research article nrf2 deficiency exaggerates doxorubicin...

Research ArticleNrf2 Deficiency Exaggerates Doxorubicin-InducedCardiotoxicity and Cardiac Dysfunction

Siying Li12 Wenjuan Wang12 Ting Niu1 Hui Wang12 Bin Li12 Lei Shao12 Yimu Lai2

Huanjie Li1 Joseph S Janicki2 Xing Li Wang1 Dongqi Tang12 and Taixing Cui12

1 Shandong University Qilu Hospital Research Center for Cell Therapy Key Laboratory of Cardiovascular Remodelingand Function Research Qilu Hospital of Shandong University Jinan 250012 China

2Department of Cell Biology and Anatomy University of South Carolina School of Medicine Columbia SC 29208 USA

Correspondence should be addressed to Dongqi Tang dongqitanguscmedscedu and Taixing Cui taixingcuiuscmedscedu

Received 16 January 2014 Revised 10 April 2014 Accepted 11 April 2014 Published 6 May 2014

Academic Editor Neelam Khaper

Copyright copy 2014 Siying Li et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The anticancer therapy of doxorubicin (Dox) has been limited by its acute and chronic cardiotoxicity In addition to a causativerole of oxidative stress autophagy appears to play an important role in the regulation of Dox-induced cardiotoxicity Howeverthe underlying mechanisms remain unclear Accordingly we explored a role of nuclear factor erythroid-2 related factor 2 (Nrf2)in Dox-induced cardiomyopathy with a focus on myocardial oxidative stress and autophagic activity In wild type (WT) micea single intraperitoneal injection of 25mgkg Dox rapidly induced cardiomyocyte necrosis and cardiac dysfunction which wereassociated with oxidative stress impaired autophagy and accumulated polyubiquitinated protein aggregates However these Dox-induced adverse effects were exaggerated in Nrf2 knockout (Nrf2minusminus) mice In cultured cardiomyocytes overexpression of Nrf2increased the steady levels of LC3-II ameliorated Dox-induced impairment of autophagic flux and accumulation of ubiquitinatedprotein aggregates and suppressed Dox-induced cytotoxicity whereas knockdown of Nrf2 exerted opposite effects Moreover theexaggerated adverse effects in Dox-intoxicated Nrf2 depleted cardiomyocytes were dramatically attenuated by forced activationof autophagy via overexpression of autophagy related gene 5 (Atg5) Thus these results suggest that Nrf2 is likely an endogenoussuppressor of Dox-induced cardiotoxicity by controlling both oxidative stress and autophagy in the heart

1 Introduction

The anticancer drug doxorubicin (Dox) (also referred to asadriamycin) is highly effective in the treatment of a broadrange of cancers however it is associated with dose-dependent acute and chronic cardiotoxicity which signifi-cantly limits its chemotherapeutic dosage [1ndash4] The cause ofDox-induced cardiotoxicity is multifactorial and includesfree radical-induced mitochondrial damage DNA damageinhibition of DNA and protein synthesis and myofiberdegeneration which cumulatively leads to myocardial apop-totic andor necrotic cell death Nevertheless the precisepathophysiology of Dox-induced cardiotoxicity is not fullyunderstood

That oxidative stress is the primary cause of Dox-inducedcardiomyopathy has been the prevailing hypothesis [3 4]

However antioxidant approaches of nonselective reactiveoxygen species (ROS) scavenging for the treatment of Dox-induced cardiomyopathy have been shown to be ineffectiveTherefore efficacious therapy may require more specifictargeting of either the source of oxidative stress or the endoge-nous antioxidant defense system However such specifictargets remain to be identified

Nuclear factor erythroid-2 related factor 2 (Nrf2) is amaster transcription factor in controlling the basal and indu-cible expression of a battery of antioxidant genes and othercytoprotective phase II detoxifying enzymes [5] We havedemonstrated that Nrf2 is a negative regulator of cardiacpathological remodeling and dysfunction via suppressingoxidative stress in diverse pathological settings [6 7] Whileit has been documented that Nrf2 plays a mediator role in

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2014 Article ID 748524 15 pageshttpdxdoiorg1011552014748524

2 Oxidative Medicine and Cellular Longevity

hydrogen sulfide-mediated suppression of oxidative stress-induced cardiac dysfunction [8] we and others have demon-strated that Nrf2 might be a drug target for the treatmentof cardiomyocyte injury and cardiac dysfunction [9 10] Ofinterest a recent report has revealed that Nrf2 is able toenhance autophagic clearance of toxic ubiquitinated proteinaggregates secondary to ROS formation suggesting a novelmediator role of Nrf2 for sufficient activation of autophagy[11] Considering the causative role of oxidative stress and theprotective effect of sufficient autophagy activation in Dox-induced cardiotoxicity as well as the Nrf2-mediated antiox-idant defense and sufficient activation of autophagy it isconceivable that Nrf2 is a negative regulator of Dox-inducedcardiomyopathy

Therefore in the present study we explored the role ofNrf2 in the regulation ofDox-induced cardiomyopathywith afocus on oxidative stress and autophagic activity in the heartWe demonstrate that loss of Nrf2 function exaggerates Dox-induced oxidative stress insufficient autophagic activitiesand cardiomyocyte necrosis as well as cardiac dysfunctionThese results indicate that Nrf2 acts as a critical negative reg-ulator of Dox-induced cardiomyopathy thereby identifying apotential novel target for the treatment of Dox-inducedcardiomyopathy

2 Results

21 Dox-Induced Cardiac Oxidative Stress Toxicity and Dys-function Considering oxidative stress to be the primarycause of Dox-induced cardiomyopathy [3 4] we first deter-mined myocardial oxidative stress and cell death in the acutemodel of Dox-induced cardiotoxicity which has been charac-terized as a rapid cardiomyocyte necrosis and reduced cardiacoutput within 4 days after a single treatment of Dox [12 13]Of note a single intraperitoneal injection of Dox (20mgkg)results in reduction of left ventricle fraction shorting (FS)in C57BL6J mice [13] Following a single intraperitonealinjection of Dox (25mgkg) there was a time dependentonset of oxidative stress in the heart which appeared at day 1and was sustained by day 4 after injection (Figure 1(a))Therewas also substantial cardiomyocyte necrosis in the heart asevidenced by cytoplasmic vacuolization and sporadic Evansblue dye uptake in cardiomyocytes [12 14] as well as theabsence of increases in myocardial cleaved 12 kDa and 17 kDacaspase 3 (Figure 1(c)) a hallmark of apoptosis [15] More-over Dox treatment for 4 days led to decreases in systolicleft ventricle posterior wall thickness (LVPW s) LV volumesduring both systole and diastole and FS (Table 1) Theseresults reveal that in the acute model of Dox-induced car-diomyopathy a rapid onset of myocardial oxidative stress isassociated with cardiomyocyte necrosis loss of myocardialmass and cardiac dysfunction

22 Dox-Induced Impairment of Autophagy and Accumu-lation of Ubiquitinated Proteins in the Heart Macroau-tophagy (commonly known as autophagy) is an evolutionar-ily conserved process that mediates the lysosome-dependentturnover of macromolecules and entire organelles [16]

The autophagy begins with formation of the autophagosomea double-membrane structure of unknownorigin that engulfscytoplasmic contents and then fuses it with a lysosome toform autolysosome (known as autophagosome mature)which then leads to proteolysis of engulfed materials Impor-tantly autophagy and the ubiquitin proteasome system (UPS)are the two major routes for the complete degradationclearance of abnormal protein products within cells [17 18]UPS is usually effective in clearing soluble misfolded ordamaged proteins via ubiquitination of the target proteinswhereas autophagy is generally efficient in clearing less sol-uble or insoluble ubiquitinated protein aggregates which areusually toxic to cellsTherefore autophagy plays a crucial rolein the maintenance of cellular homeostasis and the cellularadaptive protection against diverse stress

It has been established that during the autophagicresponse microtubule-associated protein 1 light chain 3(LC3)-I a 16-kDa homologue of Atg8 in yeast is processedand lipid conjugated resulting in LC3-II a 14-kDa activeisoform that migrates from the cytoplasm to isolation mem-branes and autophagosomes [16] The protein abundance ofLC3-II usually reflects the steady level of autophagosomeswhich is dependent on a balance between autophagosomesynthesis and autophagosome clearance via lysosomes [16]p62 is an adaptor protein for autophagosome clearance viaits fusion with lysosome and correlates inversely with auto-phagic activity [19] Therefore the impact of Dox treat-ment on myocardial autophagic activity was evaluated bydetermining expression of LC3-I and -II p62 autophagyrelated gene (Atg)5 andAtg7which are critical for autophago-some formation [16] lysosomal membrane sialoglycoprotein(LAMP)1 and lysosomal protease and cathepsinDWe foundthat Dox led to time-dependent increases in the proteinexpression of LC3-I and II and the accumulation of p62 how-ever it minimally regulated the protein expression of Atg5Atg7 LAMP and cathepsin D (Figure 2) indicating impairedautophagic activity To examine whether the accumulatedLC3-II and p62 proteins were due to impaired autophago-some degradation we measured myocardial autophagic fluxa more reliable measurement of autophagic activity [16] inthe mice treated with vehicle and Dox Treatment of chloro-quine (CQ) an inhibitor of autophagosome fusion withlysosome led to increasedmyocardial protein levels of LC3-IIand p62 (autophagic flux) in control vehicle treated mice(Figure 3(a)) demonstrating a sufficient autophagic activityin the normal heart Although Dox treatment resulted inincreased myocardial protein levels of LC3-II and p62CQ treatment had no impact on the myocardial proteinexpression of LC3-II and p62 in the mice treated with Dox(Figure 3(a)) suggesting an impaired autophagic flux in theDox-intoxicated heart In addition Dox treatment resultedin time-dependent accumulation of ubiquitinated proteins inthe heart (Figure 3(b)) Taken together these results indicatethat Dox treatment resulted in impaired autophagic activityand accumulated ubiquitinated proteins in the heart

23 Dox-Induced Nrf2 Activation in the Heart Previously wehave demonstrated that Nrf2 is a critical endogenous

Oxidative Medicine and Cellular Longevity 3

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Figure 1 Doxorubicin- (Dox-) induced oxidative stress and necrosis in the heart Male 10ndash12wk oldWTmice (C57BL6J) were subjected tosingle intraperitoneal injection of 25mgkg Dox or vehicle (Veh) control and then the hearts (119899 = 4 for each group at indicated time point)were harvested for immunochemical staining andWestern blot analysis (a) Representative images of 8-OHdG and Evans blue labelling Leftpanel brown staining indicates 8-OHdG Right panel green indicates cardiomyocyte membrane Red indicates necrotic myocardium (b)Representative HE staining of left ventricle tissue sections from the mice 4 days after treatment (c) Representative immunoblots of caspase-3and cleaved caspase-3


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(b)Figure 2 Dox-induced autophagic activity in the heart Male 10ndash12wk old WTmice (C57BL6J) were treated as in Figure 1 and then hearts(119899 = 4 for each group at indicated time point) were harvested for Western blot analysis (a) Representatives of immunoblots as indicated (b)Semiquantified analysis of protein expression as indicated (119899 = 4) lowast119875 lt 005 versus Veh in the same experimental group

inhibitor of cardiac maladaptive remodeling and dysfunctionvia suppressing oxidative stress in the heart [6] Of notea recent report has revealed that Nrf2 is able to enhanceautophagic clearance of toxic ubiquitinated protein aggre-gates secondary to ROS formation suggesting a novel medi-ator role of Nrf2 for sufficient activation of autophagy [11]Therefore these results suggest that Nrf2 may be capable ofprotecting against Dox-induced cardiomyopathy via its abil-ities to control oxidative stress and autophagic activity Toexplore the potential role of Nrf2 in Dox-induced cardiomy-opathy we then examined the Nrf2 signaling in the heart

after Dox treatment As shown in Figure 4(a) Dox treatmentenhancedmyocardial Nrf2 mRNA levels in a time dependentmanner Dox treatment also enhanced myocardial Nrf2protein levels and triggeredNrf2 nuclear translocation in car-diomyocytes 4 days after the treatment (Figure 4(a)) In addi-tion Dox treatment led to increases in myocardial mRNAlevels of NAD(P)Hquinone oxidoreductase (NQO1) andheme oxygenase-1 (HO-1) the downstream antioxidant genesof Nrf2 [6] (Figure 4(b)) These results demonstrate that Doxtreatment activates Nrf2 in the heart thus supporting thenotion that Nrf2 activation serves as a feedback mechanism


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Figure 3 Dox-induced impairment of autophagy in the heart (a) Male 10ndash12wk old WT mice (C57BL6J) were treated with Dox as inFigure 1 for 4 days and then subjected to measurement of autophagic flux in the heart (119899 = 4 for each group) Upper panel representativeimmunoblots as indicated Lower panel semiquantified analysis of LC3-II and p62 protein expression lowast119875 lt 005 versus Veh (b) LV lysatesas in Figure 2 were subjected toWestern blot analysis of polyubiquitinated proteins (Ub-proteins) Upper panel representative immunoblotsas indicated Lower panel semiquantified analysis of Ub-proteins level lowast119875 lt 005 versus Veh in the same experimental group

to suppress Dox-induced cardiomyopathy by suppression ofoxidative stress and enhancement of autophagic clearance oftoxic protein aggregates

24 Nrf2 Deficiency Exaggerates Dox-Induced MyocardialOxidative Stress and Impaired Autophagy As Well As CardiacToxicity and Dysfunction To explore the pathophysiologicalsignificance of Dox-induced Nrf2 activation in the heart wedetermined the impact of Nrf2 deficiency on Dox-inducedcardiac toxicity and dysfunction with a focus on oxidativestress and autophagic activity in the heart using littermates ofWT and Nrf2minusminus mice There was no detectable Evans blueuptake or 8-OHdG staining in the heart of WT and Nrf2minusminus

mice treated with vehicle (data not shown) As shown inFigure 5 and Table 1 knockout of Nrf2 exaggerated myocar-dial oxidative stress and necrosis as well as cardiac dysfunc-tion To further determinewhetherNrf2 regulates autophagicclearance of toxic ubiquitinated protein in Dox-intoxicatedheart we examined the levels of LC3-I and -II p62 and ubiq-uitinated proteins in both soluble and insoluble fractions ofthe hearts fromWT andNrf2minusminusmice treated with or withoutDox Loss of Nrf2 enhanced Dox-induced upregulation ofLC3-II and p62 proteins in both soluble and insoluble frac-tions (Figure 5(b)) suggesting that Nrf2 negatively regulatesDox-impaired autophagic flux Importantly Nrf2 deficiencydid not affect Dox-induced accumulation of ubiquitinated


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Figure 4 Dox-induced activation of Nrf2 in the heart Male 10ndash12wk old WT mice (C57BL6J) were treated with Dox as in Figure 1 andthe hearts (119899 = 4 for each group at indicated time point) were harvested for Q-PCR analysis Western blot analysis and immunochemicalstaining (a) Left upper panel Q-PCR analysis of Nrf2 mRNA expression 119899 = 4 lowast119875 lt 005 versus Veh in the same experimental group Rightupper panel Western blot analysis of Nrf2 protein expression in the heart from mice 4 days after treatment Lower panel immunochemicalstaining of Nrf2 protein in a heart of mouse 4 days after treatment lowast119875 lt 005 versus Veh (b) Q-PCR analysis of NQO1 and HO-1 mRNAexpression 119899 = 4 lowast119875 lt 005 versus Veh in the same experimental group


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Figure 5 Effects of Nrf2 knockout on Dox-induced myocardial necrosis oxidative stress and autophagic clearance Male 10ndash12wk oldlittermates of WT and Nrf2minusminus mice were treated with Dox as in Figure 1 for 4 days and then the hearts (119899 = 3 for each group) were harvestedfor (a) Evans blue and 8-OHdG staining and (b) Western blot analysis of LC3 p62 and polyubiquitinated proteins (Ub-proteins) in solubleand insoluble fractions lowast119875 lt 005 versus WT control in (a) lowast119875 lt 005 versus Dox (minus) control in the same experimental group


Table 1 Time-course study of echocardiography in WT and Nrf2minusminus mice after Dox treatment

WT Nrf2minusminus

(119899) Vehicle (3) Dox (5) Vehicle (3) Dox (5)Day 0

LVPW d (mm) 069 plusmn 002 065 plusmn 005 067 plusmn 004 068 plusmn 003

LVPW s (mm) 105 plusmn 002 105 plusmn 005 098 plusmn 004 098 plusmn 003

FS () 2890 plusmn 156 2831 plusmn 397 2950 plusmn 200 2853 plusmn 119

LV Vol d (120583L) 6041 plusmn 002 6086 plusmn 001 6156 plusmn 007 6053 plusmn 002

LV Vol s (120583L) 2368 plusmn 004 2378 plusmn 001 2408 plusmn 007 2381 plusmn 003

HR 46927 plusmn 5242 42362 plusmn 2997 43800 plusmn 3873 42670 plusmn 5089

Day 1LVPW d (mm) 069 plusmn 003 068 plusmn 002 066 plusmn 001 065 plusmn 003

LVPW s (mm) 107 plusmn 003 101 plusmn 002 099 plusmn 001 090 plusmn 003BC

FS () 3112 plusmn 290 2823 plusmn 110 3103 plusmn 200 2098 plusmn 194BC





LVPW s (mm) 107 plusmn 009 097 plusmn 005A

097 plusmn 006 083 plusmn 002BC

FS () 2803 plusmn 105 2300 plusmn 337A


LV Vol d (120583L) 6247 plusmn 004 6082 plusmn 001 6092 plusmn 002 5873 plusmn 007BC

LV Vol s (120583L) 2580 plusmn 003 2401 plusmn 002 2435 plusmn 002 2099 plusmn 002BC



BC





LV Vol d (120583L) 6317 plusmn 004 5888 plusmn 008A


LV Vol s (120583L) 2428 plusmn 001 2021 plusmn 001A



Male 10ndash12 wk old littermates of WT and Nrf2minusminus mice were treated with single intraperitoneal injection of 25mgkg Dox or vehicle (Veh) control for 4 daysand cardiac function was monitored daily as indicated LVPW d left ventricular posterior wall thickness diastolic LVPW s left ventricular posterior wallthickness systolic FS fractional shortening LV Vol d left ventricular volume diastolic LV Vol s left ventricular volume systolic HR heart rate Animalnumber for each group is indicated in the parentheses A119875 lt 005 versus WT Vehicle B119875 lt 005 versus Nrf2minusminus Vehicle C119875 lt 005 versus WT DOX

proteins in soluble fractions while enhancing the accu-mulation of ubiquitinated proteins in insoluble fractions(Figure 5(b)) Because the toxic ubiquitinated protein aggre-gates are usually insoluble and predominantly cleared byautophagosome fusion with lysosome the enhancement ofDox-increased levels of LC3-II p62 and ubiquitinated pro-teins in the insoluble fraction of Nrf2minusminus mice is most likelydue to an impaired autophagic clearance of toxic insolubleubiquitinated protein aggregates in the Nrf2minusminus heart Takentogether loss of Nrf2 exaggerates Dox-impaired autophago-some clearance thereby leading to the accumulated ubiquiti-nated proteins in the heart These results reveal that Nrf2 islikely a critical suppressor of Dox-induced cardiomyopathyvia its abilities to inhibit both oxidative stress and insufficientautophagic activity in the heart

25 Nrf2 Activation Ameliorates Dox-Induced AutophagyImpairment and Cell Death in Cardiomyocytes via Facilitating

Autophagic Clearance of Ubiquitinated Protein Aggregates Tofurther establish a functional link between Nrf2 activationautophagic clearance of ubiquitinated protein aggregates andcardiomyocyte survival in a setting of Dox-induced car-diotoxicity we applied adenoviral Nrf2 and autophagy relatedgene (Atg)5 gain- and loss-of-function approaches in primaryculture of rat neonatal cardiomyocytes We first determinedwhether Dox induces autophagic impairment in primary cul-ture of rat neonatal cardiomyocytes As shown in Figure 6(a)Dox at toxic dose of 1 120583M(Figure 7) increased the steady levelof LC3-II however treatment of bafilomycin A1 (BafA1) aninhibitor of autophagosome fusion with lysosome did notfurther accumulate the Dox-induced upregulation of LC3In addition the Dox (1 120583M) treatment led to accumula-tion of ubiquitinated proteins in insoluble cellular fractions(Figure 6(a)) These results revealed that Dox-induced cyto-toxicity is associated with impaired autophagic activity andaccumulated ubiquitinated protein aggregates in cardiomy-ocytes as observed in the heart afterDox treatment (Figure 3)


(fold

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lowast

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Figure 6 Effects of Nrf2 overexpression and knockdown on Dox-induced impairment of autophagy in cardiomyocytes (a) Dox-inducedimpairment of autophagic flux Rat neonatal cardiomyocytes were treated with Dox (1120583M) in serum-free DMEM for 24 h and bafilomycinA1 (BafA1 5 nM) was added during the last 4 h Left panel representative immunoblots of Dox-induced LC3-II expression with or withoutBafA1 treatment Middle panel quantified densitometric analysis of Dox-induced LC3-II expression 119899 = 4 lowast119875 lt 005 versus vehicle control(minus) Right panel representatives of immunoblots of Dox-induced accumulation of ubiquitinated proteins in soluble (Sol) and insoluble(Insol) fractions Cells were treated with or without Dox (1 120583M) for 24 h (b) Overexpression of Nrf2 on Dox-induced LC3-II expressionand accumulation of ubiquitinated proteins in rat neonatal cardiomyocytes Infected cells were subjected toWestern blot analysis of LC3 andinsoluble (Insol) ubiquitinated protein levels Cells were treated with Dox (1 120583M) for 24 h 119899 = 4 lowast119875 lt 005 versus Ad-Gfp (minus) Insertedblots confirming the infection efficiency of Ad-Nrf2 (c) Overexpression of miNrf2 on Dox-induced LC3-II expression and accumulationof ubiquitinated proteins in rat neonatal cardiomyocytes Infected cells were subjected to Western blot analysis of LC3 and insoluble (Insol)ubiquitinated protein levels Cells were treated with Dox (1120583M) for 24 h 119899 = 4 lowast119875 lt 005 versus Ad-Scramble (minus) Inserted blots confirmingthe infection efficiency of Ad-Nrf2


Atg5

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Ub-

prot

eins

120573-A

ctin

(a)

Cell

dea

th (

)

0

20

40

60

80C

ell d

eath

()

0

20

40

60

80

0 05 1 2 5 10Dox

Cel

l dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox

Ad-GfpAd-Nrf2

Ad-ScrambleAd-miNrf2

Ad-GfpAd-Atg5

Ad-miNrf2 + Ad-GfpAd-miNrf2 + Ad-Atg5

lowast

lowast

lowast

lowastlowast lowast

lowast

lowast lowast lowastlowast

lowast

lowastlowast

P lt 005

P lt 005

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastP lt 005

P lt 005

P lt 005P lt 005P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

(120583m)

(120583m)

0 05 1 2 5 10Dox(120583m)

Cell

dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox(120583m)

(b)

Figure 7 Effects of Nrf2 miNrf2 and Atg5 on Dox-induced cell death in cardiomyocytes (a) Overexpression of Atg5 on Dox-inducedaccumulation of ubiquitinated proteins in Nrf2 depleted rat neonatal cardiomyocytes Cells infected with mNrf2 were further infected withAd-Gfp or Ad-Atg5 and then treated with Dox (1 120583M) for 24 h 119899 = 4 lowast119875 lt 005 versus Ad-miNrf2 + Ad-Gfp (minus) (b) Rat neonatalcardiomyocytes infected with Ad-Gfp Nrf2 Atgr5 Ad-scramble and Ad-miNrf2 as indicated were treated with or without Dox (1120583M) for24 h and cell death was assessed by LDH cell death assay kit 119899 = 4 lowast119875 lt 005 versus Dox (0 120583M) in the same experimental group


We then examined whether Nrf2 is capable of regulating theDox-induced impairment in cultured rat neonatal cardiom-yocytes Overexpression of Nrf2 upregulated whereas Nrf2knockdown downregulated the steady levels of LC3-II(Figures 6(b) and 6(c)) indicating a potential role of Nrf2 ineither increasing autophagosome formation or decreasingautophagosome clearance Dox treatment accumulated thesteady levels of LC3-II in the Ad-Gfp control but not in Ad-Nrf2 infected cells (Figure 6(b)) suggesting a protective roleof Nrf2 inDox-induced impairment of autophagic activationIn contrast Dox accumulated the steady levels of LC3-II inboth Ad-scramble control and Ad-miNrf2 infected cells witha similar magnitude (Figure 6(c)) Importantly Nrf2 overex-pression dramatically inhibited Dox-induced accumulationof ubiquitinated protein aggregates however Nrf2 kno-ckdown enhanced basal and Dox-induced accumulationof ubiquitinated protein aggregates (Figures 6(b) and 6(c))Collectively these results suggest thatNrf2 plays a critical rolein suppressing Dox-induced accumulation of ubiquitinatedprotein aggregates most likely via enhancing autophagosomeformation and autophagic clearance of ubiquitinated proteinaggregates in cardiomyocytes Finally we investigated thefunctional significance of Nrf2-mediated enhancement ofautophagic clearance of ubiquitinated protein aggregates in asetting ofDox-induced cardiotoxicity in cultured rat neonatalcardiomyocytes As shown in Figure 7(a) the accumulationof ubiquitinated protein aggregates in Nrf2 depleted cells atbasal and Dox treatment conditions was significantly attenu-ated by forced activation of autophagy via adenoviral over-expression of Atg5 supporting a critical role of autophagyactivation for Nrf2-mediated clearance of ubiquitinated pro-tein aggregates in cardiomyocytes Moreover Dox-inducedcardiomyocyte death was inhibited or enhanced by Nrf2overexpression or knockdown respectively (Figure 7(b))Overexpression ofAtg5 potently suppressed cell death in bothNrf2 intact and depleted cardiomyocytes however the mag-nitude of cell death suppression is less in Nrf2 depleted cells(Figure 7(b)) indicating that autophagy activation may be adownstream event in Nrf2-mediated clearance of ubiquiti-nated protein aggregates in cardiomyocytes Overall thesefindings support the notion that Nrf2 is an endogenoussuppressor in Dox-induced cardiomyopathy most likely viafacilitating autophagic clearance of myocardial toxic proteinaggregates

3 Discussion

In the present study we have demonstrated for the first timethat Nrf2 is a critical inhibitor of Dox-induced cardiactoxicity and dysfunction Mechanistically it is likely that inaddition to the suppression of oxidative stress Nrf2 is capableof activating sufficientmyocardial autophagy to prevent Dox-induced cardiomyopathy These findings not only provideclarification to the controversial observations regarding therole of autophagy in Dox-induced cardiomyopathy but alsohighlight the potential of targetingNrf2 as a novel therapeuticstrategy to attenuate the Dox-induced cardiotoxicity andcardiomyopathy

31 Dox-Impaired Autophagy in the Heart The previousobservations regarding the role of autophagy in Dox-inducedcardiotoxicity are controversial [20ndash22] In a subacute modelof Dox-induced cardiomyopathy in rats treatment of 3-methyladenine (3-MA) an inhibitor of autophagy activationattenuates Dox-induced cardiotoxicity and cardiac dysfunc-tion [21] However in an acute model of Dox-inducedcardiomyopathy in mice either starvation or treatment withrapamycin an activator of autophagy suppresses theseadverse events [20 22] In addition Dox has been shown tosuppressmyocardial autophagic flux [20] whileDox-inducedincreases in autophagic flux in cultured cardiomyocytes havealso been documented [22 23] The precise reasons for thesediscrepancies are unclear However the effects of chemicalmodulators of autophagy in vivo need to be interpreted withcaution For example 3-MA has been widely used as anautophagy inhibitor based on its inhibitory effect on class IIIphosphoinositide 3-kinase (PI3K) activity which is known tobe essential for the induction of autophagy [24 25] Notablyit has also been demonstrated that 3-MApromotes autophagyin nutrient-rich conditions while it suppresses starvation-induced autophagy [26] Thus the observed cardioprotectiveeffect of 3-MA treatment in a rat model of Dox-inducedcardiotoxicity is most likely due to the activating rather thaninhibiting autophagy in the heart [21] On the other hand theincreases in autophagic activities may result in either protec-tive or detrimental consequences in the heart [27 28] Theprecise mechanisms underlying are unknown A plausibleexplanation is likely that insufficient activation of myocardialautophagy that is increased autophagosome formation with-out sufficient fusion with lysosomes andor the subsequentlysosome-mediated clearance of autophagosomes is func-tionally impaired and detrimental to the heart whereas suf-ficient activation of myocardial autophagy that is increasedautophagosome formation with sufficient fusion with lyso-somes and the subsequent lysosome-mediated clearance ofautophagosomes is functionally integrated and therefore car-dioprotective Taken together these results suggest that Doxmost likely induces insufficient activation of autophagy in theheart andmyocardial sufficient activation of autophagy couldattenuate Dox-induced cardiotoxicity In the present studywe further linked the impaired autophagy to Dox-inducedcardiotoxicity and cardiac dysfunction by demonstratingthat Dox treatment led to the accumulation of LC3-II p62and ubiquitinated proteins impaired myocardial autophagyflux and minor alternations of lysosomal function as well ascardiomyocyte necrosis and cardiac dysfunction In culturedcardiomyocytes we further demonstrated thatDox treatmentresulted in impairment of autophagic flux accumulation ofubiquitinated protein aggregates and cell death MoreoverDox-induced accumulation of ubiquitinated protein aggre-gates and cell death in cardiomyocytes were inhibited byforced activation of autophagy via adenoviral overexpressionof Atg5 These results reveal that Dox treatment instead ofactivating autophagy activity in the heart actually suppressesit thereby contributing to cardiotoxicity and cardiac dys-function The observed Dox-induced upregulation of LC3-IIsteady levels in the heart is likely due to the impairment ofautophagosome clearance rather than the activation of auto-phagosome formation


It is worthy to note that while Dox may transientlyactivate the UPS system in cardiomyocytes [29] the chronicand cumulative effect of Dox actually suppresses UPS func-tion resulting in the accumulation of damaged and toxicubiquitinated proteins in the heart [23 30] Autophagy isactivated in cultured cardiomyocytes by relative long-termtreatments (gt18 h) of Dox when it is capable of causing accu-mulation of polyubiquitinated proteins [22 23 31] It isunclear whether the observed activation of autophagy incardiomyocytes in vitro is secondary to the Dox-inducedsuppression ofUPS function or insteadDox is able to activateautophagy in cultured cardiomyocytes as a result of thespecific experimental conditions However our results andthose of others have demonstrated that Dox treatment couldimpair autophagic activity and accumulation of ubiquitinatedprotein aggregates in cardiomyocytes and in the heart Impor-tantly when UPS is functionally impaired the ubiquitinatedsoluble abnormal proteins accumulate and aggregate intoinsoluble and toxic protein aggregates As a result autophagyis activated by default and becomes the major clearanceroute of the ubiquitinated proteins [17 18] Given the Dox-induced functional insufficiency of UPS in the heart the lossof sufficient autophagic activity may be amajor cause of Dox-induced cardiotoxicity

32 An Inhibitory Role of Nrf2 in Dox-Mediated Impairmentof Myocardial Autophagy Considering the aforementionedcritical role of Nrf2 in suppressing oxidative stress and facili-tating autophagic clearance of toxic ubiquitinated proteins inthe heart we were not surprised to observe the exaggeratedcardiac necrosis and dysfunction as well as the increasedoxidative stress and enhanced impairment of autophagicclearance in Dox-intoxicated hearts due to the loss of Nrf2function Intriguingly it was unexpected that Dox-inducedcardiac oxidative stress is modestly enhanced by knockoutof Nrf2 Thus these results further support our notion thatNrf2 is able to sufficiently activate autophagy via amechanismindependent of its ability to suppress oxidative stress Indeedwe demonstrated that Nrf2 plays a critical mediator role insuppressing Dox-induced autophagy impairment and celldeath in cultured cardiomyocytes most likely via facilitatingautophagic clearance of toxic ubiquitinated protein aggre-gates which is independent of ROS formation

There are sophisticated interactions between oxidativestress and autophagy in the heart [32] Given a mediator roleof ROS in the induction of autophagy the overproduced ROScould activate autophagy to serve as a predominantly prosur-vival mechanism in a setting of oxidative stress however theautophagy machinery might also be self-destructive when itis induced insufficiently thereby exaggerating oxidative stressIndeed ROS-mediated autophagy has been shown to leadto either survival or death in cardiac cells depending onstimuli and cell types [33ndash35] Thus it is likely that ROS mayinduce either sufficient or insufficient activation of myocar-dial autophagy thereby protecting against or leading torespectively cardiac dysfunctionTheprecisemechanisms forthese opposite effects are unknown Nevertheless the obser-vations that Nrf2 deficiency exaggerated both oxidative stress

and impairment of autophagic clearance in Dox-intoxicatedhearts and Nrf2 plays a mediator role in suppressing Dox-induced autophagy impairment and cell death in culturedcardiomyocytes suggest that Nrf2 plays a critical role in theregulation of myocardial activation of sufficient and frus-trated autophagy in the setting of oxidative stress

33 Conclusion Nrf2 is likely an endogenous inhibitor ofDox-induced cardiomyopathy Mechanistically Nrf2 is capa-ble of suppressingDox-induced oxidative stress and impairedautophagy Further investigation of molecular mechanismsbywhichNrf2 regulates the interplay between oxidative stressand insufficient autophagy in Dox-induced cardiotoxicitywill provide valuable insights into the feasibility of targetingNrf2 as a novel strategy for preventing or mitigating Dox-induced cardiotoxicity and cardiac dysfunction

4 Materials and Methods

41 Animals Male C57BL6Jmice were purchased from Sha-nghai Slac Laboratory Animal Co Ltd Breeding pairs ofheterozygous Nrf2 knockout (Nrf2+minusC57BL6J) mice werepurchased from Riken BioRosource Center Japan andhoused under standard conditions in the InstitutionrsquosAAALAC approved animal facility Littermates of wild type(WT Nrf2++) and homozygous Nrf2 knockout (Nrf2minusminus)mice were generated using the Nrf2+minus breeding pairs aspreviously described [36] Mice were euthanized byintraperitoneal (IP) injection of pentobarbital sodium(100mgkg) All of the animal protocols were conducted inaccordance with the NIH Guide for Care and Use of Lab-oratory Animals and were approved by the InstitutionalAnimal Care and Use Committee at Shandong UniversityChina and the University of South Carolina USA

42 Doxorubicin-Induced Cardiomyopathy Male 10ndash12wkoldmice were subjected to single intraperitoneal injections ofeither 25mgkg doxorubicin HCl (Dox) (Aladdin ReagentInc China) dissolved in 09 NaCl or 09 NaCl vehicle aspreviously reported [12]

43 Echocardiography Echocardiography was performed onanesthetized (15 isoflurane) mice using the Vevo 770High-Resolution Imaging System (VisualSonics Inc) with a35-MHz high-frequency linear transducer as previouslydescribed [6] Left ventricle (LV) internal dimension (LVIDin mm) intraventricular septal thickness (IVS in mm) andLV posterior wall thickness (LVPW in mm) were measuredLV percent fractional shortening FS () was calculated viaVisualSonics Measurement Software

44 Measurement of Autophagic Flux In Vivo Autophagicflux in vivo was measured as previously described with aminor modification [37] Briefly mice were intraperitoneallyinjected with rapamycin (2mgkg) or rapamycin (2mgkg)plus chloroquine (10mgkg) 4 hrs prior to harvesting heartsLV tissue lysates were subjected to Western blot analysis


of LC3-I and -II The chloroquine-induced accumulation ofLC3-II was considered to be autophagic flux

45Histology and Immunochemistry Heartswere cannulatedvia the LV apex cleared by perfusion with 09 NaCl at90mmHg arrested in diastole with 60mMKCl fixed by per-fusion with 10 formalin and embedded in paraffin Paraffinsections were prepared (5 120583m Leica rotary microtome) andstored at room temperature until staining Hematoxylin-Eosin (HE) staining and immunohistochemistry were per-formed as previously described [6] Nrf2 immunohisto-chemistry was performed using a rabbit anti-mouse Nrf2polyclonal antibody (C-20 Santa Cruz Biotechnology Inc)Staining of 8-hydroxydeoxyguanosine (8-OHdG) a markerof DNA oxidization in the heart was performed usinga mouse anti-8-OHdG antibody (sc-660369 Santa CruzBiotechnology Inc)

46 Evans Blue Labelling Evans blue dye becomes insanelyred fluorescent when conjugated to albumin in the circu-lation The dyealbumin complexes are excluded from cellswith intact plasma membranes while accumulating in dam-aged myofibers when the muscle cell membrane is brokenthus providing a dye-exclusion viability test The red auto-fluorescence accumulated in myocardium has been used as ahistopathological sign of cardiomyocyte necrosis [14] Brieflymice were subjected to a single intraperitoneal injection ofEvans blue (100mgkg Solarbio science amp technology CoLtd Beijing China) 18 h prior to harvesting tissues Har-vested hearts were fixed in 4 paraformaldehyde and thenembedded in paraffin Paraffin sections were prepared (5 120583mLeica rotary microtome) and stored at room temperatureuntil staining Myocardial cellular membranes were stainedwith Wheat Germ Agglutinin Alexa Fluor 488 Conjugate(Invitrogen Corp) Sections were observed under fluores-cencemicroscope (LeicaMicrosystems Ltd) at 200xmagnifi-cation Evans blue dye-positive area (red) indicates cardiomy-ocyte necrosis

47 Virus Preparation Adenovirus of miNrf2 (Ad-miNrf2)was generated using BLOCK-iT Pol II miR RNAi ExpressionVector Kits (Gateway-adapted expression vector for theexpression ofmicroRNA (miRNA) inmammalian cells undercontrol of Pol II promoters) (Invitrogen K4936-00) as wellas ViraPower Adenoviral Promoterless Gateway ExpressionKit (Invitrogen K4940-00) Briefly double-stranded rat Nrf2miRNA (miNrf2) (51015840-TACACAGGGACAGATCACAGC-31015840)was cloned into pcDNA 62-GWEmGFP-miR expressionvector using T4DNA ligase After a BP recombination (Invit-rogen 11789020) reactionwith pcDNA62-GWEmGFP-miRand pDONR 221 (Invitrogen 12536017) vector to get a miRRNAi entry vector LR recombination (Invitrogen 12538120)reaction with entry vector and pAdCMVV5-DEST vectorwas performed to generate a pAd-miNrf2 vector Transfectionof the expression clone into 293A cells was performed toproduce Ad-miNrf2 stock Adenovirus of miScramble wasgenerated using pcDNA 62-GWEmGFP-miR-neg controlplasmidwhich is provided in the BLOCK-iT Pol IImiR RNAi

Expression Vector Kits Ad-miScramble stock was generatedas described above

Adenovirus of Nrf2 (Ad-Nrf2) and Atg5 (Ad-Atg5) wasgenerated using pENTR 1A Dual Selection Vector (Invitro-gen A10462) and ViraPower Adenoviral Promoterless Gate-way Expression Kit (Invitrogen K4940-00) Briefly humanAtg5 cDNA (pCMV-myc-Atg5 addgen 24922) and humanNrf2 cDNA (NCBI Reference Sequence NM 0061644) werecloned into a Topo blunt zero vector respectively to generatea Tope-Atg5 and a Topo-Nrf2 vectorThen the Atg5 and Nrf2coding sequences were reconstructed into the pENTR1A-GFP vector to form pENTR1A-GFP-Atg5 and pENTR1A-GFP-Nrf2 vectors LR recombination (Invitrogen 12538120)reaction with the entry vector of pENTR1A-GFP-Atg5 andpENTR1A-GFP-Nrf2 with pAdCMVV5-DEST vector wasperformed to generate pAd-Atg5 and pAd-Nrf2 vectorsAdenovirus of Gfp (Ad-Gfp) was generated using thepENTER1A-Gfp vector as described above Transfection ofthese expression vectors into 293A cells to produce theadenoviral stock

48 Cell Culture and Adenovirus Infection Rat neonatalcardiac myocytes were isolated and cultured as previouslydescribed [6] Briefly the hearts from 3-day old Wistar ratswere finely minced and digested with type II collagenase (120unitsmL Worthington Biochemical Corp Lakewood NJ)Dispersed cells were placed in culture flask for 80 minutes at37∘C in aCO

2

incubatorDuring this time only the fibroblastsbecame attached to the culture flask The purified cardiomy-ocytes the viability of which was gt85 determined bytrypan blue exclusionwere seeded onto gelatin-coated plasticculture dishes at a density of 5 times 104 cellscm2 in low glucoseDMEM supplemented with 8 horse serum 5 new-born calf serum penicillin (100UmL) and streptomycin(100mgmL) Nrf2 gain or loss-of-function approaches wereachieved by infecting the cells with adenovirus of GFP Nrf2scramble microRNA or rat Nrf2 microRNA in serum freeDMEM for 8 hours and then changed to DMEM supple-mented with 16 horse serum and 1 new-born calf serumfor totally 48 hours Ad-Atg5 infection was performed asdescribed above

Adenoviral overexpression of Nrf2 (20 MOI) or Nrf2microRNA (20 MOI) resulted in dramatic Nrf2 and down-streamNQO1 protein expression or up to 80 knockdown ofNrf2 and downstreamNQO1 protein expression in rat neona-tal cardiomyocytes without any apparent cytotoxic effects(data not shown) Ad-Atg5 (20MOI) infection sufficiently ledto increased levels of ATG5 in rat neonatal cardiomyocytesAccordingly we used adenoviral overexpression ofNrf2 Nrf2microRNA and Atg5 at a dose of 20MOI in rat neonatalcardiomyocytes

Cell deathwasmeasured using a cytotoxicity detection kit(Roche 04 744 934 001) Cell viability was calculated asfollows cell viability = (LDH activity of cell lysate in Exp)(LDH activity of cell lysate in Con) times 100 Exp indicatesexperimental groups and Con is for control

49 Western Blot Analysis Western blot analysis was per-formed as previously described [6] Regular Western blot


Table 2 Primers for real-time PCR

Genes Gene access Forward Reverse Productlength (bp)

Nrf2NQO-1HO-1GAPDH

NM 0109023NM 0087065NM 0104422XM 001479322

ATGATGGACTTGGAGTTGCCCGGTATTACGATCCTCCCTCAACAAGGAGATAGAGCGCAACAAGCAGAATGTTCCAGTATGACTCCACTCACG

TCCTGTTCCTTCTGGAGTTGAGCCTCTACAGCAGCCTCCTTCATCCAGTGAGGCCCATACCAGAAG

GAAGACACCAGTAGACTCCACGACA

200120116171

analysis was performed using LV lysates that were preparedusing RIPA buffer (50mMTris (pH 80) 150mMNaCl 1NP-40 05 sodium deoxycholate 01 SDS and a proteininhibitor mixture (Sigma-Aldrich)) In addition Westernblot analysis of protein aggregation was performed using LVdetergent-soluble and -insoluble lysates Briefly detergent-soluble fractions of LV tissues were obtained using a 2Triton X-100 lysis buffer (50mM Tris (pH 80) 150mMNaCl 1mM EDTA 10 glycerol 2 Triton X-100 anda protein inhibitor mixture (Sigma-Aldrich)) The insolu-ble fractions were further solubilized using the 2 TritonX-100 lysis buffer supplemented with 1 SDS Primaryantibodies included anti-LC3 (L7543 Sigma-Aldrich) anti-P62 (ab91526 abcam) anti-NQO1 (sc-376023 Santa CruzBiotechnology Inc) Anti-ATG5 (A2859 Sigma-Aldrich)anti-ATG7 (A2856 Sigma-Aldrich) anti-LAMP1 (ab24170Abcam) anticathepsin D (2284 CST) and anti-Ub (sc-8017Santa Cruz Biotechnology Inc) Ubiquitinated proteins withmolecular weights from the top to 35 kDa on the membranesof immunoblots were quantified

410 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Quantitative Real Time (Q-PCR) Total RNA fromthe LV was extracted using RNeasy Fibrous Tissue Minikit (Qiagen Inc Valencia CA) and reverse transcriptionreactions (SuperScript III First-Strand Synthesis System forRT-PCR Invitrogen Corp Carlsbad CA) were performedwith 05120583g ofDNase I (Qiagen)-treated RNA PCR and quan-titative real-time PCR (Q-PCR) were carried out using theCFX96 Touch Real-Time PCR Detection System (Bio-RadLaboratories Inc) Expression levels of target genes werenormalized by concurrent measurement of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels as pre-viously described [6] Primers that were used for Q-PCR aresummarized in Table 2

411 Statistical Analysis Data are shown asmeanplusmn SD Statis-tical differences were analyzed by one-way ANOVA followedby Bonferroni test for multiple comparisons using GraphPadPrism software Differences were considered significant at119875 lt 005

Conflict of Interests

Theauthors declare that they have no conflict of interests withbiotech companies financial foundations or any other thirdparties

Authorsrsquo Contribution

Siying Li and Wenjuan Wang contribute equally to the work

Acknowledgments

This work was supported by National Institute of Health KO1Grant 5KO1DK080884-05 the ShandongUniversity NationalQianren Scholar Fund Taishan Scholar Fund and theNational Natural Science Foundation of China (no81370267)

References

[1] P K Singal and N Iliskovic ldquoDoxorubicin-induced cardiomy-opathysrdquoThe New England Journal of Medicine vol 339 no 13pp 900ndash905 1998

[2] M Volkova and R Russell 3rd ldquoAnthracycline cardiotoxicityprevalence pathogenesis and treatmentrdquo Current CardiologyReviews vol 7 no 4 pp 214ndash220

[3] M Sterba O Popelova A Vavrova et al ldquoOxidative stressredox signaling andmetal chelation in anthracycline cardiotox-icity and pharmacological cardioprotectionrdquo Antioxidants ampRedox Signaling vol 18 no 8 pp 899ndash929

[4] Y Octavia C G Tocchetti K L Gabrielson S Janssens H JCrijns and A L Moens ldquoDoxorubicin-induced cardiomyopa-thy from molecular mechanisms to therapeutic strategiesrdquoJournal of Molecular and Cellular Cardiology vol 52 no 6 pp1213ndash1225 2012

[5] J Li T Ichikawa J S Janicki and T Cui ldquoTargeting the Nrf2pathway against cardiovascular diseaserdquo Expert Opinion onTherapeutic Targets vol 13 no 7 pp 785ndash794 2009

[6] J Li T Ichikawa L Villacorta et al ldquoNrf2 protects againstmaladaptive cardiac responses to hemodynamic stressrdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 29 no 11 pp1843ndash1850 2009

[7] J Li C Zhang Y Xing et al ldquoUp-regulation of p27kip1 con-tributes to Nrf2-mediated protection against angiotensin II-induced cardiac hypertrophyrdquo Cardiovascular Research vol 90no 2 pp 315ndash324 2011

[8] J W Calvert M Elston C K Nicholson et al ldquoGenetic andpharmacologic hydrogen sulfide therapy attenuates ischemia-induced heart failure in micerdquo Circulation vol 122 no 1 pp11ndash19 2010

[9] T E Sussan T Rangasamy D J Blake et al ldquoTargeting Nrf2with the triterpenoid CDDO-imidazolide attenuates cigarettesmoke-induced emphysema and cardiac dysfunction in micerdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 1 pp 250ndash255 2009


[10] Y Xing T Niu W Wang et al ldquoTriterpenoid dihydro-CDDO-trifluoroethyl amide protects against maladaptive car-diac remodeling and dysfunction inmice a critical role ofNrf2rdquoPLoS ONE vol 7 no 9 Article ID e44899

[11] K-I Fujita D Maeda Q Xiao and S M Srinivasula ldquoNrf2-mediated induction of p62 controls Toll-like receptor-4-drivenaggresome-like induced structure formation and autophagicdegradationrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 108 no 4 pp 1427ndash1432 2011

[12] K Kizaki R Ito M Okada et al ldquoEnhanced gene expression ofmyocardial matrix metalloproteinases 2 and 9 after acute treat-ment with doxorubicin in micerdquo Pharmacological Research vol53 no 4 pp 341ndash346 2006

[13] Y Zhang Y-M Kang C Tian et al ldquoOverexpression ofNrdp1 in the heart exacerbates doxorubicin-induced cardiacdysfunction inmicerdquo PLoS ONE vol 6 no 6 Article ID e211042011

[14] D L Miller P Li C Dou W F Armstrong and D GordonldquoEvans blue staining of cardiomyocytes induced by myocardialcontrast echocardiography in rats evidence for necrosis insteadof apoptosisrdquoUltrasound inMedicine and Biology vol 33 no 12pp 1988ndash1996 2007

[15] D W Nicholson A Ali N AThornberry et al ldquoIdentificationand inhibition of the ICECED-3 protease necessary for mam-malian apoptosisrdquo Nature vol 376 no 6535 pp 37ndash43 1995

[16] N Mizushima T Yoshimori and B Levine ldquoMethods in mam-malian autophagy researchrdquo Cell vol 140 no 3 pp 313ndash3262010

[17] D C Rubinsztein ldquoThe roles of intracellular protein-degrad-ation pathways in neurodegenerationrdquoNature vol 443 no 7113pp 780ndash786 2006

[18] E Wong and A M Cuervo ldquoIntegration of clearance mech-anisms the proteasome and autophagyrdquo Cold Spring HarborPerspectives in Biology vol 2 no 12 Article ID a006734 2010

[19] D J Klionsky H Abeliovich P Agostinis et al ldquoGuidelines forthe use and interpretation of assays for monitoring autophagyin higher eukaryotesrdquo Autophagy vol 4 no 2 pp 151ndash175

[20] T Kawaguchi G Takemura H Kanamori et al ldquoPrior star-vation mitigates acute doxorubicin cardiotoxicity throughrestoration of autophagy in affected cardiomyocytesrdquo Cardio-vascular Research vol 96 no 3 pp 456ndash465

[21] L Lu W Wu J Yan X Li H Yu and X Yu ldquoAdriamycin-induced autophagic cardiomyocyte death plays a pathogenicrole in a rat model of heart failurerdquo International Journal ofCardiology vol 134 no 1 pp 82ndash90 2009

[22] B J Sishi B Loos J van Rooyen and A M EngelbrechtldquoAutophagy upregulation promotes survival and attenuatesdoxorubicin-induced cardiotoxicityrdquo Biochemical Pharmacol-ogy vol 85 no 1 pp 124ndash134

[23] PDimitrakisM I Romay-Ogando F Timolati TM Suter andC Zuppinger ldquoEffects of doxorubicin cancer therapy on auto-phagy and the ubiquitin-proteasome system in long-term cul-tured adult rat cardiomyocytesrdquo Cell and Tissue Research vol350 no 2 pp 361ndash372

[24] P O Seglen and P B Gordon ldquo3-Methyladenine specificinhibitor of autophagiclysosomal protein degradation in iso-lated rat hepatocytesrdquo Proceedings of the National Academy ofSciences of the United States of America vol 79 no 6 I pp 1889ndash1892 1982

[25] A Petiot E Ogier-Denis E F C Blommaart A J Meijer andP Codogno ldquoDistinct classes of phosphatidylinositol 31015840-kinases

are involved in signaling pathways that controlmacroautophagyin HT-29 cellsrdquo Journal of Biological Chemistry vol 275 no 2pp 992ndash998 2000

[26] Y-TWuH-L TanG Shui et al ldquoDual role of 3-methyladeninein modulation of autophagy via different temporal patterns ofinhibition on class I and III phosphoinositide 3-kinaserdquo Journalof Biological Chemistry vol 285 no 14 pp 10850ndash10861 2010

[27] R A Gottlieb and R M Mentzer ldquoAutophagy during cardiacstress Joys and frustrations of autophagyrdquo Annual Review ofPhysiology vol 72 pp 45ndash59 2009

[28] ZVWang BA Rothermel and J AHill ldquoAutophagy in hyper-tensive heart diseaserdquo Journal of Biological Chemistry vol 285no 12 pp 8509ndash8514 2010

[29] J Liu H ZhengM Tang Y-C Ryu and XWang ldquoA therapeu-tic dose of doxorubicin activates ubiquitin-proteasome system-mediated proteolysis by acting on both the ubiquitinationapparatus and proteasomerdquo American Journal of PhysiologymdashHeart and Circulatory Physiology vol 295 no 6 pp H2541ndashH2550 2008

[30] B J Sishi B Loos J van Rooyen and A M Engelbrecht ldquoDox-orubicin induces protein ubiquitination and inhibits protea-some activity during cardiotoxicityrdquoToxicology vol 309 pp 23ndash29

[31] S Kobayashi P Volden D Timm K Mao X Xu and Q LiangldquoTranscription factor GATA4 inhibits doxorubicin-inducedautophagy and cardiomyocyte deathrdquo Journal of BiologicalChemistry vol 285 no 1 pp 793ndash804 2010

[32] N Gurusamy and D K Das ldquoAutophagy redox signaling andventricular remodelingrdquo Antioxidants and Redox Signaling vol11 no 8 pp 1975ndash1988 2009

[33] C W Younce and P E Kolattukudy ldquoMCP-1 causes cardiomy-oblast death via autophagy resulting from ER stress causedby oxidative stress generated by inducing a novel zinc-fingerprotein MCPIPrdquo Biochemical Journal vol 426 no 1 pp 43ndash532010

[34] HYuanCN Perry CHuang et al ldquoLPS-induced autophagy ismediated by oxidative signaling in cardiomyocytes and is asso-ciated with cytoprotectionrdquo American Journal of PhysiologymdashHeart and Circulatory Physiology vol 296 no 2 pp H470ndashH479 2009

[35] P Marambio B Toro C Sanhueza et al ldquoGlucose deprivationcauses oxidative stress and stimulates aggresome formation andautophagy in cultured cardiacmyocytesrdquoBiochimica et Biophys-ica ActamdashMolecular Basis of Disease vol 1802 no 6 pp 509ndash518 2010

[36] K Itoh T Chiba S Takahashi et al ldquoAn Nrf2small Maf het-erodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997

[37] E Iwai-Kanai H Yuan C Huang et al ldquoA method to measurecardiac autophagic flux in vivordquo Autophagy vol 4 no 3 pp322ndash329 2008

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


hydrogen sulfide-mediated suppression of oxidative stress-induced cardiac dysfunction [8] we and others have demon-strated that Nrf2 might be a drug target for the treatmentof cardiomyocyte injury and cardiac dysfunction [9 10] Ofinterest a recent report has revealed that Nrf2 is able toenhance autophagic clearance of toxic ubiquitinated proteinaggregates secondary to ROS formation suggesting a novelmediator role of Nrf2 for sufficient activation of autophagy[11] Considering the causative role of oxidative stress and theprotective effect of sufficient autophagy activation in Dox-induced cardiotoxicity as well as the Nrf2-mediated antiox-idant defense and sufficient activation of autophagy it isconceivable that Nrf2 is a negative regulator of Dox-inducedcardiomyopathy

Therefore in the present study we explored the role ofNrf2 in the regulation ofDox-induced cardiomyopathywith afocus on oxidative stress and autophagic activity in the heartWe demonstrate that loss of Nrf2 function exaggerates Dox-induced oxidative stress insufficient autophagic activitiesand cardiomyocyte necrosis as well as cardiac dysfunctionThese results indicate that Nrf2 acts as a critical negative reg-ulator of Dox-induced cardiomyopathy thereby identifying apotential novel target for the treatment of Dox-inducedcardiomyopathy

2 Results

21 Dox-Induced Cardiac Oxidative Stress Toxicity and Dys-function Considering oxidative stress to be the primarycause of Dox-induced cardiomyopathy [3 4] we first deter-mined myocardial oxidative stress and cell death in the acutemodel of Dox-induced cardiotoxicity which has been charac-terized as a rapid cardiomyocyte necrosis and reduced cardiacoutput within 4 days after a single treatment of Dox [12 13]Of note a single intraperitoneal injection of Dox (20mgkg)results in reduction of left ventricle fraction shorting (FS)in C57BL6J mice [13] Following a single intraperitonealinjection of Dox (25mgkg) there was a time dependentonset of oxidative stress in the heart which appeared at day 1and was sustained by day 4 after injection (Figure 1(a))Therewas also substantial cardiomyocyte necrosis in the heart asevidenced by cytoplasmic vacuolization and sporadic Evansblue dye uptake in cardiomyocytes [12 14] as well as theabsence of increases in myocardial cleaved 12 kDa and 17 kDacaspase 3 (Figure 1(c)) a hallmark of apoptosis [15] More-over Dox treatment for 4 days led to decreases in systolicleft ventricle posterior wall thickness (LVPW s) LV volumesduring both systole and diastole and FS (Table 1) Theseresults reveal that in the acute model of Dox-induced car-diomyopathy a rapid onset of myocardial oxidative stress isassociated with cardiomyocyte necrosis loss of myocardialmass and cardiac dysfunction

22 Dox-Induced Impairment of Autophagy and Accumu-lation of Ubiquitinated Proteins in the Heart Macroau-tophagy (commonly known as autophagy) is an evolutionar-ily conserved process that mediates the lysosome-dependentturnover of macromolecules and entire organelles [16]

The autophagy begins with formation of the autophagosomea double-membrane structure of unknownorigin that engulfscytoplasmic contents and then fuses it with a lysosome toform autolysosome (known as autophagosome mature)which then leads to proteolysis of engulfed materials Impor-tantly autophagy and the ubiquitin proteasome system (UPS)are the two major routes for the complete degradationclearance of abnormal protein products within cells [17 18]UPS is usually effective in clearing soluble misfolded ordamaged proteins via ubiquitination of the target proteinswhereas autophagy is generally efficient in clearing less sol-uble or insoluble ubiquitinated protein aggregates which areusually toxic to cellsTherefore autophagy plays a crucial rolein the maintenance of cellular homeostasis and the cellularadaptive protection against diverse stress

It has been established that during the autophagicresponse microtubule-associated protein 1 light chain 3(LC3)-I a 16-kDa homologue of Atg8 in yeast is processedand lipid conjugated resulting in LC3-II a 14-kDa activeisoform that migrates from the cytoplasm to isolation mem-branes and autophagosomes [16] The protein abundance ofLC3-II usually reflects the steady level of autophagosomeswhich is dependent on a balance between autophagosomesynthesis and autophagosome clearance via lysosomes [16]p62 is an adaptor protein for autophagosome clearance viaits fusion with lysosome and correlates inversely with auto-phagic activity [19] Therefore the impact of Dox treat-ment on myocardial autophagic activity was evaluated bydetermining expression of LC3-I and -II p62 autophagyrelated gene (Atg)5 andAtg7which are critical for autophago-some formation [16] lysosomal membrane sialoglycoprotein(LAMP)1 and lysosomal protease and cathepsinDWe foundthat Dox led to time-dependent increases in the proteinexpression of LC3-I and II and the accumulation of p62 how-ever it minimally regulated the protein expression of Atg5Atg7 LAMP and cathepsin D (Figure 2) indicating impairedautophagic activity To examine whether the accumulatedLC3-II and p62 proteins were due to impaired autophago-some degradation we measured myocardial autophagic fluxa more reliable measurement of autophagic activity [16] inthe mice treated with vehicle and Dox Treatment of chloro-quine (CQ) an inhibitor of autophagosome fusion withlysosome led to increasedmyocardial protein levels of LC3-IIand p62 (autophagic flux) in control vehicle treated mice(Figure 3(a)) demonstrating a sufficient autophagic activityin the normal heart Although Dox treatment resulted inincreased myocardial protein levels of LC3-II and p62CQ treatment had no impact on the myocardial proteinexpression of LC3-II and p62 in the mice treated with Dox(Figure 3(a)) suggesting an impaired autophagic flux in theDox-intoxicated heart In addition Dox treatment resultedin time-dependent accumulation of ubiquitinated proteins inthe heart (Figure 3(b)) Taken together these results indicatethat Dox treatment resulted in impaired autophagic activityand accumulated ubiquitinated proteins in the heart

23 Dox-Induced Nrf2 Activation in the Heart Previously wehave demonstrated that Nrf2 is a critical endogenous


1

2

4

(day

)Veh Dox Veh Dox

8-OHdG Evans blue

25120583m 25120583m

25120583m

25120583m 25120583m

25120583m

50120583m 50120583m

50120583m

50120583m 50120583m

50120583m

(a)

Veh Dox

20120583m 20120583m

(b)

15

25

35

Cleaved

Caspase-3

Caspase-3

GAPDH

Dox1 2 4 (day)

1 2 4

(kD

a)

(kD

a)

17

26


Dox(day)


120573-Actin

(c)



LC3-ILC3-II

p62

ATG5

ATG7

GAPDH

LAMP1

Cathepsin D

Dox0 1 2 4 (day)

1614

47

56

75

120

28

37

(kD

a)


(a)

LC3-II

0

2468

10

LC3-

IIG

APD

H(fo

ld ch

ange

)(fo

ld ch

ange

)

0

2468

10

(fold

chan

ge)



p62

p62

GA

PDH

ATG5

0

05

1

15

(fold

chan

ge)

0

05

1

15

(fold

chan

ge)

0

05

1

15

ATG

5G

APD

H

ATG7

ATG

7G

APD

H

0 1 2 4(day)

0 1 2 4(day)

LAMP1

LAM

P1G

APD

H

Cathepsin D

0

05

1

15

2

Cath

epsin

DG

APD

H(fo

ld ch

ange

)

lowast

lowast

lowastlowast

lowast





LC3-II

GAPDH

CQ Veh Dox

LC3-II

0

5

10

15

20

LC3-

IIG

APD

H(fo

ld ch

ange

)


ns

Veh Dox0

5

10

15

20

p62

GA

PDH

(fold

chan

ge)

ns

p62

p62

16

14

37

(kD

a)70

lowastlowast

lowastlowast

P lt 005

P lt 005

minus + minus +

(mdash)

(a)

0

05

1

15

2

Ub-

prot

eins

GA

PDH

(fold

chan

ge)

1 2 4(day)

Dox1 2 4 (day)

Ub-

prot

eins

GAPDH

VehDox

170

70

5540

130

100

35

37(k

Da)


lowast

lowast

(b)






Nrf2

0

1

2

3N

rf2G

APD

H


Relat

ive m

RNA

expr

essio

n

0 1 2 4(day)

0

02

04

06

08

Veh Dox

Nrf2

Veh Dox

0

2000

4000

6000

Veh Dox

Nrf2

expr

essio

n

Nuc

lear

Nrf2

()

Veh Dox0

80

60

40

20

43

100

(kD

a)

20120583m 20120583m

lowast

lowastlowast

lowast

P lt 005

P lt 001

n = 4 n = 4

n = 4 n = 4

n = 4 n = 4

(IO

D120583

m2)

Nrf2

120573-a

ctin

120573-Actin

(a)

0 1 2 4

NQO1

0

2

4

6

8

10

NQ

O1

GA

PDH

HO-1

0

10

20

30

40

HO

-1G

APD

H

(day)



Relat

ive m

RNA

expr

essio

n

0 1 2 4(day)

lowast

lowast

lowast

lowast

lowast

lowast

(b)



WT D

oxor

ubic

inD

oxor

ubic

in

0

20

40

Nuc

lear

8-O

HdG

()

WT

Nec

rotic

area

0

1

2

3

100120583m 100120583m

20120583m 20120583m

WT

lowast

lowast

lowast

n = 3

n = 3 n = 3

n = 3

(120583m

2pe

r sec

tiontimes1000

)

Nrf2minusminus

Nrf2minusminus

Nrf2minusminus

(a)

Dox

Sol

LC3-II

GAPDH

p62

Ub-

prot

eins

Insol

LC3-II LC3-II

05

101520253035

LC3-

IIG

APD

H(fo

ld ch

ange

)

01020304050

Sol Insol

p62

02468

1012

p62

GA

PDH

(fold

chan

ge)

02468

1012

0

05

1

15

2

25

Ub-

prot

eins

GA

PDH

(fold

chan

ge)

Ub-proteins

0

05

1

15

2

p62

Ub-proteins

Dox

16

(kD

a)

14

170

70

5540

130100

35

37

70

lowast

lowastlowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

P lt 005

P lt 005 P lt 001

P lt 001P lt 001





(b)




WT Nrf2minusminus























BC















(fold

chan

ge)

(kD

a)

(kD

a)

43

LC3-ILC3-II

16

14

DoxBafA1

0

1

2

3

4

5ns

DoxBafA1

170

70

5540

130100

35

43

Sol Insol

Dox

Ub-

prot

eins

minus minus ++

minus + minus +

minus minus ++


lowastlowast

lowast

LC3

-II120573

-act

in

120573-Actin

120573-Actin

(a)

(kD

a)

(kD

a)

(kD

a)

LC3-ILC3-II

Nrf2

Ad-N

rf2Ad

-Gfp

Ad-Nrf2Ad-Gfp

NQO143

100

31

Dox

43

1614

(fold

chan

ge)

0

05

1

15

2

25 ns

Ub-

prot

eins

170

70

5540

130100

35

43

Ub-

prot

eins

120573-a

ctin

(fold

chan

ge)

0

8

2

4

6

Dox

Insol



minus + minus +

lowastlowastlowast

P lt 001

LC3

-II120573

-act

in

120573-Actin

120573-Actin

120573-Actin

lowast

(b)

LC3-ILC3-II

120573-Actin

120573-Actin

Ad-m

iNrf2

Ad-s

cram

ble


NQO1

(kD

a)

43

1614

Dox

(fold

chan

ge)

0

1

2

Ub-

prot

eins

120573-a

ctin

Ub-

prot

eins

170

70

5540

130100

35

43 0

8

2

4

6

(fold

chan

ge)

Nrf2

Insol

(kD

a) (kD

a)

43

100

31



lowast

lowastlowast

lowast

lowast

P lt 001

P lt 005

LC3

-II120573

-act

in

120573-Actin

(c)



Atg5

43

56

Ub-

prot

eins

170

70

5540

130100

35

43

0

1

2

(fold

chan

ge)

Insol

Dox

(kD

a)

(kD

a)

minus + minus +

120573-Actin

120573-Actin

lowast

lowastP lt 005

Ad-Gfp Ad-Atg5


Dox minus + minus +


Ub-

prot

eins

120573-A

ctin

(a)

Cell

dea

th (

)

0

20

40

60

80C

ell d

eath

()

0

20

40

60

80

0 05 1 2 5 10Dox

Cel

l dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox

Ad-GfpAd-Nrf2


Ad-GfpAd-Atg5


lowast

lowast

lowast

lowastlowast lowast

lowast


lowast

lowastlowast

P lt 005

P lt 005

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastP lt 005

P lt 005

P lt 005P lt 005P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

(120583m)

(120583m)

0 05 1 2 5 10Dox(120583m)

Cell

dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox(120583m)

(b)




3 Discussion






















2








Nrf2NQO-1HO-1GAPDH

NM 0109023NM 0087065NM 0104422XM 001479322




200120116171








Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1

2

4

(day

)Veh Dox Veh Dox

8-OHdG Evans blue

25120583m 25120583m

25120583m

25120583m 25120583m

25120583m

50120583m 50120583m

50120583m

50120583m 50120583m

50120583m

(a)

Veh Dox

20120583m 20120583m

(b)

15

25

35

Cleaved

Caspase-3

Caspase-3

GAPDH

Dox1 2 4 (day)

1 2 4

(kD

a)

(kD

a)

17

26


Dox(day)


120573-Actin

(c)



LC3-ILC3-II

p62

ATG5

ATG7

GAPDH

LAMP1

Cathepsin D

Dox0 1 2 4 (day)

1614

47

56

75

120

28

37

(kD

a)


(a)

LC3-II

0

2468

10

LC3-

IIG

APD

H(fo

ld ch

ange

)(fo

ld ch

ange

)

0

2468

10

(fold

chan

ge)



p62

p62

GA

PDH

ATG5

0

05

1

15

(fold

chan

ge)

0

05

1

15

(fold

chan

ge)

0

05

1

15

ATG

5G

APD

H

ATG7

ATG

7G

APD

H

0 1 2 4(day)

0 1 2 4(day)

LAMP1

LAM

P1G

APD

H

Cathepsin D

0

05

1

15

2

Cath

epsin

DG

APD

H(fo

ld ch

ange

)

lowast

lowast

lowastlowast

lowast





LC3-II

GAPDH

CQ Veh Dox

LC3-II

0

5

10

15

20

LC3-

IIG

APD

H(fo

ld ch

ange

)


ns

Veh Dox0

5

10

15

20

p62

GA

PDH

(fold

chan

ge)

ns

p62

p62

16

14

37

(kD

a)70

lowastlowast

lowastlowast

P lt 005

P lt 005

minus + minus +

(mdash)

(a)

0

05

1

15

2

Ub-

prot

eins

GA

PDH

(fold

chan

ge)

1 2 4(day)

Dox1 2 4 (day)

Ub-

prot

eins

GAPDH

VehDox

170

70

5540

130

100

35

37(k

Da)


lowast

lowast

(b)






Nrf2

0

1

2

3N

rf2G

APD

H


Relat

ive m

RNA

expr

essio

n

0 1 2 4(day)

0

02

04

06

08

Veh Dox

Nrf2

Veh Dox

0

2000

4000

6000

Veh Dox

Nrf2

expr

essio

n

Nuc

lear

Nrf2

()

Veh Dox0

80

60

40

20

43

100

(kD

a)

20120583m 20120583m

lowast

lowastlowast

lowast

P lt 005

P lt 001

n = 4 n = 4

n = 4 n = 4

n = 4 n = 4

(IO

D120583

m2)

Nrf2

120573-a

ctin

120573-Actin

(a)

0 1 2 4

NQO1

0

2

4

6

8

10

NQ

O1

GA

PDH

HO-1

0

10

20

30

40

HO

-1G

APD

H

(day)



Relat

ive m

RNA

expr

essio

n

0 1 2 4(day)

lowast

lowast

lowast

lowast

lowast

lowast

(b)



WT D

oxor

ubic

inD

oxor

ubic

in

0

20

40

Nuc

lear

8-O

HdG

()

WT

Nec

rotic

area

0

1

2

3

100120583m 100120583m

20120583m 20120583m

WT

lowast

lowast

lowast

n = 3

n = 3 n = 3

n = 3

(120583m

2pe

r sec

tiontimes1000

)

Nrf2minusminus

Nrf2minusminus

Nrf2minusminus

(a)

Dox

Sol

LC3-II

GAPDH

p62

Ub-

prot

eins

Insol

LC3-II LC3-II

05

101520253035

LC3-

IIG

APD

H(fo

ld ch

ange

)

01020304050

Sol Insol

p62

02468

1012

p62

GA

PDH

(fold

chan

ge)

02468

1012

0

05

1

15

2

25

Ub-

prot

eins

GA

PDH

(fold

chan

ge)

Ub-proteins

0

05

1

15

2

p62

Ub-proteins

Dox

16

(kD

a)

14

170

70

5540

130100

35

37

70

lowast

lowastlowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

P lt 005

P lt 005 P lt 001

P lt 001P lt 001





(b)




WT Nrf2minusminus























BC















(fold

chan

ge)

(kD

a)

(kD

a)

43

LC3-ILC3-II

16

14

DoxBafA1

0

1

2

3

4

5ns

DoxBafA1

170

70

5540

130100

35

43

Sol Insol

Dox

Ub-

prot

eins

minus minus ++

minus + minus +

minus minus ++


lowastlowast

lowast

LC3

-II120573

-act

in

120573-Actin

120573-Actin

(a)

(kD

a)

(kD

a)

(kD

a)

LC3-ILC3-II

Nrf2

Ad-N

rf2Ad

-Gfp

Ad-Nrf2Ad-Gfp

NQO143

100

31

Dox

43

1614

(fold

chan

ge)

0

05

1

15

2

25 ns

Ub-

prot

eins

170

70

5540

130100

35

43

Ub-

prot

eins

120573-a

ctin

(fold

chan

ge)

0

8

2

4

6

Dox

Insol



minus + minus +

lowastlowastlowast

P lt 001

LC3

-II120573

-act

in

120573-Actin

120573-Actin

120573-Actin

lowast

(b)

LC3-ILC3-II

120573-Actin

120573-Actin

Ad-m

iNrf2

Ad-s

cram

ble


NQO1

(kD

a)

43

1614

Dox

(fold

chan

ge)

0

1

2

Ub-

prot

eins

120573-a

ctin

Ub-

prot

eins

170

70

5540

130100

35

43 0

8

2

4

6

(fold

chan

ge)

Nrf2

Insol

(kD

a) (kD

a)

43

100

31



lowast

lowastlowast

lowast

lowast

P lt 001

P lt 005

LC3

-II120573

-act

in

120573-Actin

(c)



Atg5

43

56

Ub-

prot

eins

170

70

5540

130100

35

43

0

1

2

(fold

chan

ge)

Insol

Dox

(kD

a)

(kD

a)

minus + minus +

120573-Actin

120573-Actin

lowast

lowastP lt 005

Ad-Gfp Ad-Atg5


Dox minus + minus +


Ub-

prot

eins

120573-A

ctin

(a)

Cell

dea

th (

)

0

20

40

60

80C

ell d

eath

()

0

20

40

60

80

0 05 1 2 5 10Dox

Cel

l dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox

Ad-GfpAd-Nrf2


Ad-GfpAd-Atg5


lowast

lowast

lowast

lowastlowast lowast

lowast


lowast

lowastlowast

P lt 005

P lt 005

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastP lt 005

P lt 005

P lt 005P lt 005P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

(120583m)

(120583m)

0 05 1 2 5 10Dox(120583m)

Cell

dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox(120583m)

(b)




3 Discussion






















2








Nrf2NQO-1HO-1GAPDH

NM 0109023NM 0087065NM 0104422XM 001479322




200120116171








Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















LC3-ILC3-II

p62

ATG5

ATG7

GAPDH

LAMP1

Cathepsin D

Dox0 1 2 4 (day)

1614

47

56

75

120

28

37

(kD

a)


(a)

LC3-II

0

2468

10

LC3-

IIG

APD

H(fo

ld ch

ange

)(fo

ld ch

ange

)

0

2468

10

(fold

chan

ge)



p62

p62

GA

PDH

ATG5

0

05

1

15

(fold

chan

ge)

0

05

1

15

(fold

chan

ge)

0

05

1

15

ATG

5G

APD

H

ATG7

ATG

7G

APD

H

0 1 2 4(day)

0 1 2 4(day)

LAMP1

LAM

P1G

APD

H

Cathepsin D

0

05

1

15

2

Cath

epsin

DG

APD

H(fo

ld ch

ange

)

lowast

lowast

lowastlowast

lowast





LC3-II

GAPDH

CQ Veh Dox

LC3-II

0

5

10

15

20

LC3-

IIG

APD

H(fo

ld ch

ange

)


ns

Veh Dox0

5

10

15

20

p62

GA

PDH

(fold

chan

ge)

ns

p62

p62

16

14

37

(kD

a)70

lowastlowast

lowastlowast

P lt 005

P lt 005

minus + minus +

(mdash)

(a)

0

05

1

15

2

Ub-

prot

eins

GA

PDH

(fold

chan

ge)

1 2 4(day)

Dox1 2 4 (day)

Ub-

prot

eins

GAPDH

VehDox

170

70

5540

130

100

35

37(k

Da)


lowast

lowast

(b)






Nrf2

0

1

2

3N

rf2G

APD

H


Relat

ive m

RNA

expr

essio

n

0 1 2 4(day)

0

02

04

06

08

Veh Dox

Nrf2

Veh Dox

0

2000

4000

6000

Veh Dox

Nrf2

expr

essio

n

Nuc

lear

Nrf2

()

Veh Dox0

80

60

40

20

43

100

(kD

a)

20120583m 20120583m

lowast

lowastlowast

lowast

P lt 005

P lt 001

n = 4 n = 4

n = 4 n = 4

n = 4 n = 4

(IO

D120583

m2)

Nrf2

120573-a

ctin

120573-Actin

(a)

0 1 2 4

NQO1

0

2

4

6

8

10

NQ

O1

GA

PDH

HO-1

0

10

20

30

40

HO

-1G

APD

H

(day)



Relat

ive m

RNA

expr

essio

n

0 1 2 4(day)

lowast

lowast

lowast

lowast

lowast

lowast

(b)



WT D

oxor

ubic

inD

oxor

ubic

in

0

20

40

Nuc

lear

8-O

HdG

()

WT

Nec

rotic

area

0

1

2

3

100120583m 100120583m

20120583m 20120583m

WT

lowast

lowast

lowast

n = 3

n = 3 n = 3

n = 3

(120583m

2pe

r sec

tiontimes1000

)

Nrf2minusminus

Nrf2minusminus

Nrf2minusminus

(a)

Dox

Sol

LC3-II

GAPDH

p62

Ub-

prot

eins

Insol

LC3-II LC3-II

05

101520253035

LC3-

IIG

APD

H(fo

ld ch

ange

)

01020304050

Sol Insol

p62

02468

1012

p62

GA

PDH

(fold

chan

ge)

02468

1012

0

05

1

15

2

25

Ub-

prot

eins

GA

PDH

(fold

chan

ge)

Ub-proteins

0

05

1

15

2

p62

Ub-proteins

Dox

16

(kD

a)

14

170

70

5540

130100

35

37

70

lowast

lowastlowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

P lt 005

P lt 005 P lt 001

P lt 001P lt 001





(b)




WT Nrf2minusminus























BC















(fold

chan

ge)

(kD

a)

(kD

a)

43

LC3-ILC3-II

16

14

DoxBafA1

0

1

2

3

4

5ns

DoxBafA1

170

70

5540

130100

35

43

Sol Insol

Dox

Ub-

prot

eins

minus minus ++

minus + minus +

minus minus ++


lowastlowast

lowast

LC3

-II120573

-act

in

120573-Actin

120573-Actin

(a)

(kD

a)

(kD

a)

(kD

a)

LC3-ILC3-II

Nrf2

Ad-N

rf2Ad

-Gfp

Ad-Nrf2Ad-Gfp

NQO143

100

31

Dox

43

1614

(fold

chan

ge)

0

05

1

15

2

25 ns

Ub-

prot

eins

170

70

5540

130100

35

43

Ub-

prot

eins

120573-a

ctin

(fold

chan

ge)

0

8

2

4

6

Dox

Insol



minus + minus +

lowastlowastlowast

P lt 001

LC3

-II120573

-act

in

120573-Actin

120573-Actin

120573-Actin

lowast

(b)

LC3-ILC3-II

120573-Actin

120573-Actin

Ad-m

iNrf2

Ad-s

cram

ble


NQO1

(kD

a)

43

1614

Dox

(fold

chan

ge)

0

1

2

Ub-

prot

eins

120573-a

ctin

Ub-

prot

eins

170

70

5540

130100

35

43 0

8

2

4

6

(fold

chan

ge)

Nrf2

Insol

(kD

a) (kD

a)

43

100

31



lowast

lowastlowast

lowast

lowast

P lt 001

P lt 005

LC3

-II120573

-act

in

120573-Actin

(c)



Atg5

43

56

Ub-

prot

eins

170

70

5540

130100

35

43

0

1

2

(fold

chan

ge)

Insol

Dox

(kD

a)

(kD

a)

minus + minus +

120573-Actin

120573-Actin

lowast

lowastP lt 005

Ad-Gfp Ad-Atg5


Dox minus + minus +


Ub-

prot

eins

120573-A

ctin

(a)

Cell

dea

th (

)

0

20

40

60

80C

ell d

eath

()

0

20

40

60

80

0 05 1 2 5 10Dox

Cel

l dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox

Ad-GfpAd-Nrf2


Ad-GfpAd-Atg5


lowast

lowast

lowast

lowastlowast lowast

lowast


lowast

lowastlowast

P lt 005

P lt 005

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastP lt 005

P lt 005

P lt 005P lt 005P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

(120583m)

(120583m)

0 05 1 2 5 10Dox(120583m)

Cell

dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox(120583m)

(b)




3 Discussion






















2








Nrf2NQO-1HO-1GAPDH

NM 0109023NM 0087065NM 0104422XM 001479322




200120116171








Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















LC3-II

GAPDH

CQ Veh Dox

LC3-II

0

5

10

15

20

LC3-

IIG

APD

H(fo

ld ch

ange

)


ns

Veh Dox0

5

10

15

20

p62

GA

PDH

(fold

chan

ge)

ns

p62

p62

16

14

37

(kD

a)70

lowastlowast

lowastlowast

P lt 005

P lt 005

minus + minus +

(mdash)

(a)

0

05

1

15

2

Ub-

prot

eins

GA

PDH

(fold

chan

ge)

1 2 4(day)

Dox1 2 4 (day)

Ub-

prot

eins

GAPDH

VehDox

170

70

5540

130

100

35

37(k

Da)


lowast

lowast

(b)






Nrf2

0

1

2

3N

rf2G

APD

H


Relat

ive m

RNA

expr

essio

n

0 1 2 4(day)

0

02

04

06

08

Veh Dox

Nrf2

Veh Dox

0

2000

4000

6000

Veh Dox

Nrf2

expr

essio

n

Nuc

lear

Nrf2

()

Veh Dox0

80

60

40

20

43

100

(kD

a)

20120583m 20120583m

lowast

lowastlowast

lowast

P lt 005

P lt 001

n = 4 n = 4

n = 4 n = 4

n = 4 n = 4

(IO

D120583

m2)

Nrf2

120573-a

ctin

120573-Actin

(a)

0 1 2 4

NQO1

0

2

4

6

8

10

NQ

O1

GA

PDH

HO-1

0

10

20

30

40

HO

-1G

APD

H

(day)



Relat

ive m

RNA

expr

essio

n

0 1 2 4(day)

lowast

lowast

lowast

lowast

lowast

lowast

(b)



WT D

oxor

ubic

inD

oxor

ubic

in

0

20

40

Nuc

lear

8-O

HdG

()

WT

Nec

rotic

area

0

1

2

3

100120583m 100120583m

20120583m 20120583m

WT

lowast

lowast

lowast

n = 3

n = 3 n = 3

n = 3

(120583m

2pe

r sec

tiontimes1000

)

Nrf2minusminus

Nrf2minusminus

Nrf2minusminus

(a)

Dox

Sol

LC3-II

GAPDH

p62

Ub-

prot

eins

Insol

LC3-II LC3-II

05

101520253035

LC3-

IIG

APD

H(fo

ld ch

ange

)

01020304050

Sol Insol

p62

02468

1012

p62

GA

PDH

(fold

chan

ge)

02468

1012

0

05

1

15

2

25

Ub-

prot

eins

GA

PDH

(fold

chan

ge)

Ub-proteins

0

05

1

15

2

p62

Ub-proteins

Dox

16

(kD

a)

14

170

70

5540

130100

35

37

70

lowast

lowastlowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

P lt 005

P lt 005 P lt 001

P lt 001P lt 001





(b)




WT Nrf2minusminus























BC















(fold

chan

ge)

(kD

a)

(kD

a)

43

LC3-ILC3-II

16

14

DoxBafA1

0

1

2

3

4

5ns

DoxBafA1

170

70

5540

130100

35

43

Sol Insol

Dox

Ub-

prot

eins

minus minus ++

minus + minus +

minus minus ++


lowastlowast

lowast

LC3

-II120573

-act

in

120573-Actin

120573-Actin

(a)

(kD

a)

(kD

a)

(kD

a)

LC3-ILC3-II

Nrf2

Ad-N

rf2Ad

-Gfp

Ad-Nrf2Ad-Gfp

NQO143

100

31

Dox

43

1614

(fold

chan

ge)

0

05

1

15

2

25 ns

Ub-

prot

eins

170

70

5540

130100

35

43

Ub-

prot

eins

120573-a

ctin

(fold

chan

ge)

0

8

2

4

6

Dox

Insol



minus + minus +

lowastlowastlowast

P lt 001

LC3

-II120573

-act

in

120573-Actin

120573-Actin

120573-Actin

lowast

(b)

LC3-ILC3-II

120573-Actin

120573-Actin

Ad-m

iNrf2

Ad-s

cram

ble


NQO1

(kD

a)

43

1614

Dox

(fold

chan

ge)

0

1

2

Ub-

prot

eins

120573-a

ctin

Ub-

prot

eins

170

70

5540

130100

35

43 0

8

2

4

6

(fold

chan

ge)

Nrf2

Insol

(kD

a) (kD

a)

43

100

31



lowast

lowastlowast

lowast

lowast

P lt 001

P lt 005

LC3

-II120573

-act

in

120573-Actin

(c)



Atg5

43

56

Ub-

prot

eins

170

70

5540

130100

35

43

0

1

2

(fold

chan

ge)

Insol

Dox

(kD

a)

(kD

a)

minus + minus +

120573-Actin

120573-Actin

lowast

lowastP lt 005

Ad-Gfp Ad-Atg5


Dox minus + minus +


Ub-

prot

eins

120573-A

ctin

(a)

Cell

dea

th (

)

0

20

40

60

80C

ell d

eath

()

0

20

40

60

80

0 05 1 2 5 10Dox

Cel

l dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox

Ad-GfpAd-Nrf2


Ad-GfpAd-Atg5


lowast

lowast

lowast

lowastlowast lowast

lowast


lowast

lowastlowast

P lt 005

P lt 005

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastP lt 005

P lt 005

P lt 005P lt 005P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

(120583m)

(120583m)

0 05 1 2 5 10Dox(120583m)

Cell

dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox(120583m)

(b)




3 Discussion






















2








Nrf2NQO-1HO-1GAPDH

NM 0109023NM 0087065NM 0104422XM 001479322




200120116171








Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Nrf2

0

1

2

3N

rf2G

APD

H


Relat

ive m

RNA

expr

essio

n

0 1 2 4(day)

0

02

04

06

08

Veh Dox

Nrf2

Veh Dox

0

2000

4000

6000

Veh Dox

Nrf2

expr

essio

n

Nuc

lear

Nrf2

()

Veh Dox0

80

60

40

20

43

100

(kD

a)

20120583m 20120583m

lowast

lowastlowast

lowast

P lt 005

P lt 001

n = 4 n = 4

n = 4 n = 4

n = 4 n = 4

(IO

D120583

m2)

Nrf2

120573-a

ctin

120573-Actin

(a)

0 1 2 4

NQO1

0

2

4

6

8

10

NQ

O1

GA

PDH

HO-1

0

10

20

30

40

HO

-1G

APD

H

(day)



Relat

ive m

RNA

expr

essio

n

0 1 2 4(day)

lowast

lowast

lowast

lowast

lowast

lowast

(b)



WT D

oxor

ubic

inD

oxor

ubic

in

0

20

40

Nuc

lear

8-O

HdG

()

WT

Nec

rotic

area

0

1

2

3

100120583m 100120583m

20120583m 20120583m

WT

lowast

lowast

lowast

n = 3

n = 3 n = 3

n = 3

(120583m

2pe

r sec

tiontimes1000

)

Nrf2minusminus

Nrf2minusminus

Nrf2minusminus

(a)

Dox

Sol

LC3-II

GAPDH

p62

Ub-

prot

eins

Insol

LC3-II LC3-II

05

101520253035

LC3-

IIG

APD

H(fo

ld ch

ange

)

01020304050

Sol Insol

p62

02468

1012

p62

GA

PDH

(fold

chan

ge)

02468

1012

0

05

1

15

2

25

Ub-

prot

eins

GA

PDH

(fold

chan

ge)

Ub-proteins

0

05

1

15

2

p62

Ub-proteins

Dox

16

(kD

a)

14

170

70

5540

130100

35

37

70

lowast

lowastlowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

P lt 005

P lt 005 P lt 001

P lt 001P lt 001





(b)




WT Nrf2minusminus























BC















(fold

chan

ge)

(kD

a)

(kD

a)

43

LC3-ILC3-II

16

14

DoxBafA1

0

1

2

3

4

5ns

DoxBafA1

170

70

5540

130100

35

43

Sol Insol

Dox

Ub-

prot

eins

minus minus ++

minus + minus +

minus minus ++


lowastlowast

lowast

LC3

-II120573

-act

in

120573-Actin

120573-Actin

(a)

(kD

a)

(kD

a)

(kD

a)

LC3-ILC3-II

Nrf2

Ad-N

rf2Ad

-Gfp

Ad-Nrf2Ad-Gfp

NQO143

100

31

Dox

43

1614

(fold

chan

ge)

0

05

1

15

2

25 ns

Ub-

prot

eins

170

70

5540

130100

35

43

Ub-

prot

eins

120573-a

ctin

(fold

chan

ge)

0

8

2

4

6

Dox

Insol



minus + minus +

lowastlowastlowast

P lt 001

LC3

-II120573

-act

in

120573-Actin

120573-Actin

120573-Actin

lowast

(b)

LC3-ILC3-II

120573-Actin

120573-Actin

Ad-m

iNrf2

Ad-s

cram

ble


NQO1

(kD

a)

43

1614

Dox

(fold

chan

ge)

0

1

2

Ub-

prot

eins

120573-a

ctin

Ub-

prot

eins

170

70

5540

130100

35

43 0

8

2

4

6

(fold

chan

ge)

Nrf2

Insol

(kD

a) (kD

a)

43

100

31



lowast

lowastlowast

lowast

lowast

P lt 001

P lt 005

LC3

-II120573

-act

in

120573-Actin

(c)



Atg5

43

56

Ub-

prot

eins

170

70

5540

130100

35

43

0

1

2

(fold

chan

ge)

Insol

Dox

(kD

a)

(kD

a)

minus + minus +

120573-Actin

120573-Actin

lowast

lowastP lt 005

Ad-Gfp Ad-Atg5


Dox minus + minus +


Ub-

prot

eins

120573-A

ctin

(a)

Cell

dea

th (

)

0

20

40

60

80C

ell d

eath

()

0

20

40

60

80

0 05 1 2 5 10Dox

Cel

l dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox

Ad-GfpAd-Nrf2


Ad-GfpAd-Atg5


lowast

lowast

lowast

lowastlowast lowast

lowast


lowast

lowastlowast

P lt 005

P lt 005

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastP lt 005

P lt 005

P lt 005P lt 005P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

(120583m)

(120583m)

0 05 1 2 5 10Dox(120583m)

Cell

dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox(120583m)

(b)




3 Discussion






















2








Nrf2NQO-1HO-1GAPDH

NM 0109023NM 0087065NM 0104422XM 001479322




200120116171








Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















WT D

oxor

ubic

inD

oxor

ubic

in

0

20

40

Nuc

lear

8-O

HdG

()

WT

Nec

rotic

area

0

1

2

3

100120583m 100120583m

20120583m 20120583m

WT

lowast

lowast

lowast

n = 3

n = 3 n = 3

n = 3

(120583m

2pe

r sec

tiontimes1000

)

Nrf2minusminus

Nrf2minusminus

Nrf2minusminus

(a)

Dox

Sol

LC3-II

GAPDH

p62

Ub-

prot

eins

Insol

LC3-II LC3-II

05

101520253035

LC3-

IIG

APD

H(fo

ld ch

ange

)

01020304050

Sol Insol

p62

02468

1012

p62

GA

PDH

(fold

chan

ge)

02468

1012

0

05

1

15

2

25

Ub-

prot

eins

GA

PDH

(fold

chan

ge)

Ub-proteins

0

05

1

15

2

p62

Ub-proteins

Dox

16

(kD

a)

14

170

70

5540

130100

35

37

70

lowast

lowastlowast

lowast

lowast lowast

lowast

lowast

lowast

lowast

lowast

lowast

P lt 005

P lt 005 P lt 001

P lt 001P lt 001





(b)




WT Nrf2minusminus























BC















(fold

chan

ge)

(kD

a)

(kD

a)

43

LC3-ILC3-II

16

14

DoxBafA1

0

1

2

3

4

5ns

DoxBafA1

170

70

5540

130100

35

43

Sol Insol

Dox

Ub-

prot

eins

minus minus ++

minus + minus +

minus minus ++


lowastlowast

lowast

LC3

-II120573

-act

in

120573-Actin

120573-Actin

(a)

(kD

a)

(kD

a)

(kD

a)

LC3-ILC3-II

Nrf2

Ad-N

rf2Ad

-Gfp

Ad-Nrf2Ad-Gfp

NQO143

100

31

Dox

43

1614

(fold

chan

ge)

0

05

1

15

2

25 ns

Ub-

prot

eins

170

70

5540

130100

35

43

Ub-

prot

eins

120573-a

ctin

(fold

chan

ge)

0

8

2

4

6

Dox

Insol



minus + minus +

lowastlowastlowast

P lt 001

LC3

-II120573

-act

in

120573-Actin

120573-Actin

120573-Actin

lowast

(b)

LC3-ILC3-II

120573-Actin

120573-Actin

Ad-m

iNrf2

Ad-s

cram

ble


NQO1

(kD

a)

43

1614

Dox

(fold

chan

ge)

0

1

2

Ub-

prot

eins

120573-a

ctin

Ub-

prot

eins

170

70

5540

130100

35

43 0

8

2

4

6

(fold

chan

ge)

Nrf2

Insol

(kD

a) (kD

a)

43

100

31



lowast

lowastlowast

lowast

lowast

P lt 001

P lt 005

LC3

-II120573

-act

in

120573-Actin

(c)



Atg5

43

56

Ub-

prot

eins

170

70

5540

130100

35

43

0

1

2

(fold

chan

ge)

Insol

Dox

(kD

a)

(kD

a)

minus + minus +

120573-Actin

120573-Actin

lowast

lowastP lt 005

Ad-Gfp Ad-Atg5


Dox minus + minus +


Ub-

prot

eins

120573-A

ctin

(a)

Cell

dea

th (

)

0

20

40

60

80C

ell d

eath

()

0

20

40

60

80

0 05 1 2 5 10Dox

Cel

l dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox

Ad-GfpAd-Nrf2


Ad-GfpAd-Atg5


lowast

lowast

lowast

lowastlowast lowast

lowast


lowast

lowastlowast

P lt 005

P lt 005

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastP lt 005

P lt 005

P lt 005P lt 005P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

(120583m)

(120583m)

0 05 1 2 5 10Dox(120583m)

Cell

dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox(120583m)

(b)




3 Discussion






















2








Nrf2NQO-1HO-1GAPDH

NM 0109023NM 0087065NM 0104422XM 001479322




200120116171








Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















WT Nrf2minusminus























BC















(fold

chan

ge)

(kD

a)

(kD

a)

43

LC3-ILC3-II

16

14

DoxBafA1

0

1

2

3

4

5ns

DoxBafA1

170

70

5540

130100

35

43

Sol Insol

Dox

Ub-

prot

eins

minus minus ++

minus + minus +

minus minus ++


lowastlowast

lowast

LC3

-II120573

-act

in

120573-Actin

120573-Actin

(a)

(kD

a)

(kD

a)

(kD

a)

LC3-ILC3-II

Nrf2

Ad-N

rf2Ad

-Gfp

Ad-Nrf2Ad-Gfp

NQO143

100

31

Dox

43

1614

(fold

chan

ge)

0

05

1

15

2

25 ns

Ub-

prot

eins

170

70

5540

130100

35

43

Ub-

prot

eins

120573-a

ctin

(fold

chan

ge)

0

8

2

4

6

Dox

Insol



minus + minus +

lowastlowastlowast

P lt 001

LC3

-II120573

-act

in

120573-Actin

120573-Actin

120573-Actin

lowast

(b)

LC3-ILC3-II

120573-Actin

120573-Actin

Ad-m

iNrf2

Ad-s

cram

ble


NQO1

(kD

a)

43

1614

Dox

(fold

chan

ge)

0

1

2

Ub-

prot

eins

120573-a

ctin

Ub-

prot

eins

170

70

5540

130100

35

43 0

8

2

4

6

(fold

chan

ge)

Nrf2

Insol

(kD

a) (kD

a)

43

100

31



lowast

lowastlowast

lowast

lowast

P lt 001

P lt 005

LC3

-II120573

-act

in

120573-Actin

(c)



Atg5

43

56

Ub-

prot

eins

170

70

5540

130100

35

43

0

1

2

(fold

chan

ge)

Insol

Dox

(kD

a)

(kD

a)

minus + minus +

120573-Actin

120573-Actin

lowast

lowastP lt 005

Ad-Gfp Ad-Atg5


Dox minus + minus +


Ub-

prot

eins

120573-A

ctin

(a)

Cell

dea

th (

)

0

20

40

60

80C

ell d

eath

()

0

20

40

60

80

0 05 1 2 5 10Dox

Cel

l dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox

Ad-GfpAd-Nrf2


Ad-GfpAd-Atg5


lowast

lowast

lowast

lowastlowast lowast

lowast


lowast

lowastlowast

P lt 005

P lt 005

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastP lt 005

P lt 005

P lt 005P lt 005P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

(120583m)

(120583m)

0 05 1 2 5 10Dox(120583m)

Cell

dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox(120583m)

(b)




3 Discussion






















2








Nrf2NQO-1HO-1GAPDH

NM 0109023NM 0087065NM 0104422XM 001479322




200120116171








Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(fold

chan

ge)

(kD

a)

(kD

a)

43

LC3-ILC3-II

16

14

DoxBafA1

0

1

2

3

4

5ns

DoxBafA1

170

70

5540

130100

35

43

Sol Insol

Dox

Ub-

prot

eins

minus minus ++

minus + minus +

minus minus ++


lowastlowast

lowast

LC3

-II120573

-act

in

120573-Actin

120573-Actin

(a)

(kD

a)

(kD

a)

(kD

a)

LC3-ILC3-II

Nrf2

Ad-N

rf2Ad

-Gfp

Ad-Nrf2Ad-Gfp

NQO143

100

31

Dox

43

1614

(fold

chan

ge)

0

05

1

15

2

25 ns

Ub-

prot

eins

170

70

5540

130100

35

43

Ub-

prot

eins

120573-a

ctin

(fold

chan

ge)

0

8

2

4

6

Dox

Insol



minus + minus +

lowastlowastlowast

P lt 001

LC3

-II120573

-act

in

120573-Actin

120573-Actin

120573-Actin

lowast

(b)

LC3-ILC3-II

120573-Actin

120573-Actin

Ad-m

iNrf2

Ad-s

cram

ble


NQO1

(kD

a)

43

1614

Dox

(fold

chan

ge)

0

1

2

Ub-

prot

eins

120573-a

ctin

Ub-

prot

eins

170

70

5540

130100

35

43 0

8

2

4

6

(fold

chan

ge)

Nrf2

Insol

(kD

a) (kD

a)

43

100

31



lowast

lowastlowast

lowast

lowast

P lt 001

P lt 005

LC3

-II120573

-act

in

120573-Actin

(c)



Atg5

43

56

Ub-

prot

eins

170

70

5540

130100

35

43

0

1

2

(fold

chan

ge)

Insol

Dox

(kD

a)

(kD

a)

minus + minus +

120573-Actin

120573-Actin

lowast

lowastP lt 005

Ad-Gfp Ad-Atg5


Dox minus + minus +


Ub-

prot

eins

120573-A

ctin

(a)

Cell

dea

th (

)

0

20

40

60

80C

ell d

eath

()

0

20

40

60

80

0 05 1 2 5 10Dox

Cel

l dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox

Ad-GfpAd-Nrf2


Ad-GfpAd-Atg5


lowast

lowast

lowast

lowastlowast lowast

lowast


lowast

lowastlowast

P lt 005

P lt 005

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastP lt 005

P lt 005

P lt 005P lt 005P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

(120583m)

(120583m)

0 05 1 2 5 10Dox(120583m)

Cell

dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox(120583m)

(b)




3 Discussion






















2








Nrf2NQO-1HO-1GAPDH

NM 0109023NM 0087065NM 0104422XM 001479322




200120116171








Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Atg5

43

56

Ub-

prot

eins

170

70

5540

130100

35

43

0

1

2

(fold

chan

ge)

Insol

Dox

(kD

a)

(kD

a)

minus + minus +

120573-Actin

120573-Actin

lowast

lowastP lt 005

Ad-Gfp Ad-Atg5


Dox minus + minus +


Ub-

prot

eins

120573-A

ctin

(a)

Cell

dea

th (

)

0

20

40

60

80C

ell d

eath

()

0

20

40

60

80

0 05 1 2 5 10Dox

Cel

l dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox

Ad-GfpAd-Nrf2


Ad-GfpAd-Atg5


lowast

lowast

lowast

lowastlowast lowast

lowast


lowast

lowastlowast

P lt 005

P lt 005

lowast

lowast

lowast

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

lowast

lowastP lt 005

P lt 005

P lt 005P lt 005P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

P lt 005

(120583m)

(120583m)

0 05 1 2 5 10Dox(120583m)

Cell

dea

th (

)

0

20

40

60

80

0 05 1 2 5 10Dox(120583m)

(b)




3 Discussion






















2








Nrf2NQO-1HO-1GAPDH

NM 0109023NM 0087065NM 0104422XM 001479322




200120116171








Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















3 Discussion






















2








Nrf2NQO-1HO-1GAPDH

NM 0109023NM 0087065NM 0104422XM 001479322




200120116171








Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































2








Nrf2NQO-1HO-1GAPDH

NM 0109023NM 0087065NM 0104422XM 001479322




200120116171








Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Nrf2NQO-1HO-1GAPDH

NM 0109023NM 0087065NM 0104422XM 001479322




200120116171








Acknowledgments


References













































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article nrf2 deficiency exaggerates doxorubicin...

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