NQO1 Activity Research Articles

The Frequency and Association of C609T and C465T Polymorphisms of NAD(P)H:Quinone Oxidoreductase Gene With Adult Acute Myeloid Leukemia

Objective: Genetic variations and mutations are the etiological factors of leukemia. NAD(P)H:quinone oxidoreductase (NQO1) plays an important role in the detoxification of quinones. C609T and C465T are 2 common polymorphisms in NQO1 resulting in lower NQO1 activity compared with wild type (CC). We assessed the frequency of C609T ( NQO1*2 ; Proline to Serine) and C465T ( NQO1*3 ; Arginine to Tryptophane) polymorphisms of the NQO1 gene among the Iranian population to determine the association between these polymorphisms and a susceptibility to adult acute myeloid leukemia (AML). Materials and Methods: Frequencies of NQO1 gene polymorphisms were determined in 140 AML patients for NQO1*2 and NQO1*3 . In addition, 160 age-sex matched healthy individuals participated in this study as a control group. Genotyping was done using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) assays. Results: No significant association was observed between these 2 polymorphisms of NQO1 and the risk of AML. Odds ratio (OR) for C609T and C465T were 0.917 (95% CI=0.513–1.639) and 1.976 (95% CI=0.549–7.121), respectively. Men showed a higher incidence of C609T and C465T NQO1 than women. The majority of patients with a mutant allele were diagnosed as M3 sub type of French-American-British (FAB) classification. Conclusions: Our findings suggest that the NQO1 C609T and C465T gene variants do not have a major influence on the susceptibility to adult AML. Interestingly, we found a higher incidence of the T allele in NQO1*2 than NQO1*3 in the control and patient groups. Further studies are required to validate these findings across different populations. * NQO1 : NAD(P)H:quinone oxidoreductase AML : acute myeloid leukemia PCR-RFLP : polymerase chain reaction and restriction fragment length polymorphism FAB : French-American-British SNP : single-nucleotide polymorphisms MTHFR : methylenetetrahydrofolate reductase CYP : cytochrome P450 GST : glutathione S-transferase TUMS : Tehran University of Medical Sciences IBTO : Iranian Blood Transfusion Organization OR : odds ratio CI : confidence intervals CC : wild type CT : heterozygous TT : homozygous HWE : Hardy-Weinberg equilibrium MPO : myeloperoxidase

Polyphenols activate Nrf2 in astrocytes via H2O2, semiquinones, and quinones

Polyphenols, which occur both in edible plants and in foodstuff, have been reported to exert a wide range of health effects; however, the mechanism of action of these molecules is not fully understood. One important cellular pathway affected by polyphenols is the activation of the transcription factor Nrf2 via the electrophile response element, which mediates generation of phase 2 detoxifying enzymes. Our study found that Nrf2 nuclear translocation and the activity of NAD(P)H quinone oxidoreductase (NQO1) were increased significantly after treatment of astrocytes with tert-butylhydroquinone (tBHQ), resveratrol, or curcumin, at 20–50μM. Incubation of tBHQ, resveratrol, and curcumin in the growth medium in the absence of astrocytes caused the accumulation of H2O2. Treatment of cells with either glutathione or metmyoglobin was found to decrease Nrf2 translocation and NQO1 activity induced by polyphenols by up to 40 and 60%, respectively. Addition of both glutathione and metmyoglobin to growth medium decreased Nrf2 translocation and NQO1 activity by up to 100 and 80%, respectively. In conclusion, because metmyoglobin, in the presence of polyphenols and glutathione, is known to interact with H2O2, semiquinones, and quinones, the up-regulation of the antioxidant defense of the cells through activation of the Nrf2 transcription factor, paradoxically, occurs via the generation of H2O2 and polyphenol-oxidized species generated from the exogenous microenvironment of the cells.

NAD(P)H: Quinone Oxidoreductase 1 is Induced by Progesterone in Cardiomyocytes

NAD(P)H: quinone oxidoreductase 1 (NQO1) is a ubiquitous flavoenzyme that catalyzes two-electron reduction of various quinones by utilizing NAD(P)H as an electron donor. Our previous study found that progesterone (PG) can protect cardiomyocytes from apoptosis induced by doxorubicin (Dox). Microarray analyses of genes induced by PG had led to the discovery of induction of NQO1 mRNA. We report here that PG induces NQO1 protein and its activity in a dose-dependent manner. Whereas NQO1 is well known as a target gene of Nrf2 transcription factor due to the presence of antioxidant response element (ARE) in the promoter, PG did not activate the ARE, suggesting Nrf2-independent induction of NQO1. To address the role of NQO1 induction in PG-induced cytoprotection, we tested the effect of NQO1 inducer β-naphthoflavone and inhibitor dicoumarol. Induction of NQO1 by β-naphthoflavone decreased Dox-induced apoptosis and potentiated the protective effect of PG as measured by caspase-3 activity. PG-induced NQO1 activity was inhibited with dicoumarol, which did not affect PG-induced cytoprotection. Dicoumarol treatment alone potentiated Dox-induced caspase-3 activity. These data suggest that while NQO1 plays a role in PG-induced cytoprotection, there are additional components contributing to PG-induced cytoprotection.

Sulforaphane protects SK‐N‐SH cells against antipsychotic‐induced oxidative stress

Adverse reactions to antipsychotic drugs (APs) have been attributed to oxidative stress. Sulforaphane (SF) is a potent antioxidant that protects against dopaminergic cell death. We examined the protective properties of SF against AP-induced oxidative stress in dopaminergic neuroblastoma cells. Human neuroblastoma SK-N-SH cells were treated with SF (0.5-5 μM), and 24 h later, haloperidol, risperidone or paliperidone (100 μM) was administered, either alone or in combination with dopamine (100 μM). To determine the antioxidant properties of SF, quinone oxidoreductase (NQO1) activity, glutathione S-transferase activity, and glutathione (GSH) levels were determined. Oxidative stress was measured by the increase in thiobarbituric acid reactive substances (TBARS) and in protein-bound quinones. Cell viability was also assessed. SF treatment increased GSH levels and induced NQO1 activity in SK-N-SH cells. Haloperidol was the only AP that increased TBARS when administered alone. When cells were cocultured with a drug in combination with dopamine, all three APs increased TBARS and protein-bound quinones and also induced neurotoxicity. In all the experimental conditions, 5 μM SF attenuated the accumulation of TBARS and protein-bound quinones and increased cell survival rates. Our results indicate that SF increases GSH levels and induces NQO1 activity and the removal of electrophilic quinones and radical oxygen species. Furthermore, SF could provide protective effects against AP-induced toxicity in dopaminergic cells.

Synthesis and biological evaluation of 8-substituted and deglucuronidated scutellarin and baicalin analogues as antioxidant responsive element activators

Flavonoids are important bioactive dietary compounds, which are proved to be antioxidant responsive element (ARE) activators to defend against electrophilic toxicants and oxidative stress. The activators induce ARE gene transcription through Nrf2 factor, a major transcriptional stimulator of cytoprotective genes, relieved from a Keap1 complex. In this report, based on the structures of two flavonoids, scutellarin and baicalin, which are extracted from two common Chinese plants Dengzhanhua (Erigeron breviscapus (vant) Hand-Mazz) and Huangqin (Scutellaria baicalensis Georigi), respectively, we synthesized several 8-substituted and deglucuronidated analogues and identified the ARE activation effects of these flavonoids. We found that the Baicalin, deglucurnonidated Baicalin and diaza cyclopenta derivative were more active. Their dose-dependent upregulation activities of ARE and NQO1 and induction effects of Nrf2 were testified. The results presented that these three analogues had good upregulation effects on ARE, and they could be potentially utilized in relieving oxidative stress, upon further neuroprotective tests.

Inhibition of Renal NQO1 Activity by Dicoumarol Suppresses Nitroreduction of Aristolochic Acid I and Attenuates its Nephrotoxicity

Aristolochic acid I (AAI) is the major toxic component of aristolochic acid that causes aristolochic acid nephropathy and Balkan endemic nephropathy. Nitroreduction is an essential metabolic process for AAI rapid clearance in different species including humans. However, which enzyme participates in AAI nitroreduction in vivo and whether this metabolic process contributes to AAI nephrotoxicity are unclear. Here, we showed that NAD(P)H:quinone oxidoreductase 1 (NQO1) was highly expressed in mouse renal tubular epithelial cells. Inhibition of NQO1 activity by dicoumarol pretreatment significantly decreased renal aristolactam I (ALI) levels, a major reductive metabolite of AAI, whereas it increased renal AAI and its major oxidative metabolite 8-hydroxy-aristolochic acid I (AAIa) levels in male C57BL/6 mice. Similar changes in renal ALI, AAI, and AAIa levels were also observed in mice pretreated with another NQO1 inhibitor, phenindione. Consistent with higher levels of renal AAI and AAIa found in dicoumarol-pretreated mice, their serum clearance was much slower compared with vehicle-pretreated mice. The survival rate of mice pretreated with dicoumarol was markedly increased when higher doses of AAI were given. Similarly, pretreatment of mice with phenindione also attenuated AAI-induced nephrotoxicity. These results indicate that NQO1 plays an important role in renal AAI nitroreduction and may thus contribute to AAI-induced nephrotoxicity.

A phase I study of amrubicin (AMR) and cyclophosphamide in patients with advanced solid organ malignancies: Correlative analysis of a trial from the Hoosier Oncology Group.

e17517 Background: AMR is an anthracycline with activity against small cell lung cancer (SCLC). Amrubicinol (AMR-OH) is an active metabolite of AMR. NAD(P)H quinone oxidoreductase 1(NQO1) participates in the metabolism of AMR and AMR-OH. NQO1 single nucleotide polymorphisms (SNPs) have been suggested as a contributing factor to AMR efficacy in Japanese patients (pts). We therefore analyzed NQO1 polymorphisms from a phase I study evaluating the combination of AMR and cyclophosphamide and their correlation with response and toxicity. Methods: Blood samples were collected from pts prior to therapy. Genomic DNA was extracted and genotyping conducted for the C to T point mutation at base pair 609 of exon 6, which codes for a proline to serine substitution in NQO1 protein. Results: Genotyping data was available for all 36 pts; CC genotype (normal enzyme activity) was detected in 23 pts (64%), CT genotype (reduced enzyme activity) in 13 pts (36%). TT genotype (associated with loss of NQO1 activity) was not detected in our pt population. Response data was available for 34 pts. No significant association (or correlation) between CC or CT polymorphisms and response or ≥ grade 4 toxicity was noted at 95% confidence level (p > 0.05). Conclusions: No correlation was noted in our study between NQO1 polymorphisms and response or toxicity to AMR. Exploratory objectives of the phase I study also include analysis of topoisomerase IIA amplifications by FISH and correlation with response to AMR. These studies are currently underway and will be presented at the ASCO Meeting.

Differential responses of targeted lung redox enzymes to rat exposure to 60 or 85% oxygen

Rat exposure to 60% O(2) (hyper-60) or 85% O(2) (hyper-85) for 7 days confers susceptibility or tolerance, respectively, of the otherwise lethal effects of exposure to 100% O(2). The objective of this study was to determine whether activities of the antioxidant cytosolic enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) and mitochondrial complex III are differentially altered in hyper-60 and hyper-85 lungs. Duroquinone (DQ), an NQO1 substrate, or its hydroquinone (DQH(2)), a complex III substrate, was infused into the arterial inflow of isolated, perfused lungs, and the venous efflux rates of DQH(2) and DQ were measured. Based on inhibitor effects and kinetic modeling, capacities of NQO1-mediated DQ reduction (V(max1)) and complex III-mediated DQH(2) oxidation (V(max2)) increased by ∼140 and ∼180% in hyper-85 lungs, respectively, compared with rates in lungs of rats exposed to room air (normoxic). In hyper-60 lungs, V(max1) increased by ∼80%, with no effect on V(max2). Additional studies revealed that mitochondrial complex I activity in hyper-60 and hyper-85 lung tissue homogenates was ∼50% lower than in normoxic lung homogenates, whereas mitochondrial complex IV activity was ∼90% higher in only hyper-85 lung tissue homogenates. Thus NQO1 activity increased in both hyper-60 and hyper-85 lungs, whereas complex III activity increased in hyper-85 lungs only. This increase, along with the increase in complex IV activity, may counter the effects the depression in complex I activity might have on tissue mitochondrial function and/or reactive oxygen species production and may be important to the tolerance of 100% O(2) observed in hyper-85 rats.

Effect of red wine polyphenol dietary supplementation on two phase II enzymes in liver of hyperhomocysteinemic mice

Hyperhomocysteinemia leads to diverse clinical manifestations, notably liver disease. The pathogenicity of homocysteine is believed to be due to its ability to produce oxidative stress. Paraoxonase-1 (Pon1), a phase I xenobiotic-metabolizing enzyme (XME) synthesized by liver with anti-oxidative properties within the circulating system is down regulated in case of hyperhomocysteinemia. In a previous study, we have shown that red wine polyphenol extract (PE) supplementation induces a decrease in plasma homocysteine level and an increase in hepatic Pon1 gene expression concomitant with an increase in hepatic and plasma Pon1 activity in a murine model of hyperhomocysteinemia. In the present study, we analyzed the effect of PE supplementation on two phase II XME: NAD(P)H:quinone oxidoreductase (Nqo1) and arylamine-N-acetyltransferase (Nat) family. We found that hyperhomocysteinemia leads to a decrease of hepatic Nqo1 gene expression and activity with a reversal effect of PE supplementation. We also found that hyperhomocysteinemia-induced decrease of peroxynitrite level is associated with an increase of hepatic total Nat activity mainly due to the Nat2 isoform with a reversal effect of PE supplementation. Our results show a beneficial effect of PE supplementation on two phase II enzymes which are altered in case of hyperhomocysteinemia.

Abstract 1850: Benzyl isothiocyanate causes proteasome-mediated degradation of aryl hydrocarbon receptor in Hepa1c1c7 cells

Abstract The biotransformation and detoxification of polycyclic aromatic carcinogens is dependent upon the activity of CYP1A1 and NQO1. Transcriptional activation of these enzymes is regulated by the aryl hydrocarbon receptor (AhR) and Nuclear Factor-Erythroid 2-Related Factor. These transcription factors reside in the cytoplasm and when activated, shed their respective chaperones and translocate into the nucleus where they interact with nuclear proteins involved with transcription of genes possessing xenobiotic (XRE) and antioxidant (ARE) response elements. We have previously reported that the phytochemical benzyl isothiocyanate (BITC) induces ligand-independent nuclear translocation of the AhR in Hepa1c1c7 cells and also induces AhR heterodimerization with ARNT. This suggests that BITC might bind the AhR and affect enzyme activities through transcriptional regulation of XRE-responsive genes. We also have observed that BITC causes rapid degradation of the AhR both alone and in combination with known receptor ligands. In the current study, we investigated whether BITC causes proteasomal degradation of the AhR and whether this degradation occurs through increased ubiqitination of the receptor. This is important because it provides a potential mechanism by which BITC affects AhR turnover and consequently its regulation of phase I and II enzymes. Hepa1 cells were treated overnight with DMSO (control), 10 µM BITC, 10 µM β-napthoflavone (BNF) or a combination of BITC and BNF both at 10 µM. AhR was immunoprecipitated and detected using western blot. AhR was degraded in samples treated with BITC alone compared to control. Additionally, BITC enhanced degradation of AhR in samples treated with both BITC and BNF compared to BNF alone. Western blots re-probed with anti-ubiquitin showed increased ubiquitination of proteins in BITC and BNF-treated samples compared to control. In additional experiments, cells were treated for 16 hours with DMSO, 10 µM BITC, 10 µM BNF or a combination of BNF (10 µM) and increasing concentrations of BITC (1-10 µM). Sub-cellular fractionation of samples treated with a combination of BITC and BNF showed a dose-dependent increase in the amount of insoluble AhR compared to either BITC or BNF alone. AhR degradation was blocked in cells pre-treated with the proteasome inhibitor MG132 but not in cells pre-treated with broad spectrum protease inhibitors or PMSF. This suggests that degradation is specific to the proteasome rather than other proteases such as serine proteases and calpains. Collectively these results show that BITC degrades the AhR and this is mediated by ubiquitin and the proteasome. AhR degradation might affect transcription of XRE responsive genes and therefore carcinogen activation. These results also provide a potential mechanism for BITC as a cancer chemopreventive agent. (Support: NIH MBRS/GM028248; RCMI RR033034) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1850. doi:10.1158/1538-7445.AM2011-1850

Abstract 2581: Indolequinone mechanism-based inhibitors of quinone reductases: Structural basis for selective inhibition of NQO2 relative to NQO1

Abstract There are two quinone reductases that occur in mammalian systems NAD(P)H:quinone oxidoreductase 1 (NQO1, EC 1.6.99.2) and NRH:quinone oxidoreductase 2 (NQO2, EC 1.10.99.2). NQO1 has been shown to be up-regulated in many types of human solid cancer; whereas NQO2 has been shown to be highly expressed in leukemias. In order to explore their potential as therapeutic targets, we have developed potent and selective mechanism-based inhibitors for both enzymes centered on the indolequinone pharmacophore. Previously, we characterized ES936 (5-methoxy-1,2-dimethyl-3-(4-nitrophenoxy)methylindole-4,7-dione) as an efficient mechanism-based inhibitor of NQO1 and we have now developed a series of indolequinones as mechanism-based inhibitors of NQO2. Substitution of the 5-methoxy group in ES936 with an aminoalkylamino side chain generated potent mechanism-based inhibitors of NQO2 that did not efficiently inhibit NQO1. NQO2 inhibition was observed with compounds with 5’-aminoalkyl chains that varied from 3 to 8 carbons in length, with maximal inhibition observed with compounds that possessed 5’-chain lengths of between 3 to 5 carbons. Compounds with substitutions at the 6-position were not efficient mechanism-based inhibitors of NQO2. As previously observed for a series of NQO1 mechanism-based inhibitors, structural requirements for efficient inhibition of NQO2 also included a substituent at the 3-position capable of leaving to generate a reactive iminium electrophile following enzymatic reduction. Selected indolequinones were tested for NQO2 inhibition using the human leukemia cell line K562 and were found to inhibit NQO2 activity in K562 cells at nanomolar concentrations while not affecting NQO1 activity. Computational-based molecular modeling simulations demonstrated the substantial difference between the active site of NQO1 and NQO2 and ESI-LC/MS provided possible explanations for selective enzyme inhibition. These indolequinones represent the first mechanism based inhibitors of NQO2 to be characterized. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2581. doi:10.1158/1538-7445.AM2011-2581

The NQO1*2/*2 polymorphism is associated with poor overall survival in patients following resection of stages II and IIIa non-small cell lung cancer

NAD(P)H:quinone oxidoreductase 1 (NQO1), is a cytosolic flavoenzyme that catalyzes the two-electron reduction of quinones into hydroquinones. A polymorphism (NQO1*2) alters enzymatic activity of NQO1 resulting in diminished NQO1 activity. Malignancies with NQO1*2 may be resistant to radiation and chemotherapy with resulting poorer survival. NQO1 allele was evaluated in subjects enrolled in ECOG 3590, a randomized comparison of radiation (RT) vs radiation and chemotherapy with cisplatin/etoposide (RCT) in patients with completely resected stages II and IIIa NSCLC. Overall survival was estimated using the Kaplan-Meier method and compared via the log-rank test. Cox models were used to assess the impact of covariates on outcomes. Among 152 patients with assessable samples, 24 (16%) had NQO1*2. Median follow-up was 139 months. The presence of NQO1*2/*2 was associated with decreased overall survival (OS) (median in the heterozygote/wild-type group 42.3 vs. 33.5 months in the variant group, p=0.04). In a multivariable Cox model, variant NQO1 (HR = 1.58, p = 0.05), age <60 (HR = 0.67, p = 0.04), PS 1 (HR = 1.47, p = 0.05), cardiovascular disease (HR = 1.93, p = 0.003) and alkaline phosphatase <100 mg/ml (HR = 0.59, p = 0.005) were all significant predictors of OS. NQO1*2/*2 may be an independent predictor of poor overall survival in individuals with resected stages II and IIIa NSCLC. Although the basis for the NQO1 association with decreased survival requires additional evaluation, NQO1 may represent a biomarker for guiding individualized therapy.

Genetic evidence for NAD(P)H:quinone oxidoreductase 1-catalyzed quinone reduction on passage through the mouse pulmonary circulation

The quinones duroquinone (DQ) and coenzyme Q(1) (CoQ(1)) and quinone reductase inhibitors have been used to identify reductases involved in quinone reduction on passage through the pulmonary circulation. In perfused rat lung, NAD(P)H:quinone oxidoreductase 1 (NQO1) was identified as the predominant DQ reductase and NQO1 and mitochondrial complex I as the CoQ(1) reductases. Since inhibitors have nonspecific effects, the goal was to use Nqo1-null (NQO1(-)/(-)) mice to evaluate DQ as an NQO1 probe in the lung. Lung homogenate cytosol NQO1 activities were 97 ± 11, 54 ± 6, and 5 ± 1 (SE) nmol dichlorophenolindophenol reduced·min(-1)·mg protein(-1) for NQO1(+/+), NQO1(+/-), and NQO1(-/-) lungs, respectively. Intact lung quinone reduction was evaluated by infusion of DQ (50 μM) or CoQ(1) (60 μM) into the pulmonary arterial inflow of the isolated perfused lung and measurement of pulmonary venous effluent hydroquinone (DQH(2) or CoQ(1)H(2)). DQH(2) efflux rates for NQO1(+/+), NQO1(+/-), and NQO1(-/-) lungs were 0.65 ± 0.08, 0.45 ± 0.04, and 0.13 ± 0.05 (SE) μmol·min(-1)·g dry lung(-1), respectively. DQ reduction in NQO1(+/+) lungs was inhibited by 90 ± 4% with dicumarol; there was no inhibition in NQO1(-/-) lungs. There was no significant difference in CoQ(1)H(2) efflux rates for NQO1(+/+) and NQO1(-/-) lungs. Differences in DQ reduction were not due to differences in lung dry weights, wet-to-dry weight ratios, perfusion pressures, perfused surface areas, or total DQ recoveries. The data provide genetic evidence implicating DQ as a specific NQO1 probe in the perfused rodent lung.

Characterization of the threshold for NAD(P)H:quinone oxidoreductase activity in intact sulforaphane-treated pulmonary arterial endothelial cells

Treatment of bovine pulmonary arterial endothelial cells in culture with the phase II enzyme inducer sulforaphane (5 μM, 24 h; sulf-treated) increased cell-lysate NAD(P)H:quinone oxidoreductase (NQO1) activity by 5.7 ± 0.6 (mean ± SEM)-fold, but intact-cell NQO1 activity by only 2.8 ± 0.1-fold compared to control cells. To evaluate the hypothesis that the threshold for sulforaphane-induced intact-cell NQO1 activity reflects a limitation in the capacity to supply NADPH at a sufficient rate to drive all the induced NQO1 to its maximum activity, total KOH-extractable pyridine nucleotides were measured in cells treated with duroquinone to stimulate maximal NQO1 activity. NQO1 activation increased NADP + in control and sulf-treated cells, with the effect more pronounced in the sulf-treated cells, in which the NADPH was also decreased. Glucose-6-phosphate dehydrogenase (G-6-PDH) inhibition partially blocked NQO1 activity in control and sulf-treated cells, but G-6-PDH overexpression via transient transfection with the human cDNA alleviated neither the restriction on intact sulf-treated cell NQO1 activity nor the impact on the NADPH/NADP + ratios. Intracellular ATP levels were not affected by NQO1 activation in control or sulf-treated cells. An increased dependence on extracellular glucose and a rightward shift in the K m for extracellular glucose were observed in NQO1-stimulated sulf-treated vs control cells. The data suggest that glucose transport in the sulf-treated cells may be insufficient to support the increased metabolic demand for pentose phosphate pathway-generated NADPH as an explanation for the NQO1 threshold.

Nuclear Factor E2–Related Factor 2–Mediated Induction of NAD(P)H:Quinone Oxidoreductase 1 by 3,5-Dimethoxy-trans-stilbene

NAD(P)H:quinone oxidoreductase 1 (NQO1), a phase II enzyme, plays an important role in the detoxification or chemoprotection of carcinogens, and induction of this enzyme is a target for the prevention of carcinogenesis. Natural stilbenoids have potential cancer chemopreventive activities, potentially through affecting NQO1 activity. Along this line, several stilbenoids were evaluated to procure more potent compounds for inducing NQO1 activity in cultured murine Hepa 1c1c7 cells. As a result, we found that 3,5-dimethoxy-trans-stilbene (DMS) possesses potent NQO1 induction activity through up-regulation of both protein and mRNA expression of NQO1 as determined by Western blot and reverse transcription-polymerase chain reaction analysis, respectively. DMS also increased protein expression of heme oxygenase-1 (HO-1), another phase II enzyme. This induction of NQO1 and HO-1 by DMS was closely related to the regulation of nuclear factor E2-related factor 2 (Nrf2). The translocation and activation of Nrf2 by DMS was also involved in the modulation of the upstream signal transduction molecule, protein kinase C δ. These findings suggest that DMS might have a cancer chemopreventive activity by inducing detoxifying enzymes such as NQO1 and HO-1.

Induction of biotransformation enzymes by the carcinogenic air-pollutant 3-nitrobenzanthrone in liver, kidney and lung, after intra-tracheal instillation in rats

3-Nitrobenzanthrone (3-NBA), a carcinogenic air pollutant, was investigated for its ability to induce cytochrome P450 (CYP) 1A1/2 and NAD(P)H:quinone oxidoreductase (NQO1) in liver, kidney and lung of rats treated by intra-tracheal instillation. The organs used were from a previous study performed to determine the persistence of 3-NBA-derived DNA adducts in target and non-target tissues (Bieler et al., Carcinogenesis 28 (2007) 1117–1121, [22]). NQO1 is the enzyme reducing 3-NBA to N-hydroxy-3-aminobenzanthrone ( N-OH-3-ABA) and CYP1A enzymes oxidize a human metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), to yield the same reactive intermediate. 3-NBA and 3-ABA are both activated to species forming DNA adducts by cytosols and/or microsomes isolated from rat lung, the target organ for 3-NBA carcinogenicity, and from liver and kidney. Each compound generated the same five DNA adducts detectable by 32P-postlabelling. When hepatic cytosols from rats treated with 0.2 or 2 mg/kg body weight of 3-NBA were incubated with 3-NBA, DNA adduct formation was 3.2- and 8.6-fold higher, respectively, than in incubations with cytosols from control animals. Likewise, cytosols isolated from lungs and kidneys of rats exposed to 3-NBA more efficiently activated 3-NBA than those of control rats. This increase corresponded to an increase in protein levels and enzymatic activities of NQO1. Incubations of hepatic, pulmonary or renal microsomes of 3-NBA-treated rats with 3-ABA led to an 9.6-fold increase in DNA-adduct formation relative to controls. The highest induction in DNA-adduct levels was found in lung. The stimulation of DNA-adduct formation correlated with expression of CYP1A1/2 induced by the intra-tracheal instillation of 3-NBA. The results demonstrate that 3-NBA induces NQO1 and CYP1A1/2 in livers, lungs and kidneys of rats after intra-tracheal instillation, thereby enhancing its own genotoxic and carcinogenic potential.

Abstract A47: Establishing candidate biomarkers for the pharmacodynamic action of sulforaphane in the breast

Abstract The goal of this study is the discovery of biomarkers reflecting the pharmacodynamic action of sulforaphane (SFN), initially using normal human mammary epithelial MCF10A cells and then in tissue obtained from healthy patients undergoing reduction mammoplasty. The isothiocyanate SFN is formed by the hydrolysis of glucoraphanin, a water soluble glucosinolate found in cruciferous vegetables with especially high levels measured in 3 day old broccoli sprouts. Chemoprevention by SFN is achieved in part through the upregulation of cytoprotective enzymes via the Keap1/Nrf2 pathway. Preliminary dose response and time course studies in MCF10A cells established that SFN upregulated cytoprotective enzymes as indicated by increased levels of NQO1 transcripts, protein and activity. NQO1 induction was dependent on the Keap1/Nrf2 pathway since NQO1 transcripts, protein and activity were enhanced by KEAP1 siRNA knockdown in MCF10As. For the biomarker discovery phase SFN treated and KEAP1 knockdown MCF10A cells were analyzed by microarray to determine transcriptomic changes in response to pharmacological and genetic stimulation of the Keap1/Nrf2 pathway. The two experimental approaches were analyzed independently and then compared. The predominant class of upregulated genes common to both SFN treatment and KEAP1 knockdown were xenobiotic metabolism and antioxidant genes including NQO1, heme oxygenase (decycling) 1, sequestosome 1, thioredoxin reductase 1 and peroxiredoxin 1. The most highly upregulated genes in this class were the aldo-keto reductase family members including AKR1B1, AKR1B10, AKR1C1 and AKR1C3. Other classes of genes with overlapping regulation were glutathione metabolism genes such as GCLC and GCLM, molecular transport, cell growth and cell death regulation genes. Some members of the xenobiotic metabolism, molecular transport, cell growth and cell death regulation classes of genes were also regulated exclusively by SFN. Additionally histone cluster and chromatin remodeling genes were downregulated by SFN. Potential biomarker genes from the microarray study include NQO1, GCLC, SQSTM1 and AKR1C1. An accompanying proteomic study for biomarker discovery using Stable Isotopic Labeling with Amino Acids in Cell Culture (SILAC) is underway. The transcriptomic and proteomic changes in response to SFN treatment and KEAP1 knockdown will be compared to determine if the changes seen on the transcriptomic level are translated to the proteomic level. Candidate biomarkers identified from microarray and SILAC studies reflecting the pharmacodynamic action of SFN in human cells will be analyzed by Western blot, qRT-PCR and enzyme assays in tissue from reduction mammoplasty patients. These women have been randomized in a clinical trial to receive either a broccoli sprout preparation containing 100 micromole SFN or a placebo beverage daily for 10 days prior to surgery. Supported by DOD grant BC073262 and NIH grants P50 CA088843 and U54 RR020839. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A47.

Modulation of rat hepatic and kidney phase II enzymes by cabbage juices: comparison with the effects of indole-3-carbinol and phenethyl isothiocyanate

The effect of raw cabbage and sauerkraut juices on the expression and activity of phase II enzymes, glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase 1 (NQO1), in the rat liver and kidney was compared with that of two commercially available products of glucosinolate degradation: indole-3-carbinol (I3C) and phenethyl isothiocyanate (PEITC). Male Wistar rats were treated by oral administration with cabbage juices, I3C or PEITC for 4, 10 and 30 d. The results showed that juices, particularly sauerkraut juice as with I3C and PEITC, significantly increased GST and NQO1 activities in the rat liver. The only exception was the 30 d time point of feeding with raw cabbage juice. Cabbage juices, I3C and PEITC affected the hepatic GST μ to the greatest extent and GST α to a lesser extent. The results of the present study also showed that the treatment of rats with juices and compounds tested caused the translocation of the NF-E2-related transcription factor (Nrf2) active subunit from the cytosol to the nucleus, providing an argument for the involvement of this transcription factor in the induction of GST and NQO1. In contrast to the liver, cabbage juices affected only the renal GST θ, while treatment with I3C and PEITC significantly increased the activity of NQO1. Thus, the results of the present study indicate that induction of the key detoxifying enzymes by cabbage juices, particularly sauerkraut, may be responsible for their chemopreventive activity demonstrated by epidemiological studies and in animal models. However, the final effects might be organ or tissue dependent.

Hyperthermia enhances the effect of β-lapachone to cause γH2AX formations and cell death in human osteosarcoma cells

Purpose: The anti-cancer effect of β-lapachone (β-lap) is positively related to the cellular activity of NAD(P)H:quinone oxidoreductase (NQO1). Heat shock has been reported to elevate cellular NQO1. The effect of heating on the NQO1 expression in human osteosarcoma cells (HOS) and the response of the cells to the combined treatment with β-lap and hyperthermia was investigated.Materials and methods: The effects of β-lap alone, hyperthermia alone and in combination to cause clonogenic death and apoptosis in HOS cells were elucidated. The effect of heating on the NQO1expression was evaluated with western blot analysis. The effect of β-lap on the cell cycle distribution was elucidated with flow cytometry and to cause DNA damage was determined by assessing the γH2AX foci formation.Results: Treatment of HOS cells with β-lap at 42°C was markedly more effective than that at 37°C in causing clonogenic cell death. Heating caused a long-lasting up-regulation of NQO1 in the cells, and sensitised the cells to β-lap. The γH2AX foci formation was increased immediately after β-lap treatment and preheating increased the β-lap-induced γH2AX foci formation.Conclusions: The sensitivity of HOS cells to β-lap was increased not only during heating but also after heating as demonstrated by the increase in the clonogenic cell death and γH2AX foci formation. The increase in β-lap sensitivity after heating appeared to be due to the heat-induced elevation of NQO1 activity.

Inhibitors of NQO1: Identification of compounds more potent than dicoumarol without associated off-target effects

The enzyme NAD(P)H quinone oxidoreductase (NQO1) can function both as a detoxifying enzyme as well as chaperone protein. The latter property has been extensively characterized by the use of dicoumarol which inhibits the chaperone properties of NQO1 in cells. However, the use of this compound is compromised by its multiple “off-target” effects. Coumarin-based compounds that are more potent than dicoumarol as inhibitors of NQO1 in cells have been identified (Nolan et al., Biochem Pharmacol 2010;80:977–81). The purpose of the work reported here is to evaluate the off-target effects of these compounds when compared to dicoumarol. A range of these substituted coumarins are identified that are significantly less toxic than dicoumarol in a panel of nine cell lines. Further a number of the compounds generate much less intracellular superoxide, and many of them also show a reduced ability to induce apoptosis when compared to dicoumarol. None of these effects correlate with the ability of the compounds to inhibit the enzymatic activity of NQO1 in cells. In conclusion, potent inhibitors of NQO1 have been identified that will be more pharmacologically useful than dicoumarol for probing the function of NQO1 in cells and tissues.