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Toxicology Letters 225 (2014) 201– 207

Contents lists available at ScienceDirect

Toxicology Letters

jou rn al hom epage: www.elsev ier .com/ locate / tox le t

elecoxib potentially inhibits metastasis of lung cancer promoted byurgery in mice, via suppression of the PGE2-modulated-catenin pathway

huai Zhang1, Liangshan Da1, Xin Yang, Dongjie Feng, Rong Yin, Ming Li,hi Zhang, Feng Jiang ∗∗, Lin Xu ∗

iangsu Key Laboratory of Tumor Molecular Biology and Translational Medicine, Thoracic Surgery Department, Jiangsu Cancer Hospital,2 Baiziting, Nanjing 210009, People’s Republic of China

i g h l i g h t s

We studied anti-metastasis effects in models with endogenous and exogenous PGE2.We firstly investigated unilateral pneumonectomy-induced A549 cells metastasis.Celecoxib inhibited PGE2-induced metastasis by suppressing GSK3�-�-catenin pathway.We provide a new rationale for the antimetastatic activity of celecoxib.

r t i c l e i n f o

rticle history:eceived 7 August 2013eceived in revised form 5 December 2013ccepted 20 December 2013vailable online 25 December 2013

eywords:elecoxibGE2ung canceretastasis

urgery

a b s t r a c t

Surgery is the major treatment method for non-small cell lung cancer. It has been reported that plasmaPGE2 level is increased following surgery and stress which promotes lung cancer metastasis. In thepresent study, two animal models were used to confirm the effects of exogenous and endogenousprostaglandin E2 (PGE2) on metastasis of lung cancer cells. We found that both PGE2 level and A549metastasis were enhanced in mice with unilateral pulmonary resection following tail vein injection oflung cancer A549 cells. Both endogenous PGE2 level and pulmonary metastatic nodules were signifi-cantly reduced by celecoxib. A549 metastases were increased in mice after exogenous PGE2 injection. Inthe animal models, celecoxib inhibited lung cancer cell metastasis induced by exogenous PGE2. There-fore, we focused on the effects of celecoxib on the downstream pathway of PGE2 in vitro and found thatcelecoxib inhibited PGE2-induced A549 migration and invasion, which were evaluated by wound heal-ing and Transwell experiments. The expression of protein and mRNA of MMP9 and E-cadherin followingtreatment with PGE2 were suppressed and increased by celecoxib, respectively; however, MMP2 showedno change. A549 cell invasion and up-regulation of the expression of MMP9 and down-regulation of E-

cadherin induced by PGE2 were inhibited by FH535, an inhibitor of �-catenin. Deletion of �-catenin bysiRNA abrogated celecoxib-induced inhibition of MMP9 up-regulation and E-cadherin down-regulationby treatment of PGE2. Furthermore, we found that the level of �-catenin together with GSK-3� phos-phorylation was inhibited by celecoxib. In conclusion, celecoxib inhibits metastasis of A549 cells in the circulation enhanced by PGE2

by suppression downstream o

∗ Corresponding author at: Jiangsu Key Laboratory of Tumor Molecular Biology and Trffiliated Cancer Hospital of Jiangsu Province, Cancer Institution of Jiangsu Province, Nan∗∗ Corresponding author at: Jiangsu Key Laboratory of Tumor Molecular Biology and Trffiliated Cancer Hospital of Jiangsu Province, Cancer Institution of Jiangsu province, Nan

E-mail addresses: jiangfeng174@sohu.com (F. Jiang), xulin.jsch@gmail.com (L. Xu).1 These authors contributed equally to this work.

378-4274/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.toxlet.2013.12.014

after surgery by not only inhibiting endogenous PGE2 expression, but alsof PGE2 via the GSK-3�-�-catenin pathway.

© 2014 Elsevier Ireland Ltd. All rights reserved.

anslational Medicine, Department of Thoracic Surgery, Nanjing Medical Universityjing 210009, People’s Republic of China. Tel.: +86 25 83283408.anslational Medicine, Department of Thoracic Surgery, Nanjing Medical Universityjing 210009, People’s Republic of China.

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. Introduction

Surgery remains an essential therapeutic approach for mostolid malignancies, including non-small cell lung cancer (NSCLC).owever, surgery is also a risk factor for the promotion ofre-existing micro-metastases and the initiation of new metas-ases through several mechanisms, including the release ofrostaglandins and stress hormones (Friedrich et al., 1999; Howe,007; Muzii et al., 1996; Singh-Ranger et al., 2008). Prostaglandinsre paracrine and endocrine lipids that mediate pain and inflamma-ion. Prostaglandin E2 (PGE2) is abundantly secreted by endothelialells in damaged tissue following mechanical trauma. PGE2 is rec-gnized by four different subtypes of G protein-coupled receptorsEP1-4), and activation of these receptors causes an elevation in PKAnd c-AMP that mediates immunocyte suppression (Su et al., 2008;ugimoto and Narumiya, 2007). It has been reported that PGE2 acti-ates PI3K and the protein kinase, Akt, by free G protein �� subunitsnd the direct association of a G protein subunit with Axin1 throughts heterotrimeric guanine nucleotide-binding protein (G protein)-oupled receptor EP2. This results in the inactivation and release ofSK3� from the destruction complex and the consequent intracel-

ular accumulation of �-catenin (Castellone et al., 2005). �-cateninccumulation eventually results in its nuclear translocation. In theucleus, �-catenin binds to members of the TCF/LEF family of tran-cription factors, thus modulating expression of a broad range ofarget genes which are associated with cell survival, proliferationnd metastasis (Fodde and Brabletz, 2007).

Elevated levels of tumor cyclooxygenase-2 (COX-2) and itsetabolite PGE2 contribute to a decrease in E-cadherin, augmen-

ation of cancer motility and invasiveness, resistance to apoptosis,nd suppression of antitumor immunity (Castelao et al., 2003;hell et al., 2006; Wang and Dubois, 2010). COX-2 has been iden-ified as one of four key ‘metastasis progression’ genes, whichollectively synergize to mediate both tumor development andetastasis to other organs (Gupta et al., 2007). Therefore, COX-2

nhibitors have been reported to inhibit metastasis via suppressionf PGE2 production. However, the influence of COX-2 inhibitorsn the PGE2-induced downstream signal pathway in NSCLC cellsemains unknown.

Celecoxib is a NSAID that specifically inhibits COX-2. It hasignificant anti-inflammatory and analgesic properties but lesseroxicity than other NSAIDs such as aspirin and ibuprofen, whichnhibit both COX-1 and COX-2 (Harris et al., 2000). In this study,

e investigated the effect of celecoxib on plasma PGE2 level andetastasis of A549 cells intravenously inoculated in mice receiving

nilateral pneumonectomy.

. Materials and methods

.1. Cell culture and reagents

The human lung cancer cell line A549 was obtained from themerican Type Culture Collection (ATCC, Manassas, VA, USA). A549ells were cultured in RPMI-1640 (Gibco, USA) supplemented with0% fetal bovine serum (FBS, Invitrogen). Cultures were maintained

n an atmosphere containing 5% CO2 (Forma Scientific). Prior tohe experiments, cell viability greater than 95% was confirmed byrypan blue staining. Antibodies were obtained as follows: anti-

MP2, anti-MMP9, anti-E-cadherin, anti-�-catenin, anti-GSK-3�nd anti-�-actin were purchased from Santa Cruz BiotechnologyCA, USA); IRDyeTM 800-conjugated secondary antibodies were

btained from Rockland, Inc. (Philadelphia, USA). Chemicals werebtained as follows: DMSO, FH535, Celecoxib for cell experimentsere purchased from Sigma Chemical (St. Louis, MO, USA); andelecoxib for animal experiments from Pfizer Pharmaceuticals Ltd.

rs 225 (2014) 201– 207

(CA, USA); and the PGE2 ELISA kit from R&D System (MN, USA).BALB/c nude mice were purchased from the Model Animal ResearchCenter of Nanjing University (Nanjing, China).

2.2. In vivo animal model and lung metastasis (Backhus et al.,2006)

Female BALB/c nude mice weighing 18–22 g were randomlyassigned to the following five treatment groups (6 mice per group):control; mice were injected with PGE2 (1 mg/kg); mice wereinjected with PGE2 (1 mg/kg) and celecoxib (100 mg/kg); micereceived pneumonectomy; mice received pneumonectomy andcelecoxib (100 mg/kg). A549 cells were prepared as a suspensionof 106 cells in 150 �L saline and injected using a 29-gauge needleinto the lateral tail vein under sterile conditions. Celecoxib adminis-tration (100 mg/kg) began one day before tumor cell injection untilthe mice were sacrificed.

After the mice were sacrificed, the lungs were rinsed with 5%picric acid solution. Lung metastasis was determined by countingthe total number of metastatic nodules in each lung. The animalstudy was carried out according to the State Food and Drug Admin-istration of China regulations on animal care.

2.3. Pneumonectomy (Gibney et al., 2011)

Surgery was carried out on the same day as A549 cell injection.After general anesthesia and intubation, the animal was maintainedon a Flexivent rodent ventilator (SCIREQ, Montreal, QC, Canada) at200 bpm, 10 mL/kg, and PEEP of 2 cm H2O with a pressure limitedconstant flow profile. Pneumonectomy was performed through thefifth intercostal space via a left thoracotomy. With minimal manip-ulation of the lung, the hilum was ligated with a 5–0 surgicalsilk tie (Ethicon, Somerville, NJ, USA) and excised. A recruitmentmaneuver involving a 3 s ramp to 30 cm H2O and a 3 s plateau wasperformed while closing the thoracotomy with a 3-0 silk stitch(Ethicon). When spontaneous muscle activity returned, the animalwas extubated and transferred to a warming cage.

2.4. Wound healing assay (Kim et al., 2010)

A549 cells were seeded in six-well plates and incubatedovernight in starvation medium. Cell monolayers were woundedusing a sterile 200-�L pipette tip, washed with starvation mediumto remove detached cells, and incubated with the indicated agentsfor 24 h. Phase contrast pictures were taken with an inverted micro-scope using a magnification of 200.

2.5. Cell proliferation assay (Yin et al., 2011)

Cells were plated on 96-well plates at 4000 cells in 100 �L perwell and detected in quadruplicate at 24 h. 10 �L Cell Counting Kit-8(Dojindo) was added to each well and viable cells were measured byabsorbance at 450 nm after incubation at 37 ◦C for 3 h. Absorbancevalues were normalized to the medium control.

2.6. Transwell assay

Pre-chilled serum-free RPMI-1640 was mixed with matrigel(1:5; BD Biosciences) for the tumor invasion assay. Prior to thisassay, cells were pretreated with PGE2 or celecoxib for 24 h. Theupper hanging millicell chambers (Millipore, Billerica, MA, USA)were filled with 100 �L of the mixture inserted into the 24-well

plates, and the matrigel was allowed to solidify at 37 ◦C for 4 h. Aftersolidification, 50,000 cells were trypsinized, washed, resuspendedin RPMI-1640 containing 0.01% FBS (Gibco), and placed evenly inthe upper chambers. The lower chambers which contained 5% FBS

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n RPMI-1640 was used as a chemoattractant. After incubation at7 ◦C in 5% CO2 for 24 h, non-invaded cells in the upper chambersere removed with a cotton swab, and invaded cells were washed

wice with PBS and fixed in 70% methanol. Fixed cells were stainedith 0.1% crystal violet for 10 min and images obtained. Four ran-om fields were analyzed for each chamber. The same protocol wassed for the cell migration assay, except the inserts were coatedith matrigel.

.7. Enzyme-linked immunosorbent assay

For examination of PGE2 in the serum, three mice were ran-omly sacrificed and blood was extracted from the eyeball in eachroup at different time points. The measurement of PGE2 was car-ied out using the PGE2 Elisa Assay kit (R&D System, MN, USA)ccording to the manufacturer’s instructions.

.8. Total RNA extraction and real-time PCR (Eramo et al., 2008)

Total RNA was extracted using the mirVana RNA Isola-ion Kit (Applied Biosystems, USA). Real-time RT-PCR waserformed using the SYBR Premix Ex Taq kit (Takara). MMP2:′-TGTGTTCTTTGCAGGGAATGAAT-3′ (forward) and 5′-TGTCTTC-TGTTTTTGCTCCAGTTA-3′ (reverse); MMP9: 5′-CCTCTGGAGGTT-GACGTGA-3′ (forward) and 5′-TAGGCTTTCTCTCGGTACTGGAA-′ (reverse); E-cadherin: 5′-GGAATCCAAAGCCTCAGGTCAT-3′

forward) and 5′-GGCAGTAAGGGCTCTTTGACC-3′ (reverse);-actin: 5′-TGACGTGGACATCCGCAAAG-3′ (forward) and 5′-TGGAAGGTGGACAGCGAGG-3′ (reverse). The expression levelsere normalized to �-actin. All protocols were carried out accord-

ng to the manufacturer’s protocols. Real-time PCR was performedsing the ABI7300 real-time PCR system (Applied Biosystems,SA). Each sample was run in triplicate. The Ct-value for each

ample was calculated using the ��Ct-method and results werexpressed as 2 − ��CT as previously described.

.9. Western blot analysis

Proteins were extracted by lysing the cells in each well in 300 �Lf lysis buffer containing protease inhibitors. After the addition ofysis buffer, the cells were then scraped off and kept on ice for0 min. Protein concentration in the lysates was measured by theCA protein assay kit (Pierce, Rockford, IL, USA) after centrifugationt 12,000 × g for 5 min. Lysates with 60 �g of protein were subjectedo SDS-PAGE for detection. After transferring to a polyvinylideneuoride membrane, the membranes were incubated overnight at◦C with appropriate primary antibodies. After washing, the mem-rane was incubated with the appropriate secondary antibody for

h at room temperature followed by the ECL kit (Pierce, Rockford,L, USA) and detected using DNR (Bio-Imaging Systems, Jerusalem,srael).

.10. siRNA transfection

�-catenin siRNA was purchased from Santa Cruz BiotechnologySanta Cruz, CA, USA) and transfection was performed using Lipo-ectamine 2000TM reagent (Invitrogen, San Diego, CA) according tohe manufacturer’s instructions provided by the vendor. The cellsere then treated with PGE2 or celecoxib and harvested for western

lot assay.

.11. Statistical analysis

All results shown represent mean ± SEM from triplicate experi-ents performed in a parallel manner unless otherwise indicated.

tatistical analyses were performed using an unpaired, two-tailed

rs 225 (2014) 201– 207 203

Student’s t-test. All comparisons are made relative to untreatedcontrols and significance of difference is indicated as *P < 0.05 and**P < 0.01.

3. Results

3.1. Effects of celecoxib on the PGE2 level and metastasis of A549cells in mice receiving unilateral pneumonectomy

A549 cells were injected via the tail vein into nude mice andthe number of metastases on the lung surface was counted. Themetastasis of A549 cells in mice receiving unilateral pneumonec-tomy was more than that in control mice, and treatment withcelecoxib inhibited this increase in metastases (Fig. 1A and B). Ourresults showed that the mean level of plasma PGE2 in mice receiv-ing unilateral pneumonectomy increased compared with controlmice. Daily oral treatment with celecoxib (100 mg/kg) significantlyinhibited the increase in PGE2 plasma level (Fig. 1C).

3.2. Effect of celecoxib on metastasis of A549 cells in mice injectedwith PGE2

A subcutaneous injection of PGE2 in nude mice increased PGE2plasma level. The results showed that the mean metastasis of A549cells in mice injected with PGE2 was more than that in control mice.In addition, treatment with celecoxib suppressed lung metastasisof A549 cells in mice with high levels of PGE2 following exogenousPGE2 administration (Fig. 1A and B).

3.3. Effects of PGE2 and celecoxib on A549 viability andmetastasis in vitro

Celecoxib inhibited lung metastasis of A549 cells which wereinjected via the tail vein in nude mice with high levels of PGE2following exogenous PGE2 administration. These results indicatedthat celecoxib not only decreased the production of PGE2 but alsosuppressed the downstream effects of PGE2. It has been reportedthat PGE2 exerts pro-metastasis effects via promotion of the �-catenin pathway. Therefore, we assessed the anti-metastasis effectsof celecoxib on A549 cells with or without treatment with exoge-nous PGE2 and FH535 a �-catenin/TCF inhibitor. It can be seenin Fig. 2A and B that celecoxib (�10 �M) and PGE2 (�12.5 �M)had no effect on the viability of A549 cells. As shown in Fig. 3,PGE2 (10 �M) increased migration and invasion of A549 cells inthe wound healing and Transwell assays, which could be reversedby FH535. Moreover, celecoxib showed similar effects to those ofFH535.

3.4. Effects of PGE2 and celecoxib on MMP2/9 and E-cadherinexpression in A549 cells

MMP2/9 and E-cadherin have been reported to be regulated byPGE2 via the �-catenin pathway. Thus, we evaluated the mRNAand protein expression of MMP2/9 and E-cadherin. As shownin Fig. 4A, MMP2 mRNA expression was not affected by PGE2,celecoxib or FH535. However, mRNA expression of MMP9 wasmarkedly increased by PGE2 and reversed by either FH535 or cele-coxib. E-cadherin mRNA expression was inhibited by PGE2, andboth celecoxib and FH535 enhanced this expression (Fig. 4A). Theresults of Western blotting showed similar effects in the proteinexpression of MMP2/9 and E-cadherin (Fig. 4B and C). We also use

siRNA of �-catenin to silence its expression, data in Fig. 4D andE showed that deletion of �-catenin abrogated celecoxib-inducedinhibition of MMP9 up-regulation and E-cadherin down-regulationby treatment of PGE2.

204 S. Zhang et al. / Toxicology Letters 225 (2014) 201– 207

Fig. 1. The effects of celecoxib on A549 metastasis in PGE2- or pneumonectomy-treated mice. A549 cells were injected intravenously into athymic nude mice. Mice weregiven celecoxib and one lung per animal was evaluated for metastatic lesions. (A) Illustration of metastatic lesions in murine lungs from different experimental groups asim

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ndicated above. (B) Number of metastatic nodules in vehicle-, PGE2-, or celecoxib-treatice. Data are means ± SEM obtained from n = 6 mice per group. Significance of differenc

ig. 2. The effects of PGE2 or celecoxib on proliferation of A549 cells. (A) The effect of PGn proliferation of A549 cells. Bars are the mean ± SEM.

ed mice. (C) Plasma PGE2 level in vehicle-, pneumonectomy-, or celecoxib-treatede is indicated as *P < 0.05.

E2 (0–100 �M) on proliferation of A549 cells. (B) The effect of celecoxib (0–10 �M)

S. Zhang et al. / Toxicology Letters 225 (2014) 201– 207 205

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ig. 3. The effects of celecoxib on PGE2-induced A549 cell metastasis. (A) The effecffect of celecoxib and FH535 on migration and invasion of A549 cells with or witho

.5. Effects of PGE2 and celecoxib on the ˇ-catenin pathway in549 cells

PGE2-induced promotion of migration and invasion, and regu-ation of MMP9 and E-cadherin expression in A549 cells could beeversed by FH535. Thus, we assessed the influence of celecoxibn the �-catenin pathway. As shown in Fig. 5A, PGE2 increased

he protein level of �-catenin in the nucleus of A549 cells, whichould be reversed by celecoxib. PGE2 has been reported to inacti-ate GSK3� and induce a consequent intracellular accumulation of-catenin. Therefore, we evaluated the effect of celecoxib on the

lecoxib and FH535 on wound healing of A549 cells with or without PGE2. (B) TheE2. Bars are the mean ± SEM. Significance of difference is indicated as *P < 0.05.

expression and activation of GSK3�. As shown in Fig. 5B, celecoxibdid not change the expression of GSK3�; however, the activationof GSK3� was significantly inhibited.

4. Discussion

It has been reported that celecoxib showed some anti-

metastasis activity in tumor cells through inhibition of PGE2 pro-duction (Li et al., 2010; Yoshinaka et al., 2006). However, the influ-ence of celecoxib on the downstream pathway of PGE2 is unknown.In our results, celecoxib showed anti-metastasis activity in two

206 S. Zhang et al. / Toxicology Letters 225 (2014) 201– 207

Fig. 4. The effects of celecoxib on mRNA and protein expression of MMP-2, MMP-9, and E-cadherin in A549 cells. (A) A549 cells were treated with PGE2, celecoxib, or FH535and total RNA was extracted. mRNA expression of MMP-2, MMP-9, and E-cadherin was determined by real-time quantitative RT-PCR and analyzed by the ��Ct method.Bars are shown as mean ± SEM and represent MMP-2, MMP-9, or E-cadherin/�-actin fold change relative to the vehicle-treated group. (B) A549 cells were treated with PGE2,celecoxib, or FH535 and total protein was extracted. Western blots were performed to detect protein levels of MMP-2, MMP-9, and E-cadherin. (C) Bars shown are mean ± SEMMMP-2, MMP-9, and E-cadherin/�-actin fold change relative to the vehicle-treated group. (D) A549 cells were transfected transiently with scrambled or �-catenin siRNA andtreated with PGE2 or celecoxib and total protein was extracted. Western blots were performed to detect protein levels of MMP-2, MMP-9, and E-cadherin. (E) Bars shownare mean ± SEM MMP-2, MMP-9, and E-cadherin/�-actin fold change relative to the vehicle-treated group. Significance of difference is indicated as *P < 0.05, **P < 0.01.

Fig. 5. The effects of celecoxib on GSK-3� phosphorylation and nuclear expression of �-catenin in A549 cells. (A) The effect of celecoxib on �-catenin level in the nucleusof A549 cells with or without PGE2. (B) The effect of celecoxib on GSK-3� phosphorylation level in A549 cells with or without PGE2. (C) Bars shown are mean ± SEM�-catenin/histone H3 and GSK-3� or p-GSK-3�/�-actin fold change relative to the vehicle-treated group. Significance of difference is indicated as *P < 0.05.

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S. Zhang et al. / Toxicolog

nimal models including mice receiving unilateral pneumonec-omy to increase PGE2 level and mice injected with exogenousGE2. The number of metastatic nodes on the lung surface in miceeceiving surgery was more than that in mice with high PGE2 level,hich was due to the complex influence of surgery on metastasis.

nterestingly, celecoxib showed marked inhibition of lung metas-asis of A549 cells in the model which received exogenous PGE2,ndicating that celecoxib exerted its activities not only via inhibitionf endogenous PGE2 production. Therefore, we studied the effectsnd mechanism of celecoxib on PGE2-induced A549 metastasis.

Our results showed that celecoxib alone reduced the migra-ion and invasion at a dosage which did not inhibit the growthf A549 cells. Following treatment with PGE2, the metastasisapacity of A549 cells was enhanced, while celecoxib signifi-antly suppressed this metastasis capacity. PGE2 is recognized toxert pro-oncogenic actions including metastasis by stimulating-catenin/TCF-mediated transcription in colorectal carcinogenesis

Shao et al., 2005). It is now recognized that in lung cancer, PGE2ctivates EP4 to enhance phosphorylation of Akt and GSK-3� andnitiates �-catenin/TCF/LEF signaling. These effects lead to upregu-ation of MIG-7 whose protein level was positively correlated withdvanced stages of human lung cancers. MIG-7 has been reportedo contribute to cancer metastases arising from aberrant activationf the cellular COX-2/PGE2 and Akt/GSK-3� signaling pathwaysHo et al., 2013). Thus, we used FH535 to investigate whetherGE2-mediated migration and invasion were dependent on the �-atenin/TCF pathway. The results showed that FH535 reversed thero-metastasis effects of PGE2 and celecoxib exhibited a similar

nfluence which was greater than that by celecoxib alone.We then evaluated the mRNA and protein level of MMP2/9 and

-cadherin following treatment with celecoxib with or withoutGE2, and found that both MMP-9 and E-cadherin were affected byelecoxib and PGE2; however, MMP2 showed no marked change.MP-9 is recognized to correlate with poor cumulative survival

n lung cancer patients (Ylisirnio et al., 2000). However, the rea-on for non-activation of MMP2 expression by PGE2 is unclear,nd requires further investigation. We also observed that celecoxibnhibited exogenous PGE2-induced �-catenin accumulation in theucleus and inhibited activation of GSK-3�; however, the mecha-ism of GSK-3� inactivation by celecoxib is unknown. We suggesthat celecoxib exerted some influence on the AKT pathway whichegulates GSK-3� activation (Lin et al., 2007).

Surgery induces many changes in cytokines level and immu-ity (Benish and Ben-Eliyahu, 2010; Ogawa et al., 2000). PGE2 isn important mediator in the promotion of metastasis by surgery.owever, other factors which affect these parameters need to be

nvestigated. In addition, more data about our surgery model areeeded. Regardless of this lack of evaluation, this report representshe first demonstration of the effects of celecoxib on metastasis in

mouse model with unilateral pneumonectomy. We also assessedhe effects of celecoxib downstream of PGE2 involving the GSK-�/�-catenin pathway, and the findings suggest that celecoxib maye a potential therapeutic option for preventing metastasis afterneumonectomy.

onflicts of interest statement

The authors declare that there are no conflicts of interest.

cknowledgments

The present work was supported by the Natural Science Key

esearch Foundation of Jiangsu Province (BK2011036) for Dr Lin Xund the National Natural Science Foundation of China (81201830)or Dr Rong Yin.

rs 225 (2014) 201– 207 207

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.toxlet.2013.12.014.

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