Research Article The Role of EGFR/PI3K/Akt/cyclinD1...

Hindawi Publishing CorporationMediators of InflammationVolume 2013 Article ID 651207 9 pageshttpdxdoiorg1011552013651207

Research ArticleThe Role of EGFRPI3KAktcyclinD1 Signaling Pathway inAcquired Middle Ear Cholesteatoma

Wei Liu1 Hongmiao Ren1 Jihao Ren1 Tuanfang Yin1 Bing Hu1 Shumin Xie1 YinghuanDai2 Weijing Wu1 Zian Xiao1 Xinming Yang1 and Dinghua Xie1

1 Department of Otolaryngology Head and Neck Surgery The Second Xiangya Hospital Central South University139 Middle Renmin Road Changsha Hunan 410011 China

2Department of Pathology The Second Xiangya Hospital Central South University Changsha Hunan 410011 China

Correspondence should be addressed to Jihao Ren jihao5114163com

Received 12 July 2013 Accepted 24 September 2013

Academic Editor PhamMy-Chan Dang

Copyright copy 2013 Wei Liu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cholesteatoma is a benign keratinizing and hyper proliferative squamous epithelial lesion of the temporal bone Epidermal growthfactor (EGF) is one of the most important cytokines which has been shown to play a critical role in cholesteatoma In thisinvestigation we studied the effects of EGF on the proliferation of keratinocytes and EGF-mediated signaling pathways underlyingthe pathogenesis of cholesteatoma We examined the expressions of phosphorylated EGF receptor (p-EGFR) phosphorylated Akt(p-Akt) cyclinD1 and proliferating cell nuclear antigen (PCNA) in 40 cholesteatoma samples and 20 samples of normal externalauditory canal (EAC) epithelium by immunohistochemical method Furthermore in vitro studies were performed to investigateEGF-induced downstream signaling pathways in primary external auditory canal keratinocytes (EACKs) The expressions of p-EGFR p-Akt cyclinD1 and PCNA in cholesteatoma epithelium were significantly increased when compared with those of controlsubjects We also demonstrated that EGF led to the activation of the EGFRPI3KAktcyclinD1 signaling pathway which playeda critical role in EGF-induced cell proliferation and cell cycle progression of EACKs Both EGFR inhibitor AG1478 and PI3Kinhibitor wortmannin inhibited the EGF-induced EGFRPI3KAktcyclinD1 signaling pathway concomitantly with inhibition ofcell proliferation and cell cycle progression of EACKs Taken together our data suggest that the EGFRPI3KAktcyclinD1 signalingpathway is active in cholesteatoma and may play a crucial role in cholesteatoma epithelial hyper-proliferation This study willfacilitate the development of potential therapeutic targets for intratympanic drug therapy for cholesteatoma

1 Introduction

Cholesteatoma is a benign keratinizing and hyperprolif-erative squamous epithelial lesion of the temporal bonewhich gradually expands and causes serious complicationsby destruction of nearby bony structures Erosion of theossicular chain and bony labyrinth may cause hearing lossfacial paralysis labyrinthine fistulae and brain abscess Ingeneral squamous epithelial cells of cholesteatoma are char-acterized by uncoordinated proliferation migration aggres-siveness and recurrence [1ndash4] However insight into itsexact underlying cellular andmolecularmechanisms remainsincomplete Clinically acquired cholesteatoma is usuallyrelated to chronic suppurative inflammation of the middleear which plays an important role in the etiopathogenesis

of cholesteatoma [5] Recently many authors have demon-strated that cytokines and mediators which are released inthe inflammatory responses may promote the formation andprogression of cholesteatoma by stimulating migration andproliferation of keratinocytes in the tympanicmembrane anddeep part of external auditory canal (EAC) [5ndash10]

Epidermal growth factor (EGF) is one of the mostimportant cytokines which has been shown to play a criticalrole in cholesteatoma [11 12] Increased EGF expression hasbeen demonstrated in cholesteatoma epithelium [13 14] andhigh EGF levels have also been shown in cholesteatomadebris and subepithelial tissue [15] However the exactmechanisms of EGF in the pathogenesis of cholesteatomaremain unclear Recently some downstream targets of EGFhave been identified and among them phosphatidylinositol

2 Mediators of Inflammation

3-kinase (PI3K)Akt signaling pathway is one of the mostimportant kinase cascades that mediates a wide range ofcellular functions such as survival proliferation migrationand differentiation [16] The EGF receptor (EGFR) is a 170-kDa transmembrane glycoprotein that consists of intracel-lular extracellular and transmembrane domains [17] Thebinding of EGF to the extracellular domain of EGFR pro-motes the activation of EGFR through autophosphorylationof their intracellular domains which subsequently triggersthe PI3KAkt signaling pathway [18] Activation of thePI3KAkt signaling pathway can lead to cell proliferation andgrowth by regulating cyclinD1 expression [19] Recent studieshave shown that EGFR immunoreactivity was significantlyincreased in cholesteatoma epithelium when compared withnormal EAC epithelium [20ndash23] These results lead us tohypothesize that EGF in the inflammatory microenviron-ment of cholesteatoma may bind with EGFR and activatethe EGFRPI3KAktcyclinD1 signaling pathway which inturn promotes the abnormal proliferation of keratinocytes incholesteatoma epithelium

To test our hypotheses firstly we examined the expres-sions of phosphorylated EGFR (p-EGFR) phosphorylatedAkt (p-Akt) cyclinD1 and proliferating cell nuclear antigen(PCNA) in middle ear cholesteatoma and normal EACskin specimens Secondly we developed a human externalauditory canal keratinocytes (EACKs) cell culture model andinvestigated EGF-induced downstream signaling in EACKs

2 Materials and Methods

21 Patients and Specimens Forty samples of acquiredcholesteatoma tissues diagnosed clinically and confirmedpathologically after middle ear surgery were obtained fromthe Department of Otolaryngology Head and Neck SurgeryThe Second Xiangya Hospital Central South Universityfrom January 2008 to October 2010 There were 23 malesand 17 females with a mean age of 385 years (range 14ndash56 years) Twenty normal EAC skin samples obtained frompatients undergoing myringoplasty for dry perforation andexploratory tympanotomy for diagnosis of middle ear diseaseserved as controls Specimens for hematoxylin-eosin (HE)staining and immunohistochemistry were immediately fixedin 10 buffered formalin and embedded in paraffin Allsamples were analyzed independently by two experiencedpathologists The study was approved by the Ethics Commit-tee of Central South University and informed consent wasobtained from all of the patients

22 Immunohistochemistry Paraffin-embedded tissues werecut into 4 120583m thick sections The sections were then deparaf-finized in xylene and rehydrated in a graded ethanol series(100 95 and 75) before incubation for 10min with 3hydrogen peroxide solution Antigen retrieval was performedin 10mmolL citrate buffer (PH 60) in a microwave ovenfor 15min at 100∘C The sections were washed in phosphate-buffered saline (PBS) and preincubated with nonimmunegoat blood serum for 15min at room temperature Thenthe sections were incubated with primary rabbit antibod-ies against p-EGFR (Tyr 1173 1 100 dilution Santa Cruz

Biotechnology USA) p-Akt (Ser 473 1 50 dilution Cellsignaling Technology USA) cyclinD1 (1 25 dilution Cellsignaling Technology USA) and PCNA (1 100 dilutionBioworld Technology USA) at 4∘C overnight in a humidifiedchamber After incubation each section was washed withPBS and incubatedwith the secondary biotinylated goat-anti-rabbit antibody (Zhongshan Goldenbridge BiotechnologyBeijing China) for 15min at room temperature After beingwashed in PBS the sections were incubated with streptavidinconjugated with horse radish peroxidase (Zhongshan Gold-enbridge Biotechnology Beijing China) Freshly prepared331015840-diaminobenzidine (DAB Sigma) was used as a substratefor peroxidase Finally the sections were counterstained withhematoxylin dehydrated and mounted For the negativecontrols PBS was used to substitute primary antibody

23 Evaluation of Immunostaining Both the percentage ofstained cells and the intensity of staining were considered[24] The percentage of positive cells was graded and scoredas 0 for negative staining 1 for lt10 2 for 10ndash50 and3 for gt50 positive cells Staining intensity was graded andscored as 0 for no staining 1 for weak staining 2 formoderatestaining and 3 for strong staining The overall score foreach specimen was obtained by multiplying the percentagescore and the staining intensity score The final results wererecorded as negative (minus) if the overall score was le2 andpositive (+) if the overall score was ge3

24 Primary Cultures of EACKs EACKs were obtainedfrom external auditory canal skin (EACS) samples duringexploratory tympanotomy surgery as described previouslyby Schmidt et al [12] and Sanjuan et al [25] Brieflytissue samples were rinsed with cold sterile PBS contain-ing 100 IUmL of penicillin and 100 ugmL of streptomycinprior to removal of connective tissue Then the sampleswere cut into small pieces and incubated in 025 trypsinsolution overnight at 4∘C Subsequently epithelial sheetswere dissected and dissociated with trypsin Dissociationwas stopped with trypsin inhibitor and cells were thencentrifuged for 5 minutes at 1000 rpm After centrifugationcells were cultured in defined keratinocyte-SFM (serum-free medium for keratinocytes Invitrogen USA) containingsupplements (bovine pituitary extract human epidermalgrowth factor Invitrogen) in humidified CO

2at 37∘C All

experiments utilized subconfluent low-passage EACKs Thestudywas approved by the EthicsCommittee ofCentral SouthUniversity and informed consent was obtained from all of thepatients

25 Identification of EACKs by Immunofluorescence Thecells were cultured onto coverslips and then fixed with 4paraformaldehyde solution followed by incubation with 02Triton X-100 for 10min to allow permeabilization Afterincubation the cells were washed three times in PBS andincubated in blocking buffer (3 bovine serum albumin)The cells were then incubated with primary mouse antibodyagainst cytokeratins (1 150 dilution Boster China) at 37∘Cfor 90min After incubation the cells were washed in PBS

Mediators of Inflammation 3

and incubated with TRITC-labeled goat-anti-mouse sec-ondary antibody (1 50 dilution Boster China) at 37∘C for60min Coverslips were finallymounted for observation PBSwas used to substitute the primary antibody (cytokeratins) asa negative control

26 Cell Treatment EACKs from passages two or threewere used for the study Defined keratinocyte-SFM withsupplements was changed to medium without supplementsfor 24 h before treatment with EGF In brief EACKs weretreated with 15 ngmL of human recombinant EGF (SigmaUSA) for the indicated periods of time For the inhibitionexperiments EACKs were cultured in defined keratinocyte-SFM containing 250 nM of the EGFR inhibitor AG1478 (Cellsignaling Technology USA) for 30min or 200 nMof the PI3Kinhibitor wortmannin (Sigma USA) for 1 h before treatmentwith EGF

27 MTT Assay The capacity for cellular proliferation wasevaluated by MTT assay Briefly cells were seeded into 96-well plates and incubated in humidified CO

2at 37∘C 24 h

later the cells were stimulated with EGF (15 ngmL) in thepresence or absence of EGFR inhibitor (AG1478 250 nmolL)and PI3K inhibitor (wortmannin 200 nmolL) for the indi-cated periods of time The cells were then incubated with20120583L of MTT solution for 4 h at 37∘C After removal ofthe culture medium 150120583L of dimethyl sulfoxide (DMSO)was added to solubilize the crystals The cell growth curvewas drawn according to the values of 490 nm wavelengthlight absorption (OD

490) Cell survival rate was () =

ODtreatODcontrol times 100

28 Cell Cycle Analysis EACKswere cultured in 6-well tissueculture plates and grown to 70 to 80 confluence At prede-termined time points after treatment EACKs were harvestedand fixed with ice-cold ethanol (70) After fixation the cellswere centrifuged and washed with ice-cold PBS Then thecells were incubated with PBS containing propidium iodide(PI 50120583gmL Sigma USA) RNase A (100 120583gmL BeyotimeChina) and 01 Triton X-100 (Beyotime China) and kept inthe dark at 4∘C for 30minTheDNA content was determinedby flow cytometry

29 Western Blotting Analysis After treatment cell lysateswere prepared and separated by SDS-PAGE gel and trans-ferred to polyvinylidene difluoride membrane Membraneswere blocked with Tris-buffered saline (TBS PH 74) con-taining 5 nonfat drymilk and then incubated with primaryantibodies overnight at 4∘C After being washed three timesin TBS the membranes were incubated with horseradishperoxidase-conjugated goat-anti-rabbit secondary antibody(1 1000 Santa Cruz Biotechnology USA) for 1 h at roomtemperature Proteins were detected by the enhanced chemi-luminescence (ECL) method (Thermo USA) The detailsabout the primary antibodies were as follows p-EGFR (Tyr1173 1 500 dilution Santa Cruz Biotechnology USA) p-Akt(Ser 473 1 2000 dilution Cell signaling Technology USA)

cyclinD1 (1 500 dilution Bioworld Technology USA) EGFR(1 500 dilution Bioworld Technology USA) and Akt (1 500dilution Bioworld Technology USA)

210 Statistical Analysis Protein expression patterns betweencholesteatoma epithelium and normal EAC epithelium wereanalyzed with the Chi-square test (1205942 test) Spearmanrsquos rankcorrelation test was used to calculate correlations We alsoanalyzed the differences between the treated cell groups andcontrol cell groups using Studentrsquos t-test Statistical analysiswas performed by SPSS 160 statistical program (SPSS IncChicago IL USA) A value of 119875 lt 005 was consideredstatistically significant

3 Results

31 Histopathological Findings HE staining showed that allcholesteatoma specimens consisted of three parts matrixperimatrix and keratin debris (Figure 1(a)) In cholesteatomaspecimens inflammatory cells and newly formed bloodvessels were always seen in the perimatrix No signs ofinflammationwere observed in the normal EAC skin sections(Figure 1(b))

32 Immunohistochemical Detection of p-EGFR p-Akt andcyclinD1 in Cholesteatoma Epithelium and Normal EACSkin Epithelium As shown in Figure 2 positive p-EGFRimmunostaining was mainly observed in the cell cytoplasmand membrane of cholesteatoma epithelium in the basal andsuprabasal layers (Figure 2(a)) p-Akt positive reactions weremainly localized in the cytoplasm of epithelium in the basaland suprabasal layers (Figure 2(c)) cyclinD1 expression wasmainly observed in the nuclei and was localized in the basallayer of the epithelium (Figure 2(e)) PCNApositive reactionswere mainly observed in the nuclei and was localized inthe basal and suprabasal layers (Figure 2(g)) The positiverate of p-EGFR p-Akt cyclinD1 and PCNA expression wassignificantly increased in cholesteatoma epithelium (650725 625 and 800 resp) when compared with normalEAC epithelium (200 350 100 and 450 resp)(Table 1 119875 lt 001) In cholesteatoma epithelium a significantpositive correlation was observed between p-EGFR andPCNA expression and between the expressions of p-Akt andPCNA cyclinD1 and PCNA respectively (119875 lt 001)

33 Establishment and Identification of Primary Cultures ofEACKs After 5ndash7 days of culture we observed cell coloniesmade up of a few cells and the cells gradually grew outwardsfrom the colonies After 20ndash22 days of culture the culturedcells grew into monolayer and appeared polygonal withldquocobblestone-likerdquo appearance (Figure 3(a)) The cells wereincubated with anti-cytokeratins antibody and showed apositive reaction to cytokeratins (Figure 3(b)) No signalwas detected for the negative controls The morpholog-ical and biological characteristics of primary cultures ofEACKs in the present study were similar to those of typicalkeratinocytes


K

P

M

(a)

EP

ST

(b)

Figure 1 HE staining of human cholesteatoma (a) and normal EAC skin (b) In the figure the keratin debris (K) matrix epithelium (M) andperimatrix subepithelial tissue (P) of cholesteatoma and the epithelium (EP) and subepithelial tissue (ST) of normal EAC skin are indicatedOriginal magnification times200

Table 1 The expressions of p-EGFR p-Akt cyclinD1 and PCNA in 40 cholesteatoma samples and 20 samples of normal EAC skin

p-EGFR p-Akt cyclinD1 PCNA+ minus P + minus P + minus P + minus P

Cholesteatoma 26 14 29 11 25 15 32 8lt001 lt001 lt001 lt001

Normal EAC skin 4 16 7 13 2 18 9 11

Table 2 Cell proliferation of EACKs responding to EGF AG1478 and wortmannin was detected by MTT assay (mean plusmn SD)

36 h 48 h 72 hSurvival rate () Survival rate () Survival rate ()

Control 1000 1000 1000EGF 1182 plusmn 23 1325 plusmn 28 1491 plusmn 32

EGF + AG1478 1078 plusmn 37 1087 plusmn 43 1151 plusmn 26

EGF + wortmannin 1104 plusmn 28 1131 plusmn 32 1186 plusmn 52

34 Inhibition of EGFRPI3KAkt Pathway DownregulatesEGF-Induced Proliferation of EACKs As shown in theFigure 4(a) EGF (15 ngmL) treatment significantlyincreased the proliferation of EACKs at all time points(Table 2 119875 lt 005) We hypothesized that the EGFRPI3KAkt survival signaling pathway may be involved in EGF-induced proliferation of EACKs Here we investigate thisby pretreatment of EACKs with EGFR inhibitor (AG1478250 nmolL) and PI3K inhibitor (wortmannin 200 nmolL)for the indicated periods of time According to the results ofthe MTT assay we observed that the pretreatment of EACKswith AG1478 or wortmannin did significantly inhibit thecell proliferation of EACKs induced by EGF stimulation(Figure 4(b) and Table 2 119875 lt 005) Our studies suggestedthat EGF might induce the cell proliferation of EACKsthrough the EGFRPI3KAkt signaling pathway

35 Inhibition of EGFRPI3KAkt Pathway DownregulatesEGF-Induced Cell Cycle Progression of EACKs from G0G1Phase to S Phase In order to further explore the mech-anism of EGF-induced cell proliferation of EACKs flowcytometry was used to monitor cell cycle changes As shown

in Figure 4(c) EGF treatment markedly increased the per-centage of EACKs cells in S+G2M phase when comparedwith the control group (4081 plusmn 69 versus 1476 plusmn 48)(119875 lt 005) However the pretreatment of cells with AG1478significantly reduced the percentage of cells in S+G2Mphasewhen compared with the EGF treatment group (1361 plusmn 34versus 4081 plusmn 69) (119875 lt 005) and the pretreatment of cellswithwortmannin significantly reduced the percentage of cellsin S+G2M phase when compared with the EGF treatmentgroup (1772plusmn42 versus 4081plusmn69) (119875 lt 005)These resultsdemonstrated that EGFmight induce cell cycle progression ofEACKs through the EGFRPI3KAkt signaling pathway

36 EGF Promotes Phosphorylation of EGFR and Akt withoutAffecting the Total Levels of EGFR and Akt in EACKsIn order to study the signaling pathways in EGF-inducedcell cycle progression of EACKs we determined activa-tionphosphorylation of EGFR and Akt by Western blottinganalysis As shown in Figure 5 EGF induced rapid phos-phorylation of EGFR and Akt within 60min with peakphosphorylation of EGFR and Akt seen at 30min and 20min(Figures 5(a) and 5(b)) respectively However the total


K

M

P

(a)

EP

ST

(b)

K

P

M

(c)

EP

ST

(d)

M

P

K

(e)

EP

ST

(f)

M

P

K

(g)

EPST

(h)

Figure 2 Immunohistochemical staining of p-EGFR ((a) (b)) p-Akt ((c) (d)) cyclinD1 ((e) (f)) and PCNA ((g) (h)) in paraffin sectionsof human cholesteatoma epithelium ((a) (c) (e) (g)) and normal EAC skin epithelium ((b) (d) (f) (h)) In the figure the keratin debris(K) matrix epithelium (M) and perimatrix subepithelial tissue (P) of cholesteatoma and the epithelium (EP) and subepithelial tissue (ST) ofnormal EAC skin are indicated Original magnification times200

(a) (b)

Figure 3 The cultured cells grew into monolayer and showed a polygonal ldquocobblestone-likerdquo appearance (a) The cultured cells showed apositive reaction to cytokeratins by immunofluorescence and the signal was detected in cytoplasm (b)


160

140

120

100

80

60

40

2036h 48h

Surv

ival

rate

()

ControlEGF

72hTime (h)

(a)

160

140

120

100

80

6036h 48h 72h

Surv

ival

rate

()

EGFEGF + AG1478

EGF + wortmannin

0hTime (h)

(b)

60

50

40

30

20

10

0Control EGF EGF + AG1478 EGF + WTM

S+

G2

M p

hase

()

(c)

Figure 4 EACKs were treated with 15 ngmL of EGF for the indicated periods of time Cells proliferation was detected by MTT assay (a)EACKs were pretreated with AG1478 or wortmannin (WTM) for 1 h and then exposed to 15 ngmL of EGF for the indicated periods of timeCells proliferation was detected by MTT assay (b) EACKs were incubated with or without AG1478 or wortmannin (WTM) for 1 h and thenexposed to 15 ngmL of EGF for 72 h Cells were harvested for cell cycle analysis by flow cytometry (c) Results were summarized as mean plusmnSD and represent at least three independent experiments

level of EGFR and Akt remained unchanged To investigatewhether EGFR activation is essential for the EGF-inducedAkt phosphorylation cells were pretreated with AG1478 for30min and then exposed to EGF Here we used the well-established PI3K inhibitor wortmannin as a positive controlfor Akt phosphorylation inhibitionThe results demonstratedthat AG1478 potently blocked EGF-induced phosphorylationof EGFR and Akt (Figures 5(c) and 5(d)) These findingsindicate that EGFR activation is essential for the activationof PI3KAkt signaling pathway in EGF-stimulated EACKs

37 EGF Induced cyclinD1 Upregulation and Cell Cycle Pro-gression through the EGFRPI3KAkt Pathway cyclinD1 is acell cycle regulatory protein which plays a crucial role in cell

proliferation and cell cycle transition from G1 to S phases[26] To determine the role of cyclinD1 in EGF-induced cellcycle progression EACKs were incubated with EGF andcyclinD1 protein was detected at different time points byWestern blotting analysis As shown in Figure 5(e) EGFinduced cyclinD1 upregulation within 24 h To investigatethe potential contribution of EGFRPI3KAkt pathway tocyclinD1 up-regulation cells were pretreated with AG1478for 30min or wortmannin for 1 h and then exposed to EGFThe results demonstrated that both AG1478 and wortmanninsignificantly inhibited EGF-induced cyclinD1 up-regulation(Figure 5(f)) These findings indicate that EGFRPI3KAktpathway is required for cyclinD1 up-regulation in EGF-stimulated EACKs


p-EGFR

EGFR

120573-Actin

0min 5min 10min 20min 40min 60min 90min

(a)

p-Akt

Akt

120573-Actin


(b)

p-EGFR

AG1478

EGF for40min

120573-Actin

++

+minusminus

minus

(c)

AG1478

EGF for20min

120573-Actin

+

+

+ + +

minus

minus

minusminus

minusminus

minus

p-Akt

Wortmannin

(d)

cyclinD1

0h 5h 12h 24h

120573-Actin

(e)

Wortmannin

AG1478

EGF for

120573-Actin

+

+

+ ++

minus minus minus

minus

+

minus

minusminus

minusminus

minus

minus

minus

cyclinD1

24h

(f)

Figure 5 EGFRPI3KAkt signaling pathway is required for cyclinD1 upregulation in EGF-stimulated EACKs Cells were treated with15 ngmL of EGF for the indicated periods of time ((a) (b) (e)) or incubated with or without AG1478 or wortmannin for 1 h and then exposedto 15 ngmL of EGF for the indicated periods of time ((c) (d) (f)) After treatment cells were harvested for Western blotting analysis usingspecific antibodies as indicated and 120573-actin was used as an equal loading control

4 Discussion

In the current study we analyzed the protein expression ofthree key components of the EGFRPI3KAktcyclinD1 path-way and demonstrated that protein expression of p-EGFRand p-Akt cyclinD1 in cholesteatoma epithelium was signif-icantly increased when compared with normal EAC epithe-lium In addition we compared the proliferative capacity ofcholesteatoma epithelium with that of normal EAC epithe-lium using PCNA as a proliferation marker We foundthat cholesteatoma epithelium had a greater proliferativecapacity than normal EAC epithelium Consistent with ourobservations previous studies using the Ki-67 proliferationmarker also showed a significantly higher proliferation indexin cholesteatoma epithelium compared with normal EACepithelium [1 27ndash29] Moreover in cholesteatoma epithe-lium we demonstrated that a significant positive correlationwas observed between p-EGFR and PCNA expression andbetween the expression levels of p-Akt and PCNA cyclinD1

and PCNA respectively These observations indicate that theEGFRPI3KAktcyclinD1 pathway is active in cholesteatomaandmay be involved in the cellular hyperplasiamechanism inacquired cholesteatoma epithelium

TheEGFRPI3KAkt pathway is now recognized as one ofthe most important pathways in regulating cell survival andproliferation [19 30] EGF is one of the most important lig-ands of the EGFR familyWhenEGF is present in the extracel-lular milieu it can bind to the extracellular domain of EGFRand activate EGFR through autophosphorylation of theintracellular domains of EGFR Once activated EGFR resultsin the activation of PI3K an intracellular signal transducerenzyme which can phosphorylate phosphatidylinositol 45-biphosphate (PIP2) at the 31015840 position of the inositol ring andconvert them into phosphatidylinositol 345-triphosphate(PIP3) As a second messenger PIP3 recruits Akt to the cellmembrane where Akt is fully activated by phosphorylationat position Ser473 [31] After activation Akt translocates to


the cytoplasm and nucleus to phosphorylate its substratesFor example Akt has been shown to promote cell proliferativeand survival signals through the upregulation of cyclinD1[32]

To investigate the regulatory mechanisms of EGF as aninducer for the activation of the downstream EGFRPI3KAktcyclinD1 pathway in vitro we stimulated EACKs withEGF and tested whether EGF induced the cell proliferationof EACKs through the EGFRPI3KAktcyclinD1 signalingpathway Our in vitro data demonstrated that the stimu-lation of EACKs with EGF led to the activation of theEGFRPI3KAkt signaling pathway which played a criticalrole in EGF-induced cell proliferation and cell cycle pro-gression through the up-regulation of cyclinD1 HoweverbothEGFR inhibitorAG1478 andPI3K inhibitorwortmanninsignificantly inhibited EGF-induced cyclinD1 up-regulationin EACKs Subsequently cell proliferation and cell cycleprogression of EACKs were also blocked These resultssuggest that EGF in the inflammatory microenvironmentcan phosphorylate and activate EGFR on EACKs Onceactivated EGFR leads to the activation of the downstreamPI3KAkt signaling pathway which subsequently induces cellproliferation and cell cycle progression of EACKs throughthe up-regulation of cyclinD1 Our observations are consis-tent with the report by Ouyang et al in which data wasprovided that cyclinD1 is a direct down-stream target of thePI3KAkt signaling pathway [33] In the study we foundthat the EGF effects on the signaling pathway mediatorswere early however the effects on proliferation were seenlater 36 hndash72 h As we know many kinase phosphorylationevents occur very quickly after exposure to a stimulus Inour investigation EGFR and subsequent PI3KAkt activationhave been described as early key events However cellularfunctions such as proliferation which needs mitosis proteinsynthesis are considered as later final events

The EGFRPI3KAkt signaling pathway is associated withvarious proliferative lesions including benign and malignantproliferative diseases Previous studies have revealed thata nonlethal dose of ultraviolet A could promote humanHaCat keratinocytes proliferation through the activationof EGFRPI3KAktcyclinD1 pathway [19] Recently it hasbeen reported that a disintegrin and metalloprotease 17(ADAM17) could induce prostate cancer cell proliferationvia EGFRPI3KAkt pathway activation [34] Our resultsare consistent with these findings and demonstrate for thefirst time that EGFRPI3KAktcyclinD1 signaling pathwayis active in cholesteatoma epithelium and involved in EGF-induced cell proliferation of EACKs Therefore inhibitionof the EGFRPI3KAktcyclinD1 signaling pathway withsmall molecule inhibitors [19 35] (eg monoclonal anti-bodies small molecule tyrosine kinase inhibitors and smallinterfering RNA) might be used as a promising target forintratympanic drug therapy for the nonsurgical patients orcomplementary treatment for preventing recurrence of thecholesteatoma after surgery In our future study we willdevelop animal models of middle ear cholesteatoma in whichpotential inhibitors of EGFRPI3KAktcyclinD1 signalingpathway might become more accessible to research

5 Conclusion

In summary our studies demonstrate that the EGFRPI3KAktcyclinD1 signaling pathway is active in cholesteatomaand may play a crucial role in cholesteatoma epithelialhyperproliferation The in vitro studies indicate that theEGFRPI3KAkt pathway may be involved in EGF-inducedcell proliferation of EACKs through the upregulation ofcyclinD1 This study may help to understand the molecularmechanisms underlying the pathogenesis of cholesteatomaand identify potential therapeutic targets for intratympanicdrug therapy for cholesteatoma

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgment

The research was supported by Hunan Provincial InnovationFoundation For Postgraduate China (CX2010B055)

References

[1] K Sikka S C Sharma A Thakar and S DattaguptaldquoEvaluation of epithelial proliferation in paediatric and adultcholesteatomas using the Ki-67 proliferation markerrdquoThe Jour-nal of Laryngology amp Otology vol 126 no 5 pp 460ndash463 2012

[2] T Helgaland B Engelen C Olsnes H J Aarstad and FS Vassbotn ldquoIn vitro cholesteatoma growth and secretion ofcytokinesrdquo Acta Oto-Laryngologica vol 130 no 7 pp 815ndash8192010

[3] Y Mallet J Nouwen M Lecomte-Houcke and A DesaultyldquoAggressiveness and quantification of epithelial proliferation ofmiddle ear cholesteatoma by MIB1rdquo Laryngoscope vol 113 no2 pp 328ndash331 2003

[4] E Olszewska M Wagner M Bernal-Sprekelsen et alldquoEtiopathogenesis of cholesteatomardquo European Archives ofOto-Rhino-Laryngology vol 261 no 1 pp 6ndash24 2004

[5] S K Juhn M K Jung M D Hoffman et al ldquoThe role ofinflammatorymediators in the pathogenesis of otitis media andsequelaerdquoClinical and Experimental Otorhinolaryngology vol 1no 3 pp 117ndash138 2008

[6] S Raffa L Leone C Scrofani S Monini M R Torrisi andM Barbara ldquoCholesteatoma-associated fibroblasts modulateepithelial growth and differentiation through KGFFGF7 secre-tionrdquo Histochemistry and Cell Biology vol 138 no 2 pp 251ndash269 2012

[7] L Louw ldquoAcquired cholesteatoma pathogenesis stepwise expla-nationsrdquo The Journal of Laryngology amp Otology vol 124 no 6pp 587ndash593 2010

[8] C S Karmody and C Northrop ldquoThe pathogenesis of acquiredcholesteatoma of the human middle ear support for themigration hypothesisrdquo Otology and Neurotology vol 33 no 1pp 42ndash47 2012

[9] D A Preciado ldquoBiology of cholesteatoma special considera-tions in pediatric patientsrdquo International Journal of PediatricOtorhinolaryngology vol 76 no 3 pp 319ndash321 2012

[10] J Y Byun T Y Yune J Y Lee S G Yeo and M S ParkldquoExpression of CYLD and NF-120581B in human cholesteatoma


epitheliumrdquo Mediators of Inflammation vol 2010 Article ID796315 6 pages 2010

[11] Y Goto ldquoEpidermal growth factor in cholesteatomamdashthe firstreport the localization in the cholesteatoma tissuerdquo NihonJibiinkoka Gakkai Kaiho vol 93 no 8 pp 1186ndash1191 1990

[12] M Schmidt P Grunsfelder and F Hoppe ldquoInduction of matrixmetalloproteinases in keratinocytes by cholesteatoma debrisand granulation tissue extractsrdquo European Archives of Oto-Rhino-Laryngology vol 257 no 8 pp 425ndash429 2000

[13] HKojimaM Shiwa YKamide andHMoriyama ldquoExpressionand localization of mRNA for epidermal growth factor andepidermal growth factor receptor in human cholesteatomardquoActa Oto-Laryngologica vol 114 no 4 pp 423ndash429 1994

[14] S Yetiser B Satar and N Aydin ldquoExpression of epidermalgrowth factor tumor necrosis factor-120572 and interleukin-1120572 inchronic otitis media with or without cholesteatomardquo Otologyand Neurotology vol 23 no 5 pp 647ndash652 2002

[15] Y Goto ldquoEpidermal growth factor in cholesteatomamdashthe sec-ond report the localization in the horny layerrdquoNihon JibiinkokaGakkai Kaiho vol 93 no 8 pp 1192ndash1199 1990

[16] B M Marte and J Downward ldquoPKBAkt connecting phos-phoinositide 3-kinase to cell survival and beyondrdquo Trends inBiochemical Sciences vol 22 no 9 pp 355ndash358 1997

[17] E Mishani G Abourbeh M Eiblmaier and C J AndersonldquoImaging of EGFR and EGFR tyrosine kinase overexpression intumors by nuclear medicine modalitiesrdquo Current Pharmaceuti-cal Design vol 14 no 28 pp 2983ndash2998 2008

[18] H Nakamura A Kawakami H Ida T Koji and K EguchildquoEGF activates PI3K-Akt and NF-120581B via distinct pathways insalivary epithelial cells in Sjogrenrsquos syndromerdquo RheumatologyInternational vol 28 no 2 pp 127ndash136 2007

[19] Y Y He S E Council L Feng and C F Chignell ldquoUVA-induced cell cycle progression is mediated by a disintegrin andmetalloproteaseepidermal growth factor receptorAKTcyclinD1 pathways in keratinocytesrdquo Cancer Research vol 68 no 10pp 3752ndash3758 2008

[20] M Barbara S Raffa C Mure et al ldquoKeratinocyte growthfactor receptor (KGF-R) in cholesteatoma tissuerdquo Acta Oto-Laryngologica vol 128 no 4 pp 360ndash364 2008

[21] B J Jin H J Min J H Jeong C W Park and S H LeeldquoExpression of EGFR and microvessel density in middle earcholesteatomardquoClinical and Experimental Otorhinolaryngologyvol 4 no 2 pp 67ndash71 2011

[22] A L Alves C S B Pereira M D F P Carvalho J H TG Fregnani and F Q Ribeiro ldquoEGFR expression in acquiredmiddle ear cholesteatoma in children and adultsrdquo EuropeanJournal of Pediatrics vol 171 no 2 pp 307ndash310 2012

[23] M Yoshikawa H Kojima Y Yaguchi N Okada H Saito andH Moriyama ldquoCholesteatoma fibroblasts promote epithelialcell proliferation through overexpression of epiregulinrdquo PLoSONE vol 8 no 6 Article ID e66725 2013

[24] Y Hiraishi T Wada K Nakatani K Negoro and S FujitaldquoImmunohistochemical expression of EGFR and p-EGFRin oral squamous cell carcinomasrdquo Pathology and OncologyResearch vol 12 no 2 pp 87ndash91 2006

[25] M Sanjuan F Sabatier L Andrac-Meyer J-P Lavieille and JMagnan ldquoEar canal keratinocyte culture clinical perspectiverdquoOtology and Neurotology vol 28 no 4 pp 504ndash509 2007

[26] M Fu C Wang Z Li T Sakamaki and R G Pestell ldquoMinire-view cyclin D1 normal and abnormal functionsrdquo Endocrinol-ogy vol 145 no 12 pp 5439ndash5447 2004

[27] T Yamamoto-Fukuda H Takahashi and T Koji ldquoExpressionof keratinocyte growth factor (KGF) and its receptor in amiddle-ear cavity problemrdquo International Journal of PediatricOtorhinolaryngology vol 76 no 1 pp 76ndash81 2012

[28] V Akdogan I Yilmaz T Canpolat and L N OzluogluldquoRole of Langerhans cells Ki-67 protein and apoptosis inacquired cholesteatoma prospective clinical studyrdquoThe Journalof Laryngology amp Otology vol 127 no 3 pp 252ndash259 2013

[29] S H Lee Y H Jang K Tae et al ldquoTelomerase activity and cellproliferation index in cholesteatomardquo Acta Oto-Laryngologicavol 125 no 7 pp 707ndash712 2005

[30] L A Bazley and W J Gullick ldquoThe epidermal growth factorreceptor familyrdquo Endocrine-Related Cancer vol 12 supplement1 pp S17ndashS27 2005

[31] L CCantley ldquoThephosphoinositide 3-kinase pathwayrdquo Sciencevol 296 no 5573 pp 1655ndash1657 2002

[32] W Ouyang J Li D Zhang B H Jiang and C Huang ldquoPI-3KAkt signal pathway plays a crucial role in arsenite-inducedcell proliferation of human keratinocytes through induction ofcyclin D1rdquo Journal of Cellular Biochemistry vol 101 no 4 pp969ndash978 2007

[33] W Ouyang W Luo D Zhang et al ldquoPI-3KAkt pathway-dependent cyclin D1 expression is responsible for arsenite-induced human keratinocyte transformationrdquo EnvironmentalHealth Perspectives vol 116 no 1 pp 1ndash6 2008

[34] P Lin X Sun T Feng et al ldquoADAM17 regulates prostate cancercell proliferation through mediating cell cycle progression byEGFRPI3KAKT pathwayrdquo Molecular and Cellular Biochem-istry vol 359 no 1-2 pp 235ndash243 2012

[35] SWeinstein R Emmanuel AM Jacobi et al ldquoRNA inhibitionhighlights cyclin D1 as a potential therapeutic target for mantlecell lymphomardquo PLoSONE vol 7 no 8 Article ID e43343 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


3-kinase (PI3K)Akt signaling pathway is one of the mostimportant kinase cascades that mediates a wide range ofcellular functions such as survival proliferation migrationand differentiation [16] The EGF receptor (EGFR) is a 170-kDa transmembrane glycoprotein that consists of intracel-lular extracellular and transmembrane domains [17] Thebinding of EGF to the extracellular domain of EGFR pro-motes the activation of EGFR through autophosphorylationof their intracellular domains which subsequently triggersthe PI3KAkt signaling pathway [18] Activation of thePI3KAkt signaling pathway can lead to cell proliferation andgrowth by regulating cyclinD1 expression [19] Recent studieshave shown that EGFR immunoreactivity was significantlyincreased in cholesteatoma epithelium when compared withnormal EAC epithelium [20ndash23] These results lead us tohypothesize that EGF in the inflammatory microenviron-ment of cholesteatoma may bind with EGFR and activatethe EGFRPI3KAktcyclinD1 signaling pathway which inturn promotes the abnormal proliferation of keratinocytes incholesteatoma epithelium

To test our hypotheses firstly we examined the expres-sions of phosphorylated EGFR (p-EGFR) phosphorylatedAkt (p-Akt) cyclinD1 and proliferating cell nuclear antigen(PCNA) in middle ear cholesteatoma and normal EACskin specimens Secondly we developed a human externalauditory canal keratinocytes (EACKs) cell culture model andinvestigated EGF-induced downstream signaling in EACKs

2 Materials and Methods

21 Patients and Specimens Forty samples of acquiredcholesteatoma tissues diagnosed clinically and confirmedpathologically after middle ear surgery were obtained fromthe Department of Otolaryngology Head and Neck SurgeryThe Second Xiangya Hospital Central South Universityfrom January 2008 to October 2010 There were 23 malesand 17 females with a mean age of 385 years (range 14ndash56 years) Twenty normal EAC skin samples obtained frompatients undergoing myringoplasty for dry perforation andexploratory tympanotomy for diagnosis of middle ear diseaseserved as controls Specimens for hematoxylin-eosin (HE)staining and immunohistochemistry were immediately fixedin 10 buffered formalin and embedded in paraffin Allsamples were analyzed independently by two experiencedpathologists The study was approved by the Ethics Commit-tee of Central South University and informed consent wasobtained from all of the patients

22 Immunohistochemistry Paraffin-embedded tissues werecut into 4 120583m thick sections The sections were then deparaf-finized in xylene and rehydrated in a graded ethanol series(100 95 and 75) before incubation for 10min with 3hydrogen peroxide solution Antigen retrieval was performedin 10mmolL citrate buffer (PH 60) in a microwave ovenfor 15min at 100∘C The sections were washed in phosphate-buffered saline (PBS) and preincubated with nonimmunegoat blood serum for 15min at room temperature Thenthe sections were incubated with primary rabbit antibod-ies against p-EGFR (Tyr 1173 1 100 dilution Santa Cruz

Biotechnology USA) p-Akt (Ser 473 1 50 dilution Cellsignaling Technology USA) cyclinD1 (1 25 dilution Cellsignaling Technology USA) and PCNA (1 100 dilutionBioworld Technology USA) at 4∘C overnight in a humidifiedchamber After incubation each section was washed withPBS and incubatedwith the secondary biotinylated goat-anti-rabbit antibody (Zhongshan Goldenbridge BiotechnologyBeijing China) for 15min at room temperature After beingwashed in PBS the sections were incubated with streptavidinconjugated with horse radish peroxidase (Zhongshan Gold-enbridge Biotechnology Beijing China) Freshly prepared331015840-diaminobenzidine (DAB Sigma) was used as a substratefor peroxidase Finally the sections were counterstained withhematoxylin dehydrated and mounted For the negativecontrols PBS was used to substitute primary antibody

23 Evaluation of Immunostaining Both the percentage ofstained cells and the intensity of staining were considered[24] The percentage of positive cells was graded and scoredas 0 for negative staining 1 for lt10 2 for 10ndash50 and3 for gt50 positive cells Staining intensity was graded andscored as 0 for no staining 1 for weak staining 2 formoderatestaining and 3 for strong staining The overall score foreach specimen was obtained by multiplying the percentagescore and the staining intensity score The final results wererecorded as negative (minus) if the overall score was le2 andpositive (+) if the overall score was ge3

24 Primary Cultures of EACKs EACKs were obtainedfrom external auditory canal skin (EACS) samples duringexploratory tympanotomy surgery as described previouslyby Schmidt et al [12] and Sanjuan et al [25] Brieflytissue samples were rinsed with cold sterile PBS contain-ing 100 IUmL of penicillin and 100 ugmL of streptomycinprior to removal of connective tissue Then the sampleswere cut into small pieces and incubated in 025 trypsinsolution overnight at 4∘C Subsequently epithelial sheetswere dissected and dissociated with trypsin Dissociationwas stopped with trypsin inhibitor and cells were thencentrifuged for 5 minutes at 1000 rpm After centrifugationcells were cultured in defined keratinocyte-SFM (serum-free medium for keratinocytes Invitrogen USA) containingsupplements (bovine pituitary extract human epidermalgrowth factor Invitrogen) in humidified CO

2at 37∘C All

experiments utilized subconfluent low-passage EACKs Thestudywas approved by the EthicsCommittee ofCentral SouthUniversity and informed consent was obtained from all of thepatients

25 Identification of EACKs by Immunofluorescence Thecells were cultured onto coverslips and then fixed with 4paraformaldehyde solution followed by incubation with 02Triton X-100 for 10min to allow permeabilization Afterincubation the cells were washed three times in PBS andincubated in blocking buffer (3 bovine serum albumin)The cells were then incubated with primary mouse antibodyagainst cytokeratins (1 150 dilution Boster China) at 37∘Cfor 90min After incubation the cells were washed in PBS





2at 37∘C 24 h








3 Results





K

P

M

(a)

EP

ST

(b)





Normal EAC skin 4 16 7 13 2 18 9 11











K

M

P

(a)

EP

ST

(b)

K

P

M

(c)

EP

ST

(d)

M

P

K

(e)

EP

ST

(f)

M

P

K

(g)

EPST

(h)


(a) (b)



160

140

120

100

80

60

40

2036h 48h

Surv

ival

rate

()

ControlEGF

72hTime (h)

(a)

160

140

120

100

80

6036h 48h 72h

Surv

ival

rate

()

EGFEGF + AG1478

EGF + wortmannin

0hTime (h)

(b)

60

50

40

30

20

10


S+

G2

M p

hase

()

(c)






p-EGFR

EGFR

120573-Actin


(a)

p-Akt

Akt

120573-Actin


(b)

p-EGFR

AG1478

EGF for40min

120573-Actin

++

+minusminus

minus

(c)

AG1478

EGF for20min

120573-Actin

+

+

+ + +

minus

minus

minusminus

minusminus

minus

p-Akt

Wortmannin

(d)

cyclinD1

0h 5h 12h 24h

120573-Actin

(e)

Wortmannin

AG1478

EGF for

120573-Actin

+

+

+ ++

minus minus minus

minus

+

minus

minusminus

minusminus

minus

minus

minus

cyclinD1

24h

(f)


4 Discussion








5 Conclusion




Acknowledgment


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















2at 37∘C 24 h








3 Results





K

P

M

(a)

EP

ST

(b)





Normal EAC skin 4 16 7 13 2 18 9 11











K

M

P

(a)

EP

ST

(b)

K

P

M

(c)

EP

ST

(d)

M

P

K

(e)

EP

ST

(f)

M

P

K

(g)

EPST

(h)


(a) (b)



160

140

120

100

80

60

40

2036h 48h

Surv

ival

rate

()

ControlEGF

72hTime (h)

(a)

160

140

120

100

80

6036h 48h 72h

Surv

ival

rate

()

EGFEGF + AG1478

EGF + wortmannin

0hTime (h)

(b)

60

50

40

30

20

10


S+

G2

M p

hase

()

(c)






p-EGFR

EGFR

120573-Actin


(a)

p-Akt

Akt

120573-Actin


(b)

p-EGFR

AG1478

EGF for40min

120573-Actin

++

+minusminus

minus

(c)

AG1478

EGF for20min

120573-Actin

+

+

+ + +

minus

minus

minusminus

minusminus

minus

p-Akt

Wortmannin

(d)

cyclinD1

0h 5h 12h 24h

120573-Actin

(e)

Wortmannin

AG1478

EGF for

120573-Actin

+

+

+ ++

minus minus minus

minus

+

minus

minusminus

minusminus

minus

minus

minus

cyclinD1

24h

(f)


4 Discussion








5 Conclusion




Acknowledgment


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















K

P

M

(a)

EP

ST

(b)





Normal EAC skin 4 16 7 13 2 18 9 11











K

M

P

(a)

EP

ST

(b)

K

P

M

(c)

EP

ST

(d)

M

P

K

(e)

EP

ST

(f)

M

P

K

(g)

EPST

(h)


(a) (b)



160

140

120

100

80

60

40

2036h 48h

Surv

ival

rate

()

ControlEGF

72hTime (h)

(a)

160

140

120

100

80

6036h 48h 72h

Surv

ival

rate

()

EGFEGF + AG1478

EGF + wortmannin

0hTime (h)

(b)

60

50

40

30

20

10


S+

G2

M p

hase

()

(c)






p-EGFR

EGFR

120573-Actin


(a)

p-Akt

Akt

120573-Actin


(b)

p-EGFR

AG1478

EGF for40min

120573-Actin

++

+minusminus

minus

(c)

AG1478

EGF for20min

120573-Actin

+

+

+ + +

minus

minus

minusminus

minusminus

minus

p-Akt

Wortmannin

(d)

cyclinD1

0h 5h 12h 24h

120573-Actin

(e)

Wortmannin

AG1478

EGF for

120573-Actin

+

+

+ ++

minus minus minus

minus

+

minus

minusminus

minusminus

minus

minus

minus

cyclinD1

24h

(f)


4 Discussion








5 Conclusion




Acknowledgment


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















K

M

P

(a)

EP

ST

(b)

K

P

M

(c)

EP

ST

(d)

M

P

K

(e)

EP

ST

(f)

M

P

K

(g)

EPST

(h)


(a) (b)



160

140

120

100

80

60

40

2036h 48h

Surv

ival

rate

()

ControlEGF

72hTime (h)

(a)

160

140

120

100

80

6036h 48h 72h

Surv

ival

rate

()

EGFEGF + AG1478

EGF + wortmannin

0hTime (h)

(b)

60

50

40

30

20

10


S+

G2

M p

hase

()

(c)






p-EGFR

EGFR

120573-Actin


(a)

p-Akt

Akt

120573-Actin


(b)

p-EGFR

AG1478

EGF for40min

120573-Actin

++

+minusminus

minus

(c)

AG1478

EGF for20min

120573-Actin

+

+

+ + +

minus

minus

minusminus

minusminus

minus

p-Akt

Wortmannin

(d)

cyclinD1

0h 5h 12h 24h

120573-Actin

(e)

Wortmannin

AG1478

EGF for

120573-Actin

+

+

+ ++

minus minus minus

minus

+

minus

minusminus

minusminus

minus

minus

minus

cyclinD1

24h

(f)


4 Discussion








5 Conclusion




Acknowledgment


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















160

140

120

100

80

60

40

2036h 48h

Surv

ival

rate

()

ControlEGF

72hTime (h)

(a)

160

140

120

100

80

6036h 48h 72h

Surv

ival

rate

()

EGFEGF + AG1478

EGF + wortmannin

0hTime (h)

(b)

60

50

40

30

20

10


S+

G2

M p

hase

()

(c)






p-EGFR

EGFR

120573-Actin


(a)

p-Akt

Akt

120573-Actin


(b)

p-EGFR

AG1478

EGF for40min

120573-Actin

++

+minusminus

minus

(c)

AG1478

EGF for20min

120573-Actin

+

+

+ + +

minus

minus

minusminus

minusminus

minus

p-Akt

Wortmannin

(d)

cyclinD1

0h 5h 12h 24h

120573-Actin

(e)

Wortmannin

AG1478

EGF for

120573-Actin

+

+

+ ++

minus minus minus

minus

+

minus

minusminus

minusminus

minus

minus

minus

cyclinD1

24h

(f)


4 Discussion








5 Conclusion




Acknowledgment


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















p-EGFR

EGFR

120573-Actin


(a)

p-Akt

Akt

120573-Actin


(b)

p-EGFR

AG1478

EGF for40min

120573-Actin

++

+minusminus

minus

(c)

AG1478

EGF for20min

120573-Actin

+

+

+ + +

minus

minus

minusminus

minusminus

minus

p-Akt

Wortmannin

(d)

cyclinD1

0h 5h 12h 24h

120573-Actin

(e)

Wortmannin

AG1478

EGF for

120573-Actin

+

+

+ ++

minus minus minus

minus

+

minus

minusminus

minusminus

minus

minus

minus

cyclinD1

24h

(f)


4 Discussion








5 Conclusion




Acknowledgment


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Conclusion




Acknowledgment


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article The Role of EGFR/PI3K/Akt/cyclinD1...

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Transcript of Research Article The Role of EGFR/PI3K/Akt/cyclinD1...