Gy of γ-radiation.Conclusions: The aqueous extract of roots of I. racemosa enhanced the survival of irradiated NKE cells and rescued C57BL/6 mice against WBI-induced mortality. The radiation modulation efficacy was mediated through cumulative activation of HO-1 and NQO1 downstream of Nrf2 translocation in NKE cells.Abbreviations:ARE: Antioxidant Response Element; FITC: Fluorescein isothiocyanate; GCS: Glutamylcysteine synthase; HO-1: Heme oxygenase-1; LPS: Lipopolysacharide; MRP: Multidrug Resistance-Associated Proteins; NQO1: NAD(P)H Quinone Dehydrogenase 1; NRH: Quinone Oxidoreductase 2 (NQO2); PBS: Phosphate Buffer Saline; PKA: Protein Kinase A; PKC: Protein Kinase C; PI3-kinase: Phosphatidylinositol 3-Kinase; SRB: Sulforhodamine B; UV: Ultra-Violet radiation

Abstract B003: NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis

Abstract Advanced-stage prostate cancer is characterized by osseous metastases whose establishment involves the dynamic interplay of factors and exchange of cellular contents by constituents of the tumor bony microenvironment. Among the factors that transport cellular cargoes and facilitate the transmission of signaling complexes for establishment of the bone metastatic lesions are large extracellular vesicles (LEVs), also known as large oncosomes. Information on the contents of LEVs and the mechanisms by which LEVs are formed and regulated is incomplete. In the course of studies aimed at elucidating the mechanisms of bony metastases using the LNCaP-C4-2B prostate cancer progression model, we show that the expression and cellular distribution of nicotinamide riboside: quinone oxidoreductase 2 (NQO2) and glutaminase (GLS, kidney type) both participate in the induction of membrane-localized oxidative stress and the formation of LEVs. The salient features of our findings are the following: The LNCaP model system exhibits gradually increasing oxidative stress levels as it progresses to the bone metastatic derivative C4-2B, while at the same time showing a progressive decrease in the expression level of NQO2. Reverse transcription PCR studies showed that NQO2 mRNA levels were unchanged between LNCaP and its metastatic derivatives C4, C4-2 and C4-2B. As a control, the expression level of the analogous enzyme NADP(H):quinone oxidoreductase 1 (NQO1) was also unchanged. These results suggested the possibility that the NQO2 enzyme in the bone metastatic C4-2B derivative could be localized elsewhere, possibly at an extracellular location. Further studies showed that a fraction of the NQO2 enzyme was associated with the detergent resistant membrane fraction of the C4-2B prostate cancer cells. Molecular modeling studies showed that the NQO2 enzyme had two caveolin-1 and NQO1 enzyme had one caveolin-1 binding sites. While the caveolin-1 binding site of NQO1 is buried inside the molecule and hence inaccessible to caveolin-1, one of the two binding sites available in NQO2 was external and accessible to caveolin-1. We have validated the specific interaction between NQO2 and caveolin-1 using deletion constructs of caveolin-1 fused with glutathione S-transferase (GST). Mitochondrial preparations of LNCaP, C4, C4-2, and C4-2B cells showed increasing level of expression of the GLS enzyme, suggesting a correlation between increased oxidative stress, glutaminase activity, and metastatic potential. Moreover, we found that the metastatic C4-2B derivative extruded LEVs in which the enzyme NQO2 and caveolae specific caveolin-1 could be visualized as cargo using double immunofluorescence studies. The extrusion of these LEVs could be inhibited by the GLS specific inhibitor BP-TES. Characteristically, curcumin was also found to inhibit the extrusion of these LEVs. This naturally occurring plant-based compound was shown earlier to inhibit prostate cancer bone metastasis in vivo using the C4-2B model. These results indicate a role for the NQO2 enzyme at the membrane level, possibly increasing the oxidative stress in a localized manner by lipid peroxidation. These results also suggest that a glutamine/GLS-mediated metabolic reprogramming in the LNCaP model system as it progresses toward the bone metastatic C4-2B is an integral component of the force driving the metastatic process. Based on the results of our studies, a combinatorial strategy using antimetastatic therapies such as nitrogen-containing bisphosphonates (NBPs) and anti-GLS therapy or, alternatively, a naturally occurring plant compound-based anti-NQO2 and anti-GLS therapy, could be considered for treating advanced prostate cancer patients with bone metastatic complications. Citation Format: Thambi Dorai, Ankeeta B. Shah, Faith Summers, Rajamma Mathew, Jing Huang, Tze-chen Hsieh, Joseph M. Wu. NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B003.

NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis.

In the course of studies aimed at the role of oxidative stress in the development of metastatic potential in the LNCaP-C4-2B prostate cancer progression model system, we found a relative decrease in the level of expression of the cytoplasmic nicotinamide riboside: quinone oxidoreductase (NQO2) and an increase in the oxidative stress in C4-2B cells compared to that in LNCaP or its derivatives C4 and C4-2. It was also found that C4-2B cells specifically shed large extracellular vesicles (LEVs) suggesting that these LEVs and their cargo could participate in the establishment of the osseous metastases. The level of expression of caveolin-1 increased as the system progresses from LNCaP to C4-2B. Since NQO2 RNA levels were not changed in LNCaP, C4, C4-2, and C4-2B, we tested an altered cellular distribution hypothesis of NQO2 being compartmentalized in the membrane fractions of C4-2B cells which are rich in lipid rafts and caveolae. This was confirmed when the detergent resistant membrane fractions were probed on immunoblots. Moreover, when the LEVs were analyzed for membrane associated caveolin-1 as possible cargo, we noticed that the enzyme NQO2 was also a component of the cargo along with caveolin-1 as seen in double immunofluorescence studies. Molecular modeling studies showed that a caveolin-1 accessible site is present in NQO2. Specific interaction between NQO2 and caveolin-1 was confirmed using deletion constructs of caveolin-1 fused with glutathione S-transferase (GST). Interestingly, whole cell lysate and mitochondrial preparations of LNCaP, C4, C4-2, and C4-2B showed an increasing expression of glutaminase (GLS, kidney type). The extrusion of LEVs appears to be a specific property of the bone metastatic C4-2B cells and this process could be inhibited by a GLS specific inhibitor BPTES, suggesting the critical role of a functioning glutamine metabolism. Our results indicate that a high level of expression of caveolin-1 in C4-2B cells contributes to an interaction between caveolin-1 and NQO2 and to their packaging as cargo in the shed LEVs. These results suggest an important role of membrane associated oxidoreductases in the establishment of osseous metastases in prostate cancer.

Inter-individual Variability in Activity of the Major Drug Metabolizing Enzymes in Liver Homogenates of 20 Individuals

Inter-individual variability in hepatic drug metabolizing enzyme (DME) activity is a major contributor to heterogeneity in drug clearance and safety. Accurate data on expression levels and activities of DMEs is an important prerequisite for in vitro-in vivo extrapolation and in silico based predictions. Characterization and assessment of inter-correlations of the major DMEs cytochrome P450s (CYPs) and UDP-glucuronosyltransferases (UGTs) have been extensively documented, but simultaneous quantification including other major DMEs has been lacking. Assessment of inter-donor variability and inter-correlations of CYPs, UGTs, sulfotransferases (SULTs), glutathione S-transferases (GSTs), NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH: quinone oxidoreductase 2 (NQO2) in a set of 20 individual liver homogenates. The main drug metabolizing isoforms of CYP and UGT have been reaction phenotype in individual liver microsomes and NQO1, NQO2, GSTT1 and GSTT2 in corresponding cytosol. In addition, we assessed overall SULT activity in liver cytosol using acetaminophen and 7-hydroxycoumarin as non-selective substrates and cytosolic GST activity using the non-selective substrate 1-chloro-2,4-dinitrobenzene (CDNB). Expression of GST isoforms was also assessed. While hepatic NQO1 activity was highly variable, NQO2 activity was more conserved. In addition, we found that of the hepatic GST isoforms, the variation in GSTM3 levels, which is poorly studied, was highest. The majority of significant correlations were found amongst CYP and UGT enzyme activities. The dataset presented provides the absolute quantification of the largest number of hepatic DME activities so far and constitute an essential resource for in silico toxicokinetic and metabolic modelling studies.

Evaluation of analogues of furan-amidines as inhibitors of NQO2

Inhibitors of the enzyme NQO2 (NRH: quinone oxidoreductase 2) are of potential use in cancer chemotherapy and malaria. We have previously reported that non-symmetrical furan amidines are potent inhibitors of NQO2 and here novel analogues are evaluated. The furan ring has been changed to other heterocycles (imidazole, N-methylimidazole, oxazole, thiophene) and the amidine group has been replaced with imidate, reversed amidine, N-arylamide and amidoxime to probe NQO2 activity, improve solubility and decrease basicity of the lead furan amidine. All compounds were fully characterised spectroscopically and the structure of the unexpected product N-hydroxy-4-(5-methyl-4-phenylfuran-2-yl)benzamidine was established by X-ray crystallography. The analogues were evaluated for inhibition of NQO2, which showed lower activity than the lead furan amidine. The observed structure-activity relationship for the furan-amidine series with NQO2 was rationalized by preliminary molecular docking and binding mode analysis. In addition, the oxazole-amidine analogue inhibited the growth of Plasmodium falciparum with an IC50 value of 0.3 μM.

Reduction and Scavenging of Chemically Reactive Drug Metabolites by NAD(P)H:Quinone Oxidoreductase 1 and NRH:Quinone Oxidoreductase 2 and Variability in Hepatic Concentrations.

Detoxicating enzymes NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2) catalyze the two-electron reduction of quinone-like compounds. The protective role of the polymorphic NQO1 and NQO2 enzymes is especially of interest in the liver as the major site of drug bioactivation to chemically reactive drug metabolites. In the current study, we quantified the concentrations of NQO1 and NQO2 in 20 human liver donors and NQO1 and NQO2 activities with quinone-like drug metabolites. Hepatic NQO1 concentrations ranged from 8 to 213 nM. Using recombinant NQO1, we showed that low nM concentrations of NQO1 are sufficient to reduce synthetic amodiaquine and carbamazepine quinone-like metabolites in vitro. Hepatic NQO2 concentrations ranged from 2 to 31 μM. NQO2 catalyzed the reduction of quinone-like metabolites derived from acetaminophen, clozapine, 4′-hydroxydiclofenac, mefenamic acid, amodiaquine, and carbamazepine. The reduction of the clozapine nitrenium ion supports association studies showing that NQO2 is a genetic risk factor for clozapine-induced agranulocytosis. The 5-hydroxydiclofenac quinone imine, which was previously shown to be reduced by NQO1, was not reduced by NQO2. Tacrine was identified as a potent NQO2 inhibitor and was applied to further confirm the catalytic activity of NQO2 in these assays. While the in vivo relevance of NQO2-catalyzed reduction of quinone-like metabolites remains to be established by identification of the physiologically relevant co-substrates, our results suggest an additional protective role of the NQO2 protein by non-enzymatic scavenging of quinone-like metabolites. Hepatic NQO1 activity in detoxication of quinone-like metabolites becomes especially important when other detoxication pathways are exhausted and NQO1 levels are induced.

NQO2 inhibition relieves reactive oxygen species effects on mouse oocyte meiotic maturation and embryo development.

NRH: quinone oxidoreductase 2 (NQO2) is a cytosolic and ubiquitously expressed flavoprotein that catalyzes the two-electron reduction of quinone to hydroquinones. Herein, we assessed the protein expression, subcellular localization, and possible functions of NQO2 in mouse oocyte meiotic maturation and embryo development. Western blot analysis detected high and stable protein expression of NQO2 in mouse oocytes during meiotic progression. Immunofluorescence illustrated NQO2 distribution on nuclear membrane, chromosomes, and meiotic spindles. Microtubule poisons treatment (nocodazole and taxol) showed that filamentous assembly of NQO2 and its co-localization with microtubules require microtubule integrity and normal dynamics. Increased levels of NQO2, reactive oxygen species (ROS), malondialdehyde (MDA), and autophagy protein Beclin1 expression were detected in oocytes cultured with ROS stimulator vitamin K3 (VK3), combined with decreased antioxidant glutathione (GSH). These oocytes were arrested at metaphase I with abnormal spindle structure and chromosome configuration. However, this impact was counteracted by melatonin or NQO2 inhibitor S29434, and the spindle configuration and first polar body extrusion were restored. Similarly, morpholino oligo-induced NQO2 knockdown suppressed ROS, MDA, and Beclin1, instead increased GSH in oocytes under VK3. Supplementary S29434 or melatonin limited changes in NQO2, ROS, MDA, Beclin1, and GSH during in vitro aging of ovulated oocytes, thereby maintaining spindle structure, as well as ordered chromosome separation and embryo development potential after parthenogenetic activation with SrCl2. Taken together, NQO2 is involved in ROS generation and subsequent cytotoxicity in oocytes, and its inhibition can restore oocyte maturation and embryo development, suggesting NQO2 as a pharmacological target for infertility cure.

Abstract 3007: Evaluating novel 4-aminoquinoline inhibitors of NQO2 in ovarian cancer

Abstract Introduction: NRH quinone oxidoreductase 2 (NQO2) is one of the mammalian enzymes responsible for the two-electron reduction of quinones to hydroquinones. NQO2 can use a variety of dihydronicotinamide analogues as co-factors. In particular, the co-factor, EP0152R has been used in Phase I clinical trials to support evaluation of the NQO2-mediated reductive activation of CB1954. As well as being a prodrug-activating enzyme, there is evidence to show that NQO2 may be involved in cancer progression as a consequence of its ability to act as a “nanny” protein, alter cyclin D expression and modulate the activity of NF-κB. Thus, we have synthesized a series of novel 4-aminoquinolines to study their ability to inhibit NQO2 in ovarian cancer cells (OVCs). We then propose to compare the results of pharmacological inhibition with those obtained by genetic down-regulation of NQO2 level. Methods: NQO2 level in a panel of OVC cell lines was determined by using western blot analysis and Cytochrome C based spectrophotometric enzyme activity assay. Novel compounds have been synthesized and assessed for their inhibitory potency against recombinant human NQO2 (rhNQO2), using DCPIP spectrophotometric assay. MTT cytotoxicity assay was utilized for several purposes: Firstly, to determine the toxicity of known and novel NQO2 inhibitors in the SKOV3 cell line; and secondly, to evaluate the sensitivity of OVC cell lines to CB1954 in the presence of EP0152R, and subsequently measure the intracellular NQO2 inhibitory potency of novel compounds, by combining them with CB1954 and EP0152R. Results and conclusion: In the OVCs, NQO2 protein level and enzymatic activity showed an excellent correlation; however, expression and activity differed widely among the OVC cell lines. The NQO2 levels were highest in SKOV3 and lowest in the TOV112D cell lines. The sensitivity of OVCs to CB1954 was significantly increased when combined with EP0152R; supporting the notion that NQO2 mediates the toxicity of CB1954. This was confirmed when a strong correlation was observed between the cellular NQO2 activity and the responsiveness of the OVC cell lines to CB1954. Further, overexpressing NQO2 in TOV112D cells resulted in a significant enhancing of its responsiveness to treatment with CB1954. The novel 4-aminoquinolines inhibited the activity of recombinant NQO2 when used in the nano-molar range. The inherent cytotoxicity of the inhibitors (as measured by the IC50 following 24 and 96 hours exposure to SKOV3 cells) ranged between 1.7 to >100μM. There was no relationship between toxicity and the ability of the compounds to inhibit the activity of rhNQO2. However, some of these compounds showed functional activity as NQO2 inhibitors in cells; this was revealed by their ability to inhibit the toxicity of CB1954. Several compounds were able to do this at sub-μM, non-toxic concentrations and therefore they may be able to act as molecular probes for the (multi-) functional activity of NQO2 in cells. Citation Format: Balqis A. Ikhmais, Buthaina Hussein, Ian Stratford, Sally Freeman, Joaana Neill. Evaluating novel 4-aminoquinoline inhibitors of NQO2 in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3007.

Abstract A11: Downregulation of NRH:quinone oxidoreductase 2(NQO2) in the LNCaP prostate cancer progression model system: Implications to metastasis

Abstract Prostate cancer is characterized by an innate capacity to produce higher concentrations of reactive oxygen species (ROS). ROS is also a hallmark for highly aggressive disease. Our laboratory has focused on the molecular mechanisms of inhibition of prostate cancer bone metastasis by the naturally occurring plant based anti-oxidant compound curcumin, arising from the rhizomes of Curcuma Longa. These studies highlight the importance of studying the mechanisms of redox homeostasis in normal cells and how they are subverted in cancer cells. The LNCaP prostate cancer progression model system as exemplified by the development of C4, C4-2 and C4-2B ( bone metastatic) sublines derived from the parental LNCaP prostate cancer cells offer an unique avenue to study the role of redox systems in the progression to metastatic disease, culminating in the establishment of osseous metastases. The mechanisms of ROS production and destruction, the cellular machinery and the enzyme systems involved, the relationship of ROS to cellular oncogene activation and the antagonistic duality of oncogenes and tumor suppressor genes with respect to ROS homeostasis have been subjects of intense investigation. How the prostate cancer cells metabolize ROS to achieve their proliferative and metastatic potential is not completely understood. We chose to study one redox system orchestrated by the expression of NADPH:Quinone Oxidoreductase1 (NQO1) and the Nicotinamide Riboside (NRH): Quinone Oxidoreductase 2 (NQO2) in the LNCaP progression model system. Particularly, the enzyme NQO2 has been shown earlier to be the target of another polyphenolic chemopreventive antioxidant resveratrol. The role of NQO2 as a phase II detoxification enzyme acting on several xenobiotics is well characterized. However, the role of NQO2 in regulating ROS and hence cancer progression is only beginning to be understood. With this background, we investigated the potential role of NQO2/NQO1 expression in the transition from androgen dependence to androgen independence and to acquiring bone metastatic potential in C4-2B prostate cancer cells. Our studies indicate that 1) there is a down regulation of NQO2 as the androgen dependent LNCaP cells progress towards androgen independence in C4 and C4-2 cells. In C4-2B cells, there is a barely detectable level of NQO2. In contrast, there is very little change in the level of expression of NQO1; 2) Characteristically, the intracellular ROS production progressively increases from LNCaP to C4-2B; 3) In parallel, there is an up regulation of caveolin-1 as the LNCaP cells progress to bone metastatic C4-2B and 4) a small fraction of NQO2 could be detected in the detergent resistant membrane (DRM) fraction consisting of lipid rafts and caveolae. Our studies suggest that caveolin-1 may play a role in the membrane sequestration of the largely cytoplasmic NQO2 in LNCaP cells and that this interaction could be lost in bone metastatic C4-2B cells. The significance of these findings in relation to the metastatic potential of C4-2B cells will be discussed. Citation Format: Ankeeta Shah, Tze-Chen Hsieh, Rajamma Mathew, Joseph M. Wu, Thambi Dorai. Downregulation of NRH:quinone oxidoreductase 2(NQO2) in the LNCaP prostate cancer progression model system: Implications to metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A11.

Quercetin Potentiates Toxicity Of Hemolytic Drugs In G6PD Deficient Human Erythrocytes

Quercetin, a flavonoid, is widely distributed in plants and a prominent constituent of dietary supplements. Quercetin-based supplements are recommended for prevention of cancer, improvement of cardiovascular functions and mental health. However, these effects have not been validated clinically. Except for limited interactions with some drugs (fluoroquinolone antibiotics and taxol/paclitaxel), there is little evidence of safety concerns, and with oral use, quercetin-containing preparations have been generally well tolerated. Quercetin inhibits NRH: quinone oxidoreductase 2 (NQO2), an enzyme involved in quinone metabolism and detoxification. Individuals with genetic deficiency of glucose 6-phosphate dehydrogenase (G6PD) are highly susceptible to hemolytic toxicity of certain oxidant drugs. Treatment of G6PD deficient human erythrocytes in vitro with quercetin caused significant increase in methemoglobin formation and selective depletion of reduced glutathione (GSH) levels. Further, when tested in combination with 5, 6-orthoquinone PQ [6] and dapsone hydroxylamine [3], the hemotoxic metabolites of primaquine [4] and dapsone [2] respectively, caused significant potentiation of their hemotoxic effect as determined through an increase in methemoglobin formation and depletion of GSH. Quercetin also augmented the hemotoxic effect of 2-amino-4-chlorophenol in vitro. The results warrant further investigation of quercetin regarding its toxic interaction with drugs clinically known to cause hemolytic toxicity.

Abstract 1388: Biological role of novel NQO2 inhibitors in cancer therapeutics

Abstract Background: NRH: quinone oxidoreductase 2 (NQO2), is a cytosolic flavoprotein that catalyses the two-electron reduction of quinones into hydroquinones. The physiological role of NQO2 in cancer biology is still not well understood. Some studies have suggested that inhibiting NQO2 could have a great potential in chemoprevention. This is based on the observation that NQO2 gene knockdown promotes protection against the toxicity of quinones such as menadione. Aim: To evaluate the biological role of novel NQO2 inhibitors in cancer therapeutics. Novel inhibitors will be used as pharmacological tools to study the physiological role of NQO2 in cancer cells, and to identify the consequences of inhibiting NQO2 on the tumor microenvironment. Methods: Novel compounds with diverse structures have been tested for their inhibition of NQO2 enzymatic activity in a cell-free system using a pure enzyme assay. The functional activity of these compounds as NQO2 inhibitors in the MDA-MB 231 breast cancer cells was also assessed. The effect of these inhibitors on the cellular proliferation of MDA-MB 231 breast cancer cells was tested using an MTT assay. Off-target (DNA binding) effects were analyzed by measuring the DNA thermal temperature. Moreover, the effect of these inhibitors on modulating TNFα-mediated NF-kB transcriptional activity was tested and the mechanism behind this modulation was also examined. Results: The novel inhibitors tested resulted in the inhibition of NQO2 activity in the cell free and the cellular system at nano-molar concentrations .Inhibition of MDA-MB 231 cell proliferation was also witnessed at low micro-molar concentrations. The inhibitors also displayed no DNA binding ability. In addition, the inhibitors were able to attenuate TNFα-mediated NF-kB transcriptional activity by blocking the phosphorylation of IkBα. This has resulted in the suppression of cell growth and promotion of cell apoptosis. Conclusions: This study has demonstrated novel potent inhibitors of NQO2 that are functionally active in the cells at nano-molar concentrations. The effect of the inhibitors on TNFα-mediated NF-kB transcriptional activity raises the possibility that inhibiting NQO2 could modulate the tumor micro-environmental factors, such as metastasis and angiogenesis. Citation Format: Elham Santina, Amy Chadwick, Soraya Al Nabulsi, Sally Freeman, Constantinos Demonacos, Ian Stratford. Biological role of novel NQO2 inhibitors in cancer therapeutics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1388. doi:10.1158/1538-7445.AM2014-1388

The two common polymorphic forms of human NRH-quinone oxidoreductase 2 (NQO2) have different biochemical properties.

There are two common forms of NRH-quinone oxidoreductase 2 (NQO2) in the human population resulting from SNP rs1143684. One has phenylalanine at position 47 (NQO2-F47) and the other leucine (NQO2-L47). Using recombinant proteins, we show that these variants have similar steady state kinetic parameters, although NQO2-L47 has a slightly lower specificity constant. NQO2-L47 is less stable towards proteolytic digestion and thermal denaturation than NQO2-F47. Both forms are inhibited by resveratrol, but NQO2-F47 shows negative cooperativity with this inhibitor. Thus these data demonstrate, for the first time, clear biochemical differences between the variants which help explain previous biomedical and epidemiological findings.

NRH:Quinone Oxidoreductase 2 (NQO2) Protein Competes with the 20 S Proteasome to Stabilize Transcription Factor CCAAT Enhancer-binding Protein α (C/EBPα), Leading to Protection against γ Radiation-induced Myeloproliferative Disease

NRH:quinone oxidoreductase 2 (NQO2) is a flavoprotein that protects cells against radiation and chemical-induced oxidative stress. Disruption of the NQO2 gene in mice leads to γ radiation-induced myeloproliferative diseases. In this report, we showed that the 20 S proteasome and NQO2 both interact with myeloid differentiation factor CCAAT-enhancer-binding protein α (C/EBPα). The interaction of the 20 S proteasome with C/EBPα led to the degradation of C/EBPα. NQO2, in the presence of its cofactor NRH, protected C/EBPα against 20 S degradation. Deletion and site-directed mutagenesis demonstrated that NQO2 and 20 S competed for the same binding region of S(268)GAGAGKAKKSV(279) in C/EBPα. Exposure of mice and HL-60 cells to γ radiation enhanced the levels of NQO2, which led to an increased NQO2 interaction with C/EBPα and decreased 20 S interaction with C/EBPα. NQO2 stabilization of C/EBPα was independent of NQO1, even though both interacted with the same C/EBPα domain. NQO2(-/-) mice, deficient in NQO2, failed to stabilize C/EBPα. This contributed to the development of γ radiation-induced myeloproliferative disease in NQO2(-/-) mice.