Research ArticleIs Hydrogen Peroxide a Suitable ApoptosisInducer for All Cell Types

Jinmei Xiang12 Chunyun Wan13 Rui Guo4 and Dingzong Guo1

1College of Veterinary Medicine Huazhong Agricultural University Wuhan Hubei 430070 China2Hubei Vocational College of Bio-Technology Wuhan Hubei 430070 China3College of Animal Science Yangtze University Jingzhou Hubei 434023 China4Hubei Academy of Agricultural Sciences Wuhan Hubei 430070 China

Correspondence should be addressed to Dingzong Guo hlgdz163com

Received 9 May 2016 Accepted 11 July 2016

Academic Editor Emanuele Marzetti

Copyright copy 2016 Jinmei Xiang 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

Hydrogen peroxide is currently themostwidely used apoptosis inducer due to its broad cytotoxic efficacy against nearly all cell typesHowever equivalent cytotoxicity is achieved over a wide range of doses although the reasons for this differential sensitivity are notalways clear In this study three kinds of cells the 293T cell line primary fibroblasts and terminally differentiated myocardial cellswere treated with a wide range of H

2O2doses Times to apoptosis initiation and endweremeasured cytochemically and the changes

in expression of caspase-9 P53 NF-120581B and RIP were determined by RT-PCR The 293T cell line was the most sensitive to H2O2

undergoing necroptosis andor apoptosis at all concentrations from 01 to 16mM At gt 04mM H2O2also caused necroptosis in

primary cells At lt 04mM however primary cells exhibited classic signs of apoptosis although they tended to survive for 36 hoursin lt 02mM H

2O2 Thus H

2O2is a broadly effective apoptosis inducer but the dose range differs by cell type For cell lines a low

dose is required and the exposure time must be reduced compared to primary cells to avoid cell death primarily by necroptosis ornecrosis

1 Introduction

Cell apoptosis was first described as a cell death pathwayunique from necrosis in 1972 [1] Thereafter a plethoraof apoptosis inducers were identified such as hydrogenperoxide (H

2O2) dithiothreitol (DTT) and oxidized LDL [2ndash

4] Among these agents H2O2has been the most widely used

and studied at the mechanistic level In many cases transientexposure to H

2O2triggers apoptosis through the mitochon-

drial pathway involving sequential loss of mitochondrialmembrane potential cytochrome c release and effectorcaspase-3 activation [5ndash7] Several factors that can antagonizeapoptosis induced by H

2O2have also been identified such

as nerve growth factor (NGF) and chlorogenic acid [8 9]Hydrogen peroxide is used as an apoptosis inducer for manytypes of cells including cell lines tumor cells primary cellsand highly differentiated cells [10ndash15] although the dosesused vary widely from 005 to 10mM [12ndash14 16 17] Asthere are several interacting but mechanistically distinct cell

death pathways that may be activated by H2O2 it is critical to

identify the ranges over which these pathways are primarilyactivated Moreover such information could yield valuableinformation on the interactions among these pathways undercell stress To date however there is still no study that sys-tematically studied different susceptibility to H

2O2-induced

apoptosis among cell types which is critical for determiningif H2O2is a suitable apoptosis inducer in a specific context

Indeed whether apoptosis or necroptosis is induced underdifferent dosages of H

2O2is often unconfirmed [18] and

apoptosis is only assumedCertain indices can reveal specific aspects of the cell death

process For example caspase-9 can be used to monitor theinitiation of apoptosis [19] activation of NF-120581B is usuallycaused by oxidative stress and may indicate DNA damage[20] and P53 can indicate dysregulation of the cell cycleand proliferation [21] RIP is a key mediator of necroptosissignaling pathways therefore RIP can be used to distinguishapoptosis from necroptosis [22]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 7343965 6 pageshttpdxdoiorg10115520167343965

2 BioMed Research International

Table 1 Primers for quantitative real-time PCR

Gene Forward Reverse Thermocycle

FC

Caspase-9 51015840TCAGACATCGTATCCTCCA 51015840AGTCACAGCAGCACA

98∘C3min + 40 times [98∘15 s 61∘C40 s]P53 51015840ACCTGCACTTACTCCCCGGT 51015840TCTTATAGACGGCCACGGCG

NF-120581B 51015840AGGACTTAAAATGGCAGGAGAG 51015840GCTGTTCGTAGTGGTAAGTCTGRIP1 51015840GAACTAGGCTTCAGCAACTCCG 51015840GCAGCCAAAGAGGGCTTTGG120573-actin 51015840GGCACCCAGCACAATGAAG 51015840CCGATCCACACGGAGTACTTG

293T

Caspase-9 51015840TGCTGAGCAGCGAGCTGTT 51015840AGCCTGCCCGCTGGAT

98∘C3min + 40 times [98∘15 s 61∘C40 s]P53 51015840CCACCATCCACTACAACTACAT 51015840CAAACACGGACAGGACCC

NF-120581B 51015840TCTCCCTGGTCACCAAGGAC 51015840TCATAGAAGCCATCCCGGCRIP1 51015840CATGGAAAAGGCGTGATACAC 51015840ACTTCCCTCAGCTCATTGTG120573-actin 51015840GGCACCCAGCACAATGAAG 51015840CCGATCCACACGGAGTACTTG

FC fibroblastscardiomyocytes

To determine if H2O2is a suitable apoptosis inducer for

a given cell type we measured cytochemical and geneticindices of apoptosis and necroptosis under a range ofH2O2doses in three cell types an immortalized cell line

primary fibroblasts and terminally differentiated cardiomy-ocytes These cells were chosen for their distinct proliferativefeatures The 293T immortalized cell line is characterizedby unlimited proliferation and passages while fibroblastsshow limited proliferation and passages and terminallydifferentiated cardiomyocytes show no further proliferationFor convenience both fibroblasts and myocardial cells werederived from chicken embryos

2 Materials and Methods

21 Cell Culture Isolation and culture of chicken embryofibroblasts and myocardial cells followed methods previouslydescribed [23 24] with some modifications Briefly WhiteLeghorn eggs were obtained from Beijing Merial Vital Lab-oratory Animal Technology (Beijing China) At embryonicday 11 (E11) embryos were removed and decapitated in a Petridish filled with Medium 199EBSS (HyClone Logan UtahUSA) supplemented with 3 fetal bovine serum (FBS GibcoGrand Island New York USA) Ventricular tissues and torsoof chicken embryo were isolated for the preparations ofmyocardial cells and CEF and treated with 005 trypsin-EDTA to obtain a cell suspension as described [14 25 26]respectively Specifically the cells of CEF andmyocardial cellswere respectively incubated at 8 times 105 per well in 24-wellplates in growth medium (Medium 199EBSS containing 10FBS) at 37∘C under a 5 CO

2atmosphere Cultures were

washed three times at 8 24 and 48 h to remove dead anddying cellsThe serum concentration in themediumwas thenchanged from growth (10) to maintenance (2) conditions

293T cells at low passage were incubated at 1 times 105cellswell in 24-well plates with Dulbeccorsquos modified Eaglemedium (DMEM) (HyClone Logan Utah USA) containing10 FBS at 37∘C under a 5 CO

2atmosphere After plating

the serum concentrationwas decreased from growth (10) tomaintenance (2) conditions

22 Apoptosis Induction and Assessment Each cell type wasdivided into three groups H

2O2treatment positive control

(DTT treatment) and negative control H2O2groups were

incubated in 01 02 04 08 or 16mM H2O2 the positive

control groups were incubated in 2mM DTT and thenegative control groups received no treatment After the startof exposure cells were examined every 05 h by staining withAOEB to monitor the initiation of apoptosis DAPI stainingwas used to determine the time to substantial apoptosisThen apoptosis times and cell survival rates were determinedby AOEB staining and DAPI staining Finally total RNAwas extracted from cells using Trizol reagent (InvitrogenCarlsbad CA USA) to assess expression levels of apoptosis-and necroptosis-associated genes Reverse transcription wasperformed using a PrimeScript II 1st Strand cDNA synthesiskit (TaKaRa Otsu Shiga Japan) Quantitative real-time PCRwas conducted to evaluate changes in caspase-9 P53 NF-120581Band RIP expression levels using the primers and thermocycleconditions shown in Table 1 Groupmeans were compared byANOVA using SPSS170 software All bar figures were createdby Graphpad Prism 5 software

3 Results

31 Initiation Time of Apoptosis Apoptosis initiation times atdifferent H

2O2doses are shown in Figure 1 for all three cell

types The time to initiation was estimated by AOEB doublestaining every 05 h under an inverted epifluorescencemicro-scope The 293T cells were very sensitive to both DTT- andH2O2-induced apoptosis as indicated by the shorter delays

until initiation compared to the other cell types Alternativelyapoptosis initiation times did not differ significantly betweenmyocardial cells and fibroblasts in response to DTT or H

2O2

32 Significant Differences in Times to Substantial Apoptosisand the End of Apoptosis among Cell Types After apoptosisstarted DAPI staining was used to determine the time atwhich significant apoptosis occurred (Figure 2) At gt 04mMH2O2 apoptosis developed rapidly with little difference

between cell types At le 04mM however the time tosubstantial apoptosis was much more sensitive to H

2O2

BioMed Research International 3Ti

mes

to ap

opto

sis in

itiat

ion

(h)

T M F

01

02

04

08

16

DTT2

10

8

6

4

2

0

Figure 1 Time to apoptosis initiation 293T cells (T) myocardialcells (M) and fibroblasts (F) were treated with a range of hydrogenperoxide concentrations (01ndash16mM) or DTT (2mM positivecontrol)

01

02

04

08

16

DTT2

T M F

Tim

e to

signi

fican

t apo

ptos

is in

duct

ion

for e

ach

cell

type

in re

spon

se to

DTT

or h

ydro

gen

pero

xide

(h)

15

10

5

0

Figure 2 Time to significant apoptosis induction for each cell typein response to DTT or hydrogen peroxide Hydrogen peroxide 01ndash16mMDTT 2mM T 293T cellsMmyocardial cells F fibroblasts

dose and cell type with markedly faster times for 293Tcells particularly at 04mM compared to fibroblasts andcardiomyocytes For the apoptosis ending test the medium(both control and medium containing H

2O2) was replaced

every 12 hours to prevent a decrease in inducer concentrationApoptosis ending was observed for up to 36 hours (Figure 3)and indicated that primary cells (fibroblasts and cardiomy-ocytes) can survive at 02mM for 36 h

33 Changes in Expression of Apoptosis Indicators Afterapoptosis became significant the RNA of each cell groupwas extracted and reverse-transcribed for real-time PCRanalysis of caspase-9 P53 NF-120581B and RIP expression levels

01

02

04

08

16

DTT2

T M F

Tim

e to

end

of ap

opto

sis in

resp

onse

to h

ydro

gen

pero

xide

and

DTT

(h)

40

30

20

10

0

Figure 3 Time to end of apoptosis in response to hydrogen peroxideand DTT Hydrogen peroxide 01ndash16mM DTT 2mM T 293Tcells M myocardial cells F fibroblasts

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

10

8

6

4

2

0

Expr

essio

n of

apop

tosis

and

necr

opto

sis in

dice

s in293

T ce

lls (h

)

Caspase-9P53

NF-120581BRIP1

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast lowast

lowastlowastlowastlowast

lowastlowast

Figure 4 Expression of apoptosis and necroptosis indices in293T cells Hydrogen peroxide 01ndash16mM DTT 2mM Each barrepresents the means plusmn SD (n = 3) lowastlowast119901 lt 001 lowast119901 lt 005 comparedwith control

In 293T cells (Figure 4) caspase-9 P53 and NF-120581B increasedat all H

2O2concentrations In general expression rose with

increasing concentration but the peak differed for caspase-9 P53 and NK-120581B (06 or 08mM) The trend for RIP wasdistinct RIP was induced at all H

2O2concentrations in

contrast to the expression pattern in fibroblasts (Figure 5) andcardiomyocytes (Figure 6)

Hydrogen peroxide- andDTT-induced changes in fibrob-last expression of apoptosis- and necroptosis-associatedgenes are shown in Figure 5 Compared to 293T cells therewere differences in themagnitude of the expression increasesbut the general trends were similar A notable exception wasthe higher threshold concentration for upregulation of thenecroptosis indicator RIP

Changes in expression also followed similar trends inmyocardial cells (Figure 6) although NF-120581B expression was

4 BioMed Research International

Caspase-9P53

NF-120581BRIP1

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

Expr

essio

n of

apop

tosis

and

necr

opto

sis in

dice

s in

fibro

blas

t (h) 20

15

10

5

0

Figure 5 Expression of apoptosis and necroptosis indices infibroblasts Hydrogen peroxide 01ndash16mM DTT 2mM Each barrepresents the means plusmn SD (n = 3) lowastlowast119901 lt 001 compared withcontrol

Caspase-9P53

NF-120581BRIP1

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

Expr

essio

n of

apop

tosis

and

necr

opto

sis

indi

ces i

n m

yoca

rdia

l cel

ls (h

) 40

30

20

10

0

Figure 6 Expression of apoptosis and necroptosis indices inmyocardial cells Hydrogen peroxide 01ndash16mMDTT 2mM Eachbar represents the means plusmn SD (n = 3) lowastlowast119901 lt 001 lowast119901 lt 005compared with control

lower with no trend for dose-dependence Moreover similarto fibroblasts and in contrast to 293T cells RIP expressionwasnot upregulated by the lowest H

2O2concentrations

4 Discussion

The three cell types examined showed clear differences insensitivity to apoptosis induction by hydrogen peroxide andDTT We speculated that highly proliferative cells wouldshow the strongest apoptosis resistance followed by primarycells and then highly differentiated cells However the exactopposite proved to be the case with 293T cells showingthe fastest apoptosis induction across H

2O2concentrations

as well as the shortest time to substantial induction andthe earliest end of induction compared to fibroblasts andcardiomyocytesMoreover the induction of apoptosis indiceswas accompanied by upregulation of RIP a gene associatedwith necroptosis at all H

2O2concentrations Alternatively

terminally differentiated myocytes with no proliferativecapacity showed minimal induction of apoptosis at concen-trations inducing substantial apoptosisnecroptosis in 293Tcells In addition internal ROS production may be differentfrom one cell type to another and H

2O2produced during

normal cell metabolism and production must be higherin rapidly proliferative cells Therefore H

2O2concentration

used should be chosen carefully according to cell model instudies of apoptosis as the induction range differs markedlyamong cell types

The necroptosis index RIP was induced by low concen-trations of H

2O2(01 and 02mM) only in 293T cells while

RIP induction required 04mM or higher in fibroblasts andcardiomyocytes Thus H

2O2concentrations of 02ndash04mM

are appropriate for studying ldquopurerdquo apoptosis in fibroblastsand cardiomyocytes In contrast very low doses may berequired to study ldquopurerdquo apoptosis in 293T cells Thereforeif an inadequate dosage was used for specific cell types theapoptosis inducing effect of H

2O2would not be observed and

the appropriate dosage for inducing the apoptosis of specificcell types should be determined firstlyThe series of measure-ments conducted here constitute a template for determiningthe optimal H

2O2concentration range for specific analysis of

apoptosis (ie in the absence of necrosis or necroptosis) fora given cell type

41 Efficiency of Hydrogen Peroxide In our experiments ahydrogen peroxide dosage was gt 04mM evoked rapid andrelatively uniform cell death while the extent of cell deathwas highly dose-sensitive le 04mM At 01 and 02mMfibroblasts and cardiomyocytes survived for 36 hours whilesubstantial death of 293T cells was observed Cell lineshave a higher cell death efficiency induced by hydrogenperoxide For all the cells the starting time of apoptosiswas dose-dependent the higher dosage inducer had a fastercell death In this experiment we first wanted to use flowcytometry for assessment but there were some interferencesespecially in primary cells for dregs can be easily stainedand thus interfere with the assessment Therefore we used afluorescence inversion microscope system

42 Factors Controlling Susceptibility to Hydrogen PeroxideIt was found that cell density in a 24-well plate and thegeneration number of 293T cells has a significant impacton the beginning and ending times of apoptosis If the cellline has a high generation or density it would be moresensitive to the induction by hydrogen peroxide So wedecreased the number of the three kinds of cells per wellduring the incubation The beginning and ending timesof apoptosis were used to determine cell susceptibility tohydrogen peroxide Unexpectedly it was found that cell linesare themost sensitive to hydrogen peroxide Even at the lowerdosage hydrogen peroxide may cause the apoptosis of 293Tcells and apoptosis can occur earlier and faster than the other2 kinds of cells

43 Cell Death Type after Induction with Hydrogen PeroxideIn the experiments 4 indices were used to monitor the

BioMed Research International 5

changes in cells after induction with hydrogen peroxide Asjudged by RIP it was found that 293T cells may be involvedin necroptosis even at a low dosage Thus if we want toinduce cell lines into apoptosis we must reduce the actiontime or use DTT to induce apoptosis For myocardial cellsand fibroblasts they tend to be involved in necroptosis only ata higher dosage From the other 3 indices it can be concludedthat 3 kinds of cells have different responses to the inductionby hydrogen peroxide which showed that fibroblasts are notinclined to necroptosis after induction from the expressionof RIP For other indices fibroblasts have a higher expressionof NF-120581B which may indicate that it has had more DNAcorrosion and handicap of transcription 293T cells havea higher expression of P53 indicating a disorder in cellcycle and proliferation The myocardial cells have a higherexpression of all indices which may be a comprehensiveeffect

In the experiments it was found that the cells underwentmorphologic changes after induction by hydrogen peroxideand it is dose-dependent After the comparison of DTTand hydrogen peroxide there are many differences in the 4indices however the trend is basically consistent It was alsofound that the hydrogen peroxide can greatly influence cellmotor ability but after the induction of DTT for 6 hoursthe impulse of some myocardial cells still exists and theapoptosis effect is less than hydrogen peroxide The existingquestion is whether or not hydrogen peroxide has a selectivityin different cell lines or if hydrogen peroxide has a speciesselectivity such as cells from rats

5 Conclusion

Hydrogen peroxide has a high efficiency leading to cell deathHydrogen peroxide causes necroptosis in 293T cells at aconcentration ranging from 01 to 16mMThe cell lines usedin this study were sensitive to hydrogen peroxide In primarycells a concentration gt 04mM may also cause necroptosisA concentration lt 04mM had a tendency to apoptosisPrimary cells can survive in hydrogen peroxide for 36 hoursat a concentration le 02mM Different cells have a differentresponse to induction by hydrogen peroxideThus hydrogenperoxide qualifies as an apoptosis inducer at a specific dosagecorresponding to the specific cell types but researchers donotpay attention to these findings

Ethical Approval

This study was approved by the Animal Care and UseCommittee of Hubei Province China All animal procedureswere performed according to the guidelines developed byChinarsquos Council on Animal Care

Disclosure

The funders had no role in the study design data collection oranalysis decision to publish or the preparation of the paper

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Jinmei Xiang Chunyun Wan and Dingzong Guo conceivedand designed the experiments Jinmei Xiang and ChunyunWan performed the experiments Jinmei Xiang ChunyunWan and Rui Guo analyzed the data Rui Guo contributedregentsmaterialsanalysis tools Jinmei Xiang and ChunyunWan wrote the paper Jinmei Xiang and Chunyun Wancontributed equally to this work

Acknowledgments

Special thanks are due to Li Liang and Li Youwen forapoptosis identification and technical assistance and toZhangGuoxing for his help in cell culture and treatment Thiswork was supported by a preliminary research project grantfrom Huazhong Agricultural University [52209-814112] andthe National Natural Sciences Foundation of China (no31502132)

References

[1] J F Kerr A H Wyllie and A R Currie ldquoApoptosis abasic biological phenomenon with wide-ranging implicationsin tissue kineticsrdquo British Journal of Cancer vol 26 no 4 pp239ndash257 1972

[2] Q Yating Y Yuan ZWei et al ldquoOxidized LDL induces apopto-sis of human retinal pigment epithelium through activation ofERK-BaxBcl-2 signaling pathwaysrdquo Current Eye Research vol40 no 4 pp 415ndash422 2015

[3] L Tartier Y L McCarey J E Biaglow I E Kochevar andK D Held ldquoApoptosis induced by dithiothreitol in HL-60cells shows early activation of caspase 3 and is independent ofmitochondriardquo Cell Death and Differentiation vol 7 no 10 pp1002ndash1010 2000

[4] E R Whittemore D T Loo and C W Cotman ldquoExposure tohydrogen peroxide induces cell death via apoptosis in culturedrat cortical neuronsrdquo NeuroReport vol 5 no 12 pp 1485ndash14881994

[5] HMViola P G Arthur and L CHool ldquoTransient exposure tohydrogen peroxide causes an increase in mitochondria-derivedsuperoxide as a result of sustained alteration in L-type Ca2+channel function in the absence of apoptosis in ventricularmyocytesrdquo Circulation Research vol 100 no 7 pp 1036ndash10442007

[6] H-Y Lin S-C Shen C-W Lin L-Y Yang and Y-C ChenldquoBaicalein inhibition of hydrogen peroxide-induced apopto-sis via ROS-dependent heme oxygenase 1 gene expressionrdquoBiochimica et Biophysica Acta (BBA)mdashMolecular Cell Researchvol 1773 no 7 pp 1073ndash1086 2007

[7] M Singh H Sharma and N Singh ldquoHydrogen peroxideinduces apoptosis in HeLa cells through mitochondrial path-wayrdquoMitochondrion vol 7 no 6 pp 367ndash373 2007

[8] T Satoh N Sakai Y Enokido Y Uchiyama and H HatanakaldquoFree radical-independent protection by nerve growth factorand Bcl-2 of PC12 cells from hydrogen peroxide-triggered

6 BioMed Research International

apoptosisrdquo Journal of Biochemistry vol 120 no 3 pp 540ndash5461996

[9] Y Y Luo M Yin Y H Wu D W He Q Q Wei and C CYin ldquoProtective effects of chlorogenic acid against hydrogenperoxide induced rat nucleus pulposus cells apoptosisrdquo ChineseTraditional Patent Medicine vol 4 pp 656ndash660 2013

[10] J Liu Y Wang W Du et al ldquoWnt1 inhibits hydrogen peroxide-induced apoptosis in mouse cardiac stem cellsrdquo PLoS ONE vol8 no 3 Article ID e58883 2013

[11] L Wu Z Xi R Guo et al ldquoExogenous ARC down-regulatescaspase-3 expression and inhibits apoptosis of broiler chickencardiomyocytes exposed to hydrogen peroxiderdquo Avian Pathol-ogy vol 42 no 1 pp 32ndash37 2013

[12] I Bejarano J Espino A M Marchena et al ldquoMelatoninenhances hydrogen peroxide-induced apoptosis in humanpromyelocytic leukaemia HL-60 cellsrdquo Molecular and CellularBiochemistry vol 353 no 1-2 pp 167ndash176 2011

[13] P Pallepati and D A Averill-Bates ldquoMild thermotoleranceinduced at 40∘C protects HeLa cells against activation ofdeath receptor-mediated apoptosis by hydrogen peroxiderdquo FreeRadical Biology and Medicine vol 50 no 6 pp 667ndash679 2011

[14] Z Li J Zhao Q Li et al ldquoKLF4 promotes hydrogen-peroxide-induced apoptosis of chronic myeloid leukemia cells involvingthe bcl-2bax pathwayrdquo Cell Stress and Chaperones vol 15 no6 pp 905ndash912 2010

[15] G F Ribeiro M Corte-Real and B Johansson ldquoCharacteriza-tion of DNA damage in yeast apoptosis induced by hydrogenperoxide acetic acid and hyperosmotic shockrdquo MolecularBiology of the Cell vol 17 no 10 pp 4584ndash4591 2006

[16] S-J Lee H Ahn K-W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules andTherapeutics vol 20no 5 pp 487ndash491 2012

[17] P Roy E Reavey M Rayne et al ldquoEnhanced sensitivity tohydrogen peroxide-induced apoptosis in Evi1 transformed Rat1fibroblasts due to repression of carbonic anhydrase IIIrdquo TheFEBS Journal vol 277 no 2 pp 441ndash452 2010

[18] A S Ghosh S Dutta and S Raha ldquoHydrogen peroxide-induced apoptosis-like cell death in Entamoeba histolyticardquoParasitology International vol 59 no 2 pp 166ndash172 2010

[19] M L Wurstle M A Laussmann and M Rehm ldquoThe centralrole of initiator caspase-9 in apoptosis signal transduction andthe regulation of its activation and activity on the apoptosomerdquoExperimental Cell Research vol 318 no 11 pp 1213ndash1220 2012

[20] K W McCool and S Miyamoto ldquoDNA damage-dependentNF-120581B activation NEMO turns nuclear signaling inside outrdquoImmunological Reviews vol 246 no 1 pp 311ndash326 2012

[21] T Li N Kon L Jiang et al ldquoTumor suppression in the absenceof p53-mediated cell-cycle arrest apoptosis and senescencerdquoCell vol 149 no 6 pp 1269ndash1283 2012

[22] A Degterev J Hitomi M Germscheid et al ldquoIdentification ofRIP1 kinase as a specific cellular target of necrostatinsrdquo NatureChemical Biology vol 4 no 5 pp 313ndash321 2008

[23] R LDeHaan ldquoDevelopment of form in the embryonic heart anexperimental approachrdquo Circulation vol 35 no 5 pp 821ndash8331967

[24] Z Xing and K A Schat ldquoExpression of cytokine genes inMarekrsquos disease virus-infected chickens and chicken embryofibroblast culturesrdquo Immunology vol 100 no 1 pp 70ndash76 2000

[25] K A Schat and H G Purchase ldquoCell-culture methodsrdquo in ALaboratory Manual for the Isolation and Identification of Avian

Pathogens D E Swayne J R Glisson M W Jackwood J EPearson and VWM Reed Eds p 223 American Associationof Avian Pathologists Kennett Square Pa USA 4th edition1998

[26] CWan J Xiang Y Li andDGuo ldquoDifferential gene expressionpatterns in chicken cardiomyocytes during hydrogen peroxide-induced apoptosisrdquo PLoS ONE vol 11 no 1 Article IDe0147950 2016

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Microbiology

2 BioMed Research International

Table 1 Primers for quantitative real-time PCR

Gene Forward Reverse Thermocycle

FC

Caspase-9 51015840TCAGACATCGTATCCTCCA 51015840AGTCACAGCAGCACA

98∘C3min + 40 times [98∘15 s 61∘C40 s]P53 51015840ACCTGCACTTACTCCCCGGT 51015840TCTTATAGACGGCCACGGCG

NF-120581B 51015840AGGACTTAAAATGGCAGGAGAG 51015840GCTGTTCGTAGTGGTAAGTCTGRIP1 51015840GAACTAGGCTTCAGCAACTCCG 51015840GCAGCCAAAGAGGGCTTTGG120573-actin 51015840GGCACCCAGCACAATGAAG 51015840CCGATCCACACGGAGTACTTG

293T

Caspase-9 51015840TGCTGAGCAGCGAGCTGTT 51015840AGCCTGCCCGCTGGAT

98∘C3min + 40 times [98∘15 s 61∘C40 s]P53 51015840CCACCATCCACTACAACTACAT 51015840CAAACACGGACAGGACCC

NF-120581B 51015840TCTCCCTGGTCACCAAGGAC 51015840TCATAGAAGCCATCCCGGCRIP1 51015840CATGGAAAAGGCGTGATACAC 51015840ACTTCCCTCAGCTCATTGTG120573-actin 51015840GGCACCCAGCACAATGAAG 51015840CCGATCCACACGGAGTACTTG

FC fibroblastscardiomyocytes

To determine if H2O2is a suitable apoptosis inducer for

a given cell type we measured cytochemical and geneticindices of apoptosis and necroptosis under a range ofH2O2doses in three cell types an immortalized cell line

primary fibroblasts and terminally differentiated cardiomy-ocytes These cells were chosen for their distinct proliferativefeatures The 293T immortalized cell line is characterizedby unlimited proliferation and passages while fibroblastsshow limited proliferation and passages and terminallydifferentiated cardiomyocytes show no further proliferationFor convenience both fibroblasts and myocardial cells werederived from chicken embryos

2 Materials and Methods

21 Cell Culture Isolation and culture of chicken embryofibroblasts and myocardial cells followed methods previouslydescribed [23 24] with some modifications Briefly WhiteLeghorn eggs were obtained from Beijing Merial Vital Lab-oratory Animal Technology (Beijing China) At embryonicday 11 (E11) embryos were removed and decapitated in a Petridish filled with Medium 199EBSS (HyClone Logan UtahUSA) supplemented with 3 fetal bovine serum (FBS GibcoGrand Island New York USA) Ventricular tissues and torsoof chicken embryo were isolated for the preparations ofmyocardial cells and CEF and treated with 005 trypsin-EDTA to obtain a cell suspension as described [14 25 26]respectively Specifically the cells of CEF andmyocardial cellswere respectively incubated at 8 times 105 per well in 24-wellplates in growth medium (Medium 199EBSS containing 10FBS) at 37∘C under a 5 CO

2atmosphere Cultures were

washed three times at 8 24 and 48 h to remove dead anddying cellsThe serum concentration in themediumwas thenchanged from growth (10) to maintenance (2) conditions

293T cells at low passage were incubated at 1 times 105cellswell in 24-well plates with Dulbeccorsquos modified Eaglemedium (DMEM) (HyClone Logan Utah USA) containing10 FBS at 37∘C under a 5 CO

2atmosphere After plating

the serum concentrationwas decreased from growth (10) tomaintenance (2) conditions

22 Apoptosis Induction and Assessment Each cell type wasdivided into three groups H

2O2treatment positive control

(DTT treatment) and negative control H2O2groups were

incubated in 01 02 04 08 or 16mM H2O2 the positive

control groups were incubated in 2mM DTT and thenegative control groups received no treatment After the startof exposure cells were examined every 05 h by staining withAOEB to monitor the initiation of apoptosis DAPI stainingwas used to determine the time to substantial apoptosisThen apoptosis times and cell survival rates were determinedby AOEB staining and DAPI staining Finally total RNAwas extracted from cells using Trizol reagent (InvitrogenCarlsbad CA USA) to assess expression levels of apoptosis-and necroptosis-associated genes Reverse transcription wasperformed using a PrimeScript II 1st Strand cDNA synthesiskit (TaKaRa Otsu Shiga Japan) Quantitative real-time PCRwas conducted to evaluate changes in caspase-9 P53 NF-120581Band RIP expression levels using the primers and thermocycleconditions shown in Table 1 Groupmeans were compared byANOVA using SPSS170 software All bar figures were createdby Graphpad Prism 5 software

3 Results

31 Initiation Time of Apoptosis Apoptosis initiation times atdifferent H

2O2doses are shown in Figure 1 for all three cell

types The time to initiation was estimated by AOEB doublestaining every 05 h under an inverted epifluorescencemicro-scope The 293T cells were very sensitive to both DTT- andH2O2-induced apoptosis as indicated by the shorter delays

until initiation compared to the other cell types Alternativelyapoptosis initiation times did not differ significantly betweenmyocardial cells and fibroblasts in response to DTT or H

2O2

32 Significant Differences in Times to Substantial Apoptosisand the End of Apoptosis among Cell Types After apoptosisstarted DAPI staining was used to determine the time atwhich significant apoptosis occurred (Figure 2) At gt 04mMH2O2 apoptosis developed rapidly with little difference

between cell types At le 04mM however the time tosubstantial apoptosis was much more sensitive to H

2O2

BioMed Research International 3Ti

mes

to ap

opto

sis in

itiat

ion

(h)

T M F

01

02

04

08

16

DTT2

10

8

6

4

2

0

Figure 1 Time to apoptosis initiation 293T cells (T) myocardialcells (M) and fibroblasts (F) were treated with a range of hydrogenperoxide concentrations (01ndash16mM) or DTT (2mM positivecontrol)

01

02

04

08

16

DTT2

T M F

Tim

e to

signi

fican

t apo

ptos

is in

duct

ion

for e

ach

cell

type

in re

spon

se to

DTT

or h

ydro

gen

pero

xide

(h)

15

10

5

0

Figure 2 Time to significant apoptosis induction for each cell typein response to DTT or hydrogen peroxide Hydrogen peroxide 01ndash16mMDTT 2mM T 293T cellsMmyocardial cells F fibroblasts

dose and cell type with markedly faster times for 293Tcells particularly at 04mM compared to fibroblasts andcardiomyocytes For the apoptosis ending test the medium(both control and medium containing H

2O2) was replaced

every 12 hours to prevent a decrease in inducer concentrationApoptosis ending was observed for up to 36 hours (Figure 3)and indicated that primary cells (fibroblasts and cardiomy-ocytes) can survive at 02mM for 36 h

33 Changes in Expression of Apoptosis Indicators Afterapoptosis became significant the RNA of each cell groupwas extracted and reverse-transcribed for real-time PCRanalysis of caspase-9 P53 NF-120581B and RIP expression levels

01

02

04

08

16

DTT2

T M F

Tim

e to

end

of ap

opto

sis in

resp

onse

to h

ydro

gen

pero

xide

and

DTT

(h)

40

30

20

10

0

Figure 3 Time to end of apoptosis in response to hydrogen peroxideand DTT Hydrogen peroxide 01ndash16mM DTT 2mM T 293Tcells M myocardial cells F fibroblasts

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

10

8

6

4

2

0

Expr

essio

n of

apop

tosis

and

necr

opto

sis in

dice

s in293

T ce

lls (h

)

Caspase-9P53

NF-120581BRIP1

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast lowast

lowastlowastlowastlowast

lowastlowast

Figure 4 Expression of apoptosis and necroptosis indices in293T cells Hydrogen peroxide 01ndash16mM DTT 2mM Each barrepresents the means plusmn SD (n = 3) lowastlowast119901 lt 001 lowast119901 lt 005 comparedwith control

In 293T cells (Figure 4) caspase-9 P53 and NF-120581B increasedat all H

2O2concentrations In general expression rose with

increasing concentration but the peak differed for caspase-9 P53 and NK-120581B (06 or 08mM) The trend for RIP wasdistinct RIP was induced at all H

2O2concentrations in

contrast to the expression pattern in fibroblasts (Figure 5) andcardiomyocytes (Figure 6)

Hydrogen peroxide- andDTT-induced changes in fibrob-last expression of apoptosis- and necroptosis-associatedgenes are shown in Figure 5 Compared to 293T cells therewere differences in themagnitude of the expression increasesbut the general trends were similar A notable exception wasthe higher threshold concentration for upregulation of thenecroptosis indicator RIP

Changes in expression also followed similar trends inmyocardial cells (Figure 6) although NF-120581B expression was

4 BioMed Research International

Caspase-9P53

NF-120581BRIP1

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

Expr

essio

n of

apop

tosis

and

necr

opto

sis in

dice

s in

fibro

blas

t (h) 20

15

10

5

0

Figure 5 Expression of apoptosis and necroptosis indices infibroblasts Hydrogen peroxide 01ndash16mM DTT 2mM Each barrepresents the means plusmn SD (n = 3) lowastlowast119901 lt 001 compared withcontrol

Caspase-9P53

NF-120581BRIP1

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

Expr

essio

n of

apop

tosis

and

necr

opto

sis

indi

ces i

n m

yoca

rdia

l cel

ls (h

) 40

30

20

10

0

Figure 6 Expression of apoptosis and necroptosis indices inmyocardial cells Hydrogen peroxide 01ndash16mMDTT 2mM Eachbar represents the means plusmn SD (n = 3) lowastlowast119901 lt 001 lowast119901 lt 005compared with control

lower with no trend for dose-dependence Moreover similarto fibroblasts and in contrast to 293T cells RIP expressionwasnot upregulated by the lowest H

2O2concentrations

4 Discussion

The three cell types examined showed clear differences insensitivity to apoptosis induction by hydrogen peroxide andDTT We speculated that highly proliferative cells wouldshow the strongest apoptosis resistance followed by primarycells and then highly differentiated cells However the exactopposite proved to be the case with 293T cells showingthe fastest apoptosis induction across H

2O2concentrations

as well as the shortest time to substantial induction andthe earliest end of induction compared to fibroblasts andcardiomyocytesMoreover the induction of apoptosis indiceswas accompanied by upregulation of RIP a gene associatedwith necroptosis at all H

2O2concentrations Alternatively

terminally differentiated myocytes with no proliferativecapacity showed minimal induction of apoptosis at concen-trations inducing substantial apoptosisnecroptosis in 293Tcells In addition internal ROS production may be differentfrom one cell type to another and H

2O2produced during

normal cell metabolism and production must be higherin rapidly proliferative cells Therefore H

2O2concentration

used should be chosen carefully according to cell model instudies of apoptosis as the induction range differs markedlyamong cell types

The necroptosis index RIP was induced by low concen-trations of H

2O2(01 and 02mM) only in 293T cells while

RIP induction required 04mM or higher in fibroblasts andcardiomyocytes Thus H

2O2concentrations of 02ndash04mM

are appropriate for studying ldquopurerdquo apoptosis in fibroblastsand cardiomyocytes In contrast very low doses may berequired to study ldquopurerdquo apoptosis in 293T cells Thereforeif an inadequate dosage was used for specific cell types theapoptosis inducing effect of H

2O2would not be observed and

the appropriate dosage for inducing the apoptosis of specificcell types should be determined firstlyThe series of measure-ments conducted here constitute a template for determiningthe optimal H

2O2concentration range for specific analysis of

apoptosis (ie in the absence of necrosis or necroptosis) fora given cell type

41 Efficiency of Hydrogen Peroxide In our experiments ahydrogen peroxide dosage was gt 04mM evoked rapid andrelatively uniform cell death while the extent of cell deathwas highly dose-sensitive le 04mM At 01 and 02mMfibroblasts and cardiomyocytes survived for 36 hours whilesubstantial death of 293T cells was observed Cell lineshave a higher cell death efficiency induced by hydrogenperoxide For all the cells the starting time of apoptosiswas dose-dependent the higher dosage inducer had a fastercell death In this experiment we first wanted to use flowcytometry for assessment but there were some interferencesespecially in primary cells for dregs can be easily stainedand thus interfere with the assessment Therefore we used afluorescence inversion microscope system

42 Factors Controlling Susceptibility to Hydrogen PeroxideIt was found that cell density in a 24-well plate and thegeneration number of 293T cells has a significant impacton the beginning and ending times of apoptosis If the cellline has a high generation or density it would be moresensitive to the induction by hydrogen peroxide So wedecreased the number of the three kinds of cells per wellduring the incubation The beginning and ending timesof apoptosis were used to determine cell susceptibility tohydrogen peroxide Unexpectedly it was found that cell linesare themost sensitive to hydrogen peroxide Even at the lowerdosage hydrogen peroxide may cause the apoptosis of 293Tcells and apoptosis can occur earlier and faster than the other2 kinds of cells

43 Cell Death Type after Induction with Hydrogen PeroxideIn the experiments 4 indices were used to monitor the

BioMed Research International 5

changes in cells after induction with hydrogen peroxide Asjudged by RIP it was found that 293T cells may be involvedin necroptosis even at a low dosage Thus if we want toinduce cell lines into apoptosis we must reduce the actiontime or use DTT to induce apoptosis For myocardial cellsand fibroblasts they tend to be involved in necroptosis only ata higher dosage From the other 3 indices it can be concludedthat 3 kinds of cells have different responses to the inductionby hydrogen peroxide which showed that fibroblasts are notinclined to necroptosis after induction from the expressionof RIP For other indices fibroblasts have a higher expressionof NF-120581B which may indicate that it has had more DNAcorrosion and handicap of transcription 293T cells havea higher expression of P53 indicating a disorder in cellcycle and proliferation The myocardial cells have a higherexpression of all indices which may be a comprehensiveeffect

In the experiments it was found that the cells underwentmorphologic changes after induction by hydrogen peroxideand it is dose-dependent After the comparison of DTTand hydrogen peroxide there are many differences in the 4indices however the trend is basically consistent It was alsofound that the hydrogen peroxide can greatly influence cellmotor ability but after the induction of DTT for 6 hoursthe impulse of some myocardial cells still exists and theapoptosis effect is less than hydrogen peroxide The existingquestion is whether or not hydrogen peroxide has a selectivityin different cell lines or if hydrogen peroxide has a speciesselectivity such as cells from rats

5 Conclusion

Hydrogen peroxide has a high efficiency leading to cell deathHydrogen peroxide causes necroptosis in 293T cells at aconcentration ranging from 01 to 16mMThe cell lines usedin this study were sensitive to hydrogen peroxide In primarycells a concentration gt 04mM may also cause necroptosisA concentration lt 04mM had a tendency to apoptosisPrimary cells can survive in hydrogen peroxide for 36 hoursat a concentration le 02mM Different cells have a differentresponse to induction by hydrogen peroxideThus hydrogenperoxide qualifies as an apoptosis inducer at a specific dosagecorresponding to the specific cell types but researchers donotpay attention to these findings

Ethical Approval

This study was approved by the Animal Care and UseCommittee of Hubei Province China All animal procedureswere performed according to the guidelines developed byChinarsquos Council on Animal Care

Disclosure

The funders had no role in the study design data collection oranalysis decision to publish or the preparation of the paper

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Jinmei Xiang Chunyun Wan and Dingzong Guo conceivedand designed the experiments Jinmei Xiang and ChunyunWan performed the experiments Jinmei Xiang ChunyunWan and Rui Guo analyzed the data Rui Guo contributedregentsmaterialsanalysis tools Jinmei Xiang and ChunyunWan wrote the paper Jinmei Xiang and Chunyun Wancontributed equally to this work

Acknowledgments

Special thanks are due to Li Liang and Li Youwen forapoptosis identification and technical assistance and toZhangGuoxing for his help in cell culture and treatment Thiswork was supported by a preliminary research project grantfrom Huazhong Agricultural University [52209-814112] andthe National Natural Sciences Foundation of China (no31502132)

References

[1] J F Kerr A H Wyllie and A R Currie ldquoApoptosis abasic biological phenomenon with wide-ranging implicationsin tissue kineticsrdquo British Journal of Cancer vol 26 no 4 pp239ndash257 1972

[2] Q Yating Y Yuan ZWei et al ldquoOxidized LDL induces apopto-sis of human retinal pigment epithelium through activation ofERK-BaxBcl-2 signaling pathwaysrdquo Current Eye Research vol40 no 4 pp 415ndash422 2015

[3] L Tartier Y L McCarey J E Biaglow I E Kochevar andK D Held ldquoApoptosis induced by dithiothreitol in HL-60cells shows early activation of caspase 3 and is independent ofmitochondriardquo Cell Death and Differentiation vol 7 no 10 pp1002ndash1010 2000

[4] E R Whittemore D T Loo and C W Cotman ldquoExposure tohydrogen peroxide induces cell death via apoptosis in culturedrat cortical neuronsrdquo NeuroReport vol 5 no 12 pp 1485ndash14881994

[5] HMViola P G Arthur and L CHool ldquoTransient exposure tohydrogen peroxide causes an increase in mitochondria-derivedsuperoxide as a result of sustained alteration in L-type Ca2+channel function in the absence of apoptosis in ventricularmyocytesrdquo Circulation Research vol 100 no 7 pp 1036ndash10442007

[6] H-Y Lin S-C Shen C-W Lin L-Y Yang and Y-C ChenldquoBaicalein inhibition of hydrogen peroxide-induced apopto-sis via ROS-dependent heme oxygenase 1 gene expressionrdquoBiochimica et Biophysica Acta (BBA)mdashMolecular Cell Researchvol 1773 no 7 pp 1073ndash1086 2007

[7] M Singh H Sharma and N Singh ldquoHydrogen peroxideinduces apoptosis in HeLa cells through mitochondrial path-wayrdquoMitochondrion vol 7 no 6 pp 367ndash373 2007

[8] T Satoh N Sakai Y Enokido Y Uchiyama and H HatanakaldquoFree radical-independent protection by nerve growth factorand Bcl-2 of PC12 cells from hydrogen peroxide-triggered

6 BioMed Research International

apoptosisrdquo Journal of Biochemistry vol 120 no 3 pp 540ndash5461996

[9] Y Y Luo M Yin Y H Wu D W He Q Q Wei and C CYin ldquoProtective effects of chlorogenic acid against hydrogenperoxide induced rat nucleus pulposus cells apoptosisrdquo ChineseTraditional Patent Medicine vol 4 pp 656ndash660 2013

[10] J Liu Y Wang W Du et al ldquoWnt1 inhibits hydrogen peroxide-induced apoptosis in mouse cardiac stem cellsrdquo PLoS ONE vol8 no 3 Article ID e58883 2013

[11] L Wu Z Xi R Guo et al ldquoExogenous ARC down-regulatescaspase-3 expression and inhibits apoptosis of broiler chickencardiomyocytes exposed to hydrogen peroxiderdquo Avian Pathol-ogy vol 42 no 1 pp 32ndash37 2013

[12] I Bejarano J Espino A M Marchena et al ldquoMelatoninenhances hydrogen peroxide-induced apoptosis in humanpromyelocytic leukaemia HL-60 cellsrdquo Molecular and CellularBiochemistry vol 353 no 1-2 pp 167ndash176 2011

[13] P Pallepati and D A Averill-Bates ldquoMild thermotoleranceinduced at 40∘C protects HeLa cells against activation ofdeath receptor-mediated apoptosis by hydrogen peroxiderdquo FreeRadical Biology and Medicine vol 50 no 6 pp 667ndash679 2011

[14] Z Li J Zhao Q Li et al ldquoKLF4 promotes hydrogen-peroxide-induced apoptosis of chronic myeloid leukemia cells involvingthe bcl-2bax pathwayrdquo Cell Stress and Chaperones vol 15 no6 pp 905ndash912 2010

[15] G F Ribeiro M Corte-Real and B Johansson ldquoCharacteriza-tion of DNA damage in yeast apoptosis induced by hydrogenperoxide acetic acid and hyperosmotic shockrdquo MolecularBiology of the Cell vol 17 no 10 pp 4584ndash4591 2006

[16] S-J Lee H Ahn K-W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules andTherapeutics vol 20no 5 pp 487ndash491 2012

[17] P Roy E Reavey M Rayne et al ldquoEnhanced sensitivity tohydrogen peroxide-induced apoptosis in Evi1 transformed Rat1fibroblasts due to repression of carbonic anhydrase IIIrdquo TheFEBS Journal vol 277 no 2 pp 441ndash452 2010

[18] A S Ghosh S Dutta and S Raha ldquoHydrogen peroxide-induced apoptosis-like cell death in Entamoeba histolyticardquoParasitology International vol 59 no 2 pp 166ndash172 2010

[19] M L Wurstle M A Laussmann and M Rehm ldquoThe centralrole of initiator caspase-9 in apoptosis signal transduction andthe regulation of its activation and activity on the apoptosomerdquoExperimental Cell Research vol 318 no 11 pp 1213ndash1220 2012

[20] K W McCool and S Miyamoto ldquoDNA damage-dependentNF-120581B activation NEMO turns nuclear signaling inside outrdquoImmunological Reviews vol 246 no 1 pp 311ndash326 2012

[21] T Li N Kon L Jiang et al ldquoTumor suppression in the absenceof p53-mediated cell-cycle arrest apoptosis and senescencerdquoCell vol 149 no 6 pp 1269ndash1283 2012

[22] A Degterev J Hitomi M Germscheid et al ldquoIdentification ofRIP1 kinase as a specific cellular target of necrostatinsrdquo NatureChemical Biology vol 4 no 5 pp 313ndash321 2008

[23] R LDeHaan ldquoDevelopment of form in the embryonic heart anexperimental approachrdquo Circulation vol 35 no 5 pp 821ndash8331967

[24] Z Xing and K A Schat ldquoExpression of cytokine genes inMarekrsquos disease virus-infected chickens and chicken embryofibroblast culturesrdquo Immunology vol 100 no 1 pp 70ndash76 2000

[25] K A Schat and H G Purchase ldquoCell-culture methodsrdquo in ALaboratory Manual for the Isolation and Identification of Avian

Pathogens D E Swayne J R Glisson M W Jackwood J EPearson and VWM Reed Eds p 223 American Associationof Avian Pathologists Kennett Square Pa USA 4th edition1998

[26] CWan J Xiang Y Li andDGuo ldquoDifferential gene expressionpatterns in chicken cardiomyocytes during hydrogen peroxide-induced apoptosisrdquo PLoS ONE vol 11 no 1 Article IDe0147950 2016

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3Ti

mes

to ap

opto

sis in

itiat

ion

(h)

T M F

01

02

04

08

16

DTT2

10

8

6

4

2

0

Figure 1 Time to apoptosis initiation 293T cells (T) myocardialcells (M) and fibroblasts (F) were treated with a range of hydrogenperoxide concentrations (01ndash16mM) or DTT (2mM positivecontrol)

01

02

04

08

16

DTT2

T M F

Tim

e to

signi

fican

t apo

ptos

is in

duct

ion

for e

ach

cell

type

in re

spon

se to

DTT

or h

ydro

gen

pero

xide

(h)

15

10

5

0

Figure 2 Time to significant apoptosis induction for each cell typein response to DTT or hydrogen peroxide Hydrogen peroxide 01ndash16mMDTT 2mM T 293T cellsMmyocardial cells F fibroblasts

dose and cell type with markedly faster times for 293Tcells particularly at 04mM compared to fibroblasts andcardiomyocytes For the apoptosis ending test the medium(both control and medium containing H

2O2) was replaced

every 12 hours to prevent a decrease in inducer concentrationApoptosis ending was observed for up to 36 hours (Figure 3)and indicated that primary cells (fibroblasts and cardiomy-ocytes) can survive at 02mM for 36 h

33 Changes in Expression of Apoptosis Indicators Afterapoptosis became significant the RNA of each cell groupwas extracted and reverse-transcribed for real-time PCRanalysis of caspase-9 P53 NF-120581B and RIP expression levels

01

02

04

08

16

DTT2

T M F

Tim

e to

end

of ap

opto

sis in

resp

onse

to h

ydro

gen

pero

xide

and

DTT

(h)

40

30

20

10

0

Figure 3 Time to end of apoptosis in response to hydrogen peroxideand DTT Hydrogen peroxide 01ndash16mM DTT 2mM T 293Tcells M myocardial cells F fibroblasts

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

10

8

6

4

2

0

Expr

essio

n of

apop

tosis

and

necr

opto

sis in

dice

s in293

T ce

lls (h

)

Caspase-9P53

NF-120581BRIP1

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowast lowast

lowastlowastlowastlowast

lowastlowast

Figure 4 Expression of apoptosis and necroptosis indices in293T cells Hydrogen peroxide 01ndash16mM DTT 2mM Each barrepresents the means plusmn SD (n = 3) lowastlowast119901 lt 001 lowast119901 lt 005 comparedwith control

In 293T cells (Figure 4) caspase-9 P53 and NF-120581B increasedat all H

2O2concentrations In general expression rose with

increasing concentration but the peak differed for caspase-9 P53 and NK-120581B (06 or 08mM) The trend for RIP wasdistinct RIP was induced at all H

2O2concentrations in

contrast to the expression pattern in fibroblasts (Figure 5) andcardiomyocytes (Figure 6)

Hydrogen peroxide- andDTT-induced changes in fibrob-last expression of apoptosis- and necroptosis-associatedgenes are shown in Figure 5 Compared to 293T cells therewere differences in themagnitude of the expression increasesbut the general trends were similar A notable exception wasthe higher threshold concentration for upregulation of thenecroptosis indicator RIP

Changes in expression also followed similar trends inmyocardial cells (Figure 6) although NF-120581B expression was

4 BioMed Research International

Caspase-9P53

NF-120581BRIP1

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

Expr

essio

n of

apop

tosis

and

necr

opto

sis in

dice

s in

fibro

blas

t (h) 20

15

10

5

0

Figure 5 Expression of apoptosis and necroptosis indices infibroblasts Hydrogen peroxide 01ndash16mM DTT 2mM Each barrepresents the means plusmn SD (n = 3) lowastlowast119901 lt 001 compared withcontrol

Caspase-9P53

NF-120581BRIP1

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

Expr

essio

n of

apop

tosis

and

necr

opto

sis

indi

ces i

n m

yoca

rdia

l cel

ls (h

) 40

30

20

10

0

Figure 6 Expression of apoptosis and necroptosis indices inmyocardial cells Hydrogen peroxide 01ndash16mMDTT 2mM Eachbar represents the means plusmn SD (n = 3) lowastlowast119901 lt 001 lowast119901 lt 005compared with control

lower with no trend for dose-dependence Moreover similarto fibroblasts and in contrast to 293T cells RIP expressionwasnot upregulated by the lowest H

2O2concentrations

4 Discussion

The three cell types examined showed clear differences insensitivity to apoptosis induction by hydrogen peroxide andDTT We speculated that highly proliferative cells wouldshow the strongest apoptosis resistance followed by primarycells and then highly differentiated cells However the exactopposite proved to be the case with 293T cells showingthe fastest apoptosis induction across H

2O2concentrations

as well as the shortest time to substantial induction andthe earliest end of induction compared to fibroblasts andcardiomyocytesMoreover the induction of apoptosis indiceswas accompanied by upregulation of RIP a gene associatedwith necroptosis at all H

2O2concentrations Alternatively

terminally differentiated myocytes with no proliferativecapacity showed minimal induction of apoptosis at concen-trations inducing substantial apoptosisnecroptosis in 293Tcells In addition internal ROS production may be differentfrom one cell type to another and H

2O2produced during

normal cell metabolism and production must be higherin rapidly proliferative cells Therefore H

2O2concentration

used should be chosen carefully according to cell model instudies of apoptosis as the induction range differs markedlyamong cell types

The necroptosis index RIP was induced by low concen-trations of H

2O2(01 and 02mM) only in 293T cells while

RIP induction required 04mM or higher in fibroblasts andcardiomyocytes Thus H

2O2concentrations of 02ndash04mM

are appropriate for studying ldquopurerdquo apoptosis in fibroblastsand cardiomyocytes In contrast very low doses may berequired to study ldquopurerdquo apoptosis in 293T cells Thereforeif an inadequate dosage was used for specific cell types theapoptosis inducing effect of H

2O2would not be observed and

the appropriate dosage for inducing the apoptosis of specificcell types should be determined firstlyThe series of measure-ments conducted here constitute a template for determiningthe optimal H

2O2concentration range for specific analysis of

apoptosis (ie in the absence of necrosis or necroptosis) fora given cell type

41 Efficiency of Hydrogen Peroxide In our experiments ahydrogen peroxide dosage was gt 04mM evoked rapid andrelatively uniform cell death while the extent of cell deathwas highly dose-sensitive le 04mM At 01 and 02mMfibroblasts and cardiomyocytes survived for 36 hours whilesubstantial death of 293T cells was observed Cell lineshave a higher cell death efficiency induced by hydrogenperoxide For all the cells the starting time of apoptosiswas dose-dependent the higher dosage inducer had a fastercell death In this experiment we first wanted to use flowcytometry for assessment but there were some interferencesespecially in primary cells for dregs can be easily stainedand thus interfere with the assessment Therefore we used afluorescence inversion microscope system

42 Factors Controlling Susceptibility to Hydrogen PeroxideIt was found that cell density in a 24-well plate and thegeneration number of 293T cells has a significant impacton the beginning and ending times of apoptosis If the cellline has a high generation or density it would be moresensitive to the induction by hydrogen peroxide So wedecreased the number of the three kinds of cells per wellduring the incubation The beginning and ending timesof apoptosis were used to determine cell susceptibility tohydrogen peroxide Unexpectedly it was found that cell linesare themost sensitive to hydrogen peroxide Even at the lowerdosage hydrogen peroxide may cause the apoptosis of 293Tcells and apoptosis can occur earlier and faster than the other2 kinds of cells

43 Cell Death Type after Induction with Hydrogen PeroxideIn the experiments 4 indices were used to monitor the

BioMed Research International 5

changes in cells after induction with hydrogen peroxide Asjudged by RIP it was found that 293T cells may be involvedin necroptosis even at a low dosage Thus if we want toinduce cell lines into apoptosis we must reduce the actiontime or use DTT to induce apoptosis For myocardial cellsand fibroblasts they tend to be involved in necroptosis only ata higher dosage From the other 3 indices it can be concludedthat 3 kinds of cells have different responses to the inductionby hydrogen peroxide which showed that fibroblasts are notinclined to necroptosis after induction from the expressionof RIP For other indices fibroblasts have a higher expressionof NF-120581B which may indicate that it has had more DNAcorrosion and handicap of transcription 293T cells havea higher expression of P53 indicating a disorder in cellcycle and proliferation The myocardial cells have a higherexpression of all indices which may be a comprehensiveeffect

In the experiments it was found that the cells underwentmorphologic changes after induction by hydrogen peroxideand it is dose-dependent After the comparison of DTTand hydrogen peroxide there are many differences in the 4indices however the trend is basically consistent It was alsofound that the hydrogen peroxide can greatly influence cellmotor ability but after the induction of DTT for 6 hoursthe impulse of some myocardial cells still exists and theapoptosis effect is less than hydrogen peroxide The existingquestion is whether or not hydrogen peroxide has a selectivityin different cell lines or if hydrogen peroxide has a speciesselectivity such as cells from rats

5 Conclusion

Hydrogen peroxide has a high efficiency leading to cell deathHydrogen peroxide causes necroptosis in 293T cells at aconcentration ranging from 01 to 16mMThe cell lines usedin this study were sensitive to hydrogen peroxide In primarycells a concentration gt 04mM may also cause necroptosisA concentration lt 04mM had a tendency to apoptosisPrimary cells can survive in hydrogen peroxide for 36 hoursat a concentration le 02mM Different cells have a differentresponse to induction by hydrogen peroxideThus hydrogenperoxide qualifies as an apoptosis inducer at a specific dosagecorresponding to the specific cell types but researchers donotpay attention to these findings

Ethical Approval

This study was approved by the Animal Care and UseCommittee of Hubei Province China All animal procedureswere performed according to the guidelines developed byChinarsquos Council on Animal Care

Disclosure

The funders had no role in the study design data collection oranalysis decision to publish or the preparation of the paper

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Jinmei Xiang Chunyun Wan and Dingzong Guo conceivedand designed the experiments Jinmei Xiang and ChunyunWan performed the experiments Jinmei Xiang ChunyunWan and Rui Guo analyzed the data Rui Guo contributedregentsmaterialsanalysis tools Jinmei Xiang and ChunyunWan wrote the paper Jinmei Xiang and Chunyun Wancontributed equally to this work

Acknowledgments

Special thanks are due to Li Liang and Li Youwen forapoptosis identification and technical assistance and toZhangGuoxing for his help in cell culture and treatment Thiswork was supported by a preliminary research project grantfrom Huazhong Agricultural University [52209-814112] andthe National Natural Sciences Foundation of China (no31502132)

References

[1] J F Kerr A H Wyllie and A R Currie ldquoApoptosis abasic biological phenomenon with wide-ranging implicationsin tissue kineticsrdquo British Journal of Cancer vol 26 no 4 pp239ndash257 1972

[2] Q Yating Y Yuan ZWei et al ldquoOxidized LDL induces apopto-sis of human retinal pigment epithelium through activation ofERK-BaxBcl-2 signaling pathwaysrdquo Current Eye Research vol40 no 4 pp 415ndash422 2015

[3] L Tartier Y L McCarey J E Biaglow I E Kochevar andK D Held ldquoApoptosis induced by dithiothreitol in HL-60cells shows early activation of caspase 3 and is independent ofmitochondriardquo Cell Death and Differentiation vol 7 no 10 pp1002ndash1010 2000

[4] E R Whittemore D T Loo and C W Cotman ldquoExposure tohydrogen peroxide induces cell death via apoptosis in culturedrat cortical neuronsrdquo NeuroReport vol 5 no 12 pp 1485ndash14881994

[5] HMViola P G Arthur and L CHool ldquoTransient exposure tohydrogen peroxide causes an increase in mitochondria-derivedsuperoxide as a result of sustained alteration in L-type Ca2+channel function in the absence of apoptosis in ventricularmyocytesrdquo Circulation Research vol 100 no 7 pp 1036ndash10442007

[6] H-Y Lin S-C Shen C-W Lin L-Y Yang and Y-C ChenldquoBaicalein inhibition of hydrogen peroxide-induced apopto-sis via ROS-dependent heme oxygenase 1 gene expressionrdquoBiochimica et Biophysica Acta (BBA)mdashMolecular Cell Researchvol 1773 no 7 pp 1073ndash1086 2007

[7] M Singh H Sharma and N Singh ldquoHydrogen peroxideinduces apoptosis in HeLa cells through mitochondrial path-wayrdquoMitochondrion vol 7 no 6 pp 367ndash373 2007

[8] T Satoh N Sakai Y Enokido Y Uchiyama and H HatanakaldquoFree radical-independent protection by nerve growth factorand Bcl-2 of PC12 cells from hydrogen peroxide-triggered

6 BioMed Research International

apoptosisrdquo Journal of Biochemistry vol 120 no 3 pp 540ndash5461996

[9] Y Y Luo M Yin Y H Wu D W He Q Q Wei and C CYin ldquoProtective effects of chlorogenic acid against hydrogenperoxide induced rat nucleus pulposus cells apoptosisrdquo ChineseTraditional Patent Medicine vol 4 pp 656ndash660 2013

[10] J Liu Y Wang W Du et al ldquoWnt1 inhibits hydrogen peroxide-induced apoptosis in mouse cardiac stem cellsrdquo PLoS ONE vol8 no 3 Article ID e58883 2013

[11] L Wu Z Xi R Guo et al ldquoExogenous ARC down-regulatescaspase-3 expression and inhibits apoptosis of broiler chickencardiomyocytes exposed to hydrogen peroxiderdquo Avian Pathol-ogy vol 42 no 1 pp 32ndash37 2013

[12] I Bejarano J Espino A M Marchena et al ldquoMelatoninenhances hydrogen peroxide-induced apoptosis in humanpromyelocytic leukaemia HL-60 cellsrdquo Molecular and CellularBiochemistry vol 353 no 1-2 pp 167ndash176 2011

[13] P Pallepati and D A Averill-Bates ldquoMild thermotoleranceinduced at 40∘C protects HeLa cells against activation ofdeath receptor-mediated apoptosis by hydrogen peroxiderdquo FreeRadical Biology and Medicine vol 50 no 6 pp 667ndash679 2011

[14] Z Li J Zhao Q Li et al ldquoKLF4 promotes hydrogen-peroxide-induced apoptosis of chronic myeloid leukemia cells involvingthe bcl-2bax pathwayrdquo Cell Stress and Chaperones vol 15 no6 pp 905ndash912 2010

[15] G F Ribeiro M Corte-Real and B Johansson ldquoCharacteriza-tion of DNA damage in yeast apoptosis induced by hydrogenperoxide acetic acid and hyperosmotic shockrdquo MolecularBiology of the Cell vol 17 no 10 pp 4584ndash4591 2006

[16] S-J Lee H Ahn K-W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules andTherapeutics vol 20no 5 pp 487ndash491 2012

[17] P Roy E Reavey M Rayne et al ldquoEnhanced sensitivity tohydrogen peroxide-induced apoptosis in Evi1 transformed Rat1fibroblasts due to repression of carbonic anhydrase IIIrdquo TheFEBS Journal vol 277 no 2 pp 441ndash452 2010

[18] A S Ghosh S Dutta and S Raha ldquoHydrogen peroxide-induced apoptosis-like cell death in Entamoeba histolyticardquoParasitology International vol 59 no 2 pp 166ndash172 2010

[19] M L Wurstle M A Laussmann and M Rehm ldquoThe centralrole of initiator caspase-9 in apoptosis signal transduction andthe regulation of its activation and activity on the apoptosomerdquoExperimental Cell Research vol 318 no 11 pp 1213ndash1220 2012

[20] K W McCool and S Miyamoto ldquoDNA damage-dependentNF-120581B activation NEMO turns nuclear signaling inside outrdquoImmunological Reviews vol 246 no 1 pp 311ndash326 2012

[21] T Li N Kon L Jiang et al ldquoTumor suppression in the absenceof p53-mediated cell-cycle arrest apoptosis and senescencerdquoCell vol 149 no 6 pp 1269ndash1283 2012

[22] A Degterev J Hitomi M Germscheid et al ldquoIdentification ofRIP1 kinase as a specific cellular target of necrostatinsrdquo NatureChemical Biology vol 4 no 5 pp 313ndash321 2008

[23] R LDeHaan ldquoDevelopment of form in the embryonic heart anexperimental approachrdquo Circulation vol 35 no 5 pp 821ndash8331967

[24] Z Xing and K A Schat ldquoExpression of cytokine genes inMarekrsquos disease virus-infected chickens and chicken embryofibroblast culturesrdquo Immunology vol 100 no 1 pp 70ndash76 2000

[25] K A Schat and H G Purchase ldquoCell-culture methodsrdquo in ALaboratory Manual for the Isolation and Identification of Avian

Pathogens D E Swayne J R Glisson M W Jackwood J EPearson and VWM Reed Eds p 223 American Associationof Avian Pathologists Kennett Square Pa USA 4th edition1998

[26] CWan J Xiang Y Li andDGuo ldquoDifferential gene expressionpatterns in chicken cardiomyocytes during hydrogen peroxide-induced apoptosisrdquo PLoS ONE vol 11 no 1 Article IDe0147950 2016

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

Caspase-9P53

NF-120581BRIP1

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowast

Expr

essio

n of

apop

tosis

and

necr

opto

sis in

dice

s in

fibro

blas

t (h) 20

15

10

5

0

Figure 5 Expression of apoptosis and necroptosis indices infibroblasts Hydrogen peroxide 01ndash16mM DTT 2mM Each barrepresents the means plusmn SD (n = 3) lowastlowast119901 lt 001 compared withcontrol

Caspase-9P53

NF-120581BRIP1

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

01 C

02

04

08

16

DTT

2

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowast

lowastlowastlowast

Expr

essio

n of

apop

tosis

and

necr

opto

sis

indi

ces i

n m

yoca

rdia

l cel

ls (h

) 40

30

20

10

0

Figure 6 Expression of apoptosis and necroptosis indices inmyocardial cells Hydrogen peroxide 01ndash16mMDTT 2mM Eachbar represents the means plusmn SD (n = 3) lowastlowast119901 lt 001 lowast119901 lt 005compared with control

lower with no trend for dose-dependence Moreover similarto fibroblasts and in contrast to 293T cells RIP expressionwasnot upregulated by the lowest H

2O2concentrations

4 Discussion

The three cell types examined showed clear differences insensitivity to apoptosis induction by hydrogen peroxide andDTT We speculated that highly proliferative cells wouldshow the strongest apoptosis resistance followed by primarycells and then highly differentiated cells However the exactopposite proved to be the case with 293T cells showingthe fastest apoptosis induction across H

2O2concentrations

as well as the shortest time to substantial induction andthe earliest end of induction compared to fibroblasts andcardiomyocytesMoreover the induction of apoptosis indiceswas accompanied by upregulation of RIP a gene associatedwith necroptosis at all H

2O2concentrations Alternatively

terminally differentiated myocytes with no proliferativecapacity showed minimal induction of apoptosis at concen-trations inducing substantial apoptosisnecroptosis in 293Tcells In addition internal ROS production may be differentfrom one cell type to another and H

2O2produced during

normal cell metabolism and production must be higherin rapidly proliferative cells Therefore H

2O2concentration

used should be chosen carefully according to cell model instudies of apoptosis as the induction range differs markedlyamong cell types

The necroptosis index RIP was induced by low concen-trations of H

2O2(01 and 02mM) only in 293T cells while

RIP induction required 04mM or higher in fibroblasts andcardiomyocytes Thus H

2O2concentrations of 02ndash04mM

are appropriate for studying ldquopurerdquo apoptosis in fibroblastsand cardiomyocytes In contrast very low doses may berequired to study ldquopurerdquo apoptosis in 293T cells Thereforeif an inadequate dosage was used for specific cell types theapoptosis inducing effect of H

2O2would not be observed and

the appropriate dosage for inducing the apoptosis of specificcell types should be determined firstlyThe series of measure-ments conducted here constitute a template for determiningthe optimal H

2O2concentration range for specific analysis of

apoptosis (ie in the absence of necrosis or necroptosis) fora given cell type

41 Efficiency of Hydrogen Peroxide In our experiments ahydrogen peroxide dosage was gt 04mM evoked rapid andrelatively uniform cell death while the extent of cell deathwas highly dose-sensitive le 04mM At 01 and 02mMfibroblasts and cardiomyocytes survived for 36 hours whilesubstantial death of 293T cells was observed Cell lineshave a higher cell death efficiency induced by hydrogenperoxide For all the cells the starting time of apoptosiswas dose-dependent the higher dosage inducer had a fastercell death In this experiment we first wanted to use flowcytometry for assessment but there were some interferencesespecially in primary cells for dregs can be easily stainedand thus interfere with the assessment Therefore we used afluorescence inversion microscope system

42 Factors Controlling Susceptibility to Hydrogen PeroxideIt was found that cell density in a 24-well plate and thegeneration number of 293T cells has a significant impacton the beginning and ending times of apoptosis If the cellline has a high generation or density it would be moresensitive to the induction by hydrogen peroxide So wedecreased the number of the three kinds of cells per wellduring the incubation The beginning and ending timesof apoptosis were used to determine cell susceptibility tohydrogen peroxide Unexpectedly it was found that cell linesare themost sensitive to hydrogen peroxide Even at the lowerdosage hydrogen peroxide may cause the apoptosis of 293Tcells and apoptosis can occur earlier and faster than the other2 kinds of cells

43 Cell Death Type after Induction with Hydrogen PeroxideIn the experiments 4 indices were used to monitor the

BioMed Research International 5

changes in cells after induction with hydrogen peroxide Asjudged by RIP it was found that 293T cells may be involvedin necroptosis even at a low dosage Thus if we want toinduce cell lines into apoptosis we must reduce the actiontime or use DTT to induce apoptosis For myocardial cellsand fibroblasts they tend to be involved in necroptosis only ata higher dosage From the other 3 indices it can be concludedthat 3 kinds of cells have different responses to the inductionby hydrogen peroxide which showed that fibroblasts are notinclined to necroptosis after induction from the expressionof RIP For other indices fibroblasts have a higher expressionof NF-120581B which may indicate that it has had more DNAcorrosion and handicap of transcription 293T cells havea higher expression of P53 indicating a disorder in cellcycle and proliferation The myocardial cells have a higherexpression of all indices which may be a comprehensiveeffect

In the experiments it was found that the cells underwentmorphologic changes after induction by hydrogen peroxideand it is dose-dependent After the comparison of DTTand hydrogen peroxide there are many differences in the 4indices however the trend is basically consistent It was alsofound that the hydrogen peroxide can greatly influence cellmotor ability but after the induction of DTT for 6 hoursthe impulse of some myocardial cells still exists and theapoptosis effect is less than hydrogen peroxide The existingquestion is whether or not hydrogen peroxide has a selectivityin different cell lines or if hydrogen peroxide has a speciesselectivity such as cells from rats

5 Conclusion

Hydrogen peroxide has a high efficiency leading to cell deathHydrogen peroxide causes necroptosis in 293T cells at aconcentration ranging from 01 to 16mMThe cell lines usedin this study were sensitive to hydrogen peroxide In primarycells a concentration gt 04mM may also cause necroptosisA concentration lt 04mM had a tendency to apoptosisPrimary cells can survive in hydrogen peroxide for 36 hoursat a concentration le 02mM Different cells have a differentresponse to induction by hydrogen peroxideThus hydrogenperoxide qualifies as an apoptosis inducer at a specific dosagecorresponding to the specific cell types but researchers donotpay attention to these findings

Ethical Approval

This study was approved by the Animal Care and UseCommittee of Hubei Province China All animal procedureswere performed according to the guidelines developed byChinarsquos Council on Animal Care

Disclosure

The funders had no role in the study design data collection oranalysis decision to publish or the preparation of the paper

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Jinmei Xiang Chunyun Wan and Dingzong Guo conceivedand designed the experiments Jinmei Xiang and ChunyunWan performed the experiments Jinmei Xiang ChunyunWan and Rui Guo analyzed the data Rui Guo contributedregentsmaterialsanalysis tools Jinmei Xiang and ChunyunWan wrote the paper Jinmei Xiang and Chunyun Wancontributed equally to this work

Acknowledgments

Special thanks are due to Li Liang and Li Youwen forapoptosis identification and technical assistance and toZhangGuoxing for his help in cell culture and treatment Thiswork was supported by a preliminary research project grantfrom Huazhong Agricultural University [52209-814112] andthe National Natural Sciences Foundation of China (no31502132)

References

[1] J F Kerr A H Wyllie and A R Currie ldquoApoptosis abasic biological phenomenon with wide-ranging implicationsin tissue kineticsrdquo British Journal of Cancer vol 26 no 4 pp239ndash257 1972

[2] Q Yating Y Yuan ZWei et al ldquoOxidized LDL induces apopto-sis of human retinal pigment epithelium through activation ofERK-BaxBcl-2 signaling pathwaysrdquo Current Eye Research vol40 no 4 pp 415ndash422 2015

[3] L Tartier Y L McCarey J E Biaglow I E Kochevar andK D Held ldquoApoptosis induced by dithiothreitol in HL-60cells shows early activation of caspase 3 and is independent ofmitochondriardquo Cell Death and Differentiation vol 7 no 10 pp1002ndash1010 2000

[4] E R Whittemore D T Loo and C W Cotman ldquoExposure tohydrogen peroxide induces cell death via apoptosis in culturedrat cortical neuronsrdquo NeuroReport vol 5 no 12 pp 1485ndash14881994

[5] HMViola P G Arthur and L CHool ldquoTransient exposure tohydrogen peroxide causes an increase in mitochondria-derivedsuperoxide as a result of sustained alteration in L-type Ca2+channel function in the absence of apoptosis in ventricularmyocytesrdquo Circulation Research vol 100 no 7 pp 1036ndash10442007

[6] H-Y Lin S-C Shen C-W Lin L-Y Yang and Y-C ChenldquoBaicalein inhibition of hydrogen peroxide-induced apopto-sis via ROS-dependent heme oxygenase 1 gene expressionrdquoBiochimica et Biophysica Acta (BBA)mdashMolecular Cell Researchvol 1773 no 7 pp 1073ndash1086 2007

[7] M Singh H Sharma and N Singh ldquoHydrogen peroxideinduces apoptosis in HeLa cells through mitochondrial path-wayrdquoMitochondrion vol 7 no 6 pp 367ndash373 2007

[8] T Satoh N Sakai Y Enokido Y Uchiyama and H HatanakaldquoFree radical-independent protection by nerve growth factorand Bcl-2 of PC12 cells from hydrogen peroxide-triggered

6 BioMed Research International

apoptosisrdquo Journal of Biochemistry vol 120 no 3 pp 540ndash5461996

[9] Y Y Luo M Yin Y H Wu D W He Q Q Wei and C CYin ldquoProtective effects of chlorogenic acid against hydrogenperoxide induced rat nucleus pulposus cells apoptosisrdquo ChineseTraditional Patent Medicine vol 4 pp 656ndash660 2013

[10] J Liu Y Wang W Du et al ldquoWnt1 inhibits hydrogen peroxide-induced apoptosis in mouse cardiac stem cellsrdquo PLoS ONE vol8 no 3 Article ID e58883 2013

[11] L Wu Z Xi R Guo et al ldquoExogenous ARC down-regulatescaspase-3 expression and inhibits apoptosis of broiler chickencardiomyocytes exposed to hydrogen peroxiderdquo Avian Pathol-ogy vol 42 no 1 pp 32ndash37 2013

[12] I Bejarano J Espino A M Marchena et al ldquoMelatoninenhances hydrogen peroxide-induced apoptosis in humanpromyelocytic leukaemia HL-60 cellsrdquo Molecular and CellularBiochemistry vol 353 no 1-2 pp 167ndash176 2011

[13] P Pallepati and D A Averill-Bates ldquoMild thermotoleranceinduced at 40∘C protects HeLa cells against activation ofdeath receptor-mediated apoptosis by hydrogen peroxiderdquo FreeRadical Biology and Medicine vol 50 no 6 pp 667ndash679 2011

[14] Z Li J Zhao Q Li et al ldquoKLF4 promotes hydrogen-peroxide-induced apoptosis of chronic myeloid leukemia cells involvingthe bcl-2bax pathwayrdquo Cell Stress and Chaperones vol 15 no6 pp 905ndash912 2010

[15] G F Ribeiro M Corte-Real and B Johansson ldquoCharacteriza-tion of DNA damage in yeast apoptosis induced by hydrogenperoxide acetic acid and hyperosmotic shockrdquo MolecularBiology of the Cell vol 17 no 10 pp 4584ndash4591 2006

[16] S-J Lee H Ahn K-W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules andTherapeutics vol 20no 5 pp 487ndash491 2012

[17] P Roy E Reavey M Rayne et al ldquoEnhanced sensitivity tohydrogen peroxide-induced apoptosis in Evi1 transformed Rat1fibroblasts due to repression of carbonic anhydrase IIIrdquo TheFEBS Journal vol 277 no 2 pp 441ndash452 2010

[18] A S Ghosh S Dutta and S Raha ldquoHydrogen peroxide-induced apoptosis-like cell death in Entamoeba histolyticardquoParasitology International vol 59 no 2 pp 166ndash172 2010

[19] M L Wurstle M A Laussmann and M Rehm ldquoThe centralrole of initiator caspase-9 in apoptosis signal transduction andthe regulation of its activation and activity on the apoptosomerdquoExperimental Cell Research vol 318 no 11 pp 1213ndash1220 2012

[20] K W McCool and S Miyamoto ldquoDNA damage-dependentNF-120581B activation NEMO turns nuclear signaling inside outrdquoImmunological Reviews vol 246 no 1 pp 311ndash326 2012

[21] T Li N Kon L Jiang et al ldquoTumor suppression in the absenceof p53-mediated cell-cycle arrest apoptosis and senescencerdquoCell vol 149 no 6 pp 1269ndash1283 2012

[22] A Degterev J Hitomi M Germscheid et al ldquoIdentification ofRIP1 kinase as a specific cellular target of necrostatinsrdquo NatureChemical Biology vol 4 no 5 pp 313ndash321 2008

[23] R LDeHaan ldquoDevelopment of form in the embryonic heart anexperimental approachrdquo Circulation vol 35 no 5 pp 821ndash8331967

[24] Z Xing and K A Schat ldquoExpression of cytokine genes inMarekrsquos disease virus-infected chickens and chicken embryofibroblast culturesrdquo Immunology vol 100 no 1 pp 70ndash76 2000

[25] K A Schat and H G Purchase ldquoCell-culture methodsrdquo in ALaboratory Manual for the Isolation and Identification of Avian

Pathogens D E Swayne J R Glisson M W Jackwood J EPearson and VWM Reed Eds p 223 American Associationof Avian Pathologists Kennett Square Pa USA 4th edition1998

[26] CWan J Xiang Y Li andDGuo ldquoDifferential gene expressionpatterns in chicken cardiomyocytes during hydrogen peroxide-induced apoptosisrdquo PLoS ONE vol 11 no 1 Article IDe0147950 2016

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

changes in cells after induction with hydrogen peroxide Asjudged by RIP it was found that 293T cells may be involvedin necroptosis even at a low dosage Thus if we want toinduce cell lines into apoptosis we must reduce the actiontime or use DTT to induce apoptosis For myocardial cellsand fibroblasts they tend to be involved in necroptosis only ata higher dosage From the other 3 indices it can be concludedthat 3 kinds of cells have different responses to the inductionby hydrogen peroxide which showed that fibroblasts are notinclined to necroptosis after induction from the expressionof RIP For other indices fibroblasts have a higher expressionof NF-120581B which may indicate that it has had more DNAcorrosion and handicap of transcription 293T cells havea higher expression of P53 indicating a disorder in cellcycle and proliferation The myocardial cells have a higherexpression of all indices which may be a comprehensiveeffect

In the experiments it was found that the cells underwentmorphologic changes after induction by hydrogen peroxideand it is dose-dependent After the comparison of DTTand hydrogen peroxide there are many differences in the 4indices however the trend is basically consistent It was alsofound that the hydrogen peroxide can greatly influence cellmotor ability but after the induction of DTT for 6 hoursthe impulse of some myocardial cells still exists and theapoptosis effect is less than hydrogen peroxide The existingquestion is whether or not hydrogen peroxide has a selectivityin different cell lines or if hydrogen peroxide has a speciesselectivity such as cells from rats

5 Conclusion

Hydrogen peroxide has a high efficiency leading to cell deathHydrogen peroxide causes necroptosis in 293T cells at aconcentration ranging from 01 to 16mMThe cell lines usedin this study were sensitive to hydrogen peroxide In primarycells a concentration gt 04mM may also cause necroptosisA concentration lt 04mM had a tendency to apoptosisPrimary cells can survive in hydrogen peroxide for 36 hoursat a concentration le 02mM Different cells have a differentresponse to induction by hydrogen peroxideThus hydrogenperoxide qualifies as an apoptosis inducer at a specific dosagecorresponding to the specific cell types but researchers donotpay attention to these findings

Ethical Approval

This study was approved by the Animal Care and UseCommittee of Hubei Province China All animal procedureswere performed according to the guidelines developed byChinarsquos Council on Animal Care

Disclosure

The funders had no role in the study design data collection oranalysis decision to publish or the preparation of the paper

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Jinmei Xiang Chunyun Wan and Dingzong Guo conceivedand designed the experiments Jinmei Xiang and ChunyunWan performed the experiments Jinmei Xiang ChunyunWan and Rui Guo analyzed the data Rui Guo contributedregentsmaterialsanalysis tools Jinmei Xiang and ChunyunWan wrote the paper Jinmei Xiang and Chunyun Wancontributed equally to this work

Acknowledgments

Special thanks are due to Li Liang and Li Youwen forapoptosis identification and technical assistance and toZhangGuoxing for his help in cell culture and treatment Thiswork was supported by a preliminary research project grantfrom Huazhong Agricultural University [52209-814112] andthe National Natural Sciences Foundation of China (no31502132)

References

[1] J F Kerr A H Wyllie and A R Currie ldquoApoptosis abasic biological phenomenon with wide-ranging implicationsin tissue kineticsrdquo British Journal of Cancer vol 26 no 4 pp239ndash257 1972

[2] Q Yating Y Yuan ZWei et al ldquoOxidized LDL induces apopto-sis of human retinal pigment epithelium through activation ofERK-BaxBcl-2 signaling pathwaysrdquo Current Eye Research vol40 no 4 pp 415ndash422 2015

[3] L Tartier Y L McCarey J E Biaglow I E Kochevar andK D Held ldquoApoptosis induced by dithiothreitol in HL-60cells shows early activation of caspase 3 and is independent ofmitochondriardquo Cell Death and Differentiation vol 7 no 10 pp1002ndash1010 2000

[4] E R Whittemore D T Loo and C W Cotman ldquoExposure tohydrogen peroxide induces cell death via apoptosis in culturedrat cortical neuronsrdquo NeuroReport vol 5 no 12 pp 1485ndash14881994

[5] HMViola P G Arthur and L CHool ldquoTransient exposure tohydrogen peroxide causes an increase in mitochondria-derivedsuperoxide as a result of sustained alteration in L-type Ca2+channel function in the absence of apoptosis in ventricularmyocytesrdquo Circulation Research vol 100 no 7 pp 1036ndash10442007

[6] H-Y Lin S-C Shen C-W Lin L-Y Yang and Y-C ChenldquoBaicalein inhibition of hydrogen peroxide-induced apopto-sis via ROS-dependent heme oxygenase 1 gene expressionrdquoBiochimica et Biophysica Acta (BBA)mdashMolecular Cell Researchvol 1773 no 7 pp 1073ndash1086 2007

[7] M Singh H Sharma and N Singh ldquoHydrogen peroxideinduces apoptosis in HeLa cells through mitochondrial path-wayrdquoMitochondrion vol 7 no 6 pp 367ndash373 2007

[8] T Satoh N Sakai Y Enokido Y Uchiyama and H HatanakaldquoFree radical-independent protection by nerve growth factorand Bcl-2 of PC12 cells from hydrogen peroxide-triggered

6 BioMed Research International

apoptosisrdquo Journal of Biochemistry vol 120 no 3 pp 540ndash5461996

[9] Y Y Luo M Yin Y H Wu D W He Q Q Wei and C CYin ldquoProtective effects of chlorogenic acid against hydrogenperoxide induced rat nucleus pulposus cells apoptosisrdquo ChineseTraditional Patent Medicine vol 4 pp 656ndash660 2013

[10] J Liu Y Wang W Du et al ldquoWnt1 inhibits hydrogen peroxide-induced apoptosis in mouse cardiac stem cellsrdquo PLoS ONE vol8 no 3 Article ID e58883 2013

[11] L Wu Z Xi R Guo et al ldquoExogenous ARC down-regulatescaspase-3 expression and inhibits apoptosis of broiler chickencardiomyocytes exposed to hydrogen peroxiderdquo Avian Pathol-ogy vol 42 no 1 pp 32ndash37 2013

[12] I Bejarano J Espino A M Marchena et al ldquoMelatoninenhances hydrogen peroxide-induced apoptosis in humanpromyelocytic leukaemia HL-60 cellsrdquo Molecular and CellularBiochemistry vol 353 no 1-2 pp 167ndash176 2011

[13] P Pallepati and D A Averill-Bates ldquoMild thermotoleranceinduced at 40∘C protects HeLa cells against activation ofdeath receptor-mediated apoptosis by hydrogen peroxiderdquo FreeRadical Biology and Medicine vol 50 no 6 pp 667ndash679 2011

[14] Z Li J Zhao Q Li et al ldquoKLF4 promotes hydrogen-peroxide-induced apoptosis of chronic myeloid leukemia cells involvingthe bcl-2bax pathwayrdquo Cell Stress and Chaperones vol 15 no6 pp 905ndash912 2010

[15] G F Ribeiro M Corte-Real and B Johansson ldquoCharacteriza-tion of DNA damage in yeast apoptosis induced by hydrogenperoxide acetic acid and hyperosmotic shockrdquo MolecularBiology of the Cell vol 17 no 10 pp 4584ndash4591 2006

[16] S-J Lee H Ahn K-W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules andTherapeutics vol 20no 5 pp 487ndash491 2012

[17] P Roy E Reavey M Rayne et al ldquoEnhanced sensitivity tohydrogen peroxide-induced apoptosis in Evi1 transformed Rat1fibroblasts due to repression of carbonic anhydrase IIIrdquo TheFEBS Journal vol 277 no 2 pp 441ndash452 2010

[18] A S Ghosh S Dutta and S Raha ldquoHydrogen peroxide-induced apoptosis-like cell death in Entamoeba histolyticardquoParasitology International vol 59 no 2 pp 166ndash172 2010

[19] M L Wurstle M A Laussmann and M Rehm ldquoThe centralrole of initiator caspase-9 in apoptosis signal transduction andthe regulation of its activation and activity on the apoptosomerdquoExperimental Cell Research vol 318 no 11 pp 1213ndash1220 2012

[20] K W McCool and S Miyamoto ldquoDNA damage-dependentNF-120581B activation NEMO turns nuclear signaling inside outrdquoImmunological Reviews vol 246 no 1 pp 311ndash326 2012

[21] T Li N Kon L Jiang et al ldquoTumor suppression in the absenceof p53-mediated cell-cycle arrest apoptosis and senescencerdquoCell vol 149 no 6 pp 1269ndash1283 2012

[22] A Degterev J Hitomi M Germscheid et al ldquoIdentification ofRIP1 kinase as a specific cellular target of necrostatinsrdquo NatureChemical Biology vol 4 no 5 pp 313ndash321 2008

[23] R LDeHaan ldquoDevelopment of form in the embryonic heart anexperimental approachrdquo Circulation vol 35 no 5 pp 821ndash8331967

[24] Z Xing and K A Schat ldquoExpression of cytokine genes inMarekrsquos disease virus-infected chickens and chicken embryofibroblast culturesrdquo Immunology vol 100 no 1 pp 70ndash76 2000

[25] K A Schat and H G Purchase ldquoCell-culture methodsrdquo in ALaboratory Manual for the Isolation and Identification of Avian

Pathogens D E Swayne J R Glisson M W Jackwood J EPearson and VWM Reed Eds p 223 American Associationof Avian Pathologists Kennett Square Pa USA 4th edition1998

[26] CWan J Xiang Y Li andDGuo ldquoDifferential gene expressionpatterns in chicken cardiomyocytes during hydrogen peroxide-induced apoptosisrdquo PLoS ONE vol 11 no 1 Article IDe0147950 2016

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

apoptosisrdquo Journal of Biochemistry vol 120 no 3 pp 540ndash5461996

[9] Y Y Luo M Yin Y H Wu D W He Q Q Wei and C CYin ldquoProtective effects of chlorogenic acid against hydrogenperoxide induced rat nucleus pulposus cells apoptosisrdquo ChineseTraditional Patent Medicine vol 4 pp 656ndash660 2013

[10] J Liu Y Wang W Du et al ldquoWnt1 inhibits hydrogen peroxide-induced apoptosis in mouse cardiac stem cellsrdquo PLoS ONE vol8 no 3 Article ID e58883 2013

[11] L Wu Z Xi R Guo et al ldquoExogenous ARC down-regulatescaspase-3 expression and inhibits apoptosis of broiler chickencardiomyocytes exposed to hydrogen peroxiderdquo Avian Pathol-ogy vol 42 no 1 pp 32ndash37 2013

[12] I Bejarano J Espino A M Marchena et al ldquoMelatoninenhances hydrogen peroxide-induced apoptosis in humanpromyelocytic leukaemia HL-60 cellsrdquo Molecular and CellularBiochemistry vol 353 no 1-2 pp 167ndash176 2011

[13] P Pallepati and D A Averill-Bates ldquoMild thermotoleranceinduced at 40∘C protects HeLa cells against activation ofdeath receptor-mediated apoptosis by hydrogen peroxiderdquo FreeRadical Biology and Medicine vol 50 no 6 pp 667ndash679 2011

[14] Z Li J Zhao Q Li et al ldquoKLF4 promotes hydrogen-peroxide-induced apoptosis of chronic myeloid leukemia cells involvingthe bcl-2bax pathwayrdquo Cell Stress and Chaperones vol 15 no6 pp 905ndash912 2010

[15] G F Ribeiro M Corte-Real and B Johansson ldquoCharacteriza-tion of DNA damage in yeast apoptosis induced by hydrogenperoxide acetic acid and hyperosmotic shockrdquo MolecularBiology of the Cell vol 17 no 10 pp 4584ndash4591 2006

[16] S-J Lee H Ahn K-W Nam K H Kim and W Mar ldquoEffectsof rutaecarpine on hydrogen peroxide-induced apoptosis inmurine hepa-1c1c7 cellsrdquo Biomolecules andTherapeutics vol 20no 5 pp 487ndash491 2012

[17] P Roy E Reavey M Rayne et al ldquoEnhanced sensitivity tohydrogen peroxide-induced apoptosis in Evi1 transformed Rat1fibroblasts due to repression of carbonic anhydrase IIIrdquo TheFEBS Journal vol 277 no 2 pp 441ndash452 2010

[18] A S Ghosh S Dutta and S Raha ldquoHydrogen peroxide-induced apoptosis-like cell death in Entamoeba histolyticardquoParasitology International vol 59 no 2 pp 166ndash172 2010

[19] M L Wurstle M A Laussmann and M Rehm ldquoThe centralrole of initiator caspase-9 in apoptosis signal transduction andthe regulation of its activation and activity on the apoptosomerdquoExperimental Cell Research vol 318 no 11 pp 1213ndash1220 2012

[20] K W McCool and S Miyamoto ldquoDNA damage-dependentNF-120581B activation NEMO turns nuclear signaling inside outrdquoImmunological Reviews vol 246 no 1 pp 311ndash326 2012

[21] T Li N Kon L Jiang et al ldquoTumor suppression in the absenceof p53-mediated cell-cycle arrest apoptosis and senescencerdquoCell vol 149 no 6 pp 1269ndash1283 2012

[22] A Degterev J Hitomi M Germscheid et al ldquoIdentification ofRIP1 kinase as a specific cellular target of necrostatinsrdquo NatureChemical Biology vol 4 no 5 pp 313ndash321 2008

[23] R LDeHaan ldquoDevelopment of form in the embryonic heart anexperimental approachrdquo Circulation vol 35 no 5 pp 821ndash8331967

[24] Z Xing and K A Schat ldquoExpression of cytokine genes inMarekrsquos disease virus-infected chickens and chicken embryofibroblast culturesrdquo Immunology vol 100 no 1 pp 70ndash76 2000

[25] K A Schat and H G Purchase ldquoCell-culture methodsrdquo in ALaboratory Manual for the Isolation and Identification of Avian

Pathogens D E Swayne J R Glisson M W Jackwood J EPearson and VWM Reed Eds p 223 American Associationof Avian Pathologists Kennett Square Pa USA 4th edition1998

[26] CWan J Xiang Y Li andDGuo ldquoDifferential gene expressionpatterns in chicken cardiomyocytes during hydrogen peroxide-induced apoptosisrdquo PLoS ONE vol 11 no 1 Article IDe0147950 2016

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology