research article 7,8-dihydroxyflavone suppresses...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 863720 10 pageshttpdxdoiorg1011552013863720

Research Article78-Dihydroxyflavone Suppresses OxidativeStress-Induced Base Modification in DNA via Inductionof the Repair Enzyme 8-Oxoguanine DNA Glycosylase-1

Ki Cheon Kim1 In Kyung Lee2 Kyoung Ah Kang1 Ji Won Cha1 Suk Ju Cho1

Soo Young Na1 Sungwook Chae3 Hye Sun Kim4 Suhkmann Kim5 and Jin Won Hyun1

1 School of Medicine and Institute for Nuclear Science and Technology Jeju National University Jeju 690-756 Republic of Korea2Department of Obstetrics and Gynecology Asan Medical Center University of Ulsan College of MedicineSeoul 138-736 Republic of Korea

3 Aging Research Center Korea Institute of Oriental Medicine Daejeon 305-811 Republic of Korea4Cancer Research Institute Seoul National University College of Medicine Seoul 110-799 Republic of Korea5 Department of Chemistry and Chemistry Institute for Functional Materials Pusan National UniversityBusan 609-735 Republic of Korea

Correspondence should be addressed to Jin Won Hyun jinwonhjejunuackr

Received 25 May 2013 Revised 23 July 2013 Accepted 15 August 2013

Academic Editor Peter Fu

Copyright copy 2013 Ki Cheon Kim et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The modified guanine base 8-oxoguanine (8-oxoG) is abundantly produced by oxidative stress can contribute to carcinogenesisand can be removed from DNA by 8-oxoguanine DNA glycosylase-1 (OGG1) which acts as an 8-oxoG glycosylase andendonuclease This study investigated the mechanism by which 78-dihydroxyflavone (DHF) inhibits oxidative stress-induced 8-oxoG formation in hamster lung fibroblasts (V79-4) DHF significantly reduced the amount of 8-oxoG induced by hydrogenperoxide (H

2O2) and elevated the levels of OGG1 mRNA and protein DHF increased the binding of nuclear factor erythroid

2-related factor 2 (Nrf2) to antioxidant response element sequences in the upstream promoter region of OGG1 Moreover DHFincreased the nuclear levels of Nrf2 small Maf proteins and the Nrf2small Maf complex all of which are decreased by H

2O2

treatment Likewise the level of phosphorylated Akt which activates Nrf2 was decreased by H2O2treatment but restored by DHF

treatment The levels of OGG1 and nuclear translocation of Nrf2 protein were decreased upon treatment with PI3K inhibitor orAkt inhibitor and DHF treatment did not restore OGG1 and nuclear Nrf2 levels in these inhibitor-treated cells FurthermorePI3K and Akt inhibitors abolished the protective effects of DHF in cells undergoing oxidative stress These data indicate that DHFinduces OGG1 expression via the PI3K-Akt pathway and protects cells against oxidative DNA base damage by activating DNArepair systems

1 Introduction

8-Oxoguanine (8-OxoG) is a major form of DNA damageproduced by oxidative stress [1]Thismodification can induceDNA mutations or alterations which can eventually lead todiseases including cancer [2ndash8] Under normal physiologicalconditions in all aerobic organisms a balance is maintainedbetween endogenous oxidants and numerous enzymatic and

nonenzymatic antioxidant defenses [9] Single-base oxida-tions such as 8-oxoG induced by extreme oxidative condi-tions are repaired by the base excision repair (BER) system[10] which recognizes and removes damaged and irrelevantbases [11]

8-Oxoguanine DNA glycosylase-1 (OGG1) a BERenzyme repairs 8-oxoG by cleaving the glycosidic bond ofthe 8-oxoG lesion and causing a strand break in the DNA

2 BioMed Research International

backbone [12 13] The OGG1 promoter region containsantioxidant response elements (ARE) that are recognizedby nuclear factor erythroid 2-related factor 2 (Nrf2) a tran-scription factor that is essential for ARE-mediated inductionof genes encoding phase II detoxification and oxidative stress-response enzymes [14ndash16] Prior to activation Nrf2 residesin the cytosol To function as a transcription factor Nrf2must first dissociate from Keap1 which degrades Nrf2 bytargeting it for ubiquitination and subsequently translocateto the nucleus [17] The primary signal that causes Nrf2to dissociate from Keap1 is phosphorylation mediated bythe phosphoinositide 3-kinase (PI3K)Akt pathway [18 19]Once in the nucleus Nrf2 must form a heterodimer with asmall Maf protein in order to bind the ARE with high affinityand activate gene expression via this response element [17]

The polyphenolic compound 78-dihydroxyflavone(DHF) is a member of a diverse class of secondary plantmetabolites [20] We recently demonstrated that DHFprevents oxidative stress-induced genotoxicity by scavengingreactive oxygen species (ROS) and enhancing Akt activity[21] However very few reports have addressed the mecha-nisms underlying the influence of DHF on DNA repairof oxidative lesions in the DNA In this study we focusedon the cytoprotective effect of DHF against H

2O2-induced

DNA base damage and its effects on the activity of the repairenzyme OGG1

2 Materials and Methods

21 Reagents 78-Dihydroxyflavone (DHF) was purchasedfrom Tokyo Chemical Industry Co (Chuo-ku Tokyo Japan)The plasmid containing the OGG1-promoter luciferaseconstruct was a kind gift from Professor Ho Jin You(Chosun University Gwangju Republic of Korea) Avidin-conjugated tetramethylrhodamine isothiocyanate (TRITC)[3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium] bro-mide (MTT) and an antibody against OGG1 were purchasedfrom Sigma-Aldrich Corporation (St Louis MO USA)Antibodies against Nrf2 smallMaf phospho-Akt Akt TATAbox-binding protein (TBP) and120573-actinwere purchased fromSanta Cruz Biotechnology (Santa Cruz CA USA) LY294002and Akt inhibitor IV were purchased from Calbiochem (SanDiego CA USA)

22 Cell Culture Lung is frequently contacted to oxygenand ROS [22 23] Chinese hamster lung fibroblasts (V79-4) were used to elucidate the protective effects of DHFagainst the oxidative stress- induced DNA base modificationV79-4 cells were obtained from the American Type CultureCollection Cells weremaintained at 37∘C in an incubator in ahumidified atmosphere of 95 air5 CO

2 Culture medium

was Dulbeccorsquos modified Eaglersquos medium containing 10heat-inactivated fetal calf serum streptomycin (100120583gmL)and penicillin (100 unitsmL)

23 Measurement of the 8-OxoG Level We previouslyreported that 40120583MDHF displayed substantial intracellularROS scavenging activity and was nontoxic to V79-4 cells [21]

and that treatment of cells with 1mMH2O2resulted in 50ndash

60viability [21 24]These concentrations ofDHF andH2O2

were considered optimum for this present studyCells were treated with 40 120583MDHF for 1 h and then

1mMH2O2was added to the medium After 24 h cellular

DNA was isolated using the DNAzol reagent (Life Technolo-gies Grand Island NY USA) and quantified using a spec-trophotometerThe level of 8-hydroxy-2-deoxyguanosine (8-ohdG a nucleoside of 8-oxoG) in DNA considered to repre-sent the 8-oxoG level was determined using the Bioxytech8-OHdG ELISA kit in which 8-ohdG is coated on wellsurface from OXIS Health Products (Portland OR USA)The 8-oxoG level was also estimated using a fluorescence-based binding assay [25] Cells were fixed and permeabilizedwith ice-cold methanol for 15min and then incubated withavidin-conjugated TRITC (fluorescent dye) for 1 h at roomtemperature avidin binds 8-oxoG and 8-ohdG with highaffinityThe resultant TRITC signal (representing the 8-oxoGlevel) was visualized using a fluorescence microscope

24 Transient Transfection and OGG1-Promoter LuciferaseAssay The nucleotide sequence region for OGG1 promoterin the plasmid containing theOGG1-promoter luciferase con-struct is fromminus1947 to +126 which contains Nrf2 binding site(from minus5 to +13) [14] Cells were transiently transfected witha luciferase-reporter plasmid under the control of the OGG1promoter using the transfection reagent DOTAP (RocheMannheim Germany) After overnight transfection cellswere treated with 40 120583MDHF for 1 h and then 1mMH

2O2

was added to the medium After 24 h cells were lysedwith reporter lysis buffer (Promega Madison WI USA)The lysate supernatant was mixed with the luciferase assayreagent and the mixture was placed in a luminometer tomeasure the light produced

25 Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Total RNA was isolated from cells using easy-BLUE(iNtRON Biotechnology Kyounggi Republic of Korea) PCRconditions were as follows 94∘C for 2min 35 cycles of 94∘Cfor 20 sec 58∘C for 30 sec and 72∘C for 1min and 72∘Cfor 5min The primer pairs (Bioneer Corporation DaejeonRepublic of Korea) were as follows mouse OGG1 sense51015840-GCAGAGCCCTGCTCACTGGA-31015840 and antisense 51015840-CGAGGATGGCTTTGGCACTG-31015840 mouse 120573-actin sense51015840-GTGGGCCGCCCTAGGCACCAGG-31015840 and antisense 51015840-GGAGGAAGAGGATGCGGCAGTG-31015840 Amplified prod-ucts were resolved by 1 agarose gel electrophoresis stainedwith ethidium bromide and photographed under ultravioletlight

26 Extraction of Total Cellular Proteins and a NucleusProtein Fraction 5 times 105 cells were seeded into 60mmdishes containing a total of 5mL of medium To extract totalcellular proteins cells were lysed on ice for 30min in 150 120583Lof PRO-PREP (iNtRON Biotechnology) and centrifuged at13000 rpm for 30min To extract nuclear proteins cellswere lysed using the Subcellular Protein Fractionation Kit(Thermo Scientific Milwaukee WI USA)

BioMed Research International 3

27 Quantitative Real-Time PCR cDNA synthesis was per-formed with 2 120583g RNA using a Superscript kit (InvitrogenCarlsbad CA USA) Quantitative real-time PCR was per-formed with 2 times SYBR Green Mastermix (Invitrogen Carls-bad CA USA) and 900 nM primers for OGG1 and 18s rRNAThe thermal cycling parameters were as follows an initialcycle of Taq activation for 1min at 94∘C 1min at 55∘C and1min at 72∘C and optimized annealing temperature 5 sec at58ndash60∘C 30 sec at 72∘C and a detection step of 8 sec at 80∘CReactions were performed in duplicate and the specificitywasmonitored usingmelting curve analysis after cyclingThefollowing primers were designed by Bioneer Co Ltd (SeoulRepublic ofKorea) (51015840ndash31015840) 18s rRNA 51015840-CAGCCACCCCAG-ATTGAGCA-31015840 and 51015840-TAGTAGCGACGGGCGGTGTG-31015840 (51015840ndash31015840) OGG1 51015840-TACCGAGGAGACAAGAGCCA-31015840and 51015840-GGCTATACAGCTGAGCCAGG-31015840 All data weretested first for normality and data with a nonnormal distri-bution were subjected to square root transformation priorto statistical analyses The comparative cycle threshold (Ct)method was used to calculate the relative changes in geneexpression in the iQ5 real-time PCR system (Bio-Rad Labo-ratories Hercules CA USA) The 2-delta-delta Ct value wascalculated after 18s rRNA normalization [26]

28 Western Blotting Cell or nuclear lysates were collectedand protein concentrations were determined using the Brad-ford reagent Aliquots of the lysates (40 120583g of protein)were boiled for 5min and electrophoresed on 10 SDS-polyacrylamide gels Gels were transferred onto nitrocellu-lose membranes (Bio-Rad CA USA) Membranes were thenincubated with the indicated primary antibodies and furtherincubated with secondary immunoglobulin G-horseradishperoxidase conjugates Protein bands were visualized bydeveloping the blots using an Enhanced Chemilumines-cence Western Blotting Detection Kit (Amersham Bucking-hamshire UK) and exposing the membranes to X-ray film

29 Immunoprecipitation Cell lysates were mixed with theanti-Nrf2 antibody and shaken at 4∘C overnight Next 30 120583Lof protein G-agarose beads was added to the lysates andthe mixture was shaken for 2 h at 4∘C The lysates werecentrifuged at 6000 rpm for 5min and the protein G-agarose beads were collected Collected beads were incubatedwith elution buffer for 30min on ice and then centrifugedat 13000 rpm for 5min Supernatants were collected andprotein concentrations were determined using the Bradfordreagent

210 Immunocytochemistry Cells plated on coverslips werefixed with 1 paraformaldehyde for 30min and permeabi-lized with 2 Triton X-100 in PBS for 25min Cells weretreated with blocking medium (1 bovine serum albuminin PBS) for 1 h and incubated with Nrf2 antibody diluted inblockingmedium for 2 h Immunoreactive primaryNrf2 anti-body was detected by a 1 200 dilution of FITC-conjugatedsecondary antibody (Jackson ImmunoResearch LaboratoriesWestGrove PAUSA) for 1 hAfterwashingwith PBS stainedcells were mounted onto microscope slides in mounting

medium with DAPI (Vector Burlingame CA USA) Imageswere collected using the LSM510 programon a Zeiss confocalmicroscope

211 Cell Viability Cells were seeded in a 24-well plate ata density of 35 times 104 cellswell and pretreated with 50 120583MLY294002 or 1 120583M Akt IV for 1 h followed by treatmentwith 40120583MDHF for 1 h and treatment with 1mMH

2O2

for an additional 24 h MTT (150 120583L of a 2mgmL stocksolution) was then added to each well to yield a total reactionvolume of 500 120583L After incubating for 4 h the plate wascentrifuged at 1500 rpm for 5min and the supernatants wereaspirated The formazan crystals in each well were dissolvedin 1mL dimethylsulfoxide and the resultant solutions weretransferred to 96-well spectrometry plates and the A

540was

read on a scanning multiwell spectrophotometer [27]

212 Statistical Analysis All measurements were made inthree independent experiments and all values are repre-sented as the mean plusmn standard error The results weresubjected to an analysis of variance using Tukeyrsquos test foranalysis of differences Statistical significance was set at 119875 lt005

3 Results

31 DHF Suppresses Formation of 8-OxoG by Oxidative Stress8-OxoG is one of the major forms of ROS-induced oxidativebase lesions in DNA and has therefore been widely usedas a biomarker for oxidative stress and carcinogenesis [28]We measured the 8-oxoG level in DNA by ELISA usingspecific antibodies against 8-oxoG The 8-oxoG level wassignificantly higher in H

2O2-treated cells than in control

cells but DHF pretreatment prior to H2O2treatment signif-

icantly suppressed 8-oxoG formation (Figure 1(a)) We alsoestimated the amount of 8-oxoG using a fluorescence-basedbinding assay with an avidin-conjugated TRITC reagent [25]The fluorescence intensity was higher in H

2O2-treated cells

than in control cells and fluorescence was reduced in H2O2-

treated cells that had been pretreated with DHF (Figure 1(b))These results indicate that DHF decreased the 8-oxoG level inH2O2-treated cells

32 DHF Increases OGG1-Promoter Transcriptional Activityand Restores OGG1 mRNA and Protein Expression That AreReduced by H

2O2Treatment Excessive oxidative stress can

overwhelm cellular repair systems OGG1 an enzyme thatrepairs oxidized DNA bases is the primary enzyme respon-sible for the excision of 8-oxoG lesions induced by oxidativestress [29]The transcriptional activity of theOGG1 promoterwas strongly suppressed in H

2O2-treated cells (Figure 2(a))

however this activity was restored by pretreatment withDHFThe level of OGG1 mRNA determined by RT-PCR andquantitative real-time PCR was lower in H

2O2-treated cells

than in control cells however DHF pretreatment partiallyrestored the OGG1 mRNA level (Figures 2(b) and 2(c))


150000

100000

50000

0

8-O

xoG

(pg

mL)

lowast

lowast

Control DHF H2O2 DHF + H2O2

(a)


(b)

Figure 1 DHF reduces H2O2-induced 8-oxoG formation (a) Cells were treated with 40 120583MDHF for 1 h and then incubated with 1mMH

2O2

for an additional 24 h lowastSignificantly different from control cells (119875 lt 005) significantly different from H2O2-treated cells (119875 lt 005) (b)

Binding of avidin-TRITC which reflects 8-oxoG levels was visualized with a fluorescence microscope

Furthermore the level of OGG1 protein was higher in H2O2-

treated cells pretreated with DHF than in cells treated withH2O2alone (Figure 2(d)) These data demonstrate that DHF

can reverse the OGG1 transcription and OGG1 proteinexpression reduced by H

2O2treatment

33 DHF Reverses Nuclear Levels of Nrf2 Small Maf- and theNrf2-Small Maf Complex Reduced by H

2O2Treatment The

OGG1 promoter region contains specific transcription factorbinding sites (ARE sequences) for Nrf2 [14 30] Nrf2 bindsto the ARE with high affinity only as a heterodimer with asmall Maf protein and the Nrf2small Maf complex activatesgene expression directly through the ARE [17] To analyzethe levels of Nrf2 and small Maf protein in the nucleus theexpression of nuclear proteins were measured by westernblotting H

2O2treatment decreased the levels of Nrf2 and

small Maf proteins in the nucleus however DHF treatmentrestored these proteins to control levels (Figure 3(a)) AndH2O2treatment decreased the Nrf2 protein translocation

from cytoplasm to nucleus however DHF treatment restoredit (Figure 3(b)) To determine whether Nrf2 was boundto small Maf protein in the nucleus nuclear lysates wereimmune-precipitated with an anti-Nrf2 antibody and sub-jected to western blotting with an antibody against small Mafprotein H

2O2treatment decreased the binding of Nrf2 to

small Maf protein and DHF treatment restored this binding(Figure 3(c)) These results indicate that DHF treatment can

increase OGG1 expression by increasing the levels of Nrf2small Maf and Nrf2small Maf complex in the nucleus

34 The Induction of OGG1 by DHF Is Mediated via thePI3KAkt Pathway The PI3KAkt pathway is a major signal-ing process critical for cell survival during oxidative stressThe OGG1 promoter region contains Nrf2-binding sitesand the PI3KAkt pathway is involved in the upregulationof OGG1 [15] The level of phospho-Akt was decreasedupon H

2O2treatment however DHF treatment reversed

the reduction in phospho-Akt (Figure 4(a)) Treatment withLY294002 (a PI3K inhibitor) or Akt inhibitor IV attenuatedthe ability of DHF treatment to induce OGG1 expression(Figure 4(b)) Both PI3K and Akt inhibitors suppressed thetranslocation of Nrf2 to the nucleus that was induced bytreatment with DHF (Figure 4(c)) Likewise cell viabilitywas inhibited by H

2O2treatment and DHF treatment

restored viability but the restoration of cell viability byDHF was blocked by treatment with PI3K or Akt inhibitor(Figure 4(d)) These results indicate that the PI3KAkt path-way partially regulates Nrf2 transcriptional activity andOGG1 expression thereby affecting cell viability

4 Discussion

In a previous study we demonstrated that DHF exertscytoprotective properties against oxidative stress-inducedgenotoxic events such as DNA base modification and DNA


20

15

10

05

00OG

G1-

luci

fera

se ac

tivity

(rel

ativ

e int

ensit

y)

lowast

lowast


(a)


OGG1

120573-actin

503 bp

603 bp

(b)

20

15

10

05

00

lowast

lowast


Relat

ive m

RNA

expr

essio

n

(c)


OGG1

120573-actin

23 kDa

42 kDa

(d)

Figure 2 DHF induces OGG1 promoter activity and OGG1 protein expression (a) After overnight transfection with the OGG1-promoterluciferase vector cells were treated with 40120583MDHF for 1 h incubated with 1mMH

2O2for an additional 24 h and then subjected to luciferase

assays lowastSignificantly different from control cells (119875 lt 005) significantly different fromH2O2-treated cells (119875 lt 005) (b) Cells were treated

with 40120583MDHF for 1 h and then incubated with 1mMH2O2for an additional 24 h OGG1 mRNA levels were detected by RT-PCR analysis

and (c) quantitative real-time PCR lowastSignificantly different from control cells (119875 lt 005) significantly different from H2O2-treated cells

(119875 lt 005) (d) OGG1 protein levels were detected by western blot analysis

breaks by scavenging intracellular ROS and enhancing Aktactivity [21] 8-OxoG lesions induced by oxidative stresscan be repaired by OGG1 enzyme under the normal cel-lular redox state During DNA synthesis in an abnormalredox state induced by large perturbations 8-oxoG formsa mismatched base pair with adenine giving rise to aG C to T A transversion mutation potentially altering genefunction and resulting in cell death [3 4 31] OGG1 is amajor glycosylase involved in BER of oxidative DNAdamageCells transfected with siRNA to OGG1 exhibited the reducedOGG1 activity which was accompanied by delayed repair ofxanthine oxidase-induced mitochondrial DNA damage andincreased apoptosis [32] And Liu et al (2011) demonstratedthat cortical neurons isolated fromOGG1-deficientmiceweremore vulnerable to oxidative stress than that of normalOGG1mice and OGG1-deficient mice developed cortical infarctsand behavior deficits after cerebral artery occlusion [33]

The OGG1 promoter contains ARE sequences that isNrf2-binding sequences which are activated under antiox-idant or electrophilic conditions in a normal redox state

[14 15] Nrf2 translocates into the nucleus and heterodimer-izes with small Maf which are also basic leucine-zippertranscription factor the resultant Nrf2small Maf complexacts as a transcription factor for ARE-driven genes [34]

Under nonstressed conditions Nrf2 is anchored in thecytoplasm by binding to Keap1 which facilitates the ubiq-uitination and subsequent proteolysis of Nrf2 [16 35] Uponexposure to various stresses Nrf2 is released from Keap1 andtranslocates into the nucleus [36 37]Nrf2-Keap1 dissociationand Nrf2 signaling are initiated by phosphorylation of Nrf2at serine 40 and at specific threonine residues as well asby the modification of cysteine residues in Keap1 [17 38]One of the signals that trigger Nrf2-Keap1 dissociation andphosphorylation of Nrf2 is mediated by various kinasesincluding mitogen-activated protein kinases protein kinaseC PI3KAkt casein kinase-2 and PKR-like endoplasmicreticulum kinase [39]

The mild ROS conditions stimulate cellular antioxidantsystem which protects against oxidative stress in general[9] but extreme ROS can destroy the cytoprotective defense



Nuclear Nrf2

Nuclear small Maf

TBP

(a)


DAPI

Nrf2

Merge

(b)


IP Nrf2 Small Maf

(c)

Figure 3 DHF induces binding of Nrf2 to small Maf transcription factor (a) Cells were treated with 40120583MDHF for 1 h and then incubatedwith 1mMH

2O2for an additional 12 h Nuclear extracts were electrophoresed and nuclear Nrf2 small Maf protein and TBP were detected

using specific antibodies (b) Confocal images of Nrf2 antibody and FITC-conjugated secondary antibody staining indicate the location ofNrf2 protein (green) DAPI staining indicates the nucleus (blue) The merged images reveal the nuclear or cytosol location of Nrf2 protein(c) Nuclear extracts were immune-precipitated with an anti-Nrf2 antibody and subjected to western blotting using an antibody against smallMaf protein

mechanism by emasculation of antioxidant andor DNArepair systems [40] In the present study H

2O2(1mM)

inhibited OGG1 expression which eliminates the oxidizedguanine This phenomenon supposes that extreme ROScan destroy the cytoprotective defense mechanism whilepretreatment of DHF may offer to enhance the antioxidantandor DNA repair systems Also a proper dose of H

2O2

can induce the Nrf2-translocalization to the nucleus [41] butextreme concentrations of H

2O2may inhibit it Our data

demonstrated that H2O2treatment reduced the level of Nrf2

small Maf and Nrf2small Maf in the nucleus but DHFpretreatment restored these levels It is estimated that DHFpretreatment can transfer Nrf2 to the nucleus and activate theNrf2 transcriptional activity in this study In H

2O2-treated



Phospho Akt

Akt

60 kDa

(a)

Control DHF LY AKT IVLY

+ DHFAKT IV+ DHF

OGG1

120573-actin

(b)

Control DHF LY AKT IV

DAPI

Nrf2

Merge

LY + DHF AKT IV + DHF

(c)

120

100

80

60

40

20

0

Cel

l via

bilit

y (

)

lowast

sect sect

Control DHF H2O2 DHF+ H2O2

LY+ DHF+ H2O2

AKT IV+ DHF+ H2O2

(d)

Figure 4 DHF induces OGG1 expression via the PI3KAkt signaling pathway and improves cell survival (a) Cells were treated with40120583MDHF for 1 h and then incubated with 1mMH

2O2for an additional 12 h Cell lysates were electrophoresed and Akt and phospho-

Akt were detected using specific antibodies (b) Cells were pretreated with 50120583MLY294002 or 1 120583MAkt inhibitor IV for 1 h and then treatedwith 40120583MDHF for an additional 24 h OGG1 was detected by western blotting (c) Confocal images of Nrf2 antibody and FITC-conjugatedsecondary antibody staining indicate the location of Nrf2 protein (green) DAPI staining indicates the nucleus (blue) The merged imagesof DHF treatment in PI3K or Akt inhibitor-treated cells reveal the cytosolic location of Nrf2 protein (d) Cells were pretreated with 50120583MLY294002 or 1 120583MAkt inhibitor IV for 1 h treated with 40120583MDHF for 1 h and then incubated with 1mMH

2O2for an additional 24 h Cell

viability was assessed by the MTT assay lowastSignificantly different from control cells (119875 lt 005) significantly different fromH2O2-treated cells

(119875 lt 005) sectsignificantly different from H2O2-treated cells pretreated with DHF (119875 lt 005)


DHF H2O2

pPI3KAkt

Cytoplasm

pNrf2

Nucleus

pNrf2 sMaf

OGG1 8-oxoG

Cell death

Figure 5 The proposed model of OGG1 induction by DHF treat-ment and its cytoprotective effect against cell death DHF activatesthe OGG1 via the PI3KAktNrf2 signaling pathway and removes 8-oxoG in response to oxidative stress resulting in prevention againstcell death induced by oxidative damage

cells DHF pretreatment restored Akt phosphorylation andOGG1 expression but this effect was diminished by inhibi-tion of the PI3KAkt pathway suggesting that restoration ofOGG1 expression by DHF treatment is partially regulated bya PI3KAkt-dependent pathway

Previous study suggested that PI3KAkt upregulatesphase II enzymeNAD(P)Hquinone oxidoreductase andHO-1 via phosphorylation of Nrf2 transcription factors [4243] Also it has reported that upstream kinases includingPI3KAkt phosphorylate specific serine or threonine residuespresent in Nrf2 resulting in facilitating the nuclear local-ization of Nrf2 [44] Furthermore hyperoxia induced ROS-EGFR-PI3K-Akt signaling cascades which phosphorylatesan Nrf2 in pulmonary epithelial cells [45] PI3KAkt whichplays a key role in multiple cellular processes regulatescellular survival and metabolism by binding and regulatingmany downstream effectors PI3KAkt signalingwas involvedin ROS-mediated self-renewal and neurogenesis in neuralstem cells [46] and in proliferation of human coronary arteryendothelial cells [47] Therefore OGG1 induction by DHFtreatment partially may be involved via PI3KAktNrf2 path-way Furthermore the cytoprotective effects of DHF duringH2O2treatment were also attenuated by PI3KAkt inhibi-

tion Taken together the data presented here demonstratethat DHF activates the BER system via the PI3KAktNrf2signaling pathway in response to oxidative stress and therebyprevents cell death induced by oxidative damage (Figure 5)

Conflicts of Interests

The authors declare that they have no conflict of interests

Acknowledgment

This work was funded by the Ministry of Education Sci-ence and Technology (MEST) of the Republic of Korea(2012R1A1A2005350)

References

[1] J Ravanat M Berger F Benard et al ldquoPhthalocyanineand naphthalocyanine photosensitized oxidation of 21015840-deoxyguanosine distinct type I and type II productsrdquoPhotochemistry and Photobiology vol 39 no 6 pp 809ndash8141984

[2] M Honda Y YamadaM Tomonaga H Ichinose and S Kami-hira ldquoCorrelation of urinary 8-hydroxy-21015840-deoxyguanosine (8-OHdG) a biomarker of oxidative DNA damage and clinicalfeatures of hematological disorders a pilot studyrdquo LeukemiaResearch vol 24 no 6 pp 461ndash468 2000

[3] JWHyun J Y ChoiHH Zeng et al ldquoLeukemic cell line KG-1has a functional loss of hOGG1 enzyme due to a point mutationand 8-hydroxydeoxyguanosine can kill KG-1rdquoOncogene vol 19no 39 pp 4476ndash4479 2000

[4] J W Hyun Y C Jung H S Kim et al ldquo8-Hydroxyde-oxyguanosine causes death of human leukemia cells deficientin 8-oxoguanine glycosylase 1 activity by inducing apoptosisrdquoMolecular Cancer Research vol 1 no 4 pp 290ndash299 2003

[5] A Kikuchi A Takeda H Onodera et al ldquoSystemic increaseof oxidative nucleic acid damage in Parkinsonrsquos disease andmultiple system atrophyrdquo Neurobiology of Disease vol 9 no 2pp 244ndash248 2002

[6] MMatsui CNishigori S Toyokuni et al ldquoThe role of oxidativeDNA damage in human arsenic carcinogenesis detection of8-hydroxy-21015840-deoxyguanosine in arsenic-related Bowenrsquos dis-easerdquo Journal of Investigative Dermatology vol 113 no 1 pp 26ndash31 1999

[7] S Sato Y Mizuno and N Hattori ldquoUrinary 8-hydroxyde-oxyguanosine levels as a biomarker for progression of Parkin-son diseaserdquo Neurology vol 64 no 6 pp 1081ndash1083 2005

[8] L L Wu C Chiou P Chang and J T Wu ldquoUrinary 8-OHdGa marker of oxidative stress to DNA and a risk factor for canceratherosclerosis anddiabeticsrdquoClinicaChimicaActa vol 339 no1-2 pp 1ndash9 2004

[9] B Halliwell ldquoReactive species and antioxidants Redox biologyis a fundamental theme of aerobic liferdquoPlant Physiology vol 141no 2 pp 312ndash322 2006

[10] S Boiteux T R OrsquoConnor F Lederer A Gouyette and JLaval ldquoHomogeneous Escherichia coli FPG protein A DNAglycosylase which excises imidazole ring-opened purines andnicks DNA at apurinicapyrimidinic sitesrdquo Journal of BiologicalChemistry vol 265 no 7 pp 3916ndash3922 1990

[11] H E Krokan H Nilsen F Skorpen M Otterlei and GSlupphaug ldquoBase excision repair of DNA in mammalian cellsrdquoFEBS Letters vol 476 no 1-2 pp 73ndash77 2000

[12] M Bogliolo E Cappelli A DrsquoOsualdo et al ldquoEffect of Scerevisiae APN1 protein on mammalian DNA base excisionrepairrdquo Anticancer Research vol 23 no 5 pp 3727ndash3734 2003

[13] N C de Souza-Pinto L Eide B A Hogue et al ldquoRepair of8-oxodeoxyguanosine lesions in mitochondrial DNA dependson the oxoguanine DNA glycosylase (OGG1) gene and 8-oxoguanine accumulates in the mitochondrial DNA of OGG1-defective micerdquo Cancer Research vol 61 no 14 pp 5378ndash53812001


[14] A Dhenaut S Boiteux and J P Radicella ldquoCharacterization ofthe hOGG1 promoter and its expression during the cell cyclerdquoMutation Research vol 461 no 2 pp 109ndash118 2000

[15] E Ueta E Sasabe Z Yang T Osaki and T YamamotoldquoEnhancement of apoptotic damage of squamous cell carci-noma cells by inhibition of the mitochondrial DNA repairingsystemrdquo Cancer Science vol 99 no 11 pp 2230ndash2237 2008

[16] Y S Keum ldquoRegulation of Nrf2-mediated phase II detoxifica-tion and anti-oxidant genesrdquo Biomolecules amp Therapeutics vol20 no 2 pp 144ndash151 2012

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

[18] K Nakaso H Yano Y Fukuhara T Takeshima K Wada-Isoe and K Nakashima ldquoPI3K is a key molecule in the Nrf2-mediated regulation of antioxidative proteins by hemin inhuman neuroblastoma cellsrdquo FEBS Letters vol 546 no 2-3 pp181ndash184 2003

[19] L Wang Y Chen P Sternberg and J Cai ldquoEssential rolesof the PI3 kinaseAkt pathway in regulating Nrf2-dependentantioxidant functions in the RPErdquo Investigative Ophthalmologyand Visual Science vol 49 no 4 pp 1671ndash1678 2008

[20] D Tasdemir M Kaiser R Brun et al ldquoAntitrypanosomal andantileishmanial activities of flavonoids and their analogues invitro in vivo structure-activity relationship and quantitativestructure-activity relationship studiesrdquo Antimicrobial Agentsand Chemotherapy vol 50 no 4 pp 1352ndash1364 2006

[21] R Zhang K A Kang M J Piao et al ldquoPreventive effect of 78-dihydroxyflavone against oxidative stress induced genotoxicityrdquoBiological and Pharmaceutical Bulletin vol 32 no 2 pp 166ndash171 2009

[22] WA Pryor K Stone L Zang and E Bermudez ldquoFractionationof aqueous cigarette tar extracts fractions that contain the tarradical cause DNA damagerdquo Chemical Research in Toxicologyvol 11 no 5 pp 441ndash448 1998

[23] J I Murray M L Whitfield N D Trinklein R M Myers P OBrown and D Botstein ldquoDiverse and specific gene expressionresponses to stresses in cultured human cellsrdquoMolecular Biologyof the Cell vol 15 no 5 pp 2361ndash2374 2004

[24] K A Kang K H Lee S Chae et al ldquoEckol isolated fromEcklonia cava attenuates oxidative stress induced cell damagein lung fibroblast cellsrdquo FEBS Letters vol 579 no 28 pp 6295ndash6304 2005

[25] L Struthers R Patel J Clark and SThomas ldquoDirect detectionof 8-oxodeoxyguanosine and 8-oxoguanine by avidin and itsanaloguesrdquo Analytical Biochemistry vol 255 no 1 pp 20ndash311998

[26] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[27] J Carmichael W G DeGraff and A F Gazdar ldquoEvaluation ofa tetrazolium-based semiautomated colorimetric assay assess-ment of chemosensitivity testingrdquo Cancer Research vol 47 no4 pp 936ndash942 1987

[28] A Valavanidis T Vlachogianni and C Fiotakis ldquo8-hydroxy-21015840-deoxyguanosine (8-OHdG) a critical biomarker of oxidativestress and carcinogenesisrdquo Journal of Environmental Science andHealth C vol 27 no 2 pp 120ndash139 2009

[29] P Fortini B Pascucci E ParlantiMDrsquoErrico V Simonelli andE Dogliotti ldquo8-Oxoguanine DNA damage at the crossroad of

alternative repair pathwaysrdquoMutation Research vol 531 no 1-2pp 127ndash139 2003

[30] J Mo Y Xia T J Wade et al ldquoChronic arsenic exposureand oxidative stress OGG1 expression and arsenic exposurenail selenium and skin hyperkeratosis in inner MongoliardquoEnvironmental Health Perspectives vol 114 no 6 pp 835ndash8412006

[31] S S David V L OrsquoShea and S Kundu ldquoBase-excision repair ofoxidative DNA damagerdquoNature vol 447 no 7147 pp 941ndash9502007

[32] M V Ruchko O M Gorodnya A Zuleta V M Pastukh andM N Gillespie ldquoThe DNA glycosylase Ogg1 defends againstoxidant-induced mtDNA damage and apoptosis in pulmonaryartery endothelial cellsrdquo Free Radical Biology and Medicine vol50 no 9 pp 1107ndash1113 2011

[33] D Liu D L Croteau N Souza-Pinto et al ldquoEvidence thatOGG1 glycosylase protects neurons against oxidative DNAdamage and cell death under ischemic conditionsrdquo Journal ofCerebral Blood Flow andMetabolism vol 31 no 2 pp 680ndash6922011

[34] H Motohashi F Katsuoka J D Engel and M YamamotoldquoSmall Maf proteins serve as transcriptional cofactors for ker-atinocyte differentiation in the Keap1-Nrf2 regulatory pathwayrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 17 pp 6379ndash6384 2004

[35] M McMahon K Itoh M Yamamoto and J D Hayes ldquoKeap1-dependent proteasomal degradation of transcription factorNrf2 contributes to the negative regulation of antioxidantresponse element-driven gene expressionrdquo Journal of BiologicalChemistry vol 278 no 24 pp 21592ndash21600 2003

[36] T W Kensler N Wakabayashi and S Biswal ldquoCell survivalresponses to environmental stresses via the Keap1-Nrf2-AREpathwayrdquo Annual Review of Pharmacology and Toxicology vol47 pp 89ndash116 2007

[37] G P Sykiotis and D Bohmann ldquoStress-activated capldquonrdquocollartranscription factors in aging and human diseaserdquo ScienceSignaling vol 3 no 112 p re3 2010

[38] K Itoh K I Tong and M Yamamoto ldquoMolecular mecha-nism activating Nrf2-Keap1 pathway in regulation of adaptiveresponse to electrophilesrdquo Free Radical Biology and Medicinevol 36 no 10 pp 1208ndash1213 2004

[39] A Boutten D Goven E Artaud-Macari J Boczkowski andM Bonay ldquoNRF2 targeting a promising therapeutic strategyin chronic obstructive pulmonary diseaserdquo Trends in MolecularMedicine vol 17 no 7 pp 363ndash371 2011

[40] V J Thannickal and B L Fanburg ldquoReactive oxygen species incell signalingrdquo American Journal of Physiology vol 279 no 6pp L1005ndashL1028 2000

[41] J Pi W Qu J M Reece Y Kumagai and M P WaalkesldquoTranscription factor Nrf2 activation by inorganic arsenic incultured keratinocytes involvement of hydrogen peroxiderdquoExperimental Cell Research vol 290 no 2 pp 234ndash245 2003

[42] J W Lee J M Hanson W A Chu and J A Johnson ldquoPhos-phatidylinositol 3-kinase not extracellular signal-regulatedkinase regulates activation of the antioxidant-responsive ele-ment in IMR-32 human neuroblastoma cellsrdquo Journal of Biolog-ical Chemistry vol 276 no 23 pp 20011ndash20016 2001

[43] D Martin A I Rojo M Salinas et al ldquoRegulation of hemeoxygenase-1 expression through the phosphatidylinositol 3-kinaseAkt pathway and the Nrf2 transcription factor inresponse to the antioxidant phytochemical carnosolrdquo Journal ofBiological Chemistry vol 279 no 10 pp 8919ndash8929 2004


[44] Y J Surh J K Kundu and H K Na ldquoNrf2 as a master redoxswitch in turning on the cellular signaling involved in theinduction of cytoprotective genes by some chemopreventivephytochemicalsrdquo Planta Medica vol 74 no 13 pp 1526ndash15392008

[45] S Papaiahgari Q Zhang S R Kleeberger H Cho and SP Reddy ldquoHyperoxia stimulates an Nrf2-ARE transcriptionalresponse via ROS-EGFR-P13K-AktERKMAP kinase signalingin pulmonary epithelial cellsrdquoAntioxidants andRedox Signalingvol 8 no 1-2 pp 43ndash52 2006

[46] J E Le Belle N M Orozco A A Paucar et al ldquoProliferativeneural stem cells have high endogenous ROS levels that regulateself-renewal and neurogenesis in a PI3KAkt-dependant man-nerrdquo Cell Stem Cell vol 8 no 1 pp 59ndash71 2011

[47] O Erdogdu D Nathanson A Sjoholm T Nystrom and QZhang ldquoExendin-4 stimulates proliferation of human coronaryartery endothelial cells through eNOS- PKA- and PI3KAkt-dependent pathways and requires GLP-1 receptorrdquo Molecularand Cellular Endocrinology vol 325 no 1-2 pp 26ndash35 2010

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


backbone [12 13] The OGG1 promoter region containsantioxidant response elements (ARE) that are recognizedby nuclear factor erythroid 2-related factor 2 (Nrf2) a tran-scription factor that is essential for ARE-mediated inductionof genes encoding phase II detoxification and oxidative stress-response enzymes [14ndash16] Prior to activation Nrf2 residesin the cytosol To function as a transcription factor Nrf2must first dissociate from Keap1 which degrades Nrf2 bytargeting it for ubiquitination and subsequently translocateto the nucleus [17] The primary signal that causes Nrf2to dissociate from Keap1 is phosphorylation mediated bythe phosphoinositide 3-kinase (PI3K)Akt pathway [18 19]Once in the nucleus Nrf2 must form a heterodimer with asmall Maf protein in order to bind the ARE with high affinityand activate gene expression via this response element [17]

The polyphenolic compound 78-dihydroxyflavone(DHF) is a member of a diverse class of secondary plantmetabolites [20] We recently demonstrated that DHFprevents oxidative stress-induced genotoxicity by scavengingreactive oxygen species (ROS) and enhancing Akt activity[21] However very few reports have addressed the mecha-nisms underlying the influence of DHF on DNA repairof oxidative lesions in the DNA In this study we focusedon the cytoprotective effect of DHF against H

2O2-induced

DNA base damage and its effects on the activity of the repairenzyme OGG1

2 Materials and Methods

21 Reagents 78-Dihydroxyflavone (DHF) was purchasedfrom Tokyo Chemical Industry Co (Chuo-ku Tokyo Japan)The plasmid containing the OGG1-promoter luciferaseconstruct was a kind gift from Professor Ho Jin You(Chosun University Gwangju Republic of Korea) Avidin-conjugated tetramethylrhodamine isothiocyanate (TRITC)[3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium] bro-mide (MTT) and an antibody against OGG1 were purchasedfrom Sigma-Aldrich Corporation (St Louis MO USA)Antibodies against Nrf2 smallMaf phospho-Akt Akt TATAbox-binding protein (TBP) and120573-actinwere purchased fromSanta Cruz Biotechnology (Santa Cruz CA USA) LY294002and Akt inhibitor IV were purchased from Calbiochem (SanDiego CA USA)

22 Cell Culture Lung is frequently contacted to oxygenand ROS [22 23] Chinese hamster lung fibroblasts (V79-4) were used to elucidate the protective effects of DHFagainst the oxidative stress- induced DNA base modificationV79-4 cells were obtained from the American Type CultureCollection Cells weremaintained at 37∘C in an incubator in ahumidified atmosphere of 95 air5 CO

2 Culture medium

was Dulbeccorsquos modified Eaglersquos medium containing 10heat-inactivated fetal calf serum streptomycin (100120583gmL)and penicillin (100 unitsmL)

23 Measurement of the 8-OxoG Level We previouslyreported that 40120583MDHF displayed substantial intracellularROS scavenging activity and was nontoxic to V79-4 cells [21]

and that treatment of cells with 1mMH2O2resulted in 50ndash

60viability [21 24]These concentrations ofDHF andH2O2

were considered optimum for this present studyCells were treated with 40 120583MDHF for 1 h and then

1mMH2O2was added to the medium After 24 h cellular

DNA was isolated using the DNAzol reagent (Life Technolo-gies Grand Island NY USA) and quantified using a spec-trophotometerThe level of 8-hydroxy-2-deoxyguanosine (8-ohdG a nucleoside of 8-oxoG) in DNA considered to repre-sent the 8-oxoG level was determined using the Bioxytech8-OHdG ELISA kit in which 8-ohdG is coated on wellsurface from OXIS Health Products (Portland OR USA)The 8-oxoG level was also estimated using a fluorescence-based binding assay [25] Cells were fixed and permeabilizedwith ice-cold methanol for 15min and then incubated withavidin-conjugated TRITC (fluorescent dye) for 1 h at roomtemperature avidin binds 8-oxoG and 8-ohdG with highaffinityThe resultant TRITC signal (representing the 8-oxoGlevel) was visualized using a fluorescence microscope

24 Transient Transfection and OGG1-Promoter LuciferaseAssay The nucleotide sequence region for OGG1 promoterin the plasmid containing theOGG1-promoter luciferase con-struct is fromminus1947 to +126 which contains Nrf2 binding site(from minus5 to +13) [14] Cells were transiently transfected witha luciferase-reporter plasmid under the control of the OGG1promoter using the transfection reagent DOTAP (RocheMannheim Germany) After overnight transfection cellswere treated with 40 120583MDHF for 1 h and then 1mMH

2O2

was added to the medium After 24 h cells were lysedwith reporter lysis buffer (Promega Madison WI USA)The lysate supernatant was mixed with the luciferase assayreagent and the mixture was placed in a luminometer tomeasure the light produced

25 Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Total RNA was isolated from cells using easy-BLUE(iNtRON Biotechnology Kyounggi Republic of Korea) PCRconditions were as follows 94∘C for 2min 35 cycles of 94∘Cfor 20 sec 58∘C for 30 sec and 72∘C for 1min and 72∘Cfor 5min The primer pairs (Bioneer Corporation DaejeonRepublic of Korea) were as follows mouse OGG1 sense51015840-GCAGAGCCCTGCTCACTGGA-31015840 and antisense 51015840-CGAGGATGGCTTTGGCACTG-31015840 mouse 120573-actin sense51015840-GTGGGCCGCCCTAGGCACCAGG-31015840 and antisense 51015840-GGAGGAAGAGGATGCGGCAGTG-31015840 Amplified prod-ucts were resolved by 1 agarose gel electrophoresis stainedwith ethidium bromide and photographed under ultravioletlight

26 Extraction of Total Cellular Proteins and a NucleusProtein Fraction 5 times 105 cells were seeded into 60mmdishes containing a total of 5mL of medium To extract totalcellular proteins cells were lysed on ice for 30min in 150 120583Lof PRO-PREP (iNtRON Biotechnology) and centrifuged at13000 rpm for 30min To extract nuclear proteins cellswere lysed using the Subcellular Protein Fractionation Kit(Thermo Scientific Milwaukee WI USA)








2O2


540was



3 Results





2O2-treated cells








2O2-treated cells



150000

100000

50000

0

8-O

xoG

(pg

mL)

lowast

lowast


(a)


(b)


2O2






2O2treatment


2O2Treatment The













4 Discussion



20

15

10

05

00OG

G1-

luci

fera

se ac

tivity

(rel

ativ

e int

ensit

y)

lowast

lowast


(a)


OGG1

120573-actin

503 bp

603 bp

(b)

20

15

10

05

00

lowast

lowast


Relat

ive m

RNA

expr

essio

n

(c)


OGG1

120573-actin

23 kDa

42 kDa

(d)














Nuclear Nrf2

Nuclear small Maf

TBP

(a)


DAPI

Nrf2

Merge

(b)


IP Nrf2 Small Maf

(c)





2O2(1mM)


2O2





2O2-treated



Phospho Akt

Akt

60 kDa

(a)


+ DHFAKT IV+ DHF

OGG1

120573-actin

(b)


DAPI

Nrf2

Merge


(c)

120

100

80

60

40

20

0

Cel

l via

bilit

y (

)

lowast

sect sect


LY+ DHF+ H2O2

AKT IV+ DHF+ H2O2

(d)








DHF H2O2

pPI3KAkt

Cytoplasm

pNrf2

Nucleus

pNrf2 sMaf

OGG1 8-oxoG

Cell death







Acknowledgment


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








2O2


540was



3 Results





2O2-treated cells








2O2-treated cells



150000

100000

50000

0

8-O

xoG

(pg

mL)

lowast

lowast


(a)


(b)


2O2






2O2treatment


2O2Treatment The













4 Discussion



20

15

10

05

00OG

G1-

luci

fera

se ac

tivity

(rel

ativ

e int

ensit

y)

lowast

lowast


(a)


OGG1

120573-actin

503 bp

603 bp

(b)

20

15

10

05

00

lowast

lowast


Relat

ive m

RNA

expr

essio

n

(c)


OGG1

120573-actin

23 kDa

42 kDa

(d)














Nuclear Nrf2

Nuclear small Maf

TBP

(a)


DAPI

Nrf2

Merge

(b)


IP Nrf2 Small Maf

(c)





2O2(1mM)


2O2





2O2-treated



Phospho Akt

Akt

60 kDa

(a)


+ DHFAKT IV+ DHF

OGG1

120573-actin

(b)


DAPI

Nrf2

Merge


(c)

120

100

80

60

40

20

0

Cel

l via

bilit

y (

)

lowast

sect sect


LY+ DHF+ H2O2

AKT IV+ DHF+ H2O2

(d)








DHF H2O2

pPI3KAkt

Cytoplasm

pNrf2

Nucleus

pNrf2 sMaf

OGG1 8-oxoG

Cell death







Acknowledgment


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


150000

100000

50000

0

8-O

xoG

(pg

mL)

lowast

lowast


(a)


(b)


2O2






2O2treatment


2O2Treatment The













4 Discussion



20

15

10

05

00OG

G1-

luci

fera

se ac

tivity

(rel

ativ

e int

ensit

y)

lowast

lowast


(a)


OGG1

120573-actin

503 bp

603 bp

(b)

20

15

10

05

00

lowast

lowast


Relat

ive m

RNA

expr

essio

n

(c)


OGG1

120573-actin

23 kDa

42 kDa

(d)














Nuclear Nrf2

Nuclear small Maf

TBP

(a)


DAPI

Nrf2

Merge

(b)


IP Nrf2 Small Maf

(c)





2O2(1mM)


2O2





2O2-treated



Phospho Akt

Akt

60 kDa

(a)


+ DHFAKT IV+ DHF

OGG1

120573-actin

(b)


DAPI

Nrf2

Merge


(c)

120

100

80

60

40

20

0

Cel

l via

bilit

y (

)

lowast

sect sect


LY+ DHF+ H2O2

AKT IV+ DHF+ H2O2

(d)








DHF H2O2

pPI3KAkt

Cytoplasm

pNrf2

Nucleus

pNrf2 sMaf

OGG1 8-oxoG

Cell death







Acknowledgment


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


20

15

10

05

00OG

G1-

luci

fera

se ac

tivity

(rel

ativ

e int

ensit

y)

lowast

lowast


(a)


OGG1

120573-actin

503 bp

603 bp

(b)

20

15

10

05

00

lowast

lowast


Relat

ive m

RNA

expr

essio

n

(c)


OGG1

120573-actin

23 kDa

42 kDa

(d)














Nuclear Nrf2

Nuclear small Maf

TBP

(a)


DAPI

Nrf2

Merge

(b)


IP Nrf2 Small Maf

(c)





2O2(1mM)


2O2





2O2-treated



Phospho Akt

Akt

60 kDa

(a)


+ DHFAKT IV+ DHF

OGG1

120573-actin

(b)


DAPI

Nrf2

Merge


(c)

120

100

80

60

40

20

0

Cel

l via

bilit

y (

)

lowast

sect sect


LY+ DHF+ H2O2

AKT IV+ DHF+ H2O2

(d)








DHF H2O2

pPI3KAkt

Cytoplasm

pNrf2

Nucleus

pNrf2 sMaf

OGG1 8-oxoG

Cell death







Acknowledgment


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Nuclear Nrf2

Nuclear small Maf

TBP

(a)


DAPI

Nrf2

Merge

(b)


IP Nrf2 Small Maf

(c)





2O2(1mM)


2O2





2O2-treated



Phospho Akt

Akt

60 kDa

(a)


+ DHFAKT IV+ DHF

OGG1

120573-actin

(b)


DAPI

Nrf2

Merge


(c)

120

100

80

60

40

20

0

Cel

l via

bilit

y (

)

lowast

sect sect


LY+ DHF+ H2O2

AKT IV+ DHF+ H2O2

(d)








DHF H2O2

pPI3KAkt

Cytoplasm

pNrf2

Nucleus

pNrf2 sMaf

OGG1 8-oxoG

Cell death







Acknowledgment


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Phospho Akt

Akt

60 kDa

(a)


+ DHFAKT IV+ DHF

OGG1

120573-actin

(b)


DAPI

Nrf2

Merge


(c)

120

100

80

60

40

20

0

Cel

l via

bilit

y (

)

lowast

sect sect


LY+ DHF+ H2O2

AKT IV+ DHF+ H2O2

(d)








DHF H2O2

pPI3KAkt

Cytoplasm

pNrf2

Nucleus

pNrf2 sMaf

OGG1 8-oxoG

Cell death







Acknowledgment


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


DHF H2O2

pPI3KAkt

Cytoplasm

pNrf2

Nucleus

pNrf2 sMaf

OGG1 8-oxoG

Cell death







Acknowledgment


References


























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article 7,8-dihydroxyflavone suppresses...

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