NQO2

Gene Summary

Gene:NQO2; NAD(P)H dehydrogenase, quinone 2
Aliases: QR2, DHQV, DIA6, NMOR2
Location:6p25.2
Summary:This gene encodes a member of the thioredoxin family of enzymes. It is a cytosolic and ubiquitously expressed flavoprotein that catalyzes the two-electron reduction of quinone substrates and uses dihydronicotinamide riboside as a reducing coenzyme. Mutations in this gene have been associated with neurodegenerative diseases and several cancers. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Mar 2014]
Databases:OMIM, VEGA, HGNC, Ensembl, GeneCard, Gene
Protein:ribosyldihydronicotinamide dehydrogenase [quinone]
HPRD
Source:NCBIAccessed: 11 August, 2015

Ontology:

What does this gene/protein do?
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Cancer Overview

Research Indicators

Publications Per Year (1990-2015)
Graph generated 11 August 2015 using data from PubMed using criteria.

Literature Analysis

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Tag cloud generated 11 August, 2015 using data from PubMed, MeSH and CancerIndex

Specific Cancers (6)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: NQO2 (cancer-related)

Hevir-Kene N, Rižner TL
The endometrial cancer cell lines Ishikawa and HEC-1A, and the control cell line HIEEC, differ in expression of estrogen biosynthetic and metabolic genes, and in androstenedione and estrone-sulfate metabolism.
Chem Biol Interact. 2015; 234:309-19 [PubMed] Related Publications
Estrogens have important roles in the pathogenesis of endometrial cancer. They can have carcinogenic effects through stimulation of cell proliferation or formation of DNA-damaging species. To characterize model cell lines of endometrial cancer, we determined the expression profiles of the estrogen receptors (ERs) ESR1, ESR2 and GPER, and 23 estrogen biosynthetic and metabolic genes, and investigated estrogen biosynthesis in the control HIEEC cell line and the Ishikawa and HEC-1A EC cell lines. HIEEC and Ishikawa expressed all ERs to different extents, while HEC-1A cells lacked expression of ESR1. Considering the estrogen biosynthetic and metabolic enzymes, these cells showed statistically significant different gene expression profiles for SULT2B1, HSD3B2, CYP19A1, AKR1C3, HSD17B1, HSD17B7, HSD17B12, CYP1B1, CYP3A5, COMT, SULT1A1, GSTP1 and NQO2. In these cells, E2 was formed from E1S and E1, while androstenedione was not converted to estrogens. HIEEC and Ishikawa had similar profiles of androstenedione and E1 metabolism, but hydrolysis of E1S to E1 was weaker in Ishikawa cells. HEC-1A cells were less efficient for activation of E1 into the potent E2, but metabolized androstenedione to other androgenic metabolites better than HIEEC and Ishikawa cells. This study reveals that HIEEC, Ishikawa, and HEC-1A cells can all form estrogens only via the sulfatase pathway. HIEEC, Ishikawa, and HEC-1A cells expressed all the major genes in the production of hydroxyestrogens and estrogen quinones, and in their conjugation. Significantly higher CYP1B1 mRNA levels in Ishikawa cells compared to HEC-1A cells, together with lack of UGT2B7 expression, indicate that Ishikawa cells can accumulate more toxic estrogen-3,4-quinones than HEC-1A cells, as also for HIEEC cells. This study provides further characterization of HIEEC, Ishikawa, and HEC-1A cells, and shows that they differ greatly in expression of the genes investigated and in their capacity for E2 formation, and thus they represent different in vitro models.

Stepanenko AA, Vassetzky YS, Kavsan VM
Antagonistic functional duality of cancer genes.
Gene. 2013; 529(2):199-207 [PubMed] Related Publications
Cancer evolution is a stochastic process both at the genome and gene levels. Most of tumors contain multiple genetic subclones, evolving in either succession or in parallel, either in a linear or branching manner, with heterogeneous genome and gene alterations, extensively rewired signaling networks, and addicted to multiple oncogenes easily switching with each other during cancer progression and medical intervention. Hundreds of discovered cancer genes are classified according to whether they function in a dominant (oncogenes) or recessive (tumor suppressor genes) manner in a cancer cell. However, there are many cancer "gene-chameleons", which behave distinctly in opposite way in the different experimental settings showing antagonistic duality. In contrast to the widely accepted view that mutant NADP(+)-dependent isocitrate dehydrogenases 1/2 (IDH1/2) and associated metabolite 2-hydroxyglutarate (R)-enantiomer are intrinsically "the drivers" of tumourigenesis, mutant IDH1/2 inhibited, promoted or had no effect on cell proliferation, growth and tumorigenicity in diverse experiments. Similar behavior was evidenced for dozens of cancer genes. Gene function is dependent on genetic network, which is defined by the genome context. The overall changes in karyotype can result in alterations of the role and function of the same genes and pathways. The diverse cell lines and tumor samples have been used in experiments for proving gene tumor promoting/suppressive activity. They all display heterogeneous individual karyotypes and disturbed signaling networks. Consequently, the effect and function of gene under investigation can be opposite and versatile in cells with different genomes that may explain antagonistic duality of cancer genes and the cell type- or the cellular genetic/context-dependent response to the same protein. Antagonistic duality of cancer genes might contribute to failure of chemotherapy. Instructive examples of unexpected activity of cancer genes and "paradoxical" effects of different anticancer drugs depending on the cellular genetic context/signaling network are discussed.

Lee J, Kim KS, Lee MH, et al.
NAD(P)H: quinone oxidoreductase 1 and NRH:quinone oxidoreductase 2 polymorphisms in papillary thyroid microcarcinoma: correlation with phenotype.
Yonsei Med J. 2013; 54(5):1158-67 [PubMed] Free Access to Full Article Related Publications
PURPOSE: NAD(P)H:Quinone Oxidoreductase 1 (NQO1) C609T missense variant (NQO1*2) and 29 basepair (bp)-insertion/deletion (I29/D) polymorphism of the NRH:Quinone Oxidoreductase 2 (NQO2) gene promoter have been proposed as predictive and prognostic factors for cancer development and progression. The purpose of this study is to investigate the relationship between NQO1/NQO2 genotype and clinico-pathological features of papillary thyroid microcarcinoma (PTMC).
MATERIALS AND METHODS: Genomic DNA was isolated from 243 patients; and clinical data were retrospectively analyzed. NQO1*2 and tri-allelic polymorphism of NQO2 were investigated by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis.
RESULTS: PTMC with NQO1*2 frequently exhibited extra-thyroidal extension as compared to PTMC with wild-type NQO1 (p=0.039). There was a significant relationship between I29/I29 homozygosity of NQO2 and lymph node metastasis (p=0.042). Multivariate analysis showed that the I29/I29 genotype was associated with an increased risk of lymph node metastasis (OR, 2.24; 95% CI, 1.10-4.56; p=0.026).
CONCLUSION: NQO1*2 and I29 allele of the NQO2 are associated with aggressive clinical phenotypes of PTMC, and the I29 allele represents a putative prognostic marker for PTMC.

Zou J, Mi L, Yu XF, Dong J
Interaction of 14-3-3σ with KCMF1 suppresses the proliferation and colony formation of human colon cancer stem cells.
World J Gastroenterol. 2013; 19(24):3770-80 [PubMed] Free Access to Full Article Related Publications
AIM: To investigate the biological function of 14-3-3σ protein and to look for proteins that interact with 14-3-3σ protein in colon cancer stem cells.
METHODS: Reverse transcription polymerase chain reaction was performed to amplify the 14-3-3σ gene from the mRNA of colon cancer stem cells. The gene was then cloned into the pGEM-T vector. After being sequenced, the target gene 14-3-3σ was cut from the pGEM-T vector and cloned into the pGBKT7 yeast expression plasmid. Then, the bait plasmid pGBKT7-14-3-3σ was transformed into the yeast strain AH109. After the expression of the pGBKT7-14-3-3σ fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening assay was performed by mating AH109 with Y187 that contained a HeLa cDNA library plasmid. The interaction between the 14-3-3σ protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing the plasmids from the positive colonies, we performed a bioinformatics analysis. A coimmunoprecipitation assay was performed to confirm the interaction between 14-3-3σ and the proteins obtained from the positive colonies. Finally, we constructed 14-3-3σ and potassium channel modulatory factor 1 (KCMF1) siRNA expression plasmids and transfected them into colon cancer stem cells.
RESULTS: The bait plasmid pGBKT7-14-3-3σ was constructed successfully, and the 14-3-3σ protein had no toxic or autonomous activation effect on the yeast. Nineteen true-positive colonies were selected and sequenced, and their full-length sequences were obtained. We searched for homologous DNA sequences for these sequences from GenBank. Among the positive colonies, four coding genes with known functions were obtained, including KCMF1, quinone oxidoreductase (NQO2), hydroxyisobutyrate dehydrogenase (HIBADH) and 14-3-3σ. For the subsequent coimmunoprecipitation assay, the plasmids PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH were successfully constructed, and the sequences were further confirmed by DNA sequencing. The Fugene 6 reagent was used to transfect the plasmids, and fluorescence-activated cell sorting analysis showed the transfection efficiency was 97.8% after 48 h. The HEK 293FT cells showed the stable expression of the PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH plasmids. After anti-Myc antibody immunoprecipitation with Myc-KCMF1, Myc-NQO2 and Myc-HIBADH from cell lysates, the presence of Flag-14-3-3σ protein in the immunoprecipitated complex was determined by western blot analysis. The knock-down expression of the 14-3-3σ and KCMF1 proteins significantly inhibited cell proliferation and colony formation of SW1116csc.
CONCLUSION: Genes of the proteins that interacted with 14-3-3σ were successfully screened from a HeLa cDNA library. KCMF1 and 14-3-3σ protein may affect the proliferation and colony formation of human colon cancer stem cells.

Shelton P, Jaiswal AK
The transcription factor NF-E2-related factor 2 (Nrf2): a protooncogene?
FASEB J. 2013; 27(2):414-23 [PubMed] Free Access to Full Article Related Publications
The transcription factor Nrf2 is responsible for regulating a battery of antioxidant and cellular protective genes, primarily in response to oxidative stress. A member of the cap 'n' collar family of transcription factors, Nrf2 activation is tightly controlled by a series of signaling events. These events can be separated into the basal state, a preinduction response, gene induction, and finally a postinduction response, culminating in the restoration of redox homeostasis. However, despite the immensely intricate level of control the cellular environment imposes on Nrf2 activity, there are many opportunities for perturbations to arise in the signaling events that favor carcinogenesis and, therefore, implicate Nrf2 as both a tumor suppressor and a protooncogene. Herein, we highlight the ways in which Nrf2 is regulated, and discuss some of the Nrf2-inducible antioxidant (NQO1, NQO2, HO-1, GCLC), antiapoptotic (Bcl-2), metabolic (G6PD, TKT, PPARγ), and drug efflux transporter (ABCG2, MRP3, MRP4) genes. In addition, we focus on how Nrf2 functions as a tumor suppressor under normal conditions and how its ability to detoxify the cellular environment makes it an attractive target for other oncogenes either via stabilization or degradation of the transcription factor. Finally, we discuss some of the ways in which Nrf2 is being considered as a therapeutic target for cancer treatment.

Mandal RK, Nissar K, Mittal RD
Genetic variants in metabolizing genes NQO1, NQO2, MTHFR and risk of prostate cancer: a study from North India.
Mol Biol Rep. 2012; 39(12):11145-52 [PubMed] Related Publications
Quinone oxidoreductases (NAD(P)H): quinone oxidoreductase 1 (NQO1) and NRH: quinone oxidoreductase 2 (NQO2) are an antioxidant enzyme, important in the detoxification of environmental carcinogens. Methylene-tetra-hydrofolate reductase (MTHFR), plays a role in folate metabolism and may have oncogenic role through disruption of normal DNA methylation pattern, synthesis, and impaired DNA repair. In a case-control study, genotyping was done in 195 PCa and 250 age matched unrelated healthy controls of similar ethnicity to determine variants in NQO1 exon 4 (C > T, rs4986998), exon 6 (C > T, rs1800566), NQO2 -3423 (G > A, rs2070999) and MTHFR exon 4 (C > T, rs1801133) by PCR-RFLP methods. Heterozygous genotype CT and variant allele career genotype (CT + TT) of NQO1 exon 4 showed increased risk of PCa (OR = 2.06, p = 0.033; OR = 2.02, p = 0.027). Variant allele T also revealed increased risk (OR = 1.87, p = 0.029). Similarly variant genotype TT (OR = 2.71, p = 0.009), combined genotype (CT + TT) (OR = 1.59, p = 0.019) and T allele (OR = 1.63, p = 0.002) of NQO1 exon 6 demonstrated significant risk for PCa. Diplotypes of NQO1 (exon 4 and 6), C-T (OR = 1.56, Pc = 0.007) and T-T (OR = 0.011, Pc = 3.86) was associated with an increased risk for PCa. NQO2 and MTHFR did not show any risk with PCa. Our results strongly support that common sequence variants and diplotypes of NQO1 exon 4 and 6 genes may have role in PCa risk in the North Indian population, indicating the importance of genes involved in metabolism with respect to PCa risk. Additional studies on larger populations are needed to clarify the role of variation in these genes in PCa carcinogenesis.

Malik MA, Zargar SA, Mittal B
Role of NQO1 609C>T and NQO2 -3423G>A gene polymorphisms in esophageal cancer risk in Kashmir valley and meta analysis.
Mol Biol Rep. 2012; 39(9):9095-104 [PubMed] Related Publications
Esophageal cancer (EC) is a complex multifactorial disorder, where environmental and genetic factors play major role. NADPH:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2) are phase II cytosolic enzymes that catalyze metabolism of quinones, important in the detoxification of environmental carcinogens. A case-control study was performed to investigated the associations of NQO1 609C>T and NQO2 -3423G>A polymorphisms with susceptibility to EC in a high-risk Kashmiri population of India in 135 EC patients and 195 unrelated healthy controls using PCR-RFLP. We also performed a meta analysis of nine published studies (1,224 cases and 1,740 controls) on NQO1 609C>T and evaluated the association between the NQO1 609C>T polymorphisms and esophageal cancer risk. A significant difference in NQO1 609C>T and NQO2 -3423G>A genotype distribution between EC cases and corresponding controls groups was observed (OR = 2.65; 95 % CI = 1.29-5.42 and OR = 1.88; 95 % CI = 1.02-3.49 respectively). Further, gene-gene interaction showed significantly increased risk for esophageal adenocarcinoma with variant genotypes of NQO1 609C>T and NQO2 -3423G>A polymorphisms and interaction with environmental risk factors revealed pronounced risk of EC with NQO1 609C>T TT genotype in high salted tea users of Kashmir valley (OR = 3.72, 95 % CI = 0.98-14.19). Meta analysis of NQO 609C>T polymorphism also suggested association of the polymorphism with EC in Asians as well as Europeans. In conclusion, NQO1 609C>T and NQO2 -3423G>A genetic variations modulate risk of EC in high-risk Kashmir population.

Janda E, Parafati M, Aprigliano S, et al.
The antidote effect of quinone oxidoreductase 2 inhibitor against paraquat-induced toxicity in vitro and in vivo.
Br J Pharmacol. 2013; 168(1):46-59 [PubMed] Free Access to Full Article Related Publications
BACKGROUND AND PURPOSE The mechanisms of paraquat (PQ)-induced toxicity are poorly understood and PQ poisoning is often fatal due to a lack of effective antidotes. In this study we report the effects of N-[2-(2-methoxy-6H-dipyrido{2,3-a:3,2-e}pyrrolizin-11-yl)ethyl]-2-furamide (NMDPEF), a melatonin-related inhibitor of quinone oxidoreductase2 (QR2) on the toxicity of PQ in vitro & in vivo. EXPERIMENTAL APPROACH Prevention of PQ-induced toxicity was tested in different cells, including primary pneumocytes and astroglial U373 cells. Cell death and reactive oxygen species (ROS) were analysed by flow cytometry and fluorescent probes. QR2 silencing was achieved by lentiviral shRNAs. PQ (30 mg·kg(-1)) and NMDPEF were administered i.p. to Wistar rats and animals were monitored for 28 days. PQ toxicity in the substantia nigra (SN) was tested by a localized microinfusion and electrocorticography. QR2 activity was measured by fluorimetry of N-benzyldihydronicotinamide oxidation. KEY RESULTS NMDPEF potently antagonized non-apoptotic PQ-induced cell death, ROS generation and inhibited cellular QR2 activity. In contrast, the cytoprotective effect of melatonin and apocynin was limited and transient compared with NMDPEF. Silencing of QR2 attenuated PQ-induced cell death and reduced the efficacy of NMDPEF. Significantly, NMDPEF (4.5 mg·kg(-1)) potently antagonized PQ-induced systemic toxicity and animal mortality. Microinfusion of NMDPEF into SN prevented severe behavioural and electrocortical effects of PQ which correlated with inhibition of malondialdehyde accumulation in cells and tissues. CONCLUSIONS AND IMPLICATIONS NMDPEF protected against PQ-induced toxicity in vitro and in vivo, suggesting a key role for QR2 in the regulation of oxidative stress.

Hsieh TC, Yang CJ, Lin CY, et al.
Control of stability of cyclin D1 by quinone reductase 2 in CWR22Rv1 prostate cancer cells.
Carcinogenesis. 2012; 33(3):670-7 [PubMed] Free Access to Full Article Related Publications
Aberrant expression of cyclin D1, frequently observed in human malignant disorders, has been linked to the control of G(1)→S cell cycle phase transition and development and progression in carcinogenesis. Cyclin D1 level changes are partially controlled by GSK-3β-dependent phosphorylation at threonine-286 (Thr286), which targets cyclin D1 for ubiquitination and proteolytic degradation. In our continuing studies on the mechanism of prostate cancer prevention by resveratrol, focusing on the role of its recently discovered target protein, quinone reductase 2 (NQO2), we generated NQO2 knockdown CWR22Rv1 using short hairpin RNA (shRNA)-mediated gene silencing approach. We found that, compared with cells expressing NQO2 (shRNA08), NQO2 knockdown cells (shRNA25) displayed slower proliferation and G(1) phase cell accumulation. Immunoblot analyses revealed a significant decrease in phosphorylation of retinoblastoma Rb and cyclin D1 in shRNA25 compared with shRNA08. Moreover, shRNA25 cells showed a 37% decrease in chymotrypsin-like proteasome activity. An increase in AKT activity was also observed in shRNA25, supported by a ∼1.5-fold elevation in phosphorylation and ∼50% reduction/deactivation of GSK-3α/β at Ser21/9, which were accompanied by a decrease in phosphorylation of cyclin D1 at T286. NQO2 knockdown cells also showed attenuation of resveratrol-induced downregulation of cyclin D1. Our results indicate a hitherto unreported role of NQO2 in the control of AKT/GSK-3β/cyclin D1 and highlight the involvement of NQO2 in degradation of cyclin D1, as part of mechanism of chemoprevention by resveratrol.

Yu KD, Huang AJ, Fan L, et al.
Genetic variants in oxidative stress-related genes predict chemoresistance in primary breast cancer: a prospective observational study and validation.
Cancer Res. 2012; 72(2):408-19 [PubMed] Related Publications
Chemotherapy response in patients with primary breast cancer is difficult to predict and the role of host genetic factors has not been thoroughly investigated. We hypothesized that polymorphisms in oxidative stress (OS)-related genes, including estrogen-quinone metabolizing enzymes NQO2 and GSTM1-5, may influence disease progression and treatment response. In this prospective observational study, nineteen polymorphisms tagging known variations in candidate genes were genotyped and analyzed in 806 patients with primary breast cancer. Three functional polymorphisms, which were shown to affect gene expression levels in experiments in vitro and ex vivo, modified the effect of chemotherapy on disease-free survival. There were significant interactions between chemotherapy and individual polymorphisms or combined genotypes (designated as genetic score). Patients harboring high genetic score had a 75% reduction in the hazard of disease progression compared with patients with low genetic score when no chemotherapy was administered (HR = 0.25, 95% CI: 0.10-0.63, P = 0.005); however, they received much less survival benefit from adjuvant chemotherapy compared with patients with low genetic score when chemotherapy was administered (HR = 4.60 for interaction, 95% CI: 1.63-13.3, P = 0.004). These findings were validated in another population (n = 339). In conclusion, germline polymorphisms in OS-related genes affect chemotherapy sensitivity in breast cancer patients. Although reduced OS levels might prevent breast cancer progression, they probably compromise the effectiveness of adjuvant chemotherapy. Our findings also indicate that host-related factors must be considered for individualized chemotherapy.

Jamieson D, Cresti N, Bray J, et al.
Two minor NQO1 and NQO2 alleles predict poor response of breast cancer patients to adjuvant doxorubicin and cyclophosphamide therapy.
Pharmacogenet Genomics. 2011; 21(12):808-19 [PubMed] Related Publications
OBJECTIVE: A SNP in the NQO1 gene has been implicated in the response of patients with breast cancer to anthracycline containing regimens. NQO1, and its homologue NQO2, share many substrates yet retain distinct functional differences, with NQO2 being a more permissive molecule for electron accepting substrates. We aimed to determine whether functional NQO2 variants are associated with altered response to adjuvant doxorubicin and cyclophosphamide therapy, with or without tamoxifen, in the treatment of breast cancer.
METHODS: Genomic DNA samples from 227 women with early breast cancer were genotyped for NQO1 and NQO2 polymorphisms. All participants were treated with an AC adjuvant therapy regimen. The functional implications of NQO2 polymorphisms were validated in in-vitro ectopic expression models.
RESULTS: The NQO1 SNP (rs1800566) was associated with a poorer outcome and a lower likelihood of having a treatment delay. Patients who had ER and PR negative disease and were wild type for both the NQO1 and an NQO2 SNP (rs1143684) had 100% 5-year overall survival compared with 88% for carriers of one minor allele and 70% for carriers of two or more minor alleles (P=0.018, log rank). Carriers of minor alleles of a triallelic NQO2 promoter polymorphism were more likely to be withdrawn from tamoxifen therapy prematurely due to intolerance (P=0.009, log rank). MCF-7 cells were sensitized to growth inhibition by doxorubicin and 4OH tamoxifen, but not cyclophosphamide, by ectopic expression of NQO2.
CONCLUSION: This study suggests that both NQO1 and NQO2 modulate the efficacy of AC therapy and that NQO2 is associated with tamoxifen toxicity.

Goode EL, White KL, Vierkant RA, et al.
Xenobiotic-Metabolizing gene polymorphisms and ovarian cancer risk.
Mol Carcinog. 2011; 50(5):397-402 [PubMed] Free Access to Full Article Related Publications
Because selected xenobiotic-metabolizing enzymes process pro-carcinogens that could initiate ovarian carcinogenesis, we hypothesized that single nucleotide polymorphisms (SNPs) in the genes encoding xenobiotic-metabolizing enzymes are associated with risk of ovarian cancer. Cases with invasive epithelial ovarian cancer (N = 1571 including 956 of serous sub-type) and controls (N = 2046) from three studies were genotyped at 11 SNPs in EPHX1, ADH4, ADH1A, NQO2, NAT2, GSTP1, CYP1A1, and NQO1, following an initial SNP screen in a subset of participants. Logistic regression analysis of genotypes obtained via Illumina GoldenGate and Sequenom iPlex technologies revealed the following age- and study-adjusted associations: EPHX1 rs1051740 with increased serous ovarian cancer risk [per-allele odds ratio (OR) 1.17, 95% confidence interval (95% CI) 1.04-1.32, P = 0.01), ADH4 r1042364 with decreased ovarian cancer risk (OR 0.90, 95% CI: 0.81-1.00, P = 0.05), and NQO1 rs291766 with increased ovarian cancer risk (OR 1.11, 95% CI: 1.00-1.23, P = 0.04). These findings are consistent with prior studies implicating these genes in carcinogenesis and suggest that this collection of variants is worthy of follow-up in additional studies.

Hubackova M, Vaclavikova R, Ehrlichova M, et al.
Association of superoxide dismutases and NAD(P)H quinone oxidoreductases with prognosis of patients with breast carcinomas.
Int J Cancer. 2012; 130(2):338-48 [PubMed] Related Publications
Associations of transcript levels of oxidative stress-modifying genes SOD2, SOD3, NQO1 and NQO2 and their functional single nucleotide polymorphisms (SNPs) rs4880, rs1799895, rs2536512, rs699473, rs1800566 and rs1143684 with prognosis of breast cancer patients were studied. SNPs were assessed by allelic discrimination in a cohort of 321 breast cancer patients from the Czech Republic. Transcript levels were determined by real-time polymerase chain reaction (PCR) with absolute quantification in tumor and adjacent non-neoplastic control tissues. Both genotypes and transcript levels were then compared with available clinical data on patients. Patients carrying low activity allele Leu in NQO2 rs1143684 had a greater incidence of stage 0 or I disease (i.e., better prognosis) than patients with the Phe/Phe genotype. This association was more evident in patients without expression of progesterone receptors (p = 0.031). Patients carrying the Thr allele in SOD3 rs2536512 SNP had a significantly greater incidence of tumors expressing estrogen receptors than patients carrying the Ala/Ala genotype (p = 0.007). SOD3 transcript level was significantly higher in grade 1 or 2 tumors than in grade 3 tumors (p = 0.006). Patients carrying T allele in SOD3 rs699473 SNP had significantly poorer progression-free survival (PFS) than patients carrying the CC genotype (p = 0.038). The same applied to the subgroup of patients treated by hormonal regimens (p = 0.021). Patients carrying the high activity Ala/Ala genotype in SOD2 (rs4880) had significantly poorer PFS than Val allele carriers in the group treated by cyclophosphamide but not hormonal regimens (p = 0.004). Our results suggest that NQO2, SOD2 and SOD3 may significantly modify prognosis of breast cancer patients and that their significance should be further characterized.

Malik MA, Zargar SA, Mittal B
Role of NQO1 609C>T and NQO2-3423G>A polymorphisms in susceptibility to gastric cancer in Kashmir valley.
DNA Cell Biol. 2011; 30(5):297-303 [PubMed] Related Publications
NADPH: quinone oxidoreductase 1 (NQO1) and dihydronicotinamide riboside: quinone oxidoreductase 2 (NQO2) are cytosolic enzymes that catalyze reductive activation of carcinogens from cigarette smoke, such as nitrosamines and heterocyclic amines. These enzymes also protect cells against oxidative damage from reactive oxygen species. The present study investigated the associations of genetic variants of NQO1 609C>T and NQO2 -3423G>A polymorphisms with susceptibility to gastric cancer (GC) as well as their interactions with known risk factors in Kashmir valley. A case control study was performed in 303 subjects (108 GC and 195 healthy controls). All subjects were genotyped using polymerase chain reaction-restriction fragment length polymorphism method. Data were statistically analyzed by chi-square test and logistic regression model. The NQO1 609C>T TT genotype and T allele were significantly associated with increased risk for GC, whereas NQO2 -3423G>A polymorphism did not show any association with GC. Also, NQO1 609C>T TT genotype showed significant association with gastric adenocarcinoma. The interaction of NQO1/NQO2 genotypes with high consumption of salted tea, a known risk factor, did not further modulate the risk of GC. In conclusion, NQO1 609C>T polymorphism shows association with GC risk in Kashmir valley.

Mohelnikova-Duchonova B, Marsakova L, Vrana D, et al.
Superoxide dismutase and nicotinamide adenine dinucleotide phosphate: quinone oxidoreductase polymorphisms and pancreatic cancer risk.
Pancreas. 2011; 40(1):72-8 [PubMed] Related Publications
OBJECTIVES: Pancreatic carcinoma etiology and molecular pathogenesis is weakly understood. According to the assumption that genetic variation in carcinogen metabolism further modifies the risk of exposure-related cancers, an association of functional polymorphisms in oxidative stress-modifying genes superoxide dismutase 2 (SOD2 [Ala16Val, rs4880]), SOD3 (Arg231Gly, rs1799895), nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase (NQO1 [Pro187Ser, rs1800566], and NQO2 (Phe47Leu, rs1143684) with pancreatic cancer risk was studied.
METHODS: Polymorphisms were studied by allelic discrimination.
RESULTS: In a hospital-based case-control study on 500 individuals (235 cases and 265 controls) of Czech white origin, SOD2, SOD3, NQO1, and NQO2 polymorphisms showed no significant association with pancreatic cancer risk. Major lifestyle factors such as smoking and alcohol, coffee, or tea consumption did not modify the effect of the studied polymorphisms.
CONCLUSIONS: The first European study of the SOD2, SOD3, NQO1, and NQO2 roles in pancreatic cancer etiology did not find significant associations. Despite this observation, other populations with different lifestyle(s) may be at risk and should be further studied.

Hsieh TC, Elangovan S, Wu JM
gamma-Tocotrienol controls proliferation, modulates expression of cell cycle regulatory proteins and up-regulates quinone reductase NQO2 in MCF-7 breast cancer cells.
Anticancer Res. 2010; 30(7):2869-74 [PubMed] Related Publications
BACKGROUND: Tocotrienols, a subgroup of the vitamin E family, have demonstrated antioxidant and anticancer properties. Differential growth responses among different types of tocotrienols have been observed in breast cancer cells; however, specific bioactivity of each individual tocotrienol remains to be elucidated.
MATERIALS AND METHODS: In this study, the effects of gamma-tocotrienol were examined with regard to its ability to suppress cell proliferation via modulation of cell cycle regulatory protein expression, and also from the perspective of control of cellular oxidoreductive status through regulation of detoxification enzymes, e.g., quinone reductase NQO2, using estrogen receptor-positive MCF-7 human breast cancer cells.
RESULTS: It was shown that treatment by gamma-tocotrienol suppressed MCF-7 cell proliferation in a dose- and time-dependent manner. Growth suppression by gamma-tocotrienol was accompanied by changes in the levels of cell cycle regulatory proteins, notably, Rb/E2F complex, cyclin D1/cdk4 and cyclin B1/cdk1, as exemplified by loss of cyclin D1, inhibition of specific Rb phosphorylation (pRb-p at Thr821), and by the time- and dose-dependent increase in the expression of NQO2.
CONCLUSION: By exerting control on expression of specific cell cycle regulatory proteins in concomitance with suppression of cell proliferation, as well as the induction of NQO2, gamma-tocotrienol offers promise as an added chemopreventive and/or chemotherapeutic agent against breast cancer carcinogenesis.

Choi JY, Barlow WE, Albain KS, et al.
Nitric oxide synthase variants and disease-free survival among treated and untreated breast cancer patients in a Southwest Oncology Group clinical trial.
Clin Cancer Res. 2009; 15(16):5258-66 [PubMed] Free Access to Full Article Related Publications
PURPOSE: Numerous chemotherapeutic agents are cytotoxic through generation of reactive species, and variability in genes related to oxidative stress may influence disease-free survival (DFS). We examined relationships between DFS and variants in NOS3, as well as NQO1, NQO2, and CBR3, among treated and untreated breast cancer patients in a Southwest Oncology Group clinical trial (S8897).
EXPERIMENTAL DESIGN: In the parent trial, women were assigned according to prognostic features; the high-risk group was randomized to cyclophosphamide, i.v. methotrexate, and 5-fluorouracil or to cyclophosphamide, i.v. doxorubicin, and 5-fluorouracil +/- tamoxifen, and the low-risk group did not receive adjuvant therapy. We extracted DNA from normal lymph node tissue and examined functional polymorphisms in NOS3, NQO1, NQO2, and CBR3, in relation to DFS, using Cox proportional hazard model.
RESULTS: There were significant interactions between DFS, adjuvant therapy, and NOS3 Glu298Asp and -786 polymorphisms, alone and in combination (P for interaction = 0.008). When NOS3 genotypes were combined, women with genotypes encoding for lower nitric oxide who received chemotherapy had a >2-fold increase in hazard of progression (hazard ratio, 2.32; 95% confidence interval, 1.26-4.25), whereas there was reduced risk for those who did not receive adjuvant therapy (hazard ratio, 0.42; 95% confidence interval, 0.19-0.95). There were no associations between the other genotypes and DFS in either group.
CONCLUSION: Variants encoding lower activity of NOS3 may affect outcomes in breast cancer patients, with the direction of risk differing depending on chemotherapy status. These results may mirror the known dual functions of nitric oxide and nitric oxide synthase, depending on oxidative environment.

Levy S, Jaiswal AK, Forman HJ
The role of c-Jun phosphorylation in EpRE activation of phase II genes.
Free Radic Biol Med. 2009; 47(8):1172-9 [PubMed] Free Access to Full Article Related Publications
The transcription factors that bind to EpRE's play a key role in the regulation of phase II genes. In this study, we examined whether c-Jun, a partner of Nrf2 in binding to EpRE's, requires phosphorylation by JNK for binding and transcriptional activation. We used chromatin immunoprecipitation assays to measure the recruitment of transcription factors to EpRE sequences in NQO2, GCLC, and GCLM; Western analysis for phosphorylation of JNK; and EpRE-driven reporters along with a JNK-specific inhibitor peptide to determine the potential importance of c-Jun phosphorylation. Human bronchial epithelial (HBE1) and human hepatoma (HepG2) cells were exposed to 4-hydroxy-2-nonenal (HNE), and differences in the regulation of the same EpRE sequences were examined. We found that binding of c-Jun to EpRE sequences increased subsequent to HNE exposure in HepG2 cells; however, in HNE-exposed HBE1 cells, the binding of only phosphorylated c-Jun to the three EpRE sequences increased. Despite the increase in binding of phosphorylated c-Jun, reporter assays for EpRE's showed that inhibition of c-Jun phosphorylation had variable effects on basal and HNE-induced transcription of GCLC and GCLM in HBE1 cells. Thus, in terms of its role in mediating HNE induction of EpRE-mediated transcription, c-Jun seems to be a partner of Nrf2 and, whereas its phosphorylated form may predominate in one cell type versus another, the effects of phosphorylation of c-Jun on transcription can vary with the gene. This contrasts markedly with the well-established requirement for phosphorylation of c-Jun in the activation of AP-1/TRE-mediated transcription.

Hsieh TC
Antiproliferative effects of resveratrol and the mediating role of resveratrol targeting protein NQO2 in androgen receptor-positive, hormone-non-responsive CWR22Rv1 cells.
Anticancer Res. 2009; 29(8):3011-7 [PubMed] Related Publications
Trans-resveratrol, a polyphenol present in red wines and various human foods, was first reported to exhibit chemopreventive properties based on studies using a mouse skin cancer model. Our laboratory and others subsequently demonstrated the antiprostate cancer (anti-CaP) activity of resveratrol, as evident in its suppression of cell proliferation, arrest of cell cycle progression, and induction of apoptosis in androgen-responsive LNCaP and androgen-non-responsive DU145 and PC-3 CaP cells. However, the molecular mechanism of action of resveratrol has not been tested in androgen receptor (AR)-positive hormone-non-responsive CWR22Rv1 cells, which mimic the transition stages of prostate carcinoma. In this study, we investigated the antiproliferative effects of resveratrol in the context of modulation of growth suppression and NF-kappaB expression as mediated by resveratrol targeting protein NQO2, using both control and NQO2 siRNA silenced CWR22Rv1 cells. Exposure to resveratrol resulted in a potent, dose-dependent inhibition of CWR22Rv1 proliferation, which was accompanied by a reduction in the expression of NF-kappaB p65. The suppression of NF-kappaB p65 expression was abrogated in NQO2 siRNA silenced CWR22Rv1 cells, suggesting that NQO2 is upstream of and integral to the regulation of NF-kappaB p65. To our knowledge, this study is the first to reveal that resveratrol targeting protein NQO2 plays a mediating role in resveratrol-induced changes of NF-kappaB p65, which may contribute to the anti-CaP activities elicited by resveratrol.

Yu KD, Di GH, Yuan WT, et al.
Functional polymorphisms, altered gene expression and genetic association link NRH:quinone oxidoreductase 2 to breast cancer with wild-type p53.
Hum Mol Genet. 2009; 18(13):2502-17 [PubMed] Related Publications
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 and its ability to stabilize p53. We performed case-control studies to investigate the contributions of genetic variants/haplotypes of the NQO2 gene to breast cancer risk. In the first hospital-based study (n = 1604), we observed significant associations between the incidence of breast cancer and a 29 bp-insertion/deletion polymorphism (29 bp-I/D) and the rs2071002 (+237A>C) polymorphism, both of which are located within the NQO2 promoter region. Decreased risk was associated with the D-allele of 29 bp-I/D [odds ratio (OR), 0.76; P = 0.0027] and the +237C-allele of rs2071002 (OR, 0.80; P = 0.0031). Specifically, the susceptibility variants within NQO2 were notably associated with breast carcinomas with wild-type p53 (the most significant P-value: 3.3 x 10(-6)). The associations were successfully replicated in an independent population set (familial/early-onset breast cancer cases and community-based controls, n = 1442). The combined P-values of the two studies (n = 3046) are 3.8 x 10(-7) for 29 bp-I/D and 2.3 x 10(-6) for rs2071002. Furthermore, we revealed potential mechanisms of pathogenesis of the two susceptibility polymorphisms. Previous work has demonstrated that the risk-allele I-29 of 29 bp-I/D introduces transcriptional-repressor Sp3 binding sites. Using promoter reporter-gene assays and electrophoretic-mobility-shift assays, our present work demonstrated that the other risk-allele, +237A-allele of rs2071002, abolishes a transcriptional-activator Sp1 binding site. Furthermore, an ex vivo study showed that normal breast tissues harboring protective genotypes expressed significantly higher levels of NQO2 mRNA than those in normal breast tissues harboring risk genotypes. Taken together, the data presented here strongly suggest that NQO2 is a susceptibility gene for breast carcinogenesis.

Wen H, Ding Q, Fang ZJ, et al.
Population study of genetic polymorphisms and superficial bladder cancer risk in Han-Chinese smokers in Shanghai.
Int Urol Nephrol. 2009; 41(4):855-64 [PubMed] Related Publications
BACKGROUND: We investigated the association between the polymorphisms of DNA repair genes, metabolic enzyme genes, and superficial bladder cancer to better understand the role of gene polymorphisms in bladder carcinogenesis for the Han-Chinese population in Shanghai.
METHODS: The SNPs in the XPC, XPG, XRCC1, NQO2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 genes were genotyped using the TaqMan Probe-based polymerase chain reaction.
RESULTS: The AC + CC genotypes of XPC Lys939Gln and the CC genotype of XPG His1104Asp were more frequent in patients of superficial bladder cancer at the initial diagnosis (adjusted OR [95% CI], 1.89 [1.21-3.24]; adjusted OR [95% CI], 1.07 [0.86-1.87], respectively). The risk for carriers of the XPC-33512C allele increased after stratifying by smoking habits (adjusted OR [95% CI], 1.95 [0.56-6.09]). There was a significant trend for an increased carcinogenesis risk with an increasing number of putative high-risk alleles. We found no significant associations between any of the ten polymorphisms and clinicopathological features of superficial bladder cancer.
CONCLUSION: These results suggest that the polymorphism in XPC Lys939Gln may modulate superficial bladder cancer risk, and these effects may preferentially affect current smokers. The data also support the possibility of an increased risk for superficial bladder cancer in individuals with a higher number of genetic variations in DNA repair and metabolic enzyme genes.

Hsieh TC, Wu JM
Ethanolic extracts of herbal supplement Equiguard suppress growth and control gene expression in CWR22Rv1 cells representing the transition of prostate cancer from androgen dependence to hormone refractory status.
Int J Oncol. 2008; 32(1):209-19 [PubMed] Related Publications
Dietary supplements and botanical products are widely used by patients diagnosed with prostate cancer (CaP) as a primary or adjuvant form of treatment for their medical conditions in the United States. Many of the available products are complex mixtures composed of extracts from foreign plants, whose mechanism of action typically is not systematically and rigorously investigated. Laboratory studies employing precisely defined conditions and referenced methodologies are essential not only for standardization and characterization of the products, but are also important requisites for providing scientific evidence and molecular insights in regard to the clinical efficacies some of these products purportedly demonstrate. In previous studies from this laboratory, we serendipitously observed that Equiguard, a dietary supplement formulated with extracts from nine Chinese herbs for preventing decline in renal functions associated with the aging process, contain 70% ethanol-extractable ingredients that displayed potent growth inhibitory activities in androgen-dependent (AD) LNCaP and androgen-independent (AI) DU-145 and PC-3 cells. Moreover, significant reduction in expression of the androgen receptor (AR) and prostate specific antigen (PSA) also occurred in Equiguard-treated LNCaP cells. Although these results offer the possibility that Equiguard confers chemoprevention for CaP, it remains undetermined whether Equiguard functions in CaP cell types that represent the transition of AD to the AI status. Further, details of its mechanism of action have not been fully elucidated. The studies described in this report focusing on CWR22Rv1 cells are intended to fill these gaps. These cells express AR and PSA, yet show weak responsiveness to androgens and largely proliferate in an AI-independent manner - features that mimic AD --> AI in clinically advanced disease. Using the CWR22Rv1 cells, we showed that 70% ethanolic extracts of Equiguard effectively suppressed colony formation, inhibited cell proliferation, reduced expression of cell cycle regulatory proteins including cyclin D1, E2F, as well as lowered AR and PSA levels. Treatment of CWR22Rv1 cells with Equiguard also decreased cyclooxygenase 2 and led to increases in quinone reductase 1 and 2. These results provide further support that Equiguard possess multiple, chemopreventive attributes capable of disrupting the transition of AD --> AI in clinically advanced CaP.

Gong X, Kole L, Iskander K, Jaiswal AK
NRH:quinone oxidoreductase 2 and NAD(P)H:quinone oxidoreductase 1 protect tumor suppressor p53 against 20s proteasomal degradation leading to stabilization and activation of p53.
Cancer Res. 2007; 67(11):5380-8 [PubMed] Related Publications
Tumor suppressor p53 is either lost or mutated in several types of cancer. MDM2 interaction with p53 results in ubiquitination and 26S proteasomal degradation of p53. Chronic DNA damage leads to inactivation of MDM2, stabilization of p53, and apoptotic cell death. Here, we present a novel MDM2/ubiquitination-independent mechanism of stabilization and transient activation of p53. The present studies show that 20S proteasomes degrade p53. The 20S degradation of p53 was observed in ubiquitin-efficient and -deficient cells, indicating that this pathway of degradation did not require ubiquitination of p53. The cytosolic quinone oxidoreductases [NRH:quinone oxidoreductase 2 (NQO2) and NAD(P)H:quinone oxidoreductase 1 (NQO1)] interacted with p53 and protected p53 against 20S proteasomal degradation. Further studies revealed that acute exposure to radiation or chemical leads to induction of NQO1 and NQO2 that stabilizes and transiently activates p53 and downstream genes. These results suggest that stress-induced NQO1 and NQO2 transiently stabilize p53, which leads to protection against adverse effects of stressors.

Fischer TW, Zmijewski MA, Zbytek B, et al.
Oncostatic effects of the indole melatonin and expression of its cytosolic and nuclear receptors in cultured human melanoma cell lines.
Int J Oncol. 2006; 29(3):665-72 [PubMed] Related Publications
Melatonin has been shown to have oncostatic effects on malignant melanoma in vitro and in vivo. We studied the growth suppressive effects of melatonin over a wide range of concentrations in four melanoma cell lines (SBCE2, WM-98, WM-164 and SKMEL-188) representative for different growth stages and phenotype. Melanoma cells were incubated with melatonin 10(-12)-10(-3) M, and proliferation and clonogenicity was assessed at 12 h and 14 days, respectively. We also determined the expression of cytosolic quinone oxidoreductases NQO1, NQO2 (known as MT3 receptor) and nuclear receptor RORalpha by RT-PCR. Melatonin at pharmacological concentrations (10(-3)-10(-7) M) suppressed proliferation in all melanoma cell lines. In SKMEL-188 cells cultured in serum-free media, melatonin at low concentrations (10(-12)-10(-10) M) also slightly attenuated the proliferation. The effects of pharmacological doses of melatonin were confirmed in the clonogenic assay. Expression of NQO1 was detected in all cell lines, whereas NQO2 and nuclear receptor RORalpha including its isoform RORalpha4 were present only in SBCE2, WM-164 and WM-98. Thus, melatonin differentially suppressed proliferation in melanoma cell lines of different behaviour. The intensity of the oncostatic response to melatonin could be related to the cell-line specific pattern of melatonin cellular receptors and cytosolic binding protein expression.

Hsieh TC, Wang Z, Hamby CV, Wu JM
Inhibition of melanoma cell proliferation by resveratrol is correlated with upregulation of quinone reductase 2 and p53.
Biochem Biophys Res Commun. 2005; 334(1):223-30 [PubMed] Related Publications
Resveratrol (trans-3,4',5-trihydroxystilbene) is a grape-derived polyphenol under intensive study for its potential in cancer prevention. In the case of cultured human melanoma cells, no one to our knowledge has investigated whether resveratrol exerts similar anti-proliferative activities in cells with different metastatic potential. Therefore, we examined the effects of this polyphenol on the growth of weakly metastatic Line IV clone 3 and on autologous, highly metastatic Line IV clone 1 cultured melanoma cells. Comparable inhibition of growth and colony formation resulted from treatment by resveratrol in both cell lines. Flow cytometric analysis revealed that resveratrol-treated clone 1 cells had a dose-dependent increase in S phase and a concomitant reduction in the G(1) phase. No detectable change in cell cycle phase distribution was found in similarly treated clone 3 cells. Western blots demonstrated a significant increase in the expression of the tumor suppressor gene p53, without a commensurate change in p21 and several other cell cycle regulatory proteins in both cell types. Chromatography of Line IV clone 3 and clone 1 cell extracts on resveratrol affinity columns revealed that the basal expression of dihydronicotinamide riboside quinone reductase 2 (NQO2) was higher in Line IV clone 1 than clone 3 cells. Levels of NQO2 but not its structural analog NQO1 were dose-dependently increased by resveratrol in both cell lines. We propose that induction of NQO2 may relate to the observed increased expression of p53 that, in turn, contributes to the observed suppression of cell growth in both melanoma cell lines.

Wang W, Jaiswal AK
Sp3 repression of polymorphic human NRH:quinone oxidoreductase 2 gene promoter.
Free Radic Biol Med. 2004; 37(8):1231-43 [PubMed] Related Publications
Human NRH:quinone oxidoreductase 2 (NQO2) gene-containing 29-bp deletion/insertion polymorphic promoters were found to be associated with susceptibility to Parkinson's disease. Here, we demonstrate that the NQO2 gene is differentially expressed by the polymorphic promoters in human fibroblasts and Hep-G2 cells transfected with NQO2 gene reporter constructs. Promoter containing the 29-bp insertion polymorphism demonstrated significantly lower NQO2 gene expression. Deletion mutagenesis and DNase I footprinting analysis of the promoter without the 29-bp insertion identified three protected regions (region A, B, and C). Band- and supershift and transfection assays showed binding of transcription factor Sp1 to regions A and B, which regulated expression of the NQO2 gene. Similar studies of the NQO2 gene promoter with the 29-bp insertion polymorphism showed that regions A and C were identical and contributed similarly as in the promoter without the 29-bp insertion to NQO2 gene expression. However, region B was found to be inserted with 29-bp DNA element and bound to both Sp1 and Sp3. Binding of Sp3 led to repression of NQO2 gene transcription by the promoter containing the 29-bp insertion polymorphism. These studies also suggest that alterations in NQO2 activity might be an important factor in susceptibility to Parkinson's disease.

Knox RJ, Burke PJ, Chen S, Kerr DJ
CB 1954: from the Walker tumor to NQO2 and VDEPT.
Curr Pharm Des. 2003; 9(26):2091-104 [PubMed] Related Publications
CB 1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] has been the subject of continued interest for over 30 years. As an anti-cancer agent, it represents one of the very few examples of a compound that shows real anti-tumor selectivity. Unfortunately, for the treatment of human disease, this anti-tumor selectivity was seen only in certain rat tumors. The basis for the anti-tumor selectivity of CB 1954 is that it is a prodrug that is enzymatically activated to generate a difunctional agent, which can form DNA-DNA interstrand crosslinks. The bioactivation of CB 1954 in rat cells involves the aerobic reduction of its 4-nitro group to a 4-hydroxylamine by the enzyme NQO1 (DT-diaphorase). The human form of NQO1 metabolizes CB 1954 much less efficiently than rat NQO1. Thus human tumors are insensitive to CB 1954. In view of the proven success of CB 1954 in the rat system, it would be highly desirable to re-create its anti-tumor activity in man. This has led to the development of CB 1954 analogs and other prodrugs activated by nitroreduction such, as those based on a self-immolative activation mechanism. A gene therapy-based approach for targeting cancer cells and making them sensitive to CB 1954 and related compounds has been developed. VDEPT (gene-directed enzyme prodrug therapy) has been used to express an E. coli nitroreductase in tumor cells and human tumor cells transduced to express this enzyme are very sensitive to prodrugs activated by nitroreduction. CB 1954 is in clinical trial for this application. Recently it has been shown that a latent nitroreductase is present in some human tumors. This is NQO2--an enzyme that requires for activity, the non-biogenic compound dihydronicotinamide riboside (NRH) as a cosubstrate. When active, NQO2 is 3000 times more effective than human DT-diaphorase in the reduction of CB 1954. NRH and reduced pyridinium derivatives that, like NRH, act as co-substrates for NQO2, produce a dramatic increase in the cytotoxicity of CB 1954 against human cell lines in vitro and its anti-tumor activity against certain human xenografts in vivo. NQO2 activity is substantially raised in tumor samples from colorectal and hepatoma patients (up to 14-fold). A phase I clinical trial of an NQO2 co-substrate with CB 1954 is scheduled.

Skelly JV, Knox RJ, Jenkins TC
Aerobic nitroreduction by flavoproteins: enzyme structure, mechanisms and role in cancer chemotherapy.
Mini Rev Med Chem. 2001; 1(3):293-306 [PubMed] Related Publications
NQO1 (DT-diaphorase) and its truncated isoenzyme, the metalloenzyme NQO2, can reduce quinone substrates by two-electron transfer. While NQO1 is a known detoxification enzyme, the function of NQO2 is less well understood. Both rat NQO1 and human NQO2 reductively bioactivate the dinitroarene CB 1954 to a cytotoxic product that behaves as a difunctional DNA-crosslinking species with potent anti-tumour activity, although human NQO1 is much less effective. A FMN-dependent nitroreductase from E. coli B also reduces quinones and reductively bioactivates CB 1954. However, this enzyme reduces CB 1954 to the 2- and 4-hydroxylamines in equivalent yield, whereas NQO1 and NQO2 generate only the 4-isomer. The reduction profile is a key factor in the development of anti-tumour prodrugs, where distinct delivery strategies are being evaluated: prodrug therapy, antibody-, macromolecule and gene-directed enzyme prodrug therapy (ADEPT, MDEPT or GDEPT). The flavoprotein enzymes are explored in terms of structure and bioreduction mechanism, particularly for use in the design of novel prodrugs with potential application as chemotherapeutic agents.

Long DJ, Jaiswal AK
NRH:quinone oxidoreductase2 (NQO2).
Chem Biol Interact. 2000; 129(1-2):99-112 [PubMed] Related Publications
The quinone oxidoreductases [NAD(P)H:quinone oxidoreductase1 (NQO1) and NRH:quinone oxidoreductase2 (NQO2)] are flavoproteins. NQO1 is known to catalyse metabolic detoxification of quinones and protect cells from redox cycling, oxidative stress and neoplasia. NQO2 is a 231 amino acid protein (25956 mw) that is 43 amino acids shorter than NQO1 at its carboxy-terminus. The human NQO2 cDNA and protein are 54 and 49% similar to the human liver cytosolic NQO1 cDNA and protein. Recent studies have revealed that NQO2 differs from NQO1 in its cofactor requirement. NQO2 uses dihydronicotinamide riboside (NRH) rather than NAD(P)H as an electron donor. Another difference between NQO1 and NQO2 is that NQO2 is resistant to typical inhibitors of NQO1, such as dicoumarol, Cibacron blue and phenindone. Flavones, including quercetin and benzo(a)pyrene, are known inhibitors of NQO2. Even though overlapping substrate specificities have been observed for NQO1 and NQO2, significant differences exist in relative affinities for the various substrates. Analysis of the crystal structure of NQO2 revealed that NQO2 contains a specific metal binding site, which is not present in NQO1. The human NQO2 gene has been precisely localized to chromosome 6p25. The human NQO2 gene locus is highly polymorphic. The NQO2 gene is ubiquitously expressed and induced in response to TCDD. Nucleotide sequence analysis of the NQO2 gene promoter revealed the presence of several cis-elements, including SP1 binding sites, CCAAT box, xenobiotic response element (XRE) and an antioxidant response element (ARE). The complement of these elements regulates tissue specific expression and induction of the NQO2 gene in response to xenobiotics and antioxidants. The in vivo role of NQO2 and its role in quinone detoxification remains unknown.

Kuehl BL, Paterson JW, Peacock JW, et al.
Presence of a heterozygous substitution and its relationship to DT-diaphorase activity.
Br J Cancer. 1995; 72(3):555-61 [PubMed] Free Access to Full Article Related Publications
A point mutation in the mRNA of NADP(H): quinone oxidoreductase 1 (NQO1, DT-diaphorase) is believed to be responsible for reduced enzyme activity in the adenocarcinoma BE cell line. The present study examined nine cultured human non-cancerous fibroblast cell strains, five of which were from members of a single cancer-prone family, which demonstrated widely varying activity levels of DT-diaphorase (41 - 3462 nmol min-1 mg-1 protein), to determine if genetic alteration of the NQO1 or NOQ2 gene was involved in determining enzyme activity. All cell strains expressed NQO1 and NQO2 mRNA as measured by a quantitative polymerase chain reaction amplification technique. No relationship was found between the level of mRNA expressed and the enzyme activity in the cells. Sequencing of the entire complementary DNA from the cell strains revealed only a single base substitution at nucleotide 609 in one allele encoding NQO1 in every cell strain from members of the cancer-prone family, except for one cell strain which expressed only the T at nucleotide 609 in both alleles. Subsequent examination of genomic DNA from 44 individuals revealed that this base substitution is present in approximately 50% of the population. The presence of the T at nucleotide 609 in the NQO1 locus does not appear to be directly causal for altered DT-diaphorase activity.

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