review article the nrf2/ho-1 axis in cancer cell growth and...

Review ArticleThe Nrf2HO-1 Axis in Cancer CellGrowth and Chemoresistance

A L Furfaro1 N Traverso2 C Domenicotti2 S Piras2 L Moretta3 U M Marinari2

M A Pronzato2 and M Nitti2

1Giannina Gaslini Institute Via Gerolamo Gaslini 5 16147 Genoa Italy2Department of Experimental Medicine University of Genoa Via L B Alberti 2 16132 Genoa Italy3Bambino Gesu Childrenrsquos Hospital IRCCS Piazza S Onofrio 4 00165 Rome Italy

Correspondence should be addressed to A L Furfaro annalisafurfarounigeit

Received 24 April 2015 Revised 13 August 2015 Accepted 18 August 2015

Academic Editor Patrıcia Alexandra Madureira

Copyright copy 2016 A L Furfaro et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The transcription factor nuclear factor erythroid 2 p45-related factor 2 (Nrf2) acts as a sensor of oxidative or electrophilicstresses and plays a pivotal role in redox homeostasis Oxidative or electrophilic agents cause a conformational change in theNrf2 inhibitory protein Keap1 inducing the nuclear translocation of the transcription factor which through its binding to theantioxidantelectrophilic response element (AREEpRE) regulates the expression of antioxidant and detoxifying genes such asheme oxygenase 1 (HO-1) Nrf2 and HO-1 are frequently upregulated in different types of tumours and correlate with tumourprogression aggressiveness resistance to therapy and poor prognosis This review focuses on the Nrf2HO-1 stress responsemechanism as a promising target for anticancer treatment which is able to overcome resistance to therapies

1 Introduction

The availability of intracellular antioxidants is essential inmaintaining redox homeostasis in living cells In aerobicconditions cells are constantly exposed to the generationof reactive oxygen species (ROS) that can impact proteinslipids and DNA playing a pathological role in the develop-ment of various human diseases such as cancer [1]Thereforecells have evolved endogenous defence mechanisms so asto counteract oxidative stress and to maintain ROS at lowphysiological levels and the redox sensitive transcriptionfactor nuclear factor erythroid 2 p45-related factor 2 (Nrf2)acts as a key regulator of antioxidant response cruciallyinvolved in the preservation of the structure and the func-tioning of normal healthy cells [2ndash4] However cancer cellsdifferently from normal cells show an increased rate of ROSgeneration as by-products of their metabolism [5] and asldquomastersrdquo of adaptation they take advantage of the overacti-vation of antioxidant defences in particular Nrf2-dependentgenes [6ndash8] This ability to adapt and survive under condi-tions of electrophilic oxidative and inflammatory stress isstrongly dependent on the expression of a complex network

comprising nearly 500 genes induced by Nrf2 encodingproteins with different antioxidant and cytoprotective func-tions [9] In particular heme oxygenase 1 (HO-1) exerts astrong antioxidant and antiapoptotic effect favouring cancercell growth and resistance to therapy In this review we focusour attention on the deleterious properties of Nrf2 and ofits target gene HO-1 in relation to cancer cell growth andchemoresistance

2 Nrf2 Structure and Regulation

The nuclear factor erythroid 2 p45-related factor 2 (Nrf2) isa transcription factor that plays a key role in the regulationof the cellular redox status Indeed Nrf2 controls not onlythe expression of antioxidants as well as phase I and phaseII drug-metabolizing enzymes [10 11] but also multidrug-resistance-associated protein transporters [12] (Table 1)

The human Nrf2 was first described cloned and charac-terised byMoi and coworkers in 1994 [13] and its cloned geneis encoded within a 22 kB transcript predicting a protein of589 amino acids with a molecular mass of 661 kDa [13]

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 1958174 14 pageshttpdxdoiorg10115520161958174

2 Oxidative Medicine and Cellular Longevity

Table 1 Genes regulated by Nrf2 in mice and humans

Symbol Name Species ReferencesAntioxidant genes

GCLC Glutamate-cysteine ligasecatalytic subunit m h [188 189]

GCLM Glutamate-cysteine ligasemodifier subunit m h [188 189]

GLRX Glutaredoxin 1 h [190]GPX2 Glutathione peroxidase 2 m h [190 191]GPX4 Glutathione peroxidase 4 m [16]GSR1 Glutathione reductase m h [188 190]SLC6A9 Glycine transporter m [16]

SLC7A11 Cysteineglutamatetransporter m h [16 188]

PRDX1-6 Peroxiredoxins 1 and 6 m h [10 16]SRXN1 Sulfiredoxin-1 m h [11 188]TXN1 Thioredoxin m h [10 16]TXNRD1 Thioredoxin reductase 1 m h [188 190]

HO-1-related genesHMOX1 Heme oxygenase 1 m h [10 188]BLVRA Biliverdin reductase A h [190]BLVRB Biliverdin reductase B m h [16 190]FECH Ferrochelatase h [188]

FTH1 Ferritin heavy polypeptide1 m h [10]

FTHL12-17 Ferritin heavypolypeptides 12 and 17 h [188 190]

FTL1 Ferritin light polypeptide m h [188 190]Detoxifying enzymes

NQO1 NAD(P)Hquinoneoxidoreductase 1 m h [188 190]

GSTA1 Glutathione S-transferaseclass Alpha 1 m [192]

GSTM1 Glutathione S-transferaseclass Mu 1 m [192]

GSTP1 Glutathione S-transferaseclass Pi 1 m [12]

UGT1A1 UDP glucuronosyl-transferase 1 family h [190]

UGT2B7 UDP glucuronosyl-transferase 2 family m h [16 190]

Drug transporters

ABCB6 ATP-binding cassettesubfamily B(MDRTAP) m h [10 11]

ABCC1 ATP-binding cassettesubfamily C(CFTRMRP) m [193]

ABCC2 ATP-binding cassettesubfamily C(CFTRMRP) m h [193]




Nrf2 has seven functional domains named Nrf2-ECHhomology (Neh) 1ndash7 (Figure 1(a)) [12 14 15] The Neh1domain has a basic region leucine zipper structure needed

for the dimerisation with small Maf and binds to antiox-idantelectrophile responsive elements (AREEpRE) [16]Neh2 is the main negative regulatory domain which bindsto the Kelch-like ECH-associated protein 1 (Keap1) via theDLG and ETGE motifs [17] Neh3 is localised in the C-terminal region of Nrf2 and acts as a transactivation domainrecruiting the chromo-ATPasehelicase DNA binding pro-tein family (CHD6) [6] whereas both Neh4 and Neh5 aretransactivation domains that recruit cAMP response elementbinding protein- (CREB-) binding protein (CBP) andorthe receptor-associated coactivator (RAC) [18] The Neh6domainmediates the Keap1-independent degradation ofNrf2through recruitment by the DSGIS and DSAPGS motifs ofthe dimeric120573-transducin repeat-containing protein (120573-TrCP)[19] Lastly the Neh7 domain recently identified by Wangand coworkers interacts with the retinoid X receptor alpha(RXR120572) a repressor of Nrf2 [20]

21 Keap1-Dependent Posttranscriptional Regulation

211 Ubiquitination and Proteasomal Degradation of Nrf2Under basal conditions Nrf2 is localised in the cytosolassociated with its negative regulator Keap1 an adaptorcomponent of Cullin 3-based ubiquitin E3 ligase complex(Cul3) that promotes Nrf2 constant ubiquitination and pro-teasomal degradation maintaining low basal levels [21 22]Nrf2 turnover is rapid less than 20 minutes and preventsthe expression of Nrf2 target genes under normal conditions[23] On exposure to oxidative or electrophilic stress Keap1 ismodified whereas the enzymatic activity of the E3 ubiquitinligase is inhibited Nrf2 is liberated from Keap1 accumulatesin the nucleus dimerises with small Maf protein and thenactivates after ARE-sequence binding the transcription ofits target genes [23 24] Thus Keap1 is the main repressorof Nrf2 having three functional domains namely the broadcomplex Tramtrack and Bric-a-Brac (BTB) domain theintervening region (IVR) domain and the double glycinerepeat (DGR)Kelch domain [25] (Figure 1(b))

Keap1 acts as a sensor for oxidative and electrophilicstress through the modification of 27 cysteine residues [26](Figure 2)Themain three cysteine residues for the regulationof Nrf2 activity are Cys151 in the BTB domain and Cys273and Cys288 in the IVR domain all of which are targets ofoxidative and electrophilic modifications [27ndash29] It has beenshown that cells expressing Keap1 Cys151 point mutant pro-tein show reduced activation of Nrf2 in response to a numberof inducers (eg sulforaphane tert-butylhydroquinone anddiethyl maleate) in comparison to the wild-type cells [30]and that Cys273 and Cys288 are critically required for thebasal repression of Nrf2 [31] In addition the modificationof a subset of cysteine residues in Keap1 by Nrf2 inducerssupports the hypothesis of a ldquocysteine coderdquo which is criticalin the activation of Nrf2 [32]

212 Autophagic Degradation of Keap1 Furthermore otherinteracting protein partners such as the sequestosome-1protein (p62SQSTM1) can modulate Nrf2 activity [33 34]p62SQSTM1 is a scaffold protein that binds to polyubiquiti-nated proteins and targets protein aggregates for autophagic

Oxidative Medicine and Cellular Longevity 3

Neh2 Neh4 Neh7Neh5 Neh6 Neh1 Neh3

DLG ETGE DSGIS DSAPGS

Keap1 binding

COOH

Transactivationdomain DNA binding domain

Transactivationdomain

NH2

120573-TrCP1 binding

RXR120572 binding

(a)

BTP domain IVR domain KelchDGR

Cys-151 Cys-273 Cys-288

COOH

Nrf2 binding Association with Cul3

NH2

(b)

Figure 1 Schematic representation of Nrf2 and Keap1 structures (a) Nrf2 contains seven domains Neh1ndashNeh7 The Neh2 domain containstwo binding motifs DLG and ETGE responsible for the interaction with Keap1 The Neh4 Neh5 and Neh3 domains are important for thetransactivation activity of Nrf2TheNeh7 domain is critical for RXR120572 bindingTheNeh6 domain regulates Nrf2 degradation by 120573-TrCP1TheNeh1 domain has a basic region leucine zipper motif for DNA binding (b) Keap1 contains three major domains The BTB domain mediatesKeap1 homodimerisation and the IVR domain contains critical cysteine residues and together they associate with Cul3 The KelchDGRdomain mediates the binding with the Neh2 domain of Nrf2

Oxidativeelectrophilic stressCul3Nrf2

Keap1

Cys-SHHS-Cys

Nrf2Keap1

Cys-SHHS-Cys26Sproteasome

Ubiquitination

Nrf2

Cul3Keap1

S-CysCys-S

Nrf2

NucleusAntioxidantdetoxifyingresponse (HO-1 GCLC GCLM MRPs p62 etc)

Keap1Nrf2 mutations

Keap1Cul3

HS-Cys

Nrf2

Cul3Keap1Cys-SH

Basal condition

ARE

Nrf2 Maf

Keap1 interaction with other proteins (eg p62 DJ-1

Keap1Cys-SHHS-Cys

Cul3p62

Release of Nrf2

Nrf2

ARE Bach1 Maf Nucleus

Release of Nrf2

Release of Nrf2

Alternative mechanisms of amplification

Cytoplasm

Cul3Ub Cul3

Cytoplasm

5998400gctgagtcac3998400


hypermethylation and p21)

of Nrf2 signalling in cancer

Figure 2 Nrf2 activity regulation In a resting state Nrf2 is sequestered in the cytoplasm through the binding with Keap1 responsible forNrf2 ubiquitination and proteasomal degradation via Cul3 Oxidativeelectrophilic stress causes a conformational change in Keap1-Cul3 byacting on specific cysteine residues in Keap1 leading to Nrf2 dissociation Thus free Nrf2 translocates to the nucleus which dimerises withsmall Maf protein and binds to AREEpRE sequence within regulatory regions of a wide variety of target genes (eg HO-1 GCLC GCLMMRPs and p62) In cancer cells (blue box) Keap1Nrf2 mutations and Keap1Cul3 aberrant hypermethylations as well as Keap1 interactionswith ETGE motif-containing proteins lead to an increased Nrf2 activation and induction of target genes


degradation p62 contains an STGE-binding motif similar tothe Nrf2 ETGE motif [33] which is needed for the directinteraction with Keap1 [35] As a consequence Keap1 issequestered in autophagosomes and Nrf2 ubiquitinationdecreases leading to a prolonged activation of Nrf2 in re-sponse to oxidative stress [34 35] (Figure 2)

22 Keap1-Independent Posttranscriptional Regulation Re-cently alternative mechanisms for the degradation of Nrf2have been identified For example a number of studies havedemonstrated that glycogen synthase kinase 3120573 (GSK-3120573)directs the ubiquitination and proteasomal degradation ofvarious transcription factors including Nrf2 through theactivation of E3 ubiquitin ligase complex (120573-TrCP-Skp1-Cul1-Rbx1) [19 36] Indeed GSK-3120573 is able to phosphorylate Serresidues located in the Neh6 domain of Nrf2 which are thenrecognised by 120573-TrCP and through the binding to Cullin1 (Cul1) scaffold protein lead to Nrf2 ubiquitination anddegradation in a redox-independent manner [19 37]

Moreover a novel E3 ubiquitin ligase namely Hrd1 hasbeen described by Wu and coworkers [36] They showedthat Hrd1 controls the Nrf2 stability by way of an interactionbetween the C-terminal domain of Hrd1 and the Neh4-5domains of Nrf2 This Hrd1-mediated ubiquitination of Nrf2is independent of both Keap1 and 120573-TrCP and compromisesthe Nrf2-mediated protection during liver cirrhosis [36]

23 Nrf2 Regulation at the Transcriptional Level Althoughstrong evidence shows that Nrf2 is primarily regulated atthe protein level it has also been demonstrated that theoncogenic KRAS transcriptionally upregulates the mRNAlevels of Nrf2 through a TPA response element (TRE) locatedwithin the Nrf2 promoter The oncogenic mutation of KRASor KRAS overexpression indeed is able to activate the Nrf2-dependent pathway [38] In addition an increase in Nrf2mRNA levels has been shown to occur in response to theoncogenic activation of BRAF and C-MYC [39]

3 Nrf2 and Chemoprevention

It is well known that Nrf2 plays a key role in the cellularadaptation and protection against oxidative stressThe abilityof Nrf2 to activate cytoprotective genes which code fordetoxifying enzymes drug transporters antioxidants andanti-inflammatory proteins plays a crucial role in reducingelectrophiles and ROS thus decreasing DNA damage andmutations and preventing genomic instability in normal cellsSeveral studies have shown that in Nrf2 null mice thereis an enhanced susceptibility to chemical carcinogens suchas benzo(a)pyrene compared to wild-type mice due to adecreased expression of phase II detoxification and antiox-idant enzymes [40] In a similar way after exposure to N-nitrosobutyl (4-hydroxybutyl) amine (BBN) Nrf2 knockoutmice develop urinary bladder carcinoma [41] and showan increased incidence of skin colorectal and mammarytumours [42ndash44] Moreover the protective role of Nrf2against carcinogenesis is highlighted from studies in humanson single-nucleotide polymorphism (SNP) in the promoterregion of the Nrf2 gene [45] As described by Suzuki and

coworkers AA homozygotes of Nrf2 rSNP-617 showed de-creased expression of Nrf2 and consequently an increasedrisk of developing lung cancers especially in the case ofindividuals with smoking habits [46]

4 The Dark Side of Nrf2 in Cancer Biology

Several studies have shown a deleterious aspect of Nrf2defined as the ldquodark side of Nrf2rdquo [47] Its high and prolongedactivation in cancer cells has been long associated withprogression metastatic invasion angiogenesis and chemo-and radio-resistance in tumours and is considered a poorprognostic factor [15 48] Indeed the stable overexpressionof Nrf2 was found in various types of tumours such as lung[49 50] breast [51] head and neck [52] ovarian [53] andendometrial cancer [54]

41 Molecular Mechanisms of Nrf2 Activation in Cancer CellsSeveral mechanisms have been shown to be involved inthe constitutive activation of Nrf2 in cancer cells (Figure 2)mainly gain-of-function mutations in Nrf2 and loss-of-functionmutations in Keap1 leading to an impairment of thebinding to Keap1 This results in the stabilisation of Nrf2 andin the activation of its target genes as identified in patientswith lung or head and neck cancer [55 56] whereas theloss-of-functionmutations of Keap1 are mainly located in theKelchDRG domain and in the IVR domain [14] as observedin gastric hepatocellular colorectal lung breast and prostatecarcinomas as reviewed by Shelton and Jaiswal [57]

In addition epigeneticmodifications inKeap1 are respon-sible for the accumulation and activation of Nrf2 Aberranthypermethylation inhibiting Keap1 gene expression resultsin the accumulation of Nrf2 as shown in lung and malig-nant glioma [58] prostate [59] and colorectal cancer [60]Moreover somaticmutations and hypermethylation in CUL3have been identified as being responsible for Nrf2 activation[53 61 62] as shown in thyroid head and neck and ovariancancers [63ndash65]

42 Nongenomic Alteration of Nrf2 in Cancer Cells Increasedlevels of Nrf2 in cancer cells can also occur in the absenceof genomic alterations In fact much evidence shows thatdifferent proteins can alter the Nrf2-Keap1 binding [14]Nrf2 activity is subject to a positive regulation by p21 [6667] which interferes with Keap1-mediated ubiquitinationinteracting with the DLG motif in Nrf2 leading to itsstabilisationTherefore Nrf2 expression is significantly lowerin the absence of p21 and conversely it is increased upon p21overexpression [67]

It is a fact that DJ-1 a protein belonging to the ThiPfpIsuperfamily is able to stabilise Nrf2 by preventing its associa-tionwithKeap1 thus reducing ubiquitination and subsequentproteasomal degradation [68 69] DJ-1 in cancer is oftenoverexpressed and leads to increased detoxification enzymessuch as NQO1 providing a survival advantage [68 70 71]

As previously described p62 sequesters Keap1 in autoph-agosomes leading to Nrf2 activation [72 73] Moreover ithas been shown that p62 contains the ARE sequence in itspromoter region which is responsible for its Nrf2-dependent


induction in response to oxidative stress thus generating apositive feedback loop [73]

The list of proteins which can interact with Nrf2 andKeap1 and therefore modulate their regulation is contin-uously expanding (eg WTX PALB2 and DPP3) [74ndash76]All of these proteins contain an ETGF motif suggesting thatthey are capable of upregulating Nrf2 by competing for Keap1binding and suppressing Keap1-mediated ubiquitination ofNrf2 [14]

43 Nrf2 Activation and Hallmarks of Cancer MalignancyDifferent Nrf2 target genes are associated with cancer cellproliferation anddeathAmong these those genes involved inthe pentose phosphate pathway such as glucose-6-phosphatedehydrogenase phosphogluconate dehydrogenase transke-tolase and transaldolase 1 are responsible for NADPH andpurine regeneration and therefore accelerate cancer cellproliferation [14] Nrf2 may also bind to the ARE sequencein the promoter region of Notch1 contributing directly toits expression and leading to a more malignant phenotypeand amore aggressive growth [77] Moreover Nrf2 is directlyinvolved in the basal expression of the p53 inhibitor Mdm2through the binding to the ARE sequence located in thefirst intron of this gene It has been demonstrated that inNrf2-deleted murine embryonic fibroblasts (MEFs) Mdm2expression is repressed and compared to wild-type cells ahigh level of p53 is accumulated favouring cell death [78]

In addition elevated levels of Nrf2 have been observedin various tumours with high metastatic potential [79]characterised by epithelial-mesenchymal transition (EMT)and the degradation of extracellular matrix (ECM) exertedbymetalloproteases (MMPs) Amajor step of EMT is the lossof E-cadherin and the gain of N-cadherin [80] It has beenshown that E-cadherin is able to bind to the C terminus ofNrf2 preventing its nuclear accumulation and that duringEMT the overexpression of N-cadherin reduces the Nrf2inhibition thus favouring its activity [81] Moreover theknockdown of the Nrf2 by short hairpin RNA (shRNA)in esophageal squamous cell carcinoma (ESCC) suppressedthe expression of MMP-2 and enhanced E-cadherin mRNAlevels resulting in a decreased invasion and migration ofcancer cells [82]

Furthermore the upregulation of Nrf2 is also related toangiogenesis which is promoted by HIF-1120572 a transcriptionfactor that senses oxygen homeostasis and is deregulated intumours in hypoxic environments [83] Under hypoxic con-ditions indeed the O

2-dependent regulator prolyl-hydrolase

domain (PHD) is catalytically inactive and increases thestability of HIF-1120572 Consequently the expression of itstarget proteins including the vascular endothelial growthfactor (VEGF) is enhanced [84] It has been shown thatNrf2 silencing blocks HIF-1120572-dependent VEGF expressionin HT29 colon cancer and suppresses tumour growth witha concomitant reduction in VEGF-induced angiogenesis inmouse xenograft models [84]

44 Nrf2 and Cancer Resistance to Therapies Several studieshave shown that cancer cells with high levels of Nrf2 areless sensitive to etoposide cisplatin and doxorubicin [14]

Doxorubicin-resistant human cancer cells such as ovarianSKOV3 and OV90 and mammary MCF-7DOX have shownhigh levels of Nrf2-ARE binding and ARE-driven luciferaseactivity as well as the upregulation of Nrf2 target genes com-pared to the respective sensitive cell lines A2780 and MCF-7[85 86] Non-small cell lung carcinoma A549 cells in whichNrf2 is strongly activated have shown a higher resistance tocisplatin compared to NCI-H292 and LC-AI cells [87] Fur-thermore Jayakumar and coworkers have demonstrated therole of Nrf2 and its dependent genes in the radioresistance ofprostate tumour cells Specifically the radioresistant DU145cells show enhanced levels of Nrf2 and a high GSHGSSGratio in comparison to the radiosensitive PC3 cells whichshow a faster depletion of GSH after radiation exposure [88]It has also been shown that radiotherapy significantly reducescancer cell survival when applied in combination with theNrf2 inhibitor 4-(2-cyclohexylethoxy)-aniline IM3829 [89]Moreover our own studies demonstrate that Nrf2 activationplays a key role in the resistance of neuroblastoma cells toGSH depletion or proteasome inhibition [90 91]

In addition Nrf2 activity is related to the upregulation ofseveral multidrug-resistant efflux pumps such as the ATP-binding cassette subfamily G member A2 (ABCG2) whichfavours drug resistance It has been shown indeed that Nrf2silencing attenuates the expression of the ABCG2 transcriptand protein and sensitises lung cancer cells to mitoxantroneand topotecan two representative chemotherapeutic drugseffluxed mainly by the presence of ABCG2 [92] Multidrug-resistant protein-3 (MRP3)MRP4 andMRP5 are all upregu-lated by Nrf2 [93 94] Upregulation of MRP3 and GSTs leadsto the increased hydrophilicity and excretion of cytotoxicagents such as cisplatin etoposide and doxorubicin [95]

Recently it has been proven that Nrf2 also plays a criticalrole in the drug resistance of cancer stem cells [96ndash98]The resistance of glioblastoma is due to the presence ofglioblastoma stem cells (GSCs) which confer tumourigenicpotential and a survival advantage against chemotherapy[99] Moreover it has been shown that while knocking downNrf2 decreases the self-renewing activity of GSCs [100 101]the enhancement of Nrf2 levels and of its downstream genesthat is HO-1 GCLM and NQO1 is related to increasedtumourigenic activity of the human mammospheres in com-parison to their adherent counterparts MCF-7 and MDA-MB-231 cells [102]

5 HO-1 as a Key Effector of Nrf2 Upregulationin Tumour Progression

Heme oxygenase 1 (HO-1) is considered one of the maineffectors of Nrf2-dependent cell responses [48] HO-1 is theinducible form of heme oxygenase the first rate-limitingenzyme in the degradation of heme into biliverdinbilirubincarbon monoxide (CO) and ferritin induced by free ironrelease [103ndash105] (Figure 3) It is a 32 kDa stress proteinpresent at low levels in most mammalian tissues [106] andits expression is induced by a wide variety of stress stimuliincluding its substrate as well as heavy metals [107] UVirradiation ROS [108] nitric oxide [109] and inflammatorycytokines [110] HO-1 and its metabolic products are involved


NADP+NADP+

HEME

NADPH

Bilirubin

NADPH

Biliverdin

Ferritinantioxidant

Antiapoptotic

Antioxidant

HO-1

O2 H2OFe2+ + CO + Biliverdin

reductase

Figure 3 Heme catabolic pathway HO-1 catalyses the degradationof heme into biliverdinbilirubin (antioxidant) carbon monoxide(antiapoptotic) and ferritin (antioxidant) induced by free ironrelease

in the maintenance of cellular homeostasis and they playa key role in the adaptive response to cellular stress aswell as in the protection of healthy cells preventing themfrombeing transformed into neoplastic cells by counteractingROS-mediated carcinogenesis [111ndash115]

However HO-1 has been widely recognised as playingan important role in the malignant transformation of cancercells High levels of HO-1 have been found in various humantumours inducing survival advantage aggressiveness andpoor outcome [116ndash122] HO-1 overexpression has been con-sidered to be involved in invasive andmetastatic mechanisms[123ndash125] and ldquoin vitrordquo and ldquoin vivordquo studies includingclinical data have shown that the inhibition or silencing ofHO-1 inhibits this behaviour [125ndash128] Moreover a proan-giogenic role of HO-1 in cancer has been reported ldquoin vitrordquoand ldquoin vivordquo [123 124 127ndash130] Finally HO-1 has beenshown to be correlated with resistance to chemo- radio- andphotodynamic therapies [116 119 131ndash136] and its inhibitionis able to sensitize cancer cells to death [90 91 137ndash139]

However the role of HO-1 in cancer biology is not com-pletely understood and some disputes in literature remainabout its role in tumour progression especially with regard todifferent types of tumours It should be taken into consider-ation that several studies have reported that HO-1 activationprevents breast cancer proliferation [140] and prostate cancerangiogenesis [141] and mediates the anticancer activity ofsome drugs such as andrographolide by reducing theMMP-9expression in breast cancer cells [142] Moreover consideringthe complex cross talk between HO-1 activity and cellularmetabolic pathways reviewed in depth by Wegiel and coau-thors [143] it would seem conceivable that HO-1 can besubject to different modulations in different tumours sincethe various metabolic statuses of cancer cells may influencehow HO-1 activity modulates tumour growth Therefore itis important to note that HO-1 expression is controlled byother transcription factors other than Nrf2 Indeed specificconsensus sequences for both NF-kB and AP-1 are presentin the promoter region of HO-1 [144ndash148] which thenmay be activated in response to different stimuli throughthe activation of different intracellular signaling pathwaysas widely reviewed in [136 149ndash151] suggesting a highlycomplex regulation which up to now is far from being fullyunderstood

However what seems most interesting is the associationbetween the upregulation of Nrf2 and the activation of

Table 2 Nrf2 and HO-1 upregulation in tumours

Type of tumor Nrf2 HO-1 ReferenceGlioblastoma stem cells(GSCs) uarr mdash [101]

Lung cancer (NSCLC) uarr mdash [92 94]Ovarian carcinoma uarr mdash [85]Bladder cancer mdash uarr [121]Chronic myeloid leukemia mdash uarr [139]Colon adenocarcinoma mdash uarr [135]Colorectal cancer mdash uarr [118]Fibrosarcoma mdash uarr [131]Gastric cancer mdash uarr [138]Hepatocellular carcinoma(HCC) mdash uarr [127]

Kaposi sarcoma mdash uarr [128]Lung cancer (NSCLC) mdash uarr [120 122 134 137]Melanoma mdash uarr [123 131]Oral squamous cellcarcinoma mdash uarr [125]

Pancreatic cancer mdash uarr [119 124 126 133]Breast cancer uarr uarr [86]Cervical cancer uarr uarr [152]Chronic myeloid leukemia uarr uarr [136]Esophageal squamouscarcinoma uarr uarr [82]

Gallbladder cancer uarr uarr [79]Glioblastoma uarr uarr [100 155]Glioblastoma stem cells(GSCs) uarr uarr [100]

Hepatoma uarr uarr [152]Lung cancer (NSCLC) uarr uarr [87 89 152]Malignant B lymphocytes uarr uarr [154]Mammosphere stem cells(MSC) uarr uarr [102]

Multiple myeloma uarr uarr [153]Neuroblastoma uarr uarr [90 91]Ovarian carcinoma cells uarr uarr [156]Prostate cancer uarr uarr [88]Renal cancer uarr uarr [117]

HO-1 in tumour progression which correlates with canceraggressiveness and malignancy Interestingly for instancein human samples from gallbladder cancers the upreg-ulation of both Nrf2 and HO-1 correlates with tumouraggressiveness and a poor clinical outcome [79] MoreoverNrf2HO-1 association has beenwidely reported for instancein non-small lung cancer cervical cancer hepatoma [152]esophageal squamous carcinoma [82] andmultiple myeloma[153] (Table 2) In particular malignant transformations havebeen associated with Nrf2-dependent HO-1 activation inB lymphocytes exposed to prostaglandin J2 [154] There-fore the gain of metastatic phenotypes is correlated withthe overexpression of Nrf2 in association with the activation


of HO-1 as shown in osteopontin-induced glioma cell inva-siveness [155] Furthermore resistance to therapies has beenrelated to the activation of Nrf2 together withHO-1 as shownby our group in neuroblastoma cells after GSH depletionor bortezomib exposure [90 91] and by others in cisplatin-treated ovarian carcinoma cells [156] and in doxorubicin-resistant breast cancer cells [86]

Therefore HO-1 activation provides tumour cells withstrong survival advantage exerted by the antioxidant andantiapoptotic properties of its metabolic products More-over when HO-1 activation is dependent on Nrf2 activitygenerally this leads to highly aggressive cancer phenotypesThis can also account for the parallel activation of otherNrf2-dependent genes which can contribute to the prosur-viving effect for instance by inducing HIF120572 [82] increasingmultidrug-resistance-related proteins [79] or increasing thesynthesis of GSH [8] Unpublished data from our laboratoryshow that HO-1-dependent bilirubin generation and increas-ing amounts of GSH are key factors in inducing resistance tobortezomib in high-risk neuroblastoma

6 Pharmacological Modulation of Nrf2HO-1Axis as a Strategy in Anticancer Treatment

The role of Nrf2HO-1 axis in protecting cells by overcomingenvironmental stresses has already been demonstrated andcompounds that are able to modulate this activity are wellworth being considered The activation of this pathwayin normal cells can prevent tumour formation while itsinhibition can be useful in improving cancer therapies

As far as cancer prevention is concerned differentactivators of Nrf2 have been proposed and have been in-tensively reviewed over the past 10 years [42 157ndash166]Many of these compounds are plant-derived phytochemicalssuch as sulforaphane curcumin epigallocatechin-3-gallateresveratrol cinnamoyl-based compounds garlic organosul-fur compounds and lycopene These molecules have beenconsidered to be chemopreventive due to their ability toinduce antioxidantdetoxification enzymes including HO-1and xenobiotic transporters through the activation of Nrf2[18 41 50 167ndash170] On the other hand the pharmacologicalinhibition of Nrf2HO-1 axis has also recently emerged as apromising approach for cancer therapy

Starting from the modulation of HO-1 the major effectorof the pathway considered several ldquoin vivordquo studies haveconfirmed the usefulness of the HO-1 competitive inhibitorzinc (II) protoporphyrin IX ZnPPIX in the reductionof hepatoma sarcoma lung cancer and B-cell lymphomagrowth in mice [129 171] Moreover the PEG conjugation ofZnPPIX which increases water solubility of the inhibitor isable to improve its clinical application [172] Another highlywater-soluble micellar form of ZnPPIX the amphiphilicstyrene maleic acid copolymer (SMA-ZnPPIX) with a potentantitumour activity both ldquoin vitrordquo and ldquoin vivordquo has beenadditionally proposed [173]

However it is important to note that pharmacologicalHO-1 inhibitors aswell asHO-1 activators are responsible forstrong HO-1 independent activities due to some nonspecificproperties of these compounds [174 175] and therefore the

employment of siRNA could be more specific Indeed thisapproach is able to induce apoptosis of colon carcinomacells [176 177] and to diminish proliferation growth andangiogenesis in orthotopic hepatocellular tumours in mice[127]

As already discussed in the previous paragraph there isalso some evidence that HO-1 activation induced by cobaltprotoporphyrin IX (Co PPIX) or heme [140] as well asthe overexpression of HO-1 can block tumour growth andinvasion in ldquoin vitrordquo studies and this seems to be dependenton the cancer cell type and experimental model used

Furthermore the inhibition of Nrf2 acting upstreamfrom HO-1 activation and involving also other downstreamtargets in addition to HO-1 could be a successful therapeuticapproach

Unfortunately only a few inhibitors of Nrf2 have beendeveloped so far Among them brusatol extracts fromBruceajavanica growing in Southeast Asia andNorthernAustralia isable to decrease Nrf2 protein levels as well as decreasing theexpression of its target genes thus enhancing the cytotoxiceffect of several chemotherapeutic agents both ldquoin vitrordquo andldquoin vivordquo [178 179]

In addition flavonoid luteolin (310158404101584057-tetrahydroxyfla-vone) found at high concentrations in celery green pepperparsley perilla leaf and chamomile tea has been shownto be another strong and selective Nrf2 inhibitor whichis able to reduce the constitutive expression of NQO1 inHepG2 Hepa1c1c7 and RL-34 cells in a time- and dose-dependent manner [180] At physiological concentrationsluteolin inhibits Nrf2 activity by enhancing Nrf2 mRNAturnover and it has been shown to sensitise NSCLC A549cells to therapeutic drugs [181] Similar results have beenobserved in the sensitisation of colorectal cancer cell lines tooxaliplatin ldquoin vitrordquo [182] and ldquoin vivordquo in the chemotherapyof non-small cell lung cancer NSCLC Moreover its oraladministration either alone or combined with an intraperi-toneal injection of cisplatin is seen to greatly inhibit thegrowth of xenograft tumours from the NSCLC cell line A549in athymic nude mice [183]

Furthermore all-trans-retinoic acid (ATRA) is able tosuppress the Nrf2 pathway [184] ATRA like other agonists ofRA receptor 120572 (RAR120572) and retinoid X receptor 120572 (RXR120572) wasshown to inhibit the basal and the inducible activity of Nrf2both ldquoin vitrordquo and ldquoin vivordquo [12 185] After ATRA treatmentNrf2 forms a complex with RAR120572 This complex is unableto bind to the ARE sequences thus decreasing the abilityof Nrf2 to activate its target genes [15] In acute promye-locytic leukaemia cells the cytotoxic drug arsenic trioxide(ATO) induces an antioxidant response characterised byNrf2nuclear translocation and enhances the transcription of itsdownstream target genes such as HO-1 NQO1 GCLM andferritin It has been shown that after cotreatment of ATOplusATRA the Nrf2 nuclear translocation is prevented and thecytotoxic effects of ATO treatment are enhanced [186]

Lastly a recent paper shows that metformin whichwas previously associated with a better survival of diabeticpatients with pancreatic cancer [187] exerts its antitumouractivity by suppressing HO-1 expression in cancer cells Inthis paper metformin is reported to inhibit Nrf2 through


a Keap1-independent mechanism by inactivating Raf andERK signaling [152] The excellent therapeutic index ofmetformin with few side-effects associated even with long-term treatment could increase the chances of its applicationin cancer therapy

7 Conclusions

The activation of Nrf2HO-1 axis plays a central role incellular adaptive responses to oxidative stress and cytotoxicinsults representing a crucial point in the prevention ofcarcinogenesis On the contrary in tumour tissues a pro-longed activation of Nrf2 and HO-1 contributes to the gain ofmalignant phenotypes Consequently theNrf2HO-1 axis canbe used by cancer cells to promote their growth advantagemetastatic potential and resistance to therapy Therefore thetherapeutic usefulness of inhibitors of Nrf2 and of its targetgene HO-1 especially in combination with conventionalantineoplastic therapies may well represent a potential andpromising approach in the fight against cancer

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors are thankful for grants from MIUR-PRIN (no20125S38FA) (M Nitti) and Genoa University

References

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[2] T Ishii K Itoh S Takahashi et al ldquoTranscription factor Nrf2coordinately regulates a group of oxidative stress-induciblegenes inmacrophagesrdquo Journal of Biological Chemistry vol 275no 21 pp 16023ndash16029 2000

[3] T Ishii K Itoh E Ruiz et al ldquoRole of Nrf2 in the regulationof CD36 and stress protein expression in murine macrophagesactivation by oxidativelymodified LDL and 4-hydroxynonenalrdquoCirculation Research vol 94 no 5 pp 609ndash616 2004

[4] K Itoh J Mimura and M Yamamoto ldquoDiscovery of the nega-tive regulator of Nrf2 keap1 a historical overviewrdquoAntioxidantsand Redox Signaling vol 13 no 11 pp 1665ndash1678 2010

[5] F Weinberg and N S Chandel ldquoReactive oxygen species-dependent signaling regulates cancerrdquo Cellular and MolecularLife Sciences vol 66 no 23 pp 3663ndash3673 2009

[6] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[7] K Taguchi H Motohashi and M Yamamoto ldquoMolecularmechanisms of the Keap1-Nrf2 pathway in stress response andcancer evolutionrdquoGenes to Cells vol 16 no 2 pp 123ndash140 2011

[8] N Traverso R Ricciarelli M Nitti et al ldquoRole of glutathionein cancer progression and chemoresistancerdquoOxidativeMedicine

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[9] H Kumar I-S Kim S V More B-W Kim and D-K ChoildquoNatural product-derived pharmacologicalmodulators ofNrf2ARE pathway for chronic diseasesrdquo Natural Product Reportsvol 31 no 1 pp 109ndash139 2014

[10] D Malhotra E Portales-Casamar A Singh et al ldquoGlobal map-ping of binding sites for Nrf2 identifies novel targets in cellsurvival response through ChIP-Seq profiling and networkanalysisrdquo Nucleic Acids Research vol 38 no 17 Article IDgkq212 pp 5718ndash5734 2010

[11] B N Chorley M R Campbell X Wang et al ldquoIdentification ofnovel NRF2-regulated genes by ChiP-Seq influence on retinoidX receptor alphardquo Nucleic Acids Research vol 40 no 15 pp7416ndash7429 2012

[12] J D Hayes and A T Dinkova-Kostova ldquoThe Nrf2 regulatorynetwork provides an interface between redox and intermediarymetabolismrdquo Trends in Biochemical Sciences vol 39 no 4 pp199ndash218 2014

[13] P Moi K Chan I Asunis A Cao and Y W Kan ldquoIsolation ofNF-E2-related factor 2 (Nrf2) a NF-E2-like basic leucine zippertranscriptional activator that binds to the tandem NF-E2AP1repeat of the beta-globin locus control regionrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 91 no 21 pp 9926ndash9930 1994

[14] M C Jaramillo and D D Zhang ldquoThe emerging role of theNrf2-Keap1 signaling pathway in cancerrdquo Genes and Develop-ment vol 27 no 20 pp 2179ndash2191 2013

[15] E J Moon and A Giaccia ldquoDual roles of NRF2 in tumorprevention and progression possible implications in cancertreatmentrdquo Free Radical Biology and Medicine vol 79 pp 292ndash299 2015

[16] Y Hirotsu F Katsuoka R Funayama et al ldquoNrf2-MafGheterodimers contribute globally to antioxidant and metabolicnetworksrdquo Nucleic Acids Research vol 40 no 20 pp 10228ndash10239 2012

[17] K I Tong Y Katoh H Kusunoki K Itoh T Tanaka and MYamamoto ldquoKeap1 recruits Neh2 through binding to ETGEand DLG motifs characterization of the two-site molecularrecognition modelrdquoMolecular and Cellular Biology vol 26 no8 pp 2887ndash2900 2006

[18] J-H Kim S Yu J D Chen andAN Kong ldquoThe nuclear cofac-tor RAC3AIB1SRC-3 enhances Nrf2 signaling by interactingwith transactivation domainsrdquoOncogene vol 32 no 4 pp 514ndash527 2013

[19] P Rada A I Rojo S Chowdhry MMcMahon J D Hayes andA Cuadrado ldquoSCF120573-TrCP promotes glycogen synthase kinase3-dependent degradation of the Nrf2 transcription factor in aKeap1-independent mannerrdquo Molecular and Cellular Biologyvol 31 no 6 pp 1121ndash1133 2011

[20] HWang K Liu M Geng et al ldquoRXR120572 inhibits the NRF2-AREsignaling pathway through a direct interaction with the Neh7domain of NRF2rdquo Cancer Research vol 73 no 10 pp 3097ndash3108 2013

[21] S B Cullinan J D Gordan J Jin J W Harper and J A DiehlldquoThe Keap1-BTB protein is an adaptor that bridges Nrf2 to aCul3-based E3 ligase oxidative stress sensing by a Cul3-Keap1ligaserdquoMolecular and Cellular Biology vol 24 no 19 pp 8477ndash8486 2004

[22] A Kobayashi M-I Kang H Okawa et al ldquoOxidative stresssensor Keap1 functions as an adaptor for Cul3-based E3 ligase


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[28] A T Dinkova-Kostova W D Holtzclaw R N Cole et alldquoDirect evidence that sulfhydryl groups of Keap1 are the sensorsregulating induction of phase 2 enzymes that protect againstcarcinogens and oxidantsrdquo Proceedings of the National Academyof Sciences of the United States of America vol 99 no 18 pp11908ndash11913 2002

[29] NWakabayashi A T Dinkova-Kostova W D Holtzclaw et alldquoProtection against electrophile and oxidant stress by inductionof the phase 2 response fate of cysteines of the Keap1 sensormodified by inducersrdquo Proceedings of the National Academy ofSciences of the United States of America vol 101 no 7 pp 2040ndash2045 2004

[30] K Takaya T Suzuki H Motohashi et al ldquoValidation of themultiple sensor mechanism of the Keap1-Nrf2 systemrdquo FreeRadical Biology and Medicine vol 53 no 4 pp 817ndash827 2012

[31] H K Bryan A Olayanju C E Goldring and B K Park ldquoTheNrf2 cell defence pathway Keap1-dependent and -independentmechanisms of regulationrdquo Biochemical Pharmacology vol 85no 6 pp 705ndash717 2013

[32] M Kobayashi L Li N Iwamoto et al ldquoThe antioxidant defensesystemKeap1-Nrf2 comprises amultiple sensingmechanism forresponding to a wide range of chemical compoundsrdquoMolecularand Cellular Biology vol 29 no 2 pp 493ndash502 2009

[33] M Komatsu H Kurokawa S Waguri et al ldquoThe selectiveautophagy substrate p62 activates the stress responsive tran-scription factor Nrf2 through inactivation of Keap1rdquoNature CellBiology vol 12 no 3 pp 213ndash223 2010

[34] Y Ichimura S Waguri Y-S Sou et al ldquoPhosphorylation of p62activates the Keap1-Nrf2 pathway during selective autophagyrdquoMolecular Cell vol 51 no 5 pp 618ndash631 2013

[35] K Taguchi N Fujikawa M Komatsu et al ldquoKeap1 degradationby autophagy for the maintenance of redox homeostasisrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 109 no 34 pp 13561ndash13566 2012

[36] T Wu F Zhao B Gao et al ldquoHrd1 suppresses Nrf2-mediatedcellular protection during liver cirrhosisrdquo Genes and Develop-ment vol 28 no 7 pp 708ndash722 2014

[37] S Chowdhry Y Zhang M McMahon C Sutherland ACuadrado and J D Hayes ldquoNrf2 is controlled by two distinctbeta-TrCP recognitionmotifs in its Neh6 domain one of which

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[38] S Tao S Wang S J Moghaddam et al ldquoOncogenic KRAS con-fers chemoresistance by upregulating NRF2rdquo Cancer Researchvol 74 no 24 pp 7430ndash7441 2014

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[40] M Ramos-Gomez M-K Kwak P M Dolan et al ldquoSensitivityto carcinogenesis is increased and chemoprotective efficacy ofenzyme inducers is lost in nrf2 transcription factor-deficientmicerdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 98 no 6 pp 3410ndash3415 2001

[41] K Iida K Itoh Y Kumagai et al ldquoNrf2 is essential for thechemopreventive efficacy of oltipraz against urinary bladdercarcinogenesisrdquo Cancer Research vol 64 no 18 pp 6424ndash64312004

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[43] T O Khor M-T Huang A Prawan et al ldquoIncreased suscep-tibility of Nrf2 knockout mice to colitis-associated colorectalcancerrdquo Cancer Prevention Research vol 1 no 3 pp 187ndash1912008

[44] L Becks M Prince H Burson et al ldquoAggressive mammarycarcinoma progression inNrf2 knockoutmice treated with 712-dimethylbenz[a]anthracenerdquo BMC Cancer vol 10 article 5402010

[45] T Yamamoto K Yoh A Kobayashi et al ldquoIdentification ofpolymorphisms in the promoter region of the human NRF2generdquo Biochemical and Biophysical Research Communicationsvol 321 no 1 pp 72ndash79 2004

[46] T Suzuki T Shibata K Takaya et al ldquoRegulatory nexus ofsynthesis and degradation deciphers cellular Nrf2 expressionlevelsrdquoMolecular and Cellular Biology vol 33 no 12 pp 2402ndash2412 2013

[47] X-J Wang Z Sun N F Villeneuve et al ldquoNrf2 enhancesresistance of cancer cells to chemotherapeutic drugs the darkside of Nrf2rdquo Carcinogenesis vol 29 no 6 pp 1235ndash1243 2008

[48] H-K Na and Y-J Surh ldquoOncogenic potential of Nrf2 and itsprincipal target protein heme oxygenase-1rdquo Free Radical Biologyand Medicine vol 67 pp 353ndash365 2014

[49] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by Keap1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008

[50] D D Zhang ldquoMechanistic studies of the Nrf2-Keap1 signalingpathwayrdquo Drug Metabolism Reviews vol 38 no 4 pp 769ndash7892006

[51] P Nioi and T Nguyen ldquoA mutation of Keap1 found in breastcancer impairs its ability to repress Nrf2 activityrdquo Biochemicaland Biophysical Research Communications vol 362 no 4 pp816ndash821 2007

[52] D R Stacy K Ely P P Massion et al ldquoIncreased expression ofnuclear factor E2 p45-related factor 2 (NRF2) in head and necksquamous cell carcinomasrdquo Head and Neck vol 28 no 9 pp813ndash818 2006


[53] M G P van der Wijst R Brown and M G Rots ldquoNrf2the master redox switch the Achillesrsquo heel of ovarian cancerrdquoBiochimica et BiophysicaActamdashReviews onCancer vol 1846 no2 pp 494ndash509 2014

[54] T Jiang N Chen F Zhao et al ldquoHigh levels of Nrf2 deter-mine chemoresistance in type II endometrial cancerrdquo CancerResearch vol 70 no 13 pp 5486ndash5496 2010

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[56] T Shibata S Saito A Kokubu T Suzuki M Yamamoto andS Hirohashi ldquoGlobal downstream pathway analysis reveals adependence of oncogenic NFmdashE2-related factor 2 mutation onthe mTOR growth signaling pathwayrdquo Cancer Research vol 70no 22 pp 9095ndash9105 2010

[57] P Shelton and A K Jaiswal ldquoThe transcription factor NF-E2-related factor 2 (nrf2) a protooncogenerdquo The FASEB Journalvol 27 no 2 pp 414ndash423 2013

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[74] N D Camp R G James D W Dawson et al ldquoWilms tumorgene on X chromosome (WTX) inhibits degradation of NRF2protein through competitive binding toKEAP1 proteinrdquo Journalof Biological Chemistry vol 287 no 9 pp 6539ndash6550 2012

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[86] Y Zhong F Zhang Z Sun et al ldquoDrug resistance associateswith activation of Nrf2 in MCF-7DOX cells and wogoninreverses it by down-regulating Nrf2-mediated cellular defenseresponserdquoMolecular Carcinogenesis vol 52 no 10 pp 824ndash8342013

[87] S Homma Y Ishii Y Morishima et al ldquoNrf2 enhances cellproliferation and resistance to anticancer drugs in human lungcancerrdquo Clinical Cancer Research vol 15 no 10 pp 3423ndash34322009

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[89] S Lee M-J Lim M-H Kim et al ldquoAn effective strategy forincreasing the radiosensitivity of Human lung Cancer cells byblocking Nrf2-dependent antioxidant responsesrdquo Free RadicalBiology and Medicine vol 53 no 4 pp 807ndash816 2012

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[95] S L Slocum and T W Kensler ldquoNrf2 control of sensitivity tocarcinogensrdquo Archives of Toxicology vol 85 no 4 pp 273ndash2842011

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[97] D M Santos M M M Santos R Moreira S Sola and CM P Rodrigues ldquoSynthetic condensed 14-naphthoquinonederivative shifts neural stem cell differentiation by regulatingredox staterdquoMolecular Neurobiology vol 47 no 1 pp 313ndash3242013

[98] S Murakami and H Motohashi ldquoRoles of NRF2 in cell prolif-eration and differentiationrdquo Free Radical Biology and Medicine2015

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[100] J Zhu H Wang Q Sun et al ldquoNrf2 is required to maintain theself-renewal of glioma stem cellsrdquo BMC Cancer vol 13 article380 2013

[101] J Zhu H Wang X Ji et al ldquoDifferential Nrf2 expressionbetween glioma stem cells and non-stem-like cells in glioblas-tomardquo Oncology Letters vol 7 no 3 pp 693ndash698 2014

[102] T Wu B G Harder P K Wong J E Lang and D D ZhangldquoOxidative stress mammospheres and Nrf2-new implicationfor breast cancer therapyrdquoMolecular Carcinogenesis 2014

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[107] J Alam S Shibahara and A Smith ldquoTranscriptional activationof the heme oxygenase gene by heme and cadmium in mousehepatoma cellsrdquo Journal of Biological Chemistry vol 264 no 11pp 6371ndash6375 1989

[108] SM Keyse and RM Tyrrell ldquoHeme oxygenase is themajor 32-kDa stress protein induced in human skin fibroblasts by UVAradiation hydrogenperoxide and sodiumarseniterdquoProceedingsof the National Academy of Sciences of the United States ofAmerica vol 86 no 1 pp 99ndash103 1989

[109] R Foresti J E Clark C J Green and RMotterlini ldquoThiol com-pounds interactwith nitric oxide in regulating hemeoxygenase-1 induction in endothelial cells involvement of superoxide andperoxynitrite anionsrdquo The Journal of Biological Chemistry vol272 no 29 pp 18411ndash18417 1997

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[111] R Stocker Y Yamamoto A F McDonagh A N Glazer and BN Ames ldquoBilirubin is an antioxidant of possible physiologicalimportancerdquo Science vol 235 no 4792 pp 1043ndash1046 1987

[112] S Brouard L EOtterbein J Anrather et al ldquoCarbonmonoxidegenerated by heme oxygenase 1 suppresses endothelial cellapoptosisrdquo Journal of Experimental Medicine vol 192 no 7 pp1015ndash1026 2000

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[114] S W Ryter and A M K Choi ldquoHeme oxygenase-1 redoxregulation of a stress protein in lung and cell culture modelsrdquoAntioxidants and Redox Signaling vol 7 no 1-2 pp 80ndash91 2005

[115] A Prawan J K Kundu andY-J Surh ldquoMolecular basis of hemeoxygenase-1 induction implications for chemoprevention andchemoprotectionrdquo Antioxidants and Redox Signaling vol 7 no11-12 pp 1688ndash1703 2005

[116] A Jozkowicz H Was and J Dulak ldquoHeme oxygenase-1 intumors Is it a false friendrdquo Antioxidants and Redox Signalingvol 9 no 12 pp 2099ndash2117 2007

[117] P Banerjee A Basu D Datta M Gasser A M Waaga-Gasserand S Pal ldquoThe heme oxygenase-1 protein is overexpressed inhuman renal cancer cells following activation of the Ras-Raf-ERK pathway and mediates anti-apoptotic signalrdquo The Journalof Biological Chemistry vol 286 no 38 pp 33580ndash33590 2011

[118] H Yin J Fang L Liao H Maeda and Q Su ldquoUpregulationof heme oxygenase-1 in colorectal cancer patients with in-creased circulation carbon monoxide levels potentially affectschemotherapeutic sensitivityrdquo BMCCancer vol 14 no 1 article436 2014

[119] P O Berberat Z Dambrauskas A Gulbinas et al ldquoInhibitionof heme oxygenase-1 increases responsiveness of pancreaticcancer cells to anticancer treatmentrdquo Clinical Cancer Researchvol 11 no 10 pp 3790ndash3798 2005

[120] M S Degese J E Mendizabal N A Gandini et al ldquoExpressionof heme oxygenase-1 in non-small cell lung cancer (NSCLC)and its correlation with clinical datardquo Lung Cancer vol 77 no1 pp 168ndash175 2012

[121] M Miyake K Fujimoto S Anai et al ldquoClinical significance ofheme oxygenase-1 expression in non-muscle-invasive bladdercancerrdquo Urologia Internationalis vol 85 no 3 pp 355ndash3632010

[122] J-R Tsai H-M Wang P-L Liu et al ldquoHigh expression ofheme oxygenase-1 is associated with tumor invasiveness andpoor clinical outcome in non-small cell lung cancer patientsrdquoCellular Oncology vol 35 no 6 pp 461ndash471 2012

[123] H Was T Cichon R Smolarczyk et al ldquoOverexpression ofheme oxygenase-1 inmurinemelanoma increased proliferationand viability of tumor cells decreased survival of micerdquo TheAmerican Journal of Pathology vol 169 no 6 pp 2181ndash21982006

[124] M Sunamura D G Duda M H Ghattas et al ldquoHemeoxygenase-1 accelerates tumor angiogenesis of human pancre-atic cancerrdquo Angiogenesis vol 6 no 1 pp 15ndash24 2003

[125] S-S Lee S-F Yang C-H Tsai M-C Chou M-Y Chou andY-C Chang ldquoUpregulation of heme oxygenase-1 expression inareca-quid-chewing-associated oral squamous cell carcinomardquoJournal of the Formosan Medical Association vol 107 no 5 pp355ndash363 2008

[126] M A Alaoui-Jamali T A Bismar A Gupta et al ldquoA novelexperimental heme oxygenase-1-targeted therapy for hormone-refractory prostate cancerrdquo Cancer Research vol 69 no 20 pp8017ndash8024 2009

[127] G Sass P Leukel V Schmitz et al ldquoInhibition of heme oxyge-nase 1 expression by small interferingRNAdecreases orthotopictumor growth in livers of micerdquo International Journal of Cancervol 123 no 6 pp 1269ndash1277 2008

[128] M J Marinissen T Tanos M Bolos M R De Sagarra OA Coso and A Cuadrado ldquoInhibition of heme oxygenase-1interferes with the transforming activity of the Kaposi sarcomaherpesvirus-encoded G protein-coupled receptorrdquo The Journalof Biological Chemistry vol 281 no 16 pp 11332ndash11346 2006

[129] H Was J Dulak and A Jozkowicz ldquoHeme oxygenase-1 intumor biology and therapyrdquo Current Drug Targets vol 11 no12 pp 1551ndash1570 2010

[130] A Loboda A Jazwa A Grochot-Przeczek et al ldquoHemeoxygenase-1 and the vascular bed frommolecular mechanismsto therapeutic opportunitiesrdquo Antioxidants amp Redox Signalingvol 10 no 10 pp 1767ndash1812 2008

[131] J Fang K Greish H Qin et al ldquoHSP32 (HO-1) inhibitorcopoly(styrene-maleic acid)-zinc protoporphyrin IX a water-soluble micelle as anticancer agent in vitro and in vivo anti-cancer effectrdquo European Journal of Pharmaceutics and Biophar-maceutics vol 81 no 3 pp 540ndash547 2012

[132] H Nakamura L Liao Y Hitaka et al ldquoMicelles of zinc proto-porphyrin conjugated to N-(2-hydroxypropyl)methacrylamide(HPMA) copolymer for imaging and light-induced antitumoreffects in vivordquo Journal of Controlled Release vol 165 no 3 pp191ndash198 2013

[133] P Nuhn B M Kunzli R Hennig et al ldquoHeme oxygenase-1 and its metabolites affect pancreatic tumor growth in vivordquoMolecular Cancer vol 8 article 37 2009

[134] W Zhang T Qiao and L Zha ldquoInhibition of heme oxygenase-1enhances the radiosensitivity in human nonsmall cell lung can-cer A549 cellsrdquo Cancer Biotherapy and Radiopharmaceuticalsvol 26 no 5 pp 639ndash645 2011

[135] D Nowis M Legat T Grzela et al ldquoHeme oxygenase-1protects tumor cells against photodynamic therapy-mediatedcytotoxicityrdquo Oncogene vol 25 no 24 pp 3365ndash3374 2006

[136] D Ma Q Fang P Wang et al ldquoInduction of heme oxygenase-1 by Na+-H+ exchanger 1 protein plays a crucial role inimatinib-resistant chronic myeloid leukemia cellsrdquoThe Journalof Biological Chemistry vol 290 no 20 pp 12558ndash12571 2015

[137] W-K Jeon H-Y Hong W-C Seo et al ldquoSmad7 sensitizesA549 lung cancer cells to cisplatin-induced apoptosis throughheme oxygenase-1 inhibitionrdquo Biochemical and BiophysicalResearch Communications vol 420 no 2 pp 288ndash292 2012

[138] Y Yin Q Liu B Wang G Chen L Xu and H Zhou ldquoEx-pression and function of heme oxygenase-1 in human gastriccancerrdquo Experimental Biology and Medicine vol 237 no 4 pp362ndash371 2012

[139] M Mayerhofer S Florian M-T Krauth et al ldquoIdentificationof heme oxygenase-1 as a novel BCRABL-dependent survivalfactor in chronic myeloid leukemiardquo Cancer Research vol 64no 9 pp 3148ndash3154 2004

[140] M Hill V Pereira C Chauveau et al ldquoHeme oxygenase-1inhibits rat and human breast cancer cell proliferation mutualcross inhibition with indoleamine 23-dioxygenaserdquoThe FASEBJournal vol 19 no 14 pp 1957ndash1968 2005

[141] M Ferrando G Gueron B Elguero et al ldquoHeme oxygenase1 (HO-1) challenges the angiogenic switch in prostate cancerrdquoAngiogenesis vol 14 no 4 pp 467ndash479 2011

[142] C-Y Chao C-K Lii Y-T Hsu et al ldquoInduction of hemeoxygenase-1 and inhibition of TPA-induced matrix metallo-proteinase-9 expression by andrographolide in MCF-7 humanbreast cancer cellsrdquo Carcinogenesis vol 34 no 8 pp 1843ndash18512013

[143] BWegiel Z Nemeth M Correa-Costa A C Bulmer and L EOtterbein ldquoHeme oxygenase-1 a metabolic nikerdquo Antioxidantsand Redox Signaling vol 20 no 11 pp 1709ndash1722 2014

[144] S W Ryter S Xi C L Hartsfield and A M K Choi ldquoMitogenactivated protein kinase (MAPK) pathway regulates hemeoxygenase-1 gene expression by hypoxia in vascular cellsrdquo


Antioxidants and Redox Signaling vol 4 no 4 pp 587ndash5922002

[145] C-C Lin L-L Chiang C-H Lin et al ldquoTransforming growthfactor-beta1 stimulates heme oxygenase-1 expression via thePI3KAkt and NF-kappaB pathways in human lung epithelialcellsrdquo European Journal of Pharmacology vol 560 no 2-3 pp101ndash109 2007

[146] Y Lavrovsky M L Schwartzman R D Levere A Kappas andN G Abraham ldquoIdentification of binding sites for transcriptionfactors NF-kappa B and AP-2 in the promoter region of thehuman heme oxygenase 1 generdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 91 no13 pp 5987ndash5991 1994

[147] S A Rushworth K M Bowles P Raninga and D J MacE-wan ldquoNF-120581B-Lnhibited acute myeloid leukemia cells are res-cued from apoptosis by heme oxygenase-1 inductionrdquo CancerResearch vol 70 no 7 pp 2973ndash2983 2010

[148] E O Farombi and Y-J Surh ldquoHeme oxygenase-1 as a potentialtherapeutic target for hepatoprotectionrdquo Journal of Biochemistryand Molecular Biology vol 39 no 5 pp 479ndash491 2006

[149] L-H Wang Y Li S-N Yang et al ldquoGambogic acid syner-gistically potentiates cisplatin-induced apoptosis in non-small-cell lung cancer through suppressing NF-120581B and MAPKHO-1signallingrdquo British Journal of Cancer vol 110 no 2 pp 341ndash3522014

[150] S Nemmiche D Chabane-Sari M Kadri and P GuiraudldquoCadmium-induced apoptosis in the BJAB human B cell lineinvolvement of PKCERK12JNK signaling pathways in HO-1expressionrdquo Toxicology vol 300 no 3 pp 103ndash111 2012

[151] P-Y Cheng Y-M Lee N-L Shih Y-C Chen and M-H YenldquoHeme oxygenase-1 contributes to the cytoprotection of alpha-lipoic acid via activation of p4442 mitogen-activated proteinkinase in vascular smooth muscle cellsrdquo Free Radical Biologyand Medicine vol 40 no 8 pp 1313ndash1322 2006

[152] M T Do H G Kim T Khanal et al ldquoMetformin inhibits hemeoxygenase-1 expression in cancer cells through inactivation ofRaf-ERK-Nrf2 signaling and AMPK-independent pathwaysrdquoToxicology and Applied Pharmacology vol 271 no 2 pp 229ndash238 2013

[153] L N Barrera S A Rushworth K M Bowles and D JMacEwan ldquoBortezomib induces heme oxygenase-1 expressionin multiple myelomardquo Cell Cycle vol 11 no 12 pp 2248ndash22522012

[154] S Bancos C J Baglole I Rahman and R P Phipps ldquoInductionof heme oxygenase-1 in normal and malignant B lymphocytesby 15-deoxy-9987791214-prostaglandin J2 requires Nrf2rdquo CellularImmunology vol 262 no 1 pp 18ndash27 2010

[155] D-Y LuW-L Yeh S-M Huang C-H Tang H-Y Lin and S-J Chou ldquoOsteopontin increases heme oxygenase-1 expressionand subsequently induces cell migration and invasion in gliomacellsrdquo Neuro-Oncology vol 14 no 11 pp 1367ndash1378 2012

[156] L-J Bao M C Jaramillo Z-B Zhang et al ldquoNrf2 inducescisplatin resistance through activation of autophagy in ovariancarcinomardquo International Journal of Clinical and ExperimentalPathology vol 7 no 4 pp 1502ndash1513 2014

[157] T W Kensler T J Curphey Y Maxiutenko and B D Roe-buck ldquoChemoprotection by organosulfur inducers of phase 2enzymes dithiolethiones and dithiinsrdquo Drug Metabolism andDrug Interactions vol 17 no 1ndash4 pp 3ndash22 2000

[158] C C Conaway C-X Wang B Pittman et al ldquoPhenethylisothiocyanate and sulforaphane and their N-acetylcysteine

conjugates inhibit malignant progression of lung adenomasinduced by tobacco carcinogens in AJ micerdquo Cancer Researchvol 65 no 18 pp 8548ndash8557 2005

[159] R Garg S Gupta and G B Maru ldquoDietary curcumin mod-ulates transcriptional regulators of phase I and phase II en-zymes in benzo[a]pyrene-treated mice mechanism of its anti-initiating actionrdquo Carcinogenesis vol 29 no 5 pp 1022ndash10322008

[160] R K Thimmulappa T Rangasamy J Alam and S BiswalldquoDibenzoylmethane activates Nrf2-dependent detoxificationpathway and inhibits benzo(a)pyrene induced DNA adducts inlungsrdquoMedicinal Chemistry vol 4 no 5 pp 473ndash481 2008

[161] B Singh R Shoulson A Chatterjee et al ldquoResveratrol inhibitsestrogen-induced breast carcinogenesis through induction ofNRF2-mediated protective pathwaysrdquo Carcinogenesis vol 35no 8 pp 1872ndash1880 2014

[162] K M Chang F P Liang I L Chen et al ldquoDiscovery of oxime-bearing naphthalene derivatives as a novel structural type ofNrf2 activatorsrdquo Bioorganic amp Medicinal Chemistry vol 23 no13 pp 3852ndash3859 2015

[163] F-F Gan H Ling X Ang et al ldquoA novel shogaol analogsuppresses cancer cell invasion and inflammation and displayscytoprotective effects through modulation of NF-kappaB andNrf2-Keap1 signaling pathwaysrdquo Toxicology and Applied Phar-macology vol 272 no 3 pp 852ndash862 2013

[164] R Hu C L-L Saw R Yu and A-N T Kong ldquoRegulation ofNF-E2-related factor 2 signaling for cancer chemopreventionantioxidant coupled with antiinflammatoryrdquo Antioxidants andRedox Signaling vol 13 no 11 pp 1679ndash1698 2010

[165] C Li X Xu X J Wang and Y Pan ldquoImine resveratrolanalogues molecular design Nrf2 activation and SAR analysisrdquoPLoS ONE vol 9 no 7 Article ID e101455 2014

[166] C R Zhao Z H Gao and X J Qu ldquoNrf2-ARE signalingpathway and natural products for cancer chemopreventionrdquoCancer Epidemiology vol 34 no 5 pp 523ndash533 2010

[167] J-S Lee and Y-J Surh ldquoNrf2 as a novel molecular target forchemopreventionrdquo Cancer Letters vol 224 no 2 pp 171ndash1842005

[168] W-S Jeong M Jun and A-N T Kong ldquoNrf2 a potentialmolecular target for cancer chemoprevention by natural com-poundsrdquo Antioxidants amp Redox Signaling vol 8 no 1-2 pp 99ndash106 2006

[169] A Lau N F Villeneuve Z Sun P K Wong and D D ZhangldquoDual roles ofNrf2 in cancerrdquoPharmacological Research vol 58no 5-6 pp 262ndash270 2008

[170] T W Kensler J-G Chen P A Egner et al ldquoEffects of glucosin-olate-rich broccoli sprouts on urinary levels of aflatoxin-DNAadducts and phenanthrene tetraols in a randomized clinicaltrial in He Zuo Township Qidong Peoplersquos Republic of ChinardquoCancer Epidemiology Biomarkers and Prevention vol 14 no 11pp 2605ndash2613 2005

[171] F Shang L Hui X An X Zhang S Guo and Z KuildquoZnPPIX inhibits peritoneal metastasis of gastric cancer via itsantiangiogenic activityrdquo Biomedicine amp Pharmacotherapy vol71 pp 240ndash246 2015

[172] S K Sahoo T Sawa J Fang et al ldquoPegylated zinc protopor-phyrin a water-soluble heme oxygenase inhibitor with tumor-targeting capacityrdquo Bioconjugate Chemistry vol 13 no 5 pp1031ndash1038 2002

[173] A K Iyer K Greish T Seki et al ldquoPolymeric micelles of zincprotoporphyrin for tumor targeted delivery based on EPR effect


and singlet oxygen generationrdquo Journal of Drug Targeting vol15 no 7-8 pp 496ndash506 2007

[174] A Jozkowicz and J Dulak ldquoEffects of protoporphyrins on pro-duction of nitric oxide and expression of vascular endothelialgrowth factor in vascular smooth muscle cells and macropha-gesrdquo Acta Biochimica Polonica vol 50 no 1 pp 69ndash79 2003

[175] A Loboda A Jazwa B Wegiel A Jozkowicz and J DulakldquoHeme oxygenase-1-dependent and -independent regulation ofangiogenic genes expression effect of cobalt protoporphyrinand cobalt chloride on VEGF and IL-8 synthesis in humanmicrovascular endothelial cellsrdquoCellular andMolecular Biologyvol 51 no 4 pp 347ndash355 2005

[176] J Busserolles J Megıas M C Terencio and M J AlcarazldquoHeme oxygenase-1 inhibits apoptosis in Caco-2 cells viaactivation of Akt pathwayrdquo International Journal of Biochemistryand Cell Biology vol 38 no 9 pp 1510ndash1517 2006

[177] J Fang T Sawa T Akaike et al ldquoIn vivo antitumor activityof pegylated zinc protoporphyrin targeted inhibition of hemeoxygenase in solid tumorrdquo Cancer Research vol 63 no 13 pp3567ndash3574 2003

[178] D Ren N F Villeneuve T Jiang et al ldquoBrusatol enhancesthe efficacy of chemotherapy by inhibiting the Nrf2-mediateddefense mechanismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 4 pp 1433ndash1438 2011

[179] A Olayanju I M Copple H K Bryan et al ldquoBrusatolprovokes a rapid and transient inhibition of Nrf2 signaling andsensitizes mammalian cells to chemical toxicity-implicationsfor therapeutic targeting of Nrf2rdquo Free Radical Biology andMedicine vol 78 pp 202ndash212 2015

[180] T Zhang Y Kimura S Jiang K Harada Y Yamashita and HAshida ldquoLuteolin modulates expression of drug-metabolizingenzymes through the AhR and Nrf2 pathways in hepatic cellsrdquoArchives of Biochemistry and Biophysics vol 557 pp 36ndash462014

[181] X TangHWang L Fan et al ldquoLuteolin inhibitsNrf2 leading tonegative regulation of the Nrf2ARE pathway and sensitizationof human lung carcinoma A549 cells to therapeutic drugsrdquo FreeRadical Biology andMedicine vol 50 no 11 pp 1599ndash1609 2011

[182] S Chian Y-Y Li X-J Wang and X-W Tang ldquoLuteolinsensitizes two oxaliplatin-resistant colorectal cancer cell linesto chemotherapeutic drugs via inhibition of the Nrf2 pathwayrdquoAsian Pacific Journal of Cancer Prevention vol 15 no 6 pp2911ndash2916 2014

[183] S Chian R Thapa Z Chi X J Wang and X Tang ldquoLuteolininhibits the Nrf2 signaling pathway and tumor growth in vivordquoBiochemical and Biophysical Research Communications vol 447no 4 pp 602ndash608 2014

[184] S Magesh Y Chen and L Hu ldquoSmall molecule modulators ofKeap1-Nrf2-ARE pathway as potential preventive and therapeu-tic agentsrdquo Medicinal Research Reviews vol 32 no 4 pp 687ndash726 2012

[185] X J Wang J D Hayes C J Henderson and C R WolfldquoIdentification of retinoic acid as an inhibitor of transcriptionfactor Nrf2 through activation of retinoic acid receptor alphardquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 104 no 49 pp 19589ndash19594 2007

[186] M Valenzuela C Glorieux J Stockis et al ldquoRetinoic acid syn-ergizes ATO-mediated cytotoxicity by precluding Nrf2 activityin AML cellsrdquo British Journal of Cancer vol 111 no 5 pp 874ndash882 2014

[187] N Sadeghi J L Abbruzzese S-C J Yeung M Hassan and DLi ldquoMetformin use is associated with better survival of diabeticpatients with pancreatic cancerrdquo Clinical Cancer Research vol18 no 10 pp 2905ndash2912 2012

[188] A KMacLeodMMcmahon SM Plummer et al ldquoCharacter-ization of the cancer chemopreventive NRF2-dependent genebattery in human keratinocytes demonstration that theKEAP1-NRF2 pathway and not the BACH1-NRF2 pathway controlscytoprotection against electrophiles as well as redox-cyclingcompoundsrdquo Carcinogenesis vol 30 no 9 pp 1571ndash1580 2009

[189] K-A Jung B-H Choi C-WNam et al ldquoIdentification of aldo-keto reductases as NRF2-target marker genes in human cellsrdquoToxicology Letters vol 218 no 1 pp 39ndash49 2013

[190] A S Agyeman R Chaerkady P G Shaw et al ldquoTranscriptomicand proteomic profiling of KEAP1 disrupted and sulforaphane-treated human breast epithelial cells reveals common expres-sion profilesrdquo Breast Cancer Research and Treatment vol 132no 1 pp 175ndash187 2012

[191] J Paek J Y Lo S D Narasimhan et al ldquoMitochondrial SKN-1Nrfmediates a conserved starvation responserdquoCellMetabolismvol 16 no 4 pp 526ndash537 2012

[192] S A Chanas Q Jiang M McMahon et al ldquoLoss of the Nrf2transcription factor causes a marked reduction in constitutiveand inducible expression of the glutathione S-transferase Gsta1Gsta2 Gstm1 Gstm2 Gstm3 and Gstm4 genes in the livers ofmale and female micerdquo Biochemical Journal vol 365 no 2 pp405ndash416 2002

[193] J M Maher M Z Dieter L M Aleksunes et al ldquoOxidativeand electrophilic stress inducesmultidrug resistance-associatedprotein transporters via the nuclear factor-E2-related factor-2transcriptional pathwayrdquo Hepatology vol 46 no 5 pp 1597ndash1610 2007

[194] M S Yates Q T Tran P M Dolan et al ldquoGenetic versuschemoprotective activation of Nrf2 signaling overlapping yetdistinct gene expression profiles between Keap1 knockout andtriterpenoid-treated micerdquo Carcinogenesis vol 30 no 6 pp1024ndash1031 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Table 1 Genes regulated by Nrf2 in mice and humans

Symbol Name Species ReferencesAntioxidant genes

GCLC Glutamate-cysteine ligasecatalytic subunit m h [188 189]

GCLM Glutamate-cysteine ligasemodifier subunit m h [188 189]

GLRX Glutaredoxin 1 h [190]GPX2 Glutathione peroxidase 2 m h [190 191]GPX4 Glutathione peroxidase 4 m [16]GSR1 Glutathione reductase m h [188 190]SLC6A9 Glycine transporter m [16]

SLC7A11 Cysteineglutamatetransporter m h [16 188]

PRDX1-6 Peroxiredoxins 1 and 6 m h [10 16]SRXN1 Sulfiredoxin-1 m h [11 188]TXN1 Thioredoxin m h [10 16]TXNRD1 Thioredoxin reductase 1 m h [188 190]

HO-1-related genesHMOX1 Heme oxygenase 1 m h [10 188]BLVRA Biliverdin reductase A h [190]BLVRB Biliverdin reductase B m h [16 190]FECH Ferrochelatase h [188]

FTH1 Ferritin heavy polypeptide1 m h [10]

FTHL12-17 Ferritin heavypolypeptides 12 and 17 h [188 190]

FTL1 Ferritin light polypeptide m h [188 190]Detoxifying enzymes

NQO1 NAD(P)Hquinoneoxidoreductase 1 m h [188 190]

GSTA1 Glutathione S-transferaseclass Alpha 1 m [192]

GSTM1 Glutathione S-transferaseclass Mu 1 m [192]

GSTP1 Glutathione S-transferaseclass Pi 1 m [12]

UGT1A1 UDP glucuronosyl-transferase 1 family h [190]

UGT2B7 UDP glucuronosyl-transferase 2 family m h [16 190]

Drug transporters

ABCB6 ATP-binding cassettesubfamily B(MDRTAP) m h [10 11]






Nrf2 has seven functional domains named Nrf2-ECHhomology (Neh) 1ndash7 (Figure 1(a)) [12 14 15] The Neh1domain has a basic region leucine zipper structure needed

for the dimerisation with small Maf and binds to antiox-idantelectrophile responsive elements (AREEpRE) [16]Neh2 is the main negative regulatory domain which bindsto the Kelch-like ECH-associated protein 1 (Keap1) via theDLG and ETGE motifs [17] Neh3 is localised in the C-terminal region of Nrf2 and acts as a transactivation domainrecruiting the chromo-ATPasehelicase DNA binding pro-tein family (CHD6) [6] whereas both Neh4 and Neh5 aretransactivation domains that recruit cAMP response elementbinding protein- (CREB-) binding protein (CBP) andorthe receptor-associated coactivator (RAC) [18] The Neh6domainmediates the Keap1-independent degradation ofNrf2through recruitment by the DSGIS and DSAPGS motifs ofthe dimeric120573-transducin repeat-containing protein (120573-TrCP)[19] Lastly the Neh7 domain recently identified by Wangand coworkers interacts with the retinoid X receptor alpha(RXR120572) a repressor of Nrf2 [20]

21 Keap1-Dependent Posttranscriptional Regulation

211 Ubiquitination and Proteasomal Degradation of Nrf2Under basal conditions Nrf2 is localised in the cytosolassociated with its negative regulator Keap1 an adaptorcomponent of Cullin 3-based ubiquitin E3 ligase complex(Cul3) that promotes Nrf2 constant ubiquitination and pro-teasomal degradation maintaining low basal levels [21 22]Nrf2 turnover is rapid less than 20 minutes and preventsthe expression of Nrf2 target genes under normal conditions[23] On exposure to oxidative or electrophilic stress Keap1 ismodified whereas the enzymatic activity of the E3 ubiquitinligase is inhibited Nrf2 is liberated from Keap1 accumulatesin the nucleus dimerises with small Maf protein and thenactivates after ARE-sequence binding the transcription ofits target genes [23 24] Thus Keap1 is the main repressorof Nrf2 having three functional domains namely the broadcomplex Tramtrack and Bric-a-Brac (BTB) domain theintervening region (IVR) domain and the double glycinerepeat (DGR)Kelch domain [25] (Figure 1(b))

Keap1 acts as a sensor for oxidative and electrophilicstress through the modification of 27 cysteine residues [26](Figure 2)Themain three cysteine residues for the regulationof Nrf2 activity are Cys151 in the BTB domain and Cys273and Cys288 in the IVR domain all of which are targets ofoxidative and electrophilic modifications [27ndash29] It has beenshown that cells expressing Keap1 Cys151 point mutant pro-tein show reduced activation of Nrf2 in response to a numberof inducers (eg sulforaphane tert-butylhydroquinone anddiethyl maleate) in comparison to the wild-type cells [30]and that Cys273 and Cys288 are critically required for thebasal repression of Nrf2 [31] In addition the modificationof a subset of cysteine residues in Keap1 by Nrf2 inducerssupports the hypothesis of a ldquocysteine coderdquo which is criticalin the activation of Nrf2 [32]

212 Autophagic Degradation of Keap1 Furthermore otherinteracting protein partners such as the sequestosome-1protein (p62SQSTM1) can modulate Nrf2 activity [33 34]p62SQSTM1 is a scaffold protein that binds to polyubiquiti-nated proteins and targets protein aggregates for autophagic




Keap1 binding

COOH



NH2


RXR120572 binding

(a)


Cys-151 Cys-273 Cys-288

COOH


NH2

(b)



Keap1

Cys-SHHS-Cys

Nrf2Keap1


Ubiquitination

Nrf2

Cul3Keap1

S-CysCys-S

Nrf2


Keap1Nrf2 mutations

Keap1Cul3

HS-Cys

Nrf2

Cul3Keap1Cys-SH

Basal condition

ARE

Nrf2 Maf


Keap1Cys-SHHS-Cys

Cul3p62

Release of Nrf2

Nrf2


Release of Nrf2

Release of Nrf2


Cytoplasm

Cul3Ub Cul3

Cytoplasm




































NADP+NADP+

HEME

NADPH

Bilirubin

NADPH

Biliverdin

Ferritinantioxidant

Antiapoptotic

Antioxidant

HO-1


reductase
































7 Conclusions




Acknowledgments


References





















































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Keap1 binding

COOH



NH2


RXR120572 binding

(a)


Cys-151 Cys-273 Cys-288

COOH


NH2

(b)



Keap1

Cys-SHHS-Cys

Nrf2Keap1


Ubiquitination

Nrf2

Cul3Keap1

S-CysCys-S

Nrf2


Keap1Nrf2 mutations

Keap1Cul3

HS-Cys

Nrf2

Cul3Keap1Cys-SH

Basal condition

ARE

Nrf2 Maf


Keap1Cys-SHHS-Cys

Cul3p62

Release of Nrf2

Nrf2


Release of Nrf2

Release of Nrf2


Cytoplasm

Cul3Ub Cul3

Cytoplasm




































NADP+NADP+

HEME

NADPH

Bilirubin

NADPH

Biliverdin

Ferritinantioxidant

Antiapoptotic

Antioxidant

HO-1


reductase
































7 Conclusions




Acknowledgments


References





















































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































NADP+NADP+

HEME

NADPH

Bilirubin

NADPH

Biliverdin

Ferritinantioxidant

Antiapoptotic

Antioxidant

HO-1


reductase
































7 Conclusions




Acknowledgments


References





















































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































7 Conclusions




Acknowledgments


References





















































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















review article the nrf2/ho-1 axis in cancer cell growth and...

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