Overexpression of NAD(P)H:quinone oxidoreductase 1 (NQO1) and genomic gain of the NQO1 locus modulates breast cancer cell sensitivity to quinones

doi:10.1016/j.lfs.2015.12.017

Life Sciences

Volume 145, 15 January 2016, Pages 57-65

https://doi.org/10.1016/j.lfs.2015.12.017 Get rights and content

Abstract

Aims

Alterations in the expression of antioxidant enzymes are associated with changes in cancer cell sensitivity to chemotherapeutic drugs (menadione and β-lapachone). Mechanisms of acquisition of resistance to pro-oxidant drugs were investigated using a model of oxidative stress-resistant MCF-7 breast cancer cells (Resox cells).

Main methods

FISH experiments were performed in tumor biopsy and breast cancer cells to characterize the pattern of the NQO1 gene. SNP-arrays were conducted to detect chromosomal imbalances. Finally, the importance of NQO1 overexpression in the putative acquisition of either drug resistance or an increased sensitivity to quinones by cancer cells was investigated by immunoblotting and cytotoxicity assays.

Key findings

Genomic gain of the chromosomal band 16q22 was detected in Resox cells compared to parental breast cancer MCF-7 cells and normal human mammary epithelial 250MK cells. This genomic gain was associated with amplification of the NQO1 gene in one tumor biopsy as well as in breast cancer cell lines. Using different breast cell models, we found that NQO1 overexpression was a main determinant for a potential chemotherapy resistance or an increased sensitivity to quinone-bearing compounds.

Significance

Because NQO1 is frequently modified in tumors at genomic and transcriptomic levels, the impact of NQO1 modulation on breast cancer cell sensitivity places NQO1 as a potential link between cancer redox alterations and resistance to chemotherapy. Thus, the NQO1 gene copy number and NQO1 activity should be considered when quinone-bearing molecules are being utilized as potential drugs against breast tumors.

Graphical abstract

Section snippets

Chemical compounds

β-Lapachone (PubChem CID:3885)

Menadione (PubChem CID: 23665888)

Dicumarol (PubChem CID: 54676038)

Chemicals

β-Lapachone (3,4-Dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione), menadione (2-methyl-1,4-naphthoquinone) and dicumarol (3,3′-methylene-bis[4-hydroxycoumarin]) were purchased from Sigma (St Louis, MO, USA). All other chemicals were of ACS reagent grade.

Cell lines, cell culture

MCF-7 breast cancer cell line was purchased from ATCC (Manassas, USA). The Resox cell line was derived from MCF-7 cells [20], which were rendered resistant by chronic exposure to an H₂O₂-generating system made by mixing a redox cycler

NQO1 protein levels and NQO1 gene copy number in human breast tumors

Because of the general consensus that NQO1 is overexpressed in tumor cells, NQO1 protein levels were measured in 10 human mammary cancer biopsies. Indeed, high levels of NQO1 gene expression have been found in liver, lung, colon and breast tumors compared to normal tissues of the same origin [34]. The NQO1 protein level was increased in 6 out of 10 tumor samples compared to levels in tissue surrounding the tumor (Fig. 1A–B). NQO1 expression was enhanced about 2-fold in tumors compared to in

Discussion

NQO1, the enzyme that reduces quinone to hydroquinone [9], is often increased in tumors compared to healthy tissues [14], [15], [16]. In this study, the NQO1 protein levels were increased in 6 out of 10 invasive mammary ductal carcinoma samples compared to levels in tissue surrounding the tumor (Fig. 1A–B) and decreased in 3 samples. The increase or decrease of NQO1 protein levels in cancer cells could be dependent on different potentially cooperative mechanisms: corresponding genomic

Conclusions

NQO1 contributes to either drug cytotoxicity or drug elimination making the cellular response to quinone-bearing compounds a complex process. The data suggest that, in addition to the expression of NQO1 per se, NQO1 activity has a major influence on the sensitivity of cancer cells to quinone-based molecules as depicted in Fig. 5. Thus, it is tempting to suggest that determining NQO1 activity and the number of copies of the chromosomal band 16q22, more specifically of NQO1 gene copies, may be

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This project was funded by FNRS-Télévie Grant (Grant no. 7.4575.12F). The authors are grateful to Pr. David Ross and Dr. Jadwiga Kepa for providing the plasmid construct containing human NQO1 cDNA and Dr. Julie Stockis for plasmid sequencing. The authors are grateful to Sandrine Nonckreman, Khadija Bahloula, Elisabeth Wyns and their colleagues at the human genetic center of Saint-Luc hospital in Brussels for their help in establishing karyotypes, SNP arrays and FISH analyses. We thank also Pr.

References (55)

C. Lind
Formation of benzo[a]pyrene-3,6-quinol mono- and diglucuronides in rat liver microsomes
Arch. Biochem. Biophys.
(1985)
V.A. Roginsky et al.
Kinetics of redox interaction between substituted quinones and ascorbate under aerobic conditions
Chem. Biol. Interact.
(1999)
P. Hochstein
Futile redox cycling: implications for oxygen radical toxicity
Fundam. Appl. Toxicol.
(1983)
C. Lind et al.
DT-diaphorase as a quinone reductase: a cellular control device against semiquinone and superoxide radical formation
Arch. Biochem. Biophys.
(1982)
H. Morrison et al.
Induction of DNA damage by menadione (2-methyl-1,4-naphthoquinone) in primary cultures of rat hepatocytes
Biochem. Pharmacol.
(1984)
D. Siegel et al.
NAD(P)H:quinone oxidoreductase 1 (NQO1) in the sensitivity and resistance to antitumor quinones
Biochem. Pharmacol.
(2012)
D. Siegel et al.
Immunodetection of NAD(P)H:quinone oxidoreductase 1 (NQO1) in human tissues
Free Radic. Biol. Med.
(2000)
T. Cresteil et al.
High levels of expression of the NAD(P)H:quinone oxidoreductase (NQO1) gene in tumor cells compared to normal cells of the same origin
Biochem. Pharmacol.
(1991)
N. Dejeans et al.
Overexpression of GRP94 in breast cancer cells resistant to oxidative stress promotes high levels of cancer cell proliferation and migration: implications for tumor recurrence
Free Radic. Biol. Med.
(2012)
C. Glorieux et al.
Catalase expression in MCF-7 breast cancer cells is mainly controlled by PI3K/Akt/mTor signaling pathway
Biochem. Pharmacol.
(2014)

J.J. Lee et al.

Cytogenetic methods for the mouse: preparation of chromosomes, karyotyping, and in situ hybridization

Anal. Biochem.

(1990)

C. Glorieux et al.

Catalase overexpression in mammary cancer cells leads to a less aggressive phenotype and an altered response to chemotherapy

Biochem. Pharmacol.

(2011)

T. Mosmann

Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays

J. Immunol. Methods

(1983)

T.E. Downing et al.

Prognostic and predictive value of 16p12.1 and 16q22.1 copy number changes in human breast cancer

Cancer Genet. Cytogenet.

(2010)

S.A. Shaposhnikov et al.

A map of nuclear matrix attachment regions within the breast cancer loss-of-heterozygosity region on human chromosome 16q22.1

Genomics

(2007)

E. Blanco et al.

Beta-lapachone-containing PEG-PLA polymer micelles as novel nanotherapeutics against NQO1-overexpressing tumor cells

J. Control. Release

(2007)

S. Knuutila et al.

DNA copy number losses in human neoplasms

Am. J. Pathol.

(1999)

D. Di Monte et al.

Alterations in intracellular thiol homeostasis during the metabolism of menadione by isolated rat hepatocytes

Arch. Biochem. Biophys.

(1984)

T.W. Gant et al.

Redox cycling and sulphydryl arylation; their relative importance in the mechanism of quinone cytotoxicity to isolated hepatocytes

Chem. Biol. Interact.

(1988)

A.C. Collier et al.

The mitochondrial uncoupler dicumarol disrupts the MTT assay

Biochem. Pharmacol.

(2003)

K. Maruyama et al.

Syntheses of alpha- and beta-lapachones and their homologues by way of photochemical side chain introduction to quinone

Chem. Lett.

(1977)

R.H. Thompson

Naturally occurring quinones III, recent advances

(1987)

P.L. Chesis et al.

Mutagenicity of quinones: pathways of metabolic activation and detoxification

Proc. Natl. Acad. Sci. U. S. A.

(1984)

H.G. Williams-Ashman et al.

Oxydation of reduced pyridine nucleotides in mammary gland and adipose tissue following treatment with polynuclear hydrocarbons

Med. Exp. Int. J. Exp. Med.

(1961)

J. Verrax et al.

Enhancement of quinone redox cycling by ascorbate induces a caspase-3 independent cell death in human leukaemia cells. An in vitro comparative study

Free Radic. Res.

(2005)

A. Strassburg et al.

Differential gene expression of NAD(P)H:quinone oxidoreductase and NRH:quinone oxidoreductase in human hepatocellular and biliary tissue

Mol. Pharmacol.

(2002)

J.J. Schlager et al.

Cytosolic NAD(P)H:(quinone-acceptor)oxidoreductase in human normal and tumor tissue: effects of cigarette smoking and alcohol

Int. J. Cancer

(1990)

Cited by (27)

Profiling the chemical nature of anti-oxytotic/ferroptotic compounds with phenotypic screening
2021, Free Radical Biology and Medicine
Citation Excerpt :
However, there are also reports showing that NQO1 is necessary to activate quinone toxicity. This property has been investigated as a potential anti-cancer approach using quinones, including β-lapachone and tanshinone, given that NQO1 is frequently overexpressed in a variety of tumors [36,82–85]. Based on these observations and studies suggesting that the antioxidant role of the QH2 form of the endogenous ubiquinone is specifically involved in preventing lipid peroxidation versus other types of ROS, it is possible that the QH2 forms generated by NQO1 and FPS1 are responsible for the anti-oxytotic/ferroptotic activity of the quinones analyzed here via prevention of lipid peroxidation while still leading to an increase in ROS [41,86–88].
Because old age is the greatest risk factor for Alzheimer's disease (AD), it is critical to target the pathological events that link aging to AD in order to develop an efficient treatment that acts upon the primary causes of the disease. One such event might be the activation of oxytosis/ferroptosis, a unique cell death mechanism characterized by mitochondrial dysfunction and lethal lipid peroxidation. Here, a comprehensive library of >900 natural compounds was screened for protection against oxytosis/ferroptosis in nerve cells with the goal of better understanding the chemical nature of inhibitors of oxytosis/ferroptosis. Although the compounds tested spanned structurally diverse chemical classes from animal, microbial, plant and synthetic origins, a small set of very potent anti-oxytotic/ferroptotic compounds was identified that was highly enriched in plant quinones. The ability of these compounds to protect against oxytosis/ferroptosis strongly correlated with their ability to protect against in vitro ischemia and intracellular amyloid-beta toxicity in nerve cells, indicating that aspects of oxytosis/ferroptosis also underly other toxicities that are relevant to AD. Importantly, the anti-oxytotic/ferroptotic character of the quinone compounds relied on their capacity to target and directly prevent lipid peroxidation in a manner that required the reducing activity of cellular redox enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1) and ferroptosis suppressor protein 1 (FSP1). Because some of the compounds increased the production of total reactive oxygen species while decreasing lipid peroxidation, it appears that the pro-oxidant character of a compound can coexist with an inhibitory effect on lipid peroxidation and, consequently, still prevent oxytosis/ferroptosis. These findings have significant implications for the understanding of oxytosis/ferroptosis and open new approaches to the development of future neurotherapies.
Activatable imaging probes for cancer-linked NAD(P)H:quinone oxidoreductase-1 (NQO1): Advances and future prospects
2020, TrAC - Trends in Analytical Chemistry
Citation Excerpt :
Studies have also found that the consumption of tumor-NQO1 significantly reduces the proportion of cancer stem cells with high aldehyde dehydrogenase (ALDH) activity, which is an indicator of lung cancer progression, metastasis and chemoresistance [18]. Overexpression of NQO1 is also considered to be one of the main factors of potential chemoresistance [19]. For instance, the cooperation of overexpressed NQO1 and γ-glutamylcysteine synthetase is involved in the resistance of hypoxic cells to cisplatin and mitomycin C [20].
NAD(P)H:quinone oxidoreductase-1 (NQO1) is attractive as a potential cancer biomarker because it is abnormally expressed in many solid tumors and is closely related to a variety of carcinogenic processes. Determining and evaluating the content/activity of NQO1 in tumors is of great significance for early cancer diagnosis and therapeutic effect evaluation. Enzyme-activated imaging probes with tailored properties, high selectivity and sensitivity, and high temporal and spatial resolution have become powerful tools for detecting and monitoring NQO1 activity. In this review, we aim to offer a rigorous but succinct overview of activatable imaging probes by reporting the significant progress made in accurately detecting and imaging NQO1 activity. The design strategies, mechanisms and applications of relevant probes are rationally analyzed and discussed. We also emphasized the potential challenges and prospects of NQO1-activated imaging probes to further advance the development of diagnosis and therapeutic methods for NQO1-related cancers.
NADPH: Quinone oxidoreductase 1 (NQO1) mediated anti-cancer effects of plumbagin in endocrine resistant MCF7 breast cancer cells
2020, Phytomedicine
Citation Excerpt :
Quinones are widely found from natural products and serve as scaffold for cancer drugs. Quinone compounds are reduced by NQO1 enzyme that uses nicotinamide adenine dinucleotide phosphate (NADH) and its reduced form, NADPH as its cofactor (Glorieux et al., 2016). The NQO1 enzyme reduces quinone compounds to either a stable form or unstable form.
PLB is a natural naphthoquinone compound isolated from the roots of Plumbago indica plant. Our previous study reported the inhibitory effect of Plumbagin (PLB) on human endocrine resistant breast cancer cell growth and cell invasion.
Since PLB is a naphthoquinone compound, it can be reduced by the cytosolic NADPH: quinone oxidoreductase 1 (NQO1) enzyme. NQO1 expression is upregulated in various types of aggressive cancer including breast cancer. This study investigated the impact of NQO1 on anti-cancer effects of PLB in endocrine-resistant breast cancer cells.
This study was an in vitro study using ER-positive cell line (MCF7) and endocrine-resistant cell lines (MCF7/LCC2 and MCF7/LCC9 cells).
The roles of NQO1 in anti-cancer activity of PLB were investigated by using NQO1 knockdown cells, NQO1 inhibitor and NQO1 overexpressed cells. To study the impact of NQO1 on the effects of PLB on cell viability, apoptosis, invasion and generation of ROS, the following assays were used: MTT assays, annexin V-PE/7-ADD staining flow cytometry, matrigel invasion assays and DCFHDA assays. To study the mechanism of how NQO1 mediated PLB effects in tamoxifen response and apoptosis, we assessed the levels of mRNA expression by using qRT-PCR.
1. In this study, NQO1 was upregulated in endocrine-resistant cells.
2. PLB did not change the expression of NQO1 but it was able to increase NQO1 activity.
3. The inhibitory effects of PLB on cell proliferation, cell invasion and expression of tamoxifen resistant gene were attenuated in NQO1 knockdown cells or in the presence of NQO1 inhibitor.
4. The effects of PLB to induce apoptosis and generate ROS were also decreased when NQO1 activity was inhibited or when the NQO1 expression was reduced.
5. The anti-cancer effects of PLB increased when NQO1 was upregulated.
The effects of PLB in endocrine-resistant breast cancer cells is dependent on NQO1’s activity.
Phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) in mammary tissue of Holstein cows during the periparturient period is associated with mRNA abundance of antioxidant gene networks
2018, Journal of Dairy Science
Citation Excerpt :
By using NADPH as the hydrogen donor, NQO1 catalyzes the reduction of a broad array of quinones to their corresponding hydroquinones, hence this protein prevents the 1-electron reduction of quinones that results in ROS production (Dinkova-Kostova et al., 2004; Dinkova-Kostova and Talalay, 2010). In addition to its catalytic role as an antioxidant enzyme in the reduction of quinines (Glorieux et al., 2016), NQO1 has been reported to protect against estrogen quinone-mediated DNA and protein damage in human mammary epithelial cells (Yang et al., 2013). Similar to the observed postpartal upregulation of NQO1 in mammary tissue, it was reported that NQO1 was upregulated in the liver of dairy cows during the transition period (Gessner et al., 2013).
Changes in the production of reactive oxygen species in the mammary gland of dairy cows during the periparturient period could lead to oxidative stress and potentially impair mammary function. Phosphorylation of the transcription factor nuclear factor erythroid 2-like 2 (NFE2L2), also known as nuclear factor-E2-related factor 2, controls mRNA abundance of genes encoding antioxidant proteins and enzymes. The hypothesis was that NFE2L2 phosphorylation status and target gene mRNA abundance in the mammary gland of dairy cows is altered around parturition. Total NFE2L2 protein, phosphorylated protein (p-NFE2L2), and ratio of p-NFE2L2 to NFE2L2 along with mRNA abundance of 24 genes related to the NFE2L2 signaling pathway, apoptosis, and cell proliferation were measured in mammary tissue samples from Holstein cows at −30, 1, 15, and 30 d relative to parturition. Although total NFE2L2 protein abundance did not differ, p-NFE2L2 and p-NFE2L2-to-NFE2L2 ratio were greater after parturition. The upregulation of DNA damage inducible transcript 3 (DDIT3) postpartum indicated a localized oxidative stress state. Among genes evaluated, thioredoxin (TXN), glutathione peroxidase 1 (GPX1), and glutathione S-transferase mu 1 (GSTM1) had the highest (37.1, 15.1, and 4.8% of total mRNA measured, respectively) abundance. The mRNA abundance of various target genes with detoxifying enzymatic functions and free radical scavenging activities [glutamate-cysteine ligase catalytic subunit (GCLC); glutathione reductase (GSR); ferrochelatase (FECH); TXN; thioredoxin reductase 1 (TXNRD1); and NAD(P)H quinone dehydrogenase 1 (NQO1)] were consistently upregulated (linear effect of time) as parturition approached and lactation began. Among the transcription regulators, NFE2L2 had the highest mRNA abundance (7.3% of total mRNA measured). Abundance of NFE2L2 and other transcription factors [nuclear factor kappa B subunit 1 (NFKB1), retinoid X receptor α (RXRA), and mitogen-activated protein kinase 14 (MAPK14)] were upregulated (linear effect of time) from −30 d to 30 d relative to parturition. Overall, NFE2L2 phosphorylation and downstream signaling leading to postpartal upregulation of genes associated with oxidative stress and inflammation in the mammary gland seem to be key components of normal cellular function to maintain proper redox homeostasis. However, if the longitudinal increases in mRNA and protein abundance of these antioxidant mechanisms are a reflection of cellular oxidative stress, then the likelihood of protein and DNA damage would be greater and might be one factor compromising cell viability and potentially lactation persistency. The actual cues coordinating these molecular responses remain to be determined.
Prodrug strategy for cancer cell-specific targeting: A recent overview
2017, European Journal of Medicinal Chemistry
Citation Excerpt :
In addition, oxidoreductases are important therapeutic targets due to their primary role in oxidative stress. NAD(P) H:quinone oxidoreductase 1 (NQO1), formerly known as DT-diaphorase, is an oxidoreductase with high levels of expression in a variety of cancers, including colon, breast, pancreas and lung cancers [68]. Anticancer prodrug molecules possessing quinone pharmacophore will undergo bioreductive activation by NQO1 to generate hydroquinones, which will exert activity through a number of pathways [69].
The increasing development of targeted cancer therapy provides extensive possibilities in clinical trials, and numerous strategies have been explored. The prodrug is one of the most promising strategies in targeted cancer therapy to improve the selectivity and efficacy of cytotoxic compounds. Compared with normal tissues, cancer cells are characterized by unique aberrant markers, thus inactive prodrugs targeting these markers are excellent therapeutics to release active drugs, killing cancer cells without damaging normal tissues. In this review, we explore an integrated view of potential prodrugs applied in targeted cancer therapy based on aberrant cancer specific markers and some examples are provided for inspiring new ideas of prodrug strategy for cancer cell-specific targeting.
Chromatin remodeling regulates catalase expression during cancer cells adaptation to chronic oxidative stress
2016, Free Radical Biology and Medicine
Citation Excerpt :
Protein probabilities were assigned by the Protein Prophet algorithm [43]. Cell metabolic status was assessed by following the reduction of MTT (3-(4,5-dimethylthia-zolyl-2)-2,5-diphenyltetrazolium bromide) to blue formazan as previously described [44,45]. Briefly, 1×104 cells/well were plated onto 96-well plates and, after confluence, they were preincubated 48 h with inhibitors, then exposed to the respective treatments for 24 h. Afterwards, cells were washed twice with PBS and incubated for 2 h with MTT (0.5 mg/ml).
Regulation of ROS metabolism plays a major role in cellular adaptation to oxidative stress in cancer cells, but the molecular mechanism that regulates catalase, a key antioxidant enzyme responsible for conversion of hydrogen peroxide to water and oxygen, remains to be elucidated. Therefore, we investigated the transcriptional regulatory mechanism controlling catalase expression in three human mammary cell lines: the normal mammary epithelial 250MK primary cells, the breast adenocarcinoma MCF-7 cells and an experimental model of MCF-7 cells resistant against oxidative stress resulting from chronic exposure to H₂O₂ (Resox), in which catalase was overexpressed. Here we identify a novel promoter region responsible for the regulation of catalase expression at −1518/−1226 locus and the key molecules that interact with this promoter and affect catalase transcription. We show that the AP-1 family member JunB and retinoic acid receptor alpha (RARα) mediate catalase transcriptional activation and repression, respectively, by controlling chromatin remodeling through a histone deacetylases-dependent mechanism. This regulatory mechanism plays an important role in redox adaptation to chronic exposure to H₂O₂ in breast cancer cells. Our study suggests that cancer adaptation to oxidative stress may be regulated by transcriptional factors through chromatin remodeling, and reveals a potential new mechanism to target cancer cells.

View all citing articles on Scopus

¹: Current address: Université Bordeaux-Segalen, Inserm U1053, Bordeaux, France.

View full text

Overexpression of NAD(P)H:quinone oxidoreductase 1 (NQO1) and genomic gain of the NQO1 locus modulates breast cancer cell sensitivity to quinones

Abstract

Aims

Main methods

Key findings

Significance

Graphical abstract

Section snippets

Chemical compounds

Chemicals

Cell lines, cell culture

NQO1 protein levels and NQO1 gene copy number in human breast tumors

Discussion

Conclusions

Conflict of interest statement

Acknowledgments

Arch. Biochem. Biophys.

Chem. Biol. Interact.

Fundam. Appl. Toxicol.

Arch. Biochem. Biophys.

Biochem. Pharmacol.

Biochem. Pharmacol.

Free Radic. Biol. Med.

Biochem. Pharmacol.

Free Radic. Biol. Med.

Biochem. Pharmacol.

Anal. Biochem.

Biochem. Pharmacol.

J. Immunol. Methods

Cancer Genet. Cytogenet.

Genomics

J. Control. Release

Am. J. Pathol.

Arch. Biochem. Biophys.

Chem. Biol. Interact.

Biochem. Pharmacol.

Syntheses of alpha- and beta-lapachones and their homologues by way of photochemical side chain introduction to quinone

Chem. Lett.

Naturally occurring quinones III, recent advances

Mutagenicity of quinones: pathways of metabolic activation and detoxification

Proc. Natl. Acad. Sci. U. S. A.

Oxydation of reduced pyridine nucleotides in mammary gland and adipose tissue following treatment with polynuclear hydrocarbons

Med. Exp. Int. J. Exp. Med.

Enhancement of quinone redox cycling by ascorbate induces a caspase-3 independent cell death in human leukaemia cells. An in vitro comparative study

Free Radic. Res.

Differential gene expression of NAD(P)H:quinone oxidoreductase and NRH:quinone oxidoreductase in human hepatocellular and biliary tissue

Mol. Pharmacol.

Cytosolic NAD(P)H:(quinone-acceptor)oxidoreductase in human normal and tumor tissue: effects of cigarette smoking and alcohol

Int. J. Cancer