research article radixin enhances colon cancer cell...

Research ArticleRadixin Enhances Colon Cancer Cell Invasion by IncreasingMMP-7 Production via Rac1-ERK Pathway

Qi-Hong Jiang Ai-Xiang Wang and Yan Chen

Department of Gastroenterology Pursquoai Hospital Tongji Medical College Huazhong University of Science and Technology50 Gu Tian San Road Wuhan 430034 China

Correspondence should be addressed to Yan Chen chenyan puai126com

Received 24 February 2014 Revised 7 June 2014 Accepted 13 June 2014 Published 21 July 2014

Academic Editor Konstantin G Birukov

Copyright copy 2014 Qi-Hong Jiang 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

As a member of the ezrin-radixin-moesin (ERM) family radixin is overexpressed in many tumor tissues However little is knownabout its role in the progression of colon cancer So we here aimed to determine the function of radixin in colon cancer cellinvasion Interestingly we found that the expression of radixin was significantly elevated in colon cancer cells Knockdown ofradixin suppressed the invasion and migration of colon cancer cells Further knockdown of radixin inhibited the activation ofRac1 and ERK12 and decreased the expression and secretion of MMP-7 In addition Rac1-ERK signaling pathway was requiredfor the radixin-promoted invasion and MMP-7 production Together our findings suggest that radixin enhances the invasionand migration of colon cancer cells Activation of Rac1-ERK pathway and consequent upregulation of MMP-7 production maycontribute to the function of radixin in the regulation of colon cancer cell invasion Thus radixin may act as a novel target for thediagnosis and treatment of colon cancer

1 Introduction

Colon cancer is one of the most common malignancieswith high incidence and mortality rate worldwide [1] Earlydiagnostic and therapeutic strategies can improve the overallsurvival of patients with colon cancer However for patientsdiagnosed with advanced stage disease colon cancer hasoften metastasized Invasion and metastasis still account forthe main cause of death in colon cancer Invasion and metas-tasis are complex processes that require the participationof multiple pathways and genes [2] Therefore it is clearthat exploration of the key players that are involved in theinvasion and metastasis processes may provide diagnosticand therapeutic targets for colon cancer

The ezrinradixinmoesin (ERM) family is composed ofezrin radixin and moesin These proteins act as linkersbetween the actin cytoskeleton and plasma membrane pro-teins and play important roles in cell adhesion polarityand migration [3] The ERM proteins belong to the widelydistributed membrane-associated band 41 superfamily [4]

Structurally the three ERM proteins share 70ndash85 aminoacid identity and all consist of three domains a conservedN-terminal domain 120572-helical region and a C-terminaldomain The N-terminal domain is called the FERM (41-ezrine-radixine-moesin) domain and can interact directlywith membrane receptors such as CD44 and CD43 The120572-helical domain links the N-terminal domain and the C-terminal domain The C-terminal domain can mediate thelink between the actin cytoskeleton and the plasma mem-brane through F-actin binding site [5] Increased evidenceshave shown that ERM proteins are involved in the regulationof tumor progression and metastasis It is reported that ezrinexpression is correlated with metastasis and poor prognosisof many cancers such as nonsmall cell lung cancer breastcancer and gastric adenocarcinoma [6ndash8] Moesin acts as apotential epithelial-mesenchymal transition (EMT) markerin breast and pancreatic cancer and the expression level ofmoesin is linked to tumor size invasion and differentiationof oral squamous cell carcinoma [9] The expression level ofradixin is found to be significantly increased in colon tumor

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 340271 9 pageshttpdxdoiorg1011552014340271

2 The Scientific World Journal

tissues [10] However little is known with regard to the roleof radixin in colon cancer

In this study we examined the expression of radixinin colon cancer cells Further we investigated the role ofradixin in colon cancer cell invasion and migration andelucidated the possible molecular mechanism of radixin inregulating cell invasion Finally we identified that radixinpromoted invasion and migration of colon cancer cells byactivating Rac1-ERK pathway and by increasing MMP-7production

2 Materials and Methods

21 Cell Culture and Reagents All cell lines were obtainedfrom American Type Culture Collection (Manassas VAUSA) The nonmalignant human colonic epithelial cell lineNCM460 was cultured in M3D media supplemented with10 fetal bovine serum (FBS) The human colon cancercell lines HT-29 Caco-2 HCT116 and LoVo were grown inDMEM with 10 FBS Antibodies against radixin ERK12and120573-actin were purchased from Santa Cruz (Camarillo CAUSA) Antibodies against Rac1 and phospho-ERK12 werepurchased from Cell Signaling Technology (Danvers MAUSA) Rac1 specific inhibitor NSC23766 and ERK12 specificinhibitor U0126 were obtained from Sigma (St Louis MOUSA)

22 Real-Time PCR Total RNA of the cultured cells wasisolated by Trizol reagent (Invitrogen Carlsbad CAUSA) To obtain cDNA 2 120583g of RNA was subjectedto reverse transcription with Quantscript cDNA kit(TIANGEN Shanghai China) Real-time PCR wasperformed with cDNA real-time PCR Master Mixand primers The following primers of radixin (forwardprimer 51015840-TATGCTGTCCAAGCCAAGTATG-31015840 reverseprimer 51015840-CGCTGGGGTAGGAGTCTATCA-31015840) MMP-7(forward primer 51015840-GAGTGAGCTACAGTGGGAACA-31015840reverse primer 51015840-CTATGACGCGGGAGTTTAACAT-31015840)and 120573-actin (forward primer 51015840-ATAGCACAGCCT-GGATAGCAACGTAC-31015840 reverse primer 51015840-CACCTT-CTACAATGAGCTGCGTGTG-31015840) were used The real-timePCR cycle parameters were 10min at 95∘C and 40 cycles of 15s at 95∘C and 1min at 60∘C Gene expression was normalizedagainst the expression of 120573-actin gene and quantified by the2minusΔΔCt method

23 Western Blot Analysis After washing with sterile PBScells were lysed in RIPA lysis buffer supplemented withprotease and phosphatase inhibitors (Applygen TechnologiesInc Beijing China) The protein concentrations were deter-mined by the BCAmethodThen 50 120583g of protein was loadedin SDS-PAGE gel and electrophoresed under denaturingand reducing conditions The resolved proteins in the gelwere transferred to PVDF membranes The membranes werefurther immunoblotted with primary antibodies overnightat 4∘C Next the membranes were immunoblotted withsecondary antibodies for 1 h at room temperature Thenthe bands were visualized using electrochemiluminescence

(ECL) reagents (Applygen Technologies Inc) and Bio-MarkMS films

24 siRNA Transfection The control siRNA (51015840-UGGUUU-ACAUGUUUUCUGA-31015840) and radixin siRNA (51015840-AUG-UUCUUCAUGCCAGUUC-31015840) were obtained from Ge-nepharma (Shanghai China) HCT116 and LoVo cells weretransfected with siRNAs by using Lipofectamine RNAi Max(Invitrogen Carlsbad CA USA) After observing the knock-down efficiency by western blotting 48 h later the transfectedcells were used in the subsequent experiments

25 Invasion and Migration Assays The upper chambers of24-well Transwell plate (Costar San Diego CA USA) werecoated with (for invasion assay) or without (for migrationassay) Matrigel (BD Franklin Lakes NJ USA) before theexperiments were carried out The cells were adjusted at2 times 105mL in each group The upper chambers were loadedwith 200120583Lof cell suspensionwhile the lower chamberswereloaded with 500120583L of DMEM containing 30 FBS Afterincubation in a CO

2incubator at 37∘C for 12 h the invaded

or migrated cells were fixed and stained with crystal violetThe number of cells was counted from five randomly visualfields Data were expressed as the percentage of the control

26 Rac1 Activity Assay Cells transfectedwith control siRNAor radixin siRNA were lysed in RIPA lysis buffer and thensubjected to Rac1 activity assay kit (Millipore MA USA)according to manufacturerrsquos protocols Briefly the lysateswere incubated with p21 activated kinase-GST fusion proteinand Glutathione-Sepharose beads at 4∘C for 1 h Then thebeads were washed and the proteins were collected Totaland GTP bound Rac1 were determined by immunoblottingagainst Rac1

27 ELISA Assay Cell supernatant was collected from eachgroup and then was centrifuged for 10min at 10 000 rpmMMP-7 ELISA kit was purchased from Sigma and used toexamine the MMP-7 secretion in colon cancer cells accord-ing to manufacturerrsquos protocols Cells were lysed in RIPAlysis buffer and total protein concentrations were calculatedMMP-7 concentration was determined by normalizing tototal protein concentrations

28 Statistical Analysis All experiments were performed intriplicates All data representedmeanplusmn SD Statistical analysiswas performed with SPSS 170 software Studentrsquos 119905-test wasused to analyze the significance between two groups and one-way analysis of variance (ANOVA) was used to analyze thesignificance between multiple groups Statistical significancewas set at 119875 lt 005

3 Results

31 Radixin Expression Is Significantly Elevated in ColonCancer Cells Using real-time PCR and western blottingwe detected the expression of radixin in nonmalignanthuman colonic epithelial NCM460 cells and colon cancer

The Scientific World Journal 3

Radixin

NCM460 Caco-2 LoVo HCT116 HT2900

05

10

15

20

25

Rela

tive m

RNA

leve

l

lowast

lowast

lowastlowast

(a)


NCM460 Caco-2 LoVo HCT116 HT-29

0

1

2

3

Relat

ive p

rote

in le

vel

lowast

lowast

lowast

lowast

Radixin

120573-Actin

(b)

Figure 1 Radixin expression was upregulated in colon cancer cells (a) Radixin mRNA level in NCM460 HT-29 Caco-2 HCT116 and LoVocells was determined by real-time PCR (b) Radixin protein level in NCM460 HT-29 Caco-2 HCT116 and LoVo cells was determined bywestern blotting lowast119875 lt 005

HCT116 LoVoHCT116 LoVo00

05

10

15

siControlsiRadixin

siCon

trol

siRad

ixin

siCon

trol

siRad

ixin

Radixin

Relat

ive p

rote

in le

vel

lowastlowast

Radixin

120573-Actin

Figure 2 Knockdown efficiency of radixinwas examined bywestern blot analysis HCT116 and LoVo cells were transfectedwith either controlsiRNA (siControl) or radixin siRNA (siRadixin) for 48 h The knockdown efficiency was examined by western blot analysis lowast119875 lt 005

HT-29 Caco-2 HCT116 and LoVo cells Interestingly wefound that compared with NCM460 cells the expression ofradixin was markedly increased in all four colon cancer cells(Figures 1(a) and 1(b)) indicating that radixin may act as animportant player in colon cancer

32 The Expression of Radixin in Colon Cancer Cells WasSilenced by siRNA To investigate the role of radixin incolon cancer cell a radixin-specific siRNA was introduced

to silence radixin expression in HCT-116 and LoVo cells Theknockdown efficiency was up to 70 as assessed by westernblotting confirming that this radixin siRNA can prominentlyattenuate the expression of radixin inHCT-116 and LoVo cells(Figure 2)

33 Radixin Promotes the Invasion and Migration of ColonCancer Cells To examine the function of radixin in coloncancer cell invasion and migration invasion and migration


HCT116 LoVo

HCT116 LoVo

0

20

40

60

80

100

120

140

siControlsiRadixin

siControl siRadixin siControl siRadixin

Invasion

Cel

l inv

asio

n (

)

lowast lowast

(a)

HCT116 LoVo0

20

40

60

80

100

120

140

siControlsiRadixin

Migration

Cel

l mig

ratio

n (

)

lowast lowast

HCT116 LoVo


(b)

Figure 3 Knockdown of radixin by siRNA suppressed the invasion andmigration of colon cancer cells After transfectionwith radixin siRNAor control siRNA cells were subjected to invasion andmigration assays (a) Effect of radixin knockdown on the invasion of HCT116 and LoVocells (b) Effect of radixin knockdown on the migration of HCT116 and LoVo cells lowast119875 lt 005

assays were performed with control siRNA cells (siControl)and radixin siRNA cells (siRadixin) The results showed thatthe invasion and migration abilities of radixin siRNA cellswere significantly suppressed compared with control siRNAcells suggesting that radixin can enhance the invasion andmigration of colon cancer cells (Figures 3(a) and 3(b))

34 Rac1 Activated by Radixin Is Involved in the Invasion andMigration of Colon Cancer Cells Rho GTPase Rac1 is closelyrelated with cell invasion andmetastasis of cancers [11] Herewe found that knockdown of radixin led to a significantdecrease in Rac1 activation suggesting the involvement ofradixin in the regulation of Rac1 activation (Figure 4(a)) Toinvestigate the role of Rac1 activation in the radixin-promotedinvasion and migration we pretreated HCT116 and LoVocells with 100 120583M NSC23766 (Rac1 inhibitor) for 1 h Thenwe subjected the treated or untreated cells to invasion andmigration assaysThe results showed that inactivation of Rac1suppressed the invasion and migration of both HCT116 andLoVo cells (Figures 4(b) and 4(c)) suggesting that Rac1 isrequired for the radixin-promoted invasion and migration

35 Rac1-ERK Pathway Contributes to the Radixin-PromotedInvasion and Migration Next using western blotting wefound that knockdown of radixin inhibited the activationof ERK12 in both HCT-116 and LoVo cells (Figure 5(a))Furthermore to analyze the effect of Rac1 on the radixin-regulated ERK12 activation we pretreated HCT-116 andLoVo cells with 100 120583M NSC23766 (Rac1 inhibitor) for 1 hand then observed the phosphorylation of ERK12 Usingwestern blotting we found that inhibition of Rac1 attenuated

the phosphorylation of ERK12 (Figure 5(b)) These resultssuggest that radixin induces ERK12 activation via regulationof Rac1 activity To determine the function of ERK pathwayin colon cancer cell invasion and migration cells weresubjected to invasion and migration assays after treatmentwith 10 120583MU0126 (ERK12 inhibitor) for 30min Our resultsdemonstrated that U0126 treatment reduced the invasionand migration of colon cancer cells (Figures 5(c) and 5(d))Together with the results shown in Figure 4 these dataindicate that radixin enhances colon cancer cell invasion andmigration by activating Rac1-ERK pathway

36 Radixin Upregulates MMP-7 Production via Rac1 andERK12 Activation MMP-7 is crucial for the invasion andmetastasis of colon cancer [12] Here using real-time PCRandELISA assay we found that comparedwith control siRNAcells the expression and secretion of MMP-7 were greatlysuppressed in radixin siRNA cells (Figures 6(a) and 6(b))Next we incubated colon cancer cells with NSC23766 orU0126 for 12 h respectivelyThenwe examined the expressionand secretion of MMP-7 by real-time PCR and ELISA assayWe found that blocking of Rac1 and ERK12 activationboth downregulated MMP-7 expression and secretion (Fig-ures 6(c)ndash6(f)) Since radixin can induce Rac1 and ERK12activation as described above these results suggest thatradixin influences MMP-7 production via Rac1 and ERK12activation in colon cancer cells



05

10

15

siControlsiRadixin

siCon

trol

siRad

ixin

siCon

trol

siRad

ixin

Rac1

Relat

ive a

ctiv

atio

n of

Rac

1

lowastlowast

GTP-Rac1

120573-Actin

Total Rac1

(a)

lowastlowast

DMSONSC23766

HCT116 LoVo0

20

40

60

80

100

120

140

Cel

l inv

asio

n (

)

Invasion

(b)

DMSONSC23766

lowastlowast

HCT116 LoVo0

20

40

60

80

100

120

140Migration

Cel

l mig

ratio

n (

)

(c)

Figure 4 Radixin enhanced the invasion and migration of HCT116 and LoVo cells through Rac1 activation (a) Cells were transfected witheither control siRNA or radixin siRNA for 48 h Then the amount of GTP binding protein Rac1 was analyzed by Rac1 activity assay kit (b)and (c) Cells were pretreated with NSC23766 or DMSO for 1 h and then were subjected to invasion and migration assays lowast119875 lt 005

4 Discussion

Among the ERM proteins radixin has been known as animportant player in cell-cell and cell-matrix contacts as wellas in the reorganization of actin cytoskeleton [13] In thepresent study we found that the expression of radixin wasmarkedly elevated in colon cancer cells Further findingsshowed that radixin induced the activation of Rac1-ERKpathway leading to an increase in MMP-7 productionthereby contributing to the invasion and migration of coloncancer cells (Figure 7) To our knowledge this study is thefirst to reveal the molecular mechanism of radixin in coloncancer cell invasion

Clinical studies have observed the expression pattern ofradixin in different cancers In breast cancer and lung cancerreduced expression of radixin has been found in tumor tissuescompared with the adjacent benign tissues [14 15] On thecontrary the expression of radixin is greatly increased in

prostate cancer colon cancer pancreatic cancer and gliomasand elevated level of radixin is closely related to the poorprognosis of cancers [10 16 17] Our results showed thatthe expression of radixin was increased in colon cancercells further confirming a role of radixin in colon cancerExperimental studies have revealed that radixin contributesto the invasion and metastasis of gastric cancer cells andglioma cells [18 19] and silencing of radixin inhibited theinvasion and growth of pancreatic cancer cells in vitro andin vivo [20] However little is known about the functionof radixin in colon cancer Here using invasion assay andmigration assay we found that knockdown of radixin bysiRNA suppressed the invasion andmigration of colon cancercells implicating that radixin may be a key mediator in theregulation of colon cancer cell invasion

Rho GTPases are a family of GTP binding proteinsthat mediate actin dynamics and act as important modu-lators in various steps of cancer progression [21] As one


HCT116 LoVo

Relat

ive p

hosp

hory

latio

n of

ERK

lowastlowast

HCT116 LoVo00

05

10

15

siControlsiRadixin

siCon

trol

siRad

ixin

siCon

trol

siRad

ixin

p-ERK

p-ERK

ERK

120573-Actin

(a)


05

10

15

DMSONSC23766

DM

SO

NSC

2376

6

DM

SO

NSC

2376

6

p-ERK

Relat

ive p

hosp

hory

latio

n of

ERK

lowastlowast

p-ERK

ERK

120573-Actin

(b)

lowastlowast

DMSOU0126

HCT116 LoVo0

20

40

60

80

100

120

140

Cel

l inv

asio

n (

)

Invasion

(c)

lowast lowast

DMSOU0126

HCT116 LoVo0

20

40

60

80

100

120

140Migration

Cel

l mig

ratio

n (

)

(d)

Figure 5 Rac1-ERK pathway was involved in the radixin-mediated invasion and migration of colon cancer cells (a) After transfected witheither control siRNA or radixin siRNA for 48 h the phosphorylation of ERK12 was detected by western blotting (b) After incubation withNSC23766 or DMSO for 1 h the phosphorylation of ERK12 was detected by western blotting (c) and (d) Cells were pretreated with U0126or DMSO for 30min and then were subjected to invasion and migration assays lowast119875 lt 005


siControlsiRadixin

HCT116 LoVo00

05

10

15Re

lativ

e MM

P-7

mRN

A ex

pres

sion

lowast lowast

(a)

siControlsiRadixin

HCT116 LoVo0

20

40

60

80

MM

P-7

(ng

mg

prot

ein)

lowastlowast

(b)

HCT116 LoVo00

05

10

15

Relat

ive M

MP-

7 m

RNA

expr

essio

n

lowast lowast

DMSONSC23766

(c)

HCT116 LoVo

lowast

lowast

0

20

40

60

80

100

MM

P-7

(ng

mg

prot

ein)

DMSONSC23766

(d)

HCT116 LoVo00

05

10

15

Relat

ive M

MP-

7 m

RNA

expr

essio

n

lowast lowast

DMSOU0126

(e)

HCT116 LoVo

lowast

lowast

0

20

40

60

80

100

MM

P-7

(ng

mg

prot

ein)

DMSOU0126

(f)

Figure 6 Rac1-ERK pathway was required for the radixin-mediated MMP-7 production (a) and (b) After knockdown of radixin by siRNAthe production of MMP-7 was observed by real-time PCR and ELISA assay (c) and (d) After incubation with NSC23766 or DMSO for 12 hMMP-7 production was determined by real-time PCR and ELISA assay (e) and (f) After incubation with U0126 or DMSO for 12 h MMP-7production was detected by real-time PCR and ELISA assay lowast119875 lt 005


Colon cancer cellinvasion and migration

MMP-7

ERK12

Rac1

Radixin

Figure 7 Diagram depicting the possible molecular mechanism ofradixin in colon cancer cell invasion and migration

member of Rho GTPases Rac1 activation can affect theactin cytoskeleton andmediate the formation of lamellipodiaand filopodia [22] In addition Rac1 activation attenuatescell-cell adhesion by downregulation of E-cadherin [23]and increases the expression and secretion of metallopro-teinases (MMPs) [24]Thus activation of Rac1 contributes tothe migration invasion and metastasis of cancers [11] It isreported that radixin can regulate cell migration and cell-cell adhesion through Rac1 activation in prostate cancer cells[25] Consistently we found that radixin was necessary forthe activation of Rac1 in colon cancer cells Selective blockadeof Rac1 activity suppressed the invasion and migration ofHCT116 and LoVo cells implying the involvement of Rac1 inthe radixin-mediated invasion andmigration of colon cancercells

ERK pathway has a pivotal role in the regulation ofcancer development such as cell proliferation invasion andmetastasis [26] Of note ezrin as well as moesin can mediatethe activation of ERK pathway in cancer cells [27 28]However the relationship between radixin and ERK pathwayremains unclear Here our findings showed that radixin wasable to induce ERK12 activation Studies have reported thatRac1 can regulate multiple intracellular signaling pathways incolon cancer such as PI3KAKT and MAPK pathways [29]Our further experiments found that Rac1 was required for theradixin-mediated activation of ERK12 Moreover we foundthat ERK pathway contributed to the invasion and migrationof colon cancer cells Since radixin can regulate the activationof Rac1 and ERK12 and Rac1-ERK signaling is essential forthe colon cancer cell invasion and migration it is possiblethat radixin promotes the invasion and migration throughactivation of Rac1-ERK pathway

Belonging to the MMP family MMP-7 is overexpressedin many cancers including colon cancer and is associatedwith the metastasis and progression of cancers [30 31] Here

our results proved that radixin increased the expression andsecretion of MMP-7 Rac1 and ERK12 activation have beenfound to be a respond to the increased expression of MMPsinmost cancer cells [32] In this study we found that blockingof Rac1-ERK signaling decreased the expression and secretionof MMP-7 As radixin can regulate the activation of Rac1and ERK12 these data strongly support the notion thatradixin mediates MMP-7 production via activation of Rac1and ERK12

In summary our present work demonstrates that radixincan promote the invasion of colon cancer cells by activatingRac1-ERK pathway and upregulating MMP-7 productionTherefore radixin may act as a promising therapeutic targetfor the treatment of colon cancer

Conflict of Interests

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

References

[1] R Siegel J Ma Z Zou and A Jemal ldquoCancer statistics 2014rdquoCA ACancer Journal for Clinicians vol 64 no 1 pp 9ndash29 2014

[2] A K Pandurangan ldquoPotential targets for prevention of colorec-tal cancer a focus on PI3KAktmTOR and Wnt pathwaysrdquoAsian Pacific Journal of Cancer Prevention vol 14 no 4 pp2201ndash2205 2013

[3] J Clucas and F Valderrama ldquoERM proteins in cancer progres-sionrdquo Journal of Cell Science vol 127 part 2 pp 267ndash275 2014

[4] P Mangeat C Roy and M Martin ldquoERM proteins in celladhesion and membrane dynamicsrdquo Trends in Cell Biology vol9 no 5 pp 187ndash192 1999

[5] RG FehonA IMcClatchey andA Bretscher ldquoOrganizing thecell cortex the role of ERM proteinsrdquoNature ReviewsMolecularCell Biology vol 11 no 4 pp 276ndash287 2010

[6] J Jin T Jin M Quan Y Piao and Z Lin ldquoEzrin overexpressionpredicts the poor prognosis of gastric adenocarcinomardquo Diag-nostic Pathology vol 7 no 1 article 135 2012

[7] H W Lee E H Kim and M H Oh ldquoClinicopathologicimplication of ezrin expression in non-small cell lung cancerrdquoKorean Journal of Pathology vol 46 no 5 pp 470ndash477 2012

[8] L Ma and T Jiang ldquoClinical implications of Ezrin and CD44co-expression in breast cancerrdquoOncology Reports vol 30 no 4pp 1899ndash1905 2013

[9] H Kobayashi J Sagara H Kurita et al ldquoClinical significance ofcellular distribution of moesin in patients with oral squamouscell carcinomardquo Clinical Cancer Research vol 10 no 2 pp 572ndash580 2004

[10] Z Kanaan M Qadan M R Eichenberger and S GalandiukldquoThe actin-cytoskeleton pathway and its potential role ininflammatory bowel disease-associated human colorectal can-cerrdquoGenetic Testing andMolecular Biomarkers vol 14 no 3 pp347ndash353 2010

[11] A A P Schmitz E Govek B Bottner and L van Aelst ldquoRhoGTPases signaling migration and invasionrdquo Experimental CellResearch vol 261 no 1 pp 1ndash12 2000

[12] F Gao Y Zhang F Yang et al ldquoSurvivin promotes the invasionof human colon carcinoma cells by regulating the expression of


MMP7rdquo Molecular Medicine Reports vol 9 no 3 pp 825ndash8302014

[13] M Arpin D Chirivino A Naba and I Zwaenepoel ldquoEmergingrole for ERM proteins in cell adhesion and migrationrdquo CellAdhesion and Migration vol 5 no 2 pp 199ndash206 2011

[14] H Fernando T A Martin A Douglas-Jones H G KynastonR E Mansel and W G Jiang ldquoExpression of the ERMfamily members (ezrin radixin and moesin) in breast cancerrdquoExperimental andTherapeuticMedicine vol 1 no 1 pp 153ndash1602010

[15] M Tokunou T Niki Y Saitoh H Imamura M Sakamoto andS Hirohashi ldquoAltered expression of the ERM proteins in lungadenocarcinomardquo Laboratory Investigation vol 80 no 11 pp1643ndash1650 2000

[16] Y Cui J Wu M Zong et al ldquoProteomic profiling in pancreaticcancer with and without lymph node metastasisrdquo InternationalJournal of Cancer vol 124 no 7 pp 1614ndash1621 2009

[17] SWang B Jiao S Geng J Song Z Liang and S Lu ldquoConcomi-tant microRNA-31 downregulation and radixin upregulationpredicts advanced tumor progression and unfavorable progno-sis in patients with gliomasrdquo Journal of the Neurological Sciencesvol 338 no 1-2 pp 71ndash76 2013

[18] B Zheng L Liang S Huang et al ldquoMicroRNA-409 suppressestumour cell invasion and metastasis by directly targetingradixin in gastric cancersrdquo Oncogene vol 31 no 42 pp 4509ndash4516 2012

[19] D Hua D Ding X Han et al ldquoHuman miR-31 targets radixinand inhibits migration and invasion of glioma cellsrdquo OncologyReports vol 27 no 3 pp 700ndash706 2012

[20] S-D Chen M-M Song Z-Q Zhong et al ldquoKnockdown ofradixin by RNA interference suppresses the growth of humanpancreatic cancer cells in vitro and in vivordquoAsian Pacific Journalof Cancer Prevention vol 13 no 3 pp 753ndash759 2012

[21] FMVega andA J Ridley ldquoRhoGTPases in cancer cell biologyrdquoFEBS Letters vol 582 no 14 pp 2093ndash2101 2008

[22] N Tapon and A Hall ldquoRho Rac and Cdc42 GTPases regulatethe organization of the actin cytoskeletonrdquo Current Opinion inCell Biology vol 9 no 1 pp 86ndash92 1997

[23] B Hage K Meinel I Baum K Giehl and A Menke ldquoRac1activation inhibits E-cadherin-mediated adherens junctionsvia binding to IQGAP1 in pancreatic carcinoma cellsrdquo CellCommunication and Signaling vol 7 p 23 2009

[24] D C Radisky D D Levy L E Littlepage et al ldquoRac1b andreactive oxygen species mediate MMP-3-induced EMT andgenomic instabilityrdquo Nature vol 436 no 7047 pp 123ndash1272005

[25] F Valderrama SThevapala and A J Ridley ldquoRadixin regulatescell migration and cell-cell adhesion through Rac1rdquo Journal ofCell Science vol 125 no 14 pp 3310ndash3319 2012

[26] K B Reddy S M Nabha and N Atanaskova ldquoRole ofMAP kinase in tumor progression and invasionrdquo Cancer andMetastasis Reviews vol 22 no 4 pp 395ndash403 2003

[27] X Zhu F C Morales N K Agarwal T Dogruluk M Gageaand M M Georgescu ldquoMoesin is a glioma progression markerthat induces proliferation and Wnt120573-Catenin pathway activa-tion via interaction with CD44rdquo Cancer Research vol 73 no 3pp 1142ndash1155 2013

[28] J J Xie L Y Xu Y M Xie et al ldquoRoles of ezrin in thegrowth and invasiveness of esophageal squamous carcinomacellsrdquo International Journal of Cancer vol 124 no 11 pp 2549ndash2558 2009

[29] O Zugasti W Rul P Roux et al ldquoRaf-MEK-Erk cascade inanoikis is controlled by Rac1 and Cdc42 via AktrdquoMolecular andCellular Biology vol 21 no 19 pp 6706ndash6717 2001

[30] Z S Zeng W S Shu A M Cohen and J G GuillemldquoMatrix metalloproteinase-7 expression in colorectal cancerliver metastases evidence for involvement ofMMP-7 activationin human cancer metastasesrdquo Clinical Cancer Research vol 8no 1 pp 144ndash148 2002

[31] M Yoshimoto F Itoh H Yamamoto Y Hinoda K Imai andA Yachi ldquoExpression of MMP-7 (pump-1) mRNA in humancolorectal cancersrdquo International Journal of Cancer vol 54 no4 pp 614ndash618 1993

[32] J C Su J C Moon K S Bong K K Han and A KimldquoAkt- and MAPK-mediated activation and secretion of MMP-9 into stroma in breast cancer cells upon heregulin treatmentrdquoMolecular Medicine Reports vol 1 no 1 pp 83ndash88 2008

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


tissues [10] However little is known with regard to the roleof radixin in colon cancer

In this study we examined the expression of radixinin colon cancer cells Further we investigated the role ofradixin in colon cancer cell invasion and migration andelucidated the possible molecular mechanism of radixin inregulating cell invasion Finally we identified that radixinpromoted invasion and migration of colon cancer cells byactivating Rac1-ERK pathway and by increasing MMP-7production

2 Materials and Methods

21 Cell Culture and Reagents All cell lines were obtainedfrom American Type Culture Collection (Manassas VAUSA) The nonmalignant human colonic epithelial cell lineNCM460 was cultured in M3D media supplemented with10 fetal bovine serum (FBS) The human colon cancercell lines HT-29 Caco-2 HCT116 and LoVo were grown inDMEM with 10 FBS Antibodies against radixin ERK12and120573-actin were purchased from Santa Cruz (Camarillo CAUSA) Antibodies against Rac1 and phospho-ERK12 werepurchased from Cell Signaling Technology (Danvers MAUSA) Rac1 specific inhibitor NSC23766 and ERK12 specificinhibitor U0126 were obtained from Sigma (St Louis MOUSA)

22 Real-Time PCR Total RNA of the cultured cells wasisolated by Trizol reagent (Invitrogen Carlsbad CAUSA) To obtain cDNA 2 120583g of RNA was subjectedto reverse transcription with Quantscript cDNA kit(TIANGEN Shanghai China) Real-time PCR wasperformed with cDNA real-time PCR Master Mixand primers The following primers of radixin (forwardprimer 51015840-TATGCTGTCCAAGCCAAGTATG-31015840 reverseprimer 51015840-CGCTGGGGTAGGAGTCTATCA-31015840) MMP-7(forward primer 51015840-GAGTGAGCTACAGTGGGAACA-31015840reverse primer 51015840-CTATGACGCGGGAGTTTAACAT-31015840)and 120573-actin (forward primer 51015840-ATAGCACAGCCT-GGATAGCAACGTAC-31015840 reverse primer 51015840-CACCTT-CTACAATGAGCTGCGTGTG-31015840) were used The real-timePCR cycle parameters were 10min at 95∘C and 40 cycles of 15s at 95∘C and 1min at 60∘C Gene expression was normalizedagainst the expression of 120573-actin gene and quantified by the2minusΔΔCt method

23 Western Blot Analysis After washing with sterile PBScells were lysed in RIPA lysis buffer supplemented withprotease and phosphatase inhibitors (Applygen TechnologiesInc Beijing China) The protein concentrations were deter-mined by the BCAmethodThen 50 120583g of protein was loadedin SDS-PAGE gel and electrophoresed under denaturingand reducing conditions The resolved proteins in the gelwere transferred to PVDF membranes The membranes werefurther immunoblotted with primary antibodies overnightat 4∘C Next the membranes were immunoblotted withsecondary antibodies for 1 h at room temperature Thenthe bands were visualized using electrochemiluminescence

(ECL) reagents (Applygen Technologies Inc) and Bio-MarkMS films

24 siRNA Transfection The control siRNA (51015840-UGGUUU-ACAUGUUUUCUGA-31015840) and radixin siRNA (51015840-AUG-UUCUUCAUGCCAGUUC-31015840) were obtained from Ge-nepharma (Shanghai China) HCT116 and LoVo cells weretransfected with siRNAs by using Lipofectamine RNAi Max(Invitrogen Carlsbad CA USA) After observing the knock-down efficiency by western blotting 48 h later the transfectedcells were used in the subsequent experiments

25 Invasion and Migration Assays The upper chambers of24-well Transwell plate (Costar San Diego CA USA) werecoated with (for invasion assay) or without (for migrationassay) Matrigel (BD Franklin Lakes NJ USA) before theexperiments were carried out The cells were adjusted at2 times 105mL in each group The upper chambers were loadedwith 200120583Lof cell suspensionwhile the lower chamberswereloaded with 500120583L of DMEM containing 30 FBS Afterincubation in a CO

2incubator at 37∘C for 12 h the invaded

or migrated cells were fixed and stained with crystal violetThe number of cells was counted from five randomly visualfields Data were expressed as the percentage of the control

26 Rac1 Activity Assay Cells transfectedwith control siRNAor radixin siRNA were lysed in RIPA lysis buffer and thensubjected to Rac1 activity assay kit (Millipore MA USA)according to manufacturerrsquos protocols Briefly the lysateswere incubated with p21 activated kinase-GST fusion proteinand Glutathione-Sepharose beads at 4∘C for 1 h Then thebeads were washed and the proteins were collected Totaland GTP bound Rac1 were determined by immunoblottingagainst Rac1

27 ELISA Assay Cell supernatant was collected from eachgroup and then was centrifuged for 10min at 10 000 rpmMMP-7 ELISA kit was purchased from Sigma and used toexamine the MMP-7 secretion in colon cancer cells accord-ing to manufacturerrsquos protocols Cells were lysed in RIPAlysis buffer and total protein concentrations were calculatedMMP-7 concentration was determined by normalizing tototal protein concentrations

28 Statistical Analysis All experiments were performed intriplicates All data representedmeanplusmn SD Statistical analysiswas performed with SPSS 170 software Studentrsquos 119905-test wasused to analyze the significance between two groups and one-way analysis of variance (ANOVA) was used to analyze thesignificance between multiple groups Statistical significancewas set at 119875 lt 005

3 Results

31 Radixin Expression Is Significantly Elevated in ColonCancer Cells Using real-time PCR and western blottingwe detected the expression of radixin in nonmalignanthuman colonic epithelial NCM460 cells and colon cancer


Radixin


05

10

15

20

25

Rela

tive m

RNA

leve

l

lowast

lowast

lowastlowast

(a)



0

1

2

3

Relat

ive p

rote

in le

vel

lowast

lowast

lowast

lowast

Radixin

120573-Actin

(b)



05

10

15

siControlsiRadixin

siCon

trol

siRad

ixin

siCon

trol

siRad

ixin

Radixin

Relat

ive p

rote

in le

vel

lowastlowast

Radixin

120573-Actin







HCT116 LoVo

HCT116 LoVo

0

20

40

60

80

100

120

140

siControlsiRadixin


Invasion

Cel

l inv

asio

n (

)

lowast lowast

(a)

HCT116 LoVo0

20

40

60

80

100

120

140

siControlsiRadixin

Migration

Cel

l mig

ratio

n (

)

lowast lowast

HCT116 LoVo


(b)









05

10

15

siControlsiRadixin

siCon

trol

siRad

ixin

siCon

trol

siRad

ixin

Rac1

Relat

ive a

ctiv

atio

n of

Rac

1

lowastlowast

GTP-Rac1

120573-Actin

Total Rac1

(a)

lowastlowast

DMSONSC23766

HCT116 LoVo0

20

40

60

80

100

120

140

Cel

l inv

asio

n (

)

Invasion

(b)

DMSONSC23766

lowastlowast

HCT116 LoVo0

20

40

60

80

100

120

140Migration

Cel

l mig

ratio

n (

)

(c)


4 Discussion






HCT116 LoVo

Relat

ive p

hosp

hory

latio

n of

ERK

lowastlowast

HCT116 LoVo00

05

10

15

siControlsiRadixin

siCon

trol

siRad

ixin

siCon

trol

siRad

ixin

p-ERK

p-ERK

ERK

120573-Actin

(a)


05

10

15

DMSONSC23766

DM

SO

NSC

2376

6

DM

SO

NSC

2376

6

p-ERK

Relat

ive p

hosp

hory

latio

n of

ERK

lowastlowast

p-ERK

ERK

120573-Actin

(b)

lowastlowast

DMSOU0126

HCT116 LoVo0

20

40

60

80

100

120

140

Cel

l inv

asio

n (

)

Invasion

(c)

lowast lowast

DMSOU0126

HCT116 LoVo0

20

40

60

80

100

120

140Migration

Cel

l mig

ratio

n (

)

(d)



siControlsiRadixin

HCT116 LoVo00

05

10

15Re

lativ

e MM

P-7

mRN

A ex

pres

sion

lowast lowast

(a)

siControlsiRadixin

HCT116 LoVo0

20

40

60

80

MM

P-7

(ng

mg

prot

ein)

lowastlowast

(b)

HCT116 LoVo00

05

10

15

Relat

ive M

MP-

7 m

RNA

expr

essio

n

lowast lowast

DMSONSC23766

(c)

HCT116 LoVo

lowast

lowast

0

20

40

60

80

100

MM

P-7

(ng

mg

prot

ein)

DMSONSC23766

(d)

HCT116 LoVo00

05

10

15

Relat

ive M

MP-

7 m

RNA

expr

essio

n

lowast lowast

DMSOU0126

(e)

HCT116 LoVo

lowast

lowast

0

20

40

60

80

100

MM

P-7

(ng

mg

prot

ein)

DMSOU0126

(f)




MMP-7

ERK12

Rac1

Radixin









References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Radixin


05

10

15

20

25

Rela

tive m

RNA

leve

l

lowast

lowast

lowastlowast

(a)



0

1

2

3

Relat

ive p

rote

in le

vel

lowast

lowast

lowast

lowast

Radixin

120573-Actin

(b)



05

10

15

siControlsiRadixin

siCon

trol

siRad

ixin

siCon

trol

siRad

ixin

Radixin

Relat

ive p

rote

in le

vel

lowastlowast

Radixin

120573-Actin







HCT116 LoVo

HCT116 LoVo

0

20

40

60

80

100

120

140

siControlsiRadixin


Invasion

Cel

l inv

asio

n (

)

lowast lowast

(a)

HCT116 LoVo0

20

40

60

80

100

120

140

siControlsiRadixin

Migration

Cel

l mig

ratio

n (

)

lowast lowast

HCT116 LoVo


(b)









05

10

15

siControlsiRadixin

siCon

trol

siRad

ixin

siCon

trol

siRad

ixin

Rac1

Relat

ive a

ctiv

atio

n of

Rac

1

lowastlowast

GTP-Rac1

120573-Actin

Total Rac1

(a)

lowastlowast

DMSONSC23766

HCT116 LoVo0

20

40

60

80

100

120

140

Cel

l inv

asio

n (

)

Invasion

(b)

DMSONSC23766

lowastlowast

HCT116 LoVo0

20

40

60

80

100

120

140Migration

Cel

l mig

ratio

n (

)

(c)


4 Discussion






HCT116 LoVo

Relat

ive p

hosp

hory

latio

n of

ERK

lowastlowast

HCT116 LoVo00

05

10

15

siControlsiRadixin

siCon

trol

siRad

ixin

siCon

trol

siRad

ixin

p-ERK

p-ERK

ERK

120573-Actin

(a)


05

10

15

DMSONSC23766

DM

SO

NSC

2376

6

DM

SO

NSC

2376

6

p-ERK

Relat

ive p

hosp

hory

latio

n of

ERK

lowastlowast

p-ERK

ERK

120573-Actin

(b)

lowastlowast

DMSOU0126

HCT116 LoVo0

20

40

60

80

100

120

140

Cel

l inv

asio

n (

)

Invasion

(c)

lowast lowast

DMSOU0126

HCT116 LoVo0

20

40

60

80

100

120

140Migration

Cel

l mig

ratio

n (

)

(d)



siControlsiRadixin

HCT116 LoVo00

05

10

15Re

lativ

e MM

P-7

mRN

A ex

pres

sion

lowast lowast

(a)

siControlsiRadixin

HCT116 LoVo0

20

40

60

80

MM

P-7

(ng

mg

prot

ein)

lowastlowast

(b)

HCT116 LoVo00

05

10

15

Relat

ive M

MP-

7 m

RNA

expr

essio

n

lowast lowast

DMSONSC23766

(c)

HCT116 LoVo

lowast

lowast

0

20

40

60

80

100

MM

P-7

(ng

mg

prot

ein)

DMSONSC23766

(d)

HCT116 LoVo00

05

10

15

Relat

ive M

MP-

7 m

RNA

expr

essio

n

lowast lowast

DMSOU0126

(e)

HCT116 LoVo

lowast

lowast

0

20

40

60

80

100

MM

P-7

(ng

mg

prot

ein)

DMSOU0126

(f)




MMP-7

ERK12

Rac1

Radixin









References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HCT116 LoVo

HCT116 LoVo

0

20

40

60

80

100

120

140

siControlsiRadixin


Invasion

Cel

l inv

asio

n (

)

lowast lowast

(a)

HCT116 LoVo0

20

40

60

80

100

120

140

siControlsiRadixin

Migration

Cel

l mig

ratio

n (

)

lowast lowast

HCT116 LoVo


(b)









05

10

15

siControlsiRadixin

siCon

trol

siRad

ixin

siCon

trol

siRad

ixin

Rac1

Relat

ive a

ctiv

atio

n of

Rac

1

lowastlowast

GTP-Rac1

120573-Actin

Total Rac1

(a)

lowastlowast

DMSONSC23766

HCT116 LoVo0

20

40

60

80

100

120

140

Cel

l inv

asio

n (

)

Invasion

(b)

DMSONSC23766

lowastlowast

HCT116 LoVo0

20

40

60

80

100

120

140Migration

Cel

l mig

ratio

n (

)

(c)


4 Discussion






HCT116 LoVo

Relat

ive p

hosp

hory

latio

n of

ERK

lowastlowast

HCT116 LoVo00

05

10

15

siControlsiRadixin

siCon

trol

siRad

ixin

siCon

trol

siRad

ixin

p-ERK

p-ERK

ERK

120573-Actin

(a)


05

10

15

DMSONSC23766

DM

SO

NSC

2376

6

DM

SO

NSC

2376

6

p-ERK

Relat

ive p

hosp

hory

latio

n of

ERK

lowastlowast

p-ERK

ERK

120573-Actin

(b)

lowastlowast

DMSOU0126

HCT116 LoVo0

20

40

60

80

100

120

140

Cel

l inv

asio

n (

)

Invasion

(c)

lowast lowast

DMSOU0126

HCT116 LoVo0

20

40

60

80

100

120

140Migration

Cel

l mig

ratio

n (

)

(d)



siControlsiRadixin

HCT116 LoVo00

05

10

15Re

lativ

e MM

P-7

mRN

A ex

pres

sion

lowast lowast

(a)

siControlsiRadixin

HCT116 LoVo0

20

40

60

80

MM

P-7

(ng

mg

prot

ein)

lowastlowast

(b)

HCT116 LoVo00

05

10

15

Relat

ive M

MP-

7 m

RNA

expr

essio

n

lowast lowast

DMSONSC23766

(c)

HCT116 LoVo

lowast

lowast

0

20

40

60

80

100

MM

P-7

(ng

mg

prot

ein)

DMSONSC23766

(d)

HCT116 LoVo00

05

10

15

Relat

ive M

MP-

7 m

RNA

expr

essio

n

lowast lowast

DMSOU0126

(e)

HCT116 LoVo

lowast

lowast

0

20

40

60

80

100

MM

P-7

(ng

mg

prot

ein)

DMSOU0126

(f)




MMP-7

ERK12

Rac1

Radixin









References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















05

10

15

siControlsiRadixin

siCon

trol

siRad

ixin

siCon

trol

siRad

ixin

Rac1

Relat

ive a

ctiv

atio

n of

Rac

1

lowastlowast

GTP-Rac1

120573-Actin

Total Rac1

(a)

lowastlowast

DMSONSC23766

HCT116 LoVo0

20

40

60

80

100

120

140

Cel

l inv

asio

n (

)

Invasion

(b)

DMSONSC23766

lowastlowast

HCT116 LoVo0

20

40

60

80

100

120

140Migration

Cel

l mig

ratio

n (

)

(c)


4 Discussion






HCT116 LoVo

Relat

ive p

hosp

hory

latio

n of

ERK

lowastlowast

HCT116 LoVo00

05

10

15

siControlsiRadixin

siCon

trol

siRad

ixin

siCon

trol

siRad

ixin

p-ERK

p-ERK

ERK

120573-Actin

(a)


05

10

15

DMSONSC23766

DM

SO

NSC

2376

6

DM

SO

NSC

2376

6

p-ERK

Relat

ive p

hosp

hory

latio

n of

ERK

lowastlowast

p-ERK

ERK

120573-Actin

(b)

lowastlowast

DMSOU0126

HCT116 LoVo0

20

40

60

80

100

120

140

Cel

l inv

asio

n (

)

Invasion

(c)

lowast lowast

DMSOU0126

HCT116 LoVo0

20

40

60

80

100

120

140Migration

Cel

l mig

ratio

n (

)

(d)



siControlsiRadixin

HCT116 LoVo00

05

10

15Re

lativ

e MM

P-7

mRN

A ex

pres

sion

lowast lowast

(a)

siControlsiRadixin

HCT116 LoVo0

20

40

60

80

MM

P-7

(ng

mg

prot

ein)

lowastlowast

(b)

HCT116 LoVo00

05

10

15

Relat

ive M

MP-

7 m

RNA

expr

essio

n

lowast lowast

DMSONSC23766

(c)

HCT116 LoVo

lowast

lowast

0

20

40

60

80

100

MM

P-7

(ng

mg

prot

ein)

DMSONSC23766

(d)

HCT116 LoVo00

05

10

15

Relat

ive M

MP-

7 m

RNA

expr

essio

n

lowast lowast

DMSOU0126

(e)

HCT116 LoVo

lowast

lowast

0

20

40

60

80

100

MM

P-7

(ng

mg

prot

ein)

DMSOU0126

(f)




MMP-7

ERK12

Rac1

Radixin









References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HCT116 LoVo

Relat

ive p

hosp

hory

latio

n of

ERK

lowastlowast

HCT116 LoVo00

05

10

15

siControlsiRadixin

siCon

trol

siRad

ixin

siCon

trol

siRad

ixin

p-ERK

p-ERK

ERK

120573-Actin

(a)


05

10

15

DMSONSC23766

DM

SO

NSC

2376

6

DM

SO

NSC

2376

6

p-ERK

Relat

ive p

hosp

hory

latio

n of

ERK

lowastlowast

p-ERK

ERK

120573-Actin

(b)

lowastlowast

DMSOU0126

HCT116 LoVo0

20

40

60

80

100

120

140

Cel

l inv

asio

n (

)

Invasion

(c)

lowast lowast

DMSOU0126

HCT116 LoVo0

20

40

60

80

100

120

140Migration

Cel

l mig

ratio

n (

)

(d)



siControlsiRadixin

HCT116 LoVo00

05

10

15Re

lativ

e MM

P-7

mRN

A ex

pres

sion

lowast lowast

(a)

siControlsiRadixin

HCT116 LoVo0

20

40

60

80

MM

P-7

(ng

mg

prot

ein)

lowastlowast

(b)

HCT116 LoVo00

05

10

15

Relat

ive M

MP-

7 m

RNA

expr

essio

n

lowast lowast

DMSONSC23766

(c)

HCT116 LoVo

lowast

lowast

0

20

40

60

80

100

MM

P-7

(ng

mg

prot

ein)

DMSONSC23766

(d)

HCT116 LoVo00

05

10

15

Relat

ive M

MP-

7 m

RNA

expr

essio

n

lowast lowast

DMSOU0126

(e)

HCT116 LoVo

lowast

lowast

0

20

40

60

80

100

MM

P-7

(ng

mg

prot

ein)

DMSOU0126

(f)




MMP-7

ERK12

Rac1

Radixin









References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















siControlsiRadixin

HCT116 LoVo00

05

10

15Re

lativ

e MM

P-7

mRN

A ex

pres

sion

lowast lowast

(a)

siControlsiRadixin

HCT116 LoVo0

20

40

60

80

MM

P-7

(ng

mg

prot

ein)

lowastlowast

(b)

HCT116 LoVo00

05

10

15

Relat

ive M

MP-

7 m

RNA

expr

essio

n

lowast lowast

DMSONSC23766

(c)

HCT116 LoVo

lowast

lowast

0

20

40

60

80

100

MM

P-7

(ng

mg

prot

ein)

DMSONSC23766

(d)

HCT116 LoVo00

05

10

15

Relat

ive M

MP-

7 m

RNA

expr

essio

n

lowast lowast

DMSOU0126

(e)

HCT116 LoVo

lowast

lowast

0

20

40

60

80

100

MM

P-7

(ng

mg

prot

ein)

DMSOU0126

(f)




MMP-7

ERK12

Rac1

Radixin









References








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















MMP-7

ERK12

Rac1

Radixin









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 radixin enhances colon cancer cell...

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