Life Sciences 91 (2012) 789–799

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AEE788 potentiates celecoxib-induced growth inhibition and apoptosis in humancolon cancer cells

P. Venkatesan a,d, Sujit K. Bhutia b, Abhay K. Singh a, Swadesh K. Das b, Rupesh Dash b, Koel Chaudhury a,Devanand Sarkar b,c, Paul B. Fisher b,c, Mahitosh Mandal a,⁎a School of Medical Science and Technology, Indian Institute of Technology, Kharagpur 721302, Indiab Department of Human and Molecular Genetics, School of Medicine, Richmond, VA 23298, USAc VCU Institute of Molecular Medicine and VCU Massey Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, VA 23298, USAd Nanotech Research Facility, PSG Institute of Advanced Studies (PSGIAS), Coimbatore 641 004, India

⁎ Corresponding author. Tel.: +91 3222 283578; fax:E-mail addresses: mahitosh@smst.iitkgp.ernet.in, ma

(M. Mandal).

0024-3205/$ – see front matter © 2012 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.lfs.2012.08.024

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 16 April 2012Accepted 14 August 2012

Keywords:AEE788AngiogenesisCelecoxibColon cancerChemotherapyInhibitionPotentiationApoptosis

Aims: Combinatorial therapies that target multiple signaling pathways may provide improved therapeuticresponses over monotherapies. In the present study, we evaluated the effect of celecoxib and AEE788alone and in combination on cell proliferation, invasion, migration, angiogenesis, morphological changes,actin filament organization and apoptosis induction in the human colon cancer cell lines.Main methods: Effect of celecoxib and AEE788 alone and in combination on colon cancer cell lines wasevaluated by cell proliferation assay, morphological analysis, cell cycle analysis, scratch-wound healing andchorioallantoic membrane assays, zymography, nuclear fragmentation and western blot analyses.Key findings: Either drug alone or in combination inhibited human colonic adenocarcinoma cell lines HCT 15and HT 29 in a dose-dependent manner. Microscopic analysis revealed inhibition of cell membrane exten-sions, cell shrinkage, and disorganization of actin filaments. Additionally, either drug alone or in combinationinhibited HCT 15 migration, invasion and angiogenesis by suppressing matrix metalloproteinase-2 and -9activities. Increased reactive oxygen generation, loss of mitochondrial membrane potential, cleavage of PARP,

caspase-3 activation and DNA ladder formation characterized the induction of apoptosis by celecoxib and/orAEE788 treatment. Either drug individually induced apoptosis via down-regulation of the anti-apoptotic proteinsBcl2 and Bcl-xL, and up-regulation of pro-apoptotic protein Bax, cleavage of PARP, activation of caspase-3 andinhibition of vascular endothelial growth factor receptor signaling pathways.Significance: Results indicate that AEE788 potentiates celecoxib-mediated inhibition of proliferation andangiogenesis in HCT 15 colon cancer cells and may prove useful for developing a combinatorial therapy forcolon cancer.

© 2012 Elsevier Inc. All rights reserved.

Introduction

Colon cancer is the second leading cause of cancer-related deathworldwide (Lin et al., 2005). Based on a WHO report, colon canceraccounts for about 677,000 deaths per year (Yokoi et al., 2005). Globally,multiple research groups are focused on developing effective treatmentsfor colon cancer (Lin et al., 2005; Sakoguchi-Okada et al., 2007). Recur-rence and resistance to these multiple chemotherapies necessitatebetter treatment approaches to improve the outcome of this deadlydisease. Drug therapies targeting receptormolecules (epidermal growth

+91 3222 28222.hitoshm@gmail.com

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factor receptor, vascular endothelial growth factor receptor, etc.) thatare crucial to maintain the malignant phenotype of several cancer celllines are currently being developed (Kim et al., 2005).

Among the small molecule dual tyrosine kinase inhibitors, NVP-AEE788, a novel, 7H-pyrrolo [2, 3-d] class of pyrimidines, has advantagesof being well-tolerated, target-specific and displaying dose-dependentinhibition of multiple kinases (Traxler et al., 2004; Kim et al., 2005).AEE788 displays antiproliferative activity against several solid tumorswith remarkable anti-tumor effects in both in vitro and in vivo models(Makrilia et al., 2009; Grzmil and Hemmings, 2010). In general, combi-nation therapy is preferred over monotherapy, becausemultiple surviv-al pathways are activated in transformed cells (Yokoi et al., 2005; Qian etal., 2006; Gaikwad and Prchal, 2007). AEE788 in combinationwith otheranticancer agents additively or synergistically induces growth inhibitionand apoptosis in various cancer models (Yokoi et al., 2005; Qian et al.,2006; Gaikwad and Prchal, 2007; Yu et al., 2007; Grzmil and

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Hemmings, 2010). Accordingly, AEE788 represents an alternative orcomplement to cytotoxic anticancer agents to potentially improve thera-peutic response in patients with colon cancer.

In order to inhibit these survival pathways, multi-targeted, com-bination therapies are essential. Additionally, combination therapyfrequently leads to a reduction in dose and toxicity, and decreasesdevelopment of acquired drug resistance. Preclinical studies haveconfirmed anticancer activity of Nonsteroidal Anti-inflammatoryDrugs (NSAID's) on colorectal and several other tumors (Schiffmannet al., 2008). Within the family of NSAID's, celecoxib, a selectiveCOX-2 inhibitor, has been most frequently evaluated for its anticanceractivity in different cancer models (Venkatesan et al., 2010). Severalgroups have confirmed that COX-2 inhibitors enhance the cytotoxicityof chemotherapeutic agents (doxorubicin, bleomycin, vincristine and5-fluorouracil) and radiotherapy (Lin et al., 2005; Liu et al., 2008;Schiffmann et al., 2008; Qin et al., 2009). Strategies designed to selec-tively induce apoptosis in cancer cells offers significant potential foreliciting beneficial therapeutic effects. Cell death can be initiatedthrough either the extrinsic or intrinsic pathway (Ma et al., 2008).

To the best of our knowledge, no studies have previously reportedthe effect of the combination of celecoxib and AEE788 on coloncancer. This has prompted us to analyze the effect of AEE788 oncelecoxib-mediated inhibition of colon cancer cells HCT 15 and HT29 in vitro. Our results confirm and provide mechanistic insightsinto the prominent antiproliferative and antiangiogenic activities ofcelecoxib and/or AEE788 on HCT 15 colon cancer cells, which providea rationale for further detailed preclinical and potentially clinicalstudies of this combination for the therapy of colon cancer.

Materials and methods

Materials

Celecoxib and AEE788 were generously provided by Aarthi DrugLtd., India and Novartis Pharma, respectively. Both celecoxib andAEE788 were dissolved in DMSO to produce stock concentrations of1 M and 100 mM, respectively, and stored at−20 °C. The stock concen-trations were further diluted to appropriate concentrations in RPMI1640medium just before use. The concentration of DMSO in thefinal di-lution did not exceed 0.1%v/v. For Western blot analysis, the followingantibodies were used: polyclonal rabbit anti-VEGFR-2, anti-pVEGFR-2,monoclonal rabbit anti-Akt, anti-pAkt, anti-MAPK and anti-pMAPK(Cell Signaling Technology, Beverly, MA), monoclonal rabbit anti-PARP,anti-Bax, anti-caspase-3, anti-Bcl2, HRP-conjugated goat anti-rabbitIgG and goat anti-mouse IgG, AP-conjugated goat anti-rabbit IgGand goat anti-mouse IgG (Santa Cruz Biotechnology, CA) and mousemonoclonal anti-β-actin (Sigma-Aldrich, USA). Propidium iodide(PI), Hochest 33258, RNase (Ribonuclease), rhodamine-phalloidin,FITC (fluorescein isothiocyanate), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent, DAPI (4′,6-diamidino-2-phenylindole), chemiluminescent peroxidase substrate and DMSOwere obtained from Sigma-Aldrich, USA. RPMI 1640 medium, gluta-mine, penicillin, streptomycin and FBS were purchased from Himediaand Gibco® Invitrogen, India, respectively. All other chemicals used inthis study were of analytical grade. Stock solutions of MTT, PI andDAPI were prepared by dissolving 1 mg of each compound in 1 mlPBS. The solutions were protected from light, stored at 4 °C and usedwithin 1 month. Stock concentrations of 10 mg/ml RNase A wereprepared and kept at−20 °C.

Cell culture conditions

Human colon cancer cell lines HCT 15 andHT29 (National Centre forCell Science (NCCS), Pune, India) were grown as adherent cultures inRPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine,100 units/ml penicillin and 0.1 mg/ml streptomycin at 37 °C and 5%

CO2 in air. After cultures became 80% confluent (usually after 3 days),cells were trypsinized (0.25% Trypsin+0.1% EDTA), centrifuged(Heraeus Table Top Centrifuge E003, Germany) and suspended inRPMI 1640 medium. Cells used for experiments displayed >95% via-bility. For subsequent experiments, the cells were seeded in 96-wellplates, cover-slips and 60-mm petridishes.

Cytotoxicity

Conventional MTT assays were performed to determine celecoxib-and/or AEE788-induced cytotoxicity on HCT 15 and HT 29 cells asdescribed previously (Yuan et al., 2010). Celecoxib and AEE788were diluted to appropriate concentrations 0–200 μM and 0–20 μM,respectively. Briefly, cells in the exponential growth phase wereseeded in 96-well flat-bottom culture plates at a density of 3×103

(HCT 15) and 5×103 (HT 29) cells per well in 0.1 ml RPMI 1640complete medium. The cells were allowed to adhere and grow for24 h at 37 °C in an incubator (Heraeus Hera Cell, Germany), afterwhich the medium was aspirated and replaced with 0.1 ml freshmedium containing various concentrations of either celecoxib orAEE788 alone or in combination (various concentrations of celecoxibwith AEE788 at 3.8 μM). After 72 h of incubation, the culture mediumwas removed and 100 μl of 1 mg/ml MTT reagents dissolved in PBSwas added to each well. After 4–5 h of incubation, the unreducedMTT solution was discarded. Then, DMSO (100 μl) was added intoeach well to dissolve the purple formazan precipitate which wasreduced from MTT by active mitochondria of viable cells. Plateswere shaken and formazan dyewasmeasured spectrophotometricallyusing a Biorad microplate reader (Model 550, Japan). The assay wasperformed in triplicates. Cytotoxicity of each treatmentwas expressedas a percentage of cell viability relative to the untreated controlcells (% control) defined as: [[OD 550 nm treated cells] / [OD 550 nmcontrol cells]]×100, where optical density is abbreviated as OD.Unless mentioned, all the following experiments were performedwith concentrations of celecoxib (IC50), AEE788 (IC50) and combination(celecoxib (IC50)+AEE788 (3.8 μM)). As the inhibitory concentrationof AEE788 on HT 29 cell was high, we have chosen HCT cells withlow inhibitory concentration for a detailed study of effects of AEE788on celecoxib induced apoptosis.

Morphological analysis

HCT 15 cells seeded at a density of 6×103 were grown on sterilepoly-L-lysine coated cover glasses and treated without (control), orwith celecoxib (IC50), AEE788 (IC50) and celecoxib (IC50)+AEE788(3.8 μM) for 48 h. After incubation, the cells were observed underan optical microscope (20×, Leica, Germany)(Li et al., 2009). Further,morphological analyses using a scanning electron microscope (SEM)and an atomic force microscope (AFM) were performed to obtaininformation regarding the effect of celecoxib and/or AEE788 oncell membrane extensions (filopodia and lamellipodia) (Venkatesanet al., 2010). After treatment with celecoxib (IC50), AEE788 (IC50)and celecoxib (IC50)+AEE788 (3.8 μM) the cells were washed threetimes in 0.1 M cacodylate buffer (pH 7.4) and then fixed in ice-cold1% OsO4 for 1 h. The cells were then dehydrated with ethanol (50%,70%, 95% and 100%). The samples were placed in HMDS (1,1,1,3,3,3-hexamethyl disilazane) for 5 min to overcome drying effects. Sampleswere then air dried at room temperature and mounted on a stub.Next, they were placed in a vacuum chamber of SEM gold coatingapparatus and gold was coated at 2.5 kV, 20–25 mA for 120 s. Themorphogram of the HCT 15 cells was then observed using SEM(JEOL JSM5800, Japan) using 20 kV acceleration voltage.

For AFM analysis, celecoxib (IC50), AEE788 (IC50) and celecoxib(IC50)+AEE788 (3.8 μM) treated HCT 15 cells on cover slips werefixed with formaldehyde, washed with PBS and dehydrated in a seriesof alcohol gradients (50%, 70%, 90%, 95% and 100% each) for 10 min

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each. After dehydration, samples were dipped in HMDS and air-dried.Slides were then mounted and thorough examination of cell surfacewas performed using tapping mode AFM (Veeco CPII, USA) (Li et al.,2008; Venkatesan et al., 2010). The tapping mode settings were asfollows: 0.5 Hz scan rate with a resolution of 256×256 data pointsper scan. AV-shaped silicon nitride cantilever (MMP-11123, Veeco In-struments Inc., USA) having spring constant 40 N/m, length115–135 μm and radii of curvature less than 10 nm were used.

Cytoskeleton and apoptosis study

Cytoskeletal analysis of celecoxib and/or AEE788 treated HCT 15cells was performed using a confocal laser scanning electron micro-scope (CLSM) (Venkatesan et al., 2010). Briefly, HCT 15 cells weregrown on poly-L-lysine-coated cover glass slides and treated witheither celecoxib (IC50), AEE788 (IC50) and celecoxib (IC50)+AEE788(3.8 μM) for 48 h. The slides were removed, washed three timeswith PBS (pH 7.4), fixed with ice-cold 4% paraformaldehyde in PBS(pH 7.4) and permeabilized with 0.1% Triton X-100. Then, non-specific binding sites were blocked using PBS containing 10% FBS for1 h. The cells were stained with the blue fluorescent DNA stain(Hoechst 33258) and the red fluorescent dye (rhodamine-phalloidin)to visualize nuclei and cytoskeletal actin, respectively. After staining,the adhered cells were washed with PBS, air dried and mounted onslides. Fluorescent images from the stained constructs were obtainedusing a CLSM (40×, Olympus FV 1000, Japan) equipped with argon(488 nm) and He–Ne (534 nm) lasers.

Cell cycle analysis

HCT 15 cells at a density of 2×105 were cultured in 60-mmpetridishes for 24 h and then treated with celecoxib (IC50), AEE788(IC50) and celecoxib (IC50)+AEE788 (3.8 μM) for 48 h. After incuba-tion, the cells were trypsinized and centrifuged at 1200 rpm for 5 minat 4 °C. The pellet was suspended in 5 ml of PBS and then centrifugedat 1200 rpm for 10 min at 4 °C. The supernatant was discarded andthe pellet was fixed with 2 ml of ice-cold ethanol solution (70%v/vin PBS) at 4 °C overnight. Fixed cells were centrifuged at 1200 rpmfor 10 min at 4 °C and the pellet was incubated with PI mixture(10 mg/ml RNase and 20 μg/ml PI dissolved in cold PBS) for 30 minat 37 °C. DNA content analysis was carried out on a FACSCalibur(BD Bioscience, USA) flow cytometer (10,000 events were acquiredfor each sample). The data obtained were processed for cell cycledistribution with the cell quest pro software package (Tharakan etal., 2010). The amount of PI intercalating to DNA was used as aparameter to determine the cell cycle distribution phases. The apo-ptosis fraction was considered as DNA loss resulting in a sub-G1 peak.

ROS and ψm measurement

Intracellular production of ROS was measured using the oxidationsensitive dye 5-(and 6)-chloromethyl-2,7-dichlorodihydrofluoresceindiacetate (H2DCFDA) (Yu et al., 2007; Wang et al., 2010). Briefly, HCT15 cells were treated with celecoxib (IC50), AEE788 (IC50) andcelecoxib (IC50)+AEE788 (3.8 μM) for 24 h. After treatment, thecells were trypsinized, pelleted and resuspended in PBS containing10 μmol/l of H2DCFDA. After 1 h of incubation at 37 °C, the cellswere washed with PBS to remove unreacted dye and re-incubated inmedium at 37 °C for 10 min. ROS generation was estimated on FL-1channel of FACSCalibur (BD, Bioscience, USA) and analyzed usingCellquest software with excitation at 480 nm and emission at530 nm. The effects of celecoxib and/or AEE788 on ROS generationof HCT 15 cells were plotted and comparedwith fluorescence intensityof the control. Chemotherapeutic agent-induced ROS generationwhich is related to apoptosis induction was confirmed by Wright–Giemsa staining. In brief, after treatment with celecoxib (IC50),

AEE788 (IC50) and celecoxib (IC50)+AEE788 (3.8 μM) for 24 h, theadherent cells on slides were fixed, stained and evaluated under lightmicroscope for apoptosis. Apoptotic cells were identified by classicmorphological characteristics (cell shrinkage, nuclear condensationand apoptotic bodies). Five randomly selected fields comprising a totalof 200 cells per slide were calculated for the percentage of apoptoticcells. In order to analyze the role of mitochondria in celecoxib- and/orAEE788-mediated apoptosis, changes in ψm were measured usingthe cationic fluorescence dye rhodamine 123 (Rh 123) (Wang et al.,2008a; Cao et al., 2010). Briefly, HCT 15 cells were plated at a densityof 1×105/ml in a 60-mm petridish for 24 h and treated with celecoxib(IC50), AEE788 (IC50) and celecoxib (IC50)+AEE788 (3.8 μM) for 0, 12and 24 h. The cells were washed, harvested and incubated with Rh123 (10 μg/ml) in chilled PBS at 37 °C for 30 min in the dark. The cellswere then washed twice with PBS and ψm (fluorescence intensity)was measured on FL-1 channel of FACSCalibur at 530 nm with 10,000cells.

Scratch-wound healing and chick chorioallantoic membrane (CAM) assays

Effect of celecoxib and/or AEE788 on wound healing was analyzedby the scratch-wound healing assay with some modifications fromthe published protocol (Yee et al., 2010). HCT 15 cells were grownon coverslips to form a nearly confluent monolayer. Then the cover-slips were incubated in serum-free RPMI 1640 medium for 24 h toreduce the influence of hormones and growth factors. After theperiod of serum starvation, the confluent monolayers were scratchedto form a “wound” using a 200 μl sterile pipette tips. Cellular debriswere removed by washing with PBS. The cells were then incubatedin the absence or presence of celecoxib (IC50), AEE788 (IC50) andcelecoxib (IC50)+AEE788 (3.8 μM) in serum-free media for 24 h.Slides were washed with PBS, fixed with 4% glutaraldehyde andstained with hematoxylin–eosin. The images were recorded at 0 and24 h to monitor the migration of cells into the wounded area usinga photomicroscope (20×, Leica DMR, Germany). To quantitate theanalysis, the percentage of wound (scratch area) at 0 h (control)was arbitrarily assigned as 100% and the percentage of wound healingat 24 h (untreated control and treated) was the rate compared to thatat 0 h (control). Each assay was performed in triplicate.

To determine in vivo antiangiogenic activity of celecoxib and/orAEE788 onHCT 15 cells, CAM assayswere performed as described pre-viously with some modifications (Emdad et al., 2009). Two-day oldfertilized eggs were incubated at 37 °C in 60–70% relative humidity.After 5 days of incubation, a 1–2 cm2 window was opened and asterile round filter paper (5-mm in diameter, Whatman qualitativefilter papers, Sigma-Aldrich) containing PBS or celecoxib and orAEE788 (IC50 concentrations/filter paper) was applied onto the CAMof individual embryos. After 2 days of incubation, the upper eggshellwas removed and capillaries within 2.5-mm around the filter paperwere observed and photographed under a stereomicroscope (Olympus,SZX16, USA).

Gelatin zymography

Effect of celecoxib and/or AEE788 on MMP-2 and -9 was deter-mined by gelatin zymography (Yun et al., 2010). Briefly, HCT 15 cellswere seeded in 90-mm petri plates at a density of 6×105cells/ml.Cells were allowed to adhere for 24 h and then treated with varyingconcentrations of celecoxib (IC50), AEE788 (IC50) and celecoxib(IC50)+AEE788 (3.8 μM) in conditioned media for 24 h. After treat-ment, protein quantification in conditioned media was performedwith a standard protein assay (Bio-Rad, India). Proteins were loadedat 30 μg per lane under non-reducing conditions on 10% polyacryl-amide gels containing 1 mg/ml gelatin and separated at 50 V. Thegel was washed 3 times at room temperature in a solution containing2.5%v/v Triton X-100 in H2O and incubated at 37 °C for 24 h in

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zymogram developing buffer (50 mM Tris/HCl, pH 7.5, 0.2 M NaCl,5 mM CaCl2, 1 mM ZnCl2 and 0.02% Brij 35). Areas of gelatinhydrolysed by MMPs were visualized as clear zones against a bluebackground after Coomassie blue staining and the intensities of thebands were estimated by densitometry using an electrophoresisdocumentation and analysis system (EDAS 290 Kodak, Rochester, NY).

DNA fragmentation analysis

To determine DNA fragmentation, HCT 15 cells were seeded in90-mm petri plates and treated with celecoxib (IC50), AEE788 (IC50)and celecoxib (IC50)+AEE788 (3.8 μM) for 48 h (Liu et al., 2008).Then, both adherent and floating cells were collected and rinsedtwice with PBS. The pellet was resuspended in lysis buffer containing1% Nonidet P-40, 2 mMEDTA and 50 mMTris (pH 7.5) for 1 h. SolubleDNA was extracted with phenol/chloroform. After centrifugation, thesoluble DNA fragments were precipitated by the addition of 0.5 mlof 7.5 M ammonium acetate and 2 ml of ethanol. Theywere incubatedwith RNase A and proteinase K at 37 °C for 60 min. The DNA pellet wasdissolved in TE buffer, loaded onto a 1.5% agarose gel and separatedat 50 V for 90 min. DNA fragments were visualized after stainingwith ethidium bromide by transillumination under UV light.

Western blot analysis of apoptotic and growth regulatory proteins

Cells were treated with celecoxib (IC50) or AEE788 (IC50) for 48 h.The cells were then scraped and lysed in Nonidet P-40 lysis buffer(50 mM Tris HCl–pH 8.0, 137 mM sodium chloride, 10% glycerol, 1%Nonidet P-40, 50 mM sodium fluoride and 10 mM EDTA) containing1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride andprotease cocktail inhibitor. Cell extracts were separated on SDS-PAGE,transferred to nitrocellulose membranes, blocked with 2% BSA andprobed with the appropriate antibodies. Membranes were then devel-oped using enhanced chemiluminescence or alkaline phosphatase-based colorimetric methods (Sarkar et al., 2010, 2011).

Inhibition of PVEGFR-2/VEGFR-2, PMAPK/MAPK and PAKT/AKT

Cells were seeded in a six-well plate at a density of 4×105 cellsper well and incubated in 10% FBS medium overnight. The next day,the cells were washed and incubated in serum free medium for24 h. The experimental wells were treated with celecoxib (IC50) orAEE788 (IC50) for 1 h, whereas the control wells were treated with0.1% DMSO for 1 h. Then, cells were activated with recombinanthuman EGF (25 ng/ml) for 30 min, washed with PBS and scrapedin lysis buffer as described previously (Sarkar et al., 2010, 2011).The proteins (50 μg) were resolved on SDS-PAGE and Western blotanalysis was performed.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 5software. Datawere presented as themean±S.D. Statistical significancewas determined by one-way analysis of variance (ANOVA). ***Pb0.001and **pb0.05 were considered significant.

Results

Celecoxib and AEE788 cooperatively inhibit cell proliferation

AEE788 inhibited growth of HCT 15 and HT 29 cells at an IC50of 7.62±1.2 μM and 12.07±0.8 μM, respectively (Fig. 1a). HCT 15cells exhibited celecoxib-mediated growth inhibition at an IC50 of102.71±0.9 μM (Fig. 1b — solid line), which was further reduced bythe addition of 3.8 μM AEE788 (reducing the IC50 of celecoxib from102.71±0.9 μM to 37.65±1.8 μM). Treatment of celecoxib (IC50) in

combination with AEE788 (3.8 μM) resulted in a leftward shift ofthe concentration–response curve (Fig. 1b — dashed line), indicatingenhanced cytotoxicity. Data represent the mean±S.D. of three inde-pendent experiments. As the inhibitory concentration of AEE788on HT 29 cell was high, we have chosen HCT cells with low inhibitoryconcentration for a detailed study of effects of AEE788 on celecoxibinduced apoptosis.

Morphological analysis using advanced microscopic techniques

Optical and SEM analyses were used to visualize the effectsof celecoxib and/or AEE788 on the morphology of HCT 15 cells.In optical microscopy, untreated control HCT 15 cells displayed awell-spread and flattened morphology (Fig. 1c). Conversely, eithercelecoxib or AEE788 treated HCT 15 cells displayed morphologicalchanges consisting of cell rounding, reduced spreading, shrinkageand retraction of cellular processes.

SEM was used to analyze the morphology of HCT 15 cells treatedwith celecoxib (IC50), AEE788 (IC50) and celecoxib (IC50)+AEE788(3.8 μM) (Fig. 1d). Control cells appeared flat with a large numberof smooth, slender and filamentous lateral cell membrane extensions(arrowheads). This observation supports the highly motile nature ofHCT 15 cells. However, after treatment of HCT 15 cells with eitherdrug the cells displayed a thickenedmorphology, small ruffles, irregu-lar retraction of cytoplasm from the substratum and truncated cellmembrane extensions (arrows). These changes weremore prominentin HCT 15 cells treated with the combination of celecoxib (IC50) andAEE788 (3.8 μM).

AFManalysis of cancer cells provides valuable information regardingcytoskeleton and morphology. In this study, morphological features ofcelecoxib- and/or AEE788-treated HCT 15 cells were analyzed usingAFM (Fig. 2a). Control HCT 15 cells displayed smooth and elongatedshapes with dense peripheral cell membrane extensions, especiallyat the terminal parts (arrows). AEE788-treated HCT 15 cells displayedrough and shrunken cell morphology with truncated filopodia andlamellipodia at their terminal parts. However, less damage in mor-phology (cell shrinkage) and effects on cell membrane extensionswere observed in HCT 15 cells treated with celecoxib as comparedto AEE788 (arrowheads). It is worth noting that, there are some intactcell membrane extensions at both the body and terminal parts of thecelecoxib-treated HCT 15 cells. AFM images of combination-treatedHCT 15 cells indicate rough and shrunken cell morphology with acomplete loss of cell membrane extensions.

Actin filament organization and apoptosis analysis

In these experiments, we analyzed cell morphology and actinfilament organization of celecoxib- and/or AEE788-treated HCT 15cells using CLSM(Fig. 2b). The control HCT 15 cells displayed a stretchedmorphology with smooth, continuous dense networks of actin formingorganized parallel filamentous structures in the cytoplasm. AEE788-treated cells displayed a round shape with a sparse and irregular actinfilament organization and without striations. Celecoxib (IC50) treat-ment resulted in insignificant changes in cell morphology and actinfilament organization. AEE788 (IC50) and celecoxib (IC50)+AEE788(3.8 μM) resulted in a complete loss of cell membrane extensions. Thecombination-treated cells had a rounded morphology with a moreirregular actin filament organization. In contrast, celecoxib-treatedHCT 15 cells displayed partially intact filopodia and lamellipodia ontheir cell surface.

Cell cycle analysis

The effect of celecoxib and/or AEE788 on HCT 15 cell cycle wasanalyzed (Fig. 3a). Celecoxib and/or AEE788 treated HCT 15 cellshad an increased percentage of apoptotic cells (sub-G1 phase)

Fig. 1. Cytotoxicity of (a) AEE788 (IC50) on HCT 15 and HT 29 cells, (b — solid line) celecoxib (IC50) and, (b — dashed line) Combination of celecoxib (IC50) and AEE788 (3.8 μM) onHCT 15 cells was measured using MTT assays. (c) Phase contrast microscopic images (20×) of celecoxib (IC50), AEE788 (IC50) and celecoxib (IC50)+AEE788 (3.8 μM) treated HCT15 cells for 48 h. Shrunken and apoptotic cells are marked with arrows. All images were taken under identical instrument conditions and are presented at the same intensity scale.(d) Scanning electron photomicrograph of HCT 15 cells treated with celecoxib (IC50), AEE788 (IC50) and celecoxib (IC50)+AEE788 (3.8 μM) for 48 h. In the photomicrograph,healthy filopodia and lamellipodia are marked with arrowheads and the truncated lamellipodia and filopodia are marked with arrows.

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compared to untreated control (0.27±1.6%). In particular, a signifi-cantly higher percentage of apoptotic cells was observed followingcelecoxib and AEE788 combination treatment (54.9±1.1) than witheither drug alone (celecoxib: 18.36±1.7%; AEE788: 34.3±1.3%)(Fig. 3b).

ROS generation and loss of ψm by treatment with celecoxib and/or AEE788

Studies were conducted to investigate whether generation of in-tracellular ROS is part of the mechanism by which celecoxib and/orAEE788 induce apoptosis in HCT 15 cells. A dramatic ROS burst wasevident upon treatment of HCT 15 cells with celecoxib and/or AEE788as compared to untreated cells. It is important to note that, HCT 15cells treated with AEE788 (IC50) resulted in significantly higher genera-tion of ROS than celecoxib (IC50) treatment (Fig. 3c and d). In the case ofHCT 15 cells treated with the combination of celecoxib (IC50)+AEE788(3.8 μM), a marked increase in ROS generation was induced which wasassociated with enhanced apoptosis (Fig. 3e and f).

From ψm analysis, control HCT 15 cells elicited maximal Rh 123fluorescence suggesting intact and functional mitochondria. Celecoxiband AEE788 alone or in combination resulted in a rapid time-dependent diminution of ψm (Fig. 4a). In particular, a combination

treatment with celecoxib (IC50) and AEE78 (3.8 μM) resulted in agreater reduction of ψm as compared to either drug used alone.

Inhibition of HCT 15 cell migration, invasion and angiogenesis by celecoxiband/or AEE788

A scratch wound cell culture monolayer model was used to investi-gate the effect of celecoxib and/or AEE788 on HCT 15 cell migration. Inthis assay, the extent of wound closure can be considered as a directmeasure of cell migration. Representative views of the mid portion ofthe scratch lanes are shown in Fig. 4b. Closure of thewoundwas almostcomplete in untreated control HCT 15 cells within 24 h of incubation. Incontrast, the extent of wounding (%) and a significantly decreased cellmigration into the scratch lane was observed in HCT 15 cells treatedwith celecoxib (IC50), AEE788 (IC50) and celecoxib (IC50)+AEE788(3.8 μM) for 24 h (Fig. 4c). CAM model was used to investigate the ef-fect of celecoxib and/or AEE788 on angiogenesis in vivo (Emdad et al.,2009). As shown in Fig. 4d, the chorioallantoic membranes in the PBSgroupdo not showany observable avascular zone around the implantedfilter paper. However, celecoxib and/or AEE788 inhibited the develop-ment of new embryonic capillaries and produced an avascular zonearound the implanted filter papers. The inhibition of angiogenesis wasmore prominent following celecoxib/AEE788 combination treatment

Fig. 2. (a) Tapping mode AFM images of HCT 15 cells untreated control or treated with celecoxib (IC50), AEE788 (IC50) and celecoxib (IC50)+AEE788 (3.8 μM) for 48 h. Here, I, II andIII are 2D, phase and 3D images, respectively. In the images, healthy filopodia and lamellipodia are marked with arrows and, truncated filopodia and lamellipodia are marked witharrowheads. (b) Confocal laser fluorescence microscopic images (40×) of HCT 15 cells, untreated control or treated with celecoxib (IC50), AEE788 (IC50) and celecoxib(IC50)+AEE788 (3.8 μM) for 48 h and processed for staining. Red stain = rhodamine-conjugated phalloidin and blue stain = 4,-6-diamidino-2-phenylindole-DAPI. In the images,healthy filopodia and lamellipodia are marked with arrows, and truncated filopodia and lamellipodia are marked with arrowheads. Scale bar=5 μm.

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than either drug alone. However, no apparent toxicity was observed inthe embryos used in this experiment.

Inhibition of MMP-2 and MMP-9

Gelatin zymography was performed to investigate the effect ofcelecoxib and/or AEE788 on secreted MMP-2 and MMP-9 in HCT 15cells. Celecoxib and/or AEE788 markedly suppressed the amount ofsecreted MMP-2 and MMP-9 in a dose-dependent manner (Fig. 5a).Inhibition of MMPs was greatest when HCT 15 cells were treatedwith the combination of celecoxib (IC50) and AEE788 (3.8 μM) asopposed to treatment with either drug alone (IC50) (Fig. 5b).

DNA ladder formation

DNA laddering, a marker of apoptosis, was used to determine theeffect of celecoxib and/or AEE788 on induction of apoptosis in HCT15 cells. Both celecoxib and AEE788 induced DNA ladder formationin HCT 15 cells (Fig. 5c). However, celecoxib-induced apoptosis inHCT 15 cells was potentiated by the addition of 3.8 μM of AEE788.

Stimulation of intracellular apoptotic signaling pathways by celecoxiband AEE788

Celecoxib inhibited COX-2 protein in HCT 15 cells. Apoptosis is alsoregulated by Bcl-2 gene family members [57]. Either celecoxib orAEE788 induced apoptosis in HCT 15 cells by up-regulating expressionof pro-apoptotic Bax and down regulating the expression of Bcl-2, thus

shifting the equilibrium from survival to apoptosis (Fig. 5d). Celecoxiband AEE788 also decreased the expression of Bcl-xL in HCT 15 coloncancer cells. Caspase-3 is a cysteine–aspartate protease that cleavesto form two subunits, which dimerize to produce active caspase. Adecrease in the pro-enzyme in Western blot analysis reflects theformation of activated caspase-3 (Fig. 5d). The activated caspase-3processes other caspases and death substrates such as PARP, an inhibi-tor of caspase-activated deoxyribonuclease (ICAD) (Sarkar et al., 2010,2011). Treatment of HCT 15 cells with celecoxib or AEE788 resultedin a decrease in the 116-kDa fragments and an increase in the 85-kDafragment of PARP (Fig. 5d), indicating enhanced apoptosis.

Inhibition of PVEGFR/PMAPK signaling in HCT 15 cells by celecoxib andAEE78

Western blotting was used to investigate the potential mecha-nism involved in celecoxib- and AEE788-mediated inhibition of HCT15 cell proliferation. HCT 15 cells were grown in serum free mediumfor 24 h, treated with celecoxib or AEE788 (IC50) for 1 h, and thenactivated with recombinant human EGF (25 ng/ml) for 30 minfollowed by resolving proteins on SDS-PAGE and Western blots(Materials and methods). Both AEE788 and to a greater extentcelecoxib inhibited VEGFR-2 phosphorylation (Fig. 5e). Additionally,phosphorylated forms of both MAPK and Akt were also downmodulated in cells treated with celecoxib (IC50) or AEE788 (IC50)with EGF. In contrast, total levels of VEGFR-2, Akt andMAPK proteinsremained unaltered following celecoxib (IC50) or AEE788 (IC50)treatment (Fig. 5e).

Fig. 3. (a) Cell cycle analysis was performed to determine apoptotic activity of celecoxib (IC50), AEE788 (IC50) and celecoxib (IC50)+AEE788 (3.8 μM) for 48 h treatment on HCT 15cells using FACS calibur. (b) Percent (%) of apoptotic cells are indicated as the proportion of cells that contained sub-G0/G1 phase. Values shown here represent±S.D. (n=3).(c) ROS generation is associated with induction of apoptosis. Here, HCT 15 cells were treated with control (I), AEE788 (IC50) (II), celecoxib (IC50) (III) and celecoxib(IC50)+AEE788 (3.8 μM) (IV) for 24 h. ROS generation was determined by flow cytometry and reflected by the rightward shift of the histogram. (d) The % of ROS generation wascompared with control and data represent the mean fluorescence intensity (MFI)±S.D. (n=3, ***pb0.001). (e) HCT 15 cells were treated with celecoxib (IC50), AEE788 (IC50)and celecoxib (IC50)+AEE788 (3.8 μM) for 24 h and apoptosis was determined morphologically by Wright–Giemsa staining. All images were taken under identical instrumentconditions and presented at the same intensity scale. (f) The percentage of cell death was compared with control and data represent the mean±S.D. (n=3, ***pb0.001).

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Discussion

It is well established that cancer is a disease manifested by multipledysregulated signaling pathways, which can involve more than 500genes some of which can serve as targets for developing anticancertherapies (Gupta et al., 2011). Inhibition of a single signaling pathwaycan provide transient responses, but in most instances is ineffective inthe treatment of cancer. Despite this inevitable reality, the majority ofexisting anticancer therapies are based on a single targeting strategy.Drugs that interact with multiple targets are attractive in cancer drugdevelopment (imatinib, sunitinib, or sorafenib) (Gupta et al., 2010b).Accordingly, current therapeutic approaches for cancer are exploitingmulti-targeting drugs or a combination of drugs to target multiple cellsignaling pathways (Gupta et al., 2010a).

Previous work from our laboratory reported that nanoparticle-mediated delivery of celecoxib suppressed colon cancer cell growthwith reduced toxicity both in in vitro and in vivo animal models(Venkatesan et al., 2011). Both AEE788 and celecoxib have beenreported to inhibit various cancers through suppression of prolifera-tion and angiogenesis (Traxler et al., 2004; Klenke et al., 2006; Qianet al., 2006; Younes et al., 2006; Yu et al., 2007; Wang et al., 2008b;Venkatesan et al., 2010). In the present study, we analyzed theeffect of AEE788 on celecoxib-mediated inhibition of colon cancer.Cell proliferation assays documented a dose-dependent growth inhib-itory effect of celecoxib and/or AEE788 on HCT 15 and HT 29 cells.

Additionally, AEE788 potentiated celecoxib-mediated cytotoxicitytowards HCT 15 cells. Venkatesan et al. have already demonstratedthat AEE788 and/or celecoxib mediate morphological changes andantimetastatic activity on colon cancer cells, although the mechanismunderlying these effects has not been elucidated (Venkatesan et al.,2010). Phase contrast microscopy has limitations in viewing changesin cellular architecture. Therefore, it is difficult to observe finemorphological changes in cancer cells that are mediated by anticanceragents (Lv et al., 2008). In a prior study, we reported that AEE788potentiated celecoxib-mediated cytotoxicity in HCT 15 cells, asmonitored by changes in morphology using advanced microscopy(Venkatesan et al., 2010). Optical microscopic images displayed moreprominent morphological changes in HCT 15 cells upon combinationtreatment, which might be attributed to the combined cytotoxic effectof both drugs.

Morphological analysis of cell membrane extensions providesinformation relative to invasion and motility of cells. Cell membraneextensions are very fine locomotory structures that become evidentin stimulated migratory cells (cancer) and act as motors to pull lead-ing edges of a cell forward (migration and invasion) (Venkatesan etal., 2010). Additionally, the morphological features and submicronstructures of cells obtained from AFM and CLSM images provideadvantages over traditional light microscopic techniques. Informationprovided by these images includes, but are not restricted to, changesin cell morphology, cytoskeletal elements, organelles and cell volumes.

Fig. 4. (a) Loss of mitochondrial membrane potential (Δψm) in HCT 15 cells induced by celecoxib (IC50), AEE788 (IC50) and celecoxib (IC50)+AEE788 (3.8 μM) for the indicatedperiods. Δψm was evaluated by the uptake of a membrane potential-sensitive fluorescence dye Rh 123. The data are expressed as the relative levels of Δψm compared to the control(0 h). Data represent the mean±S.D. (n=3, **pb0.05, ***pb0.001). (b) Scratch wound healing assay. Confluent HCT 15 cells were scratched by 200 μl pipette tips and allowed toheal in RPMI 1640 with or without celecoxib (IC50), AEE788 (IC50) and celecoxib (IC50)+AEE788 (3.8 μM) for 24 h. Wound closure was photographed immediately at 0 or 24 hafter wounding (20×, scale bar=20 μm). Micrographs represent one of three samples performed in each experiment. (c) Statistical analysis of the percentage of wound healingby celecoxib and/or AEE788 compared to the control (24 h). The scratch area at control (0 h) was arbitrarily assigned as 100%. Values are expressed as mean±S.D. (n=3,***pb0.001). (d) Inhibition of angiogenesis in chick chorioallantoic membranes. Filter papers containing celecoxib (IC50), AEE788 (IC50) and celecoxib (IC50)+AEE788 (3.8 μM)were applied to the CAMs. After 48 h, the avascular zone (2.5-mm around the filter paper) of each CAM was examined. Figure represents appearances of chick CAMs implantedwith a filter paper containing PBS, AEE788 (IC50), celecoxib (IC50) or celecoxib (IC50)+AEE788 (3.8 μM) (magnification 10×). All images were taken under identical instrumentconditions and presented at the same intensity scale.

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Accordingly, some studies have utilized both AFM and CLSM to examinestructure–function relationships in biological systems (Li et al., 2008;Zhou et al., 2008). AFM analysis of control cells suggests invasiveproperties of the proliferating HCT 15 cells. Drug treatment experi-ments indicated less cytotoxicity of celecoxib vs. AEE788. Interestingly,combination treatment produced an additive damaging effect on cellmorphology and membrane extensions. In cytoskeleton architecture,actin is a large filamentous structure extending from the plasmamembrane through the nuclear envelope to the interior of the nucle-us. The cytoskeleton is involved in fundamental cellular processesincluding maintenance of cell shape, cell movement, cell replication,cell differentiation, apoptosis, cell communication and signal transduc-tion (Zhou et al., 2008). Either AEE788 (IC50) alone or in combination(celecoxib (IC50)+AEE788 (3.8 μM)) induced potential antimetastaticactivity in HCT 15 cells by disorganization of actin filaments and inhibi-tion of cell membrane extensions (Roh et al., 2004; Yokoi et al., 2005;Venkatesan et al., 2010).

Cell cycle is a dynamic process by which a cell acquires and inte-grates different growth control signals at various checkpoints in celldivision. Interference in the cell cycle represents a potentially effec-tive approach for inhibiting cancer growth by chemotherapeuticagents (Wang et al., 2008a). Frequently, induction of apoptosis is

associated with changes in cell cycle distribution. Cell cycle analysissuggests that AEE788 potentiates celecoxib-induced apoptosis inHCT 15 cells. It has been reported previously that both celecoxib andAEE788 induce apoptosis in various human cancer models (Parket al., 2005; Sakoguchi-Okada et al., 2007). In addition, DNA fragmen-tation assays suggest that celecoxib (IC50) in combination (celecoxib(IC50) with AEE788 (3.8 μM)) additively promotes cytotoxicity inHCT 15 cells.

ROS is produced intracellularly as a second messenger in varioussignal transduction pathways or in response to environmental stress(Deeb et al., 2010). Chemotherapeutic agents or ionizing radiationinhibits cancer cell growth by generating ROS (Yu et al., 2007). Amajority of anticancer agents act in part by inducing ROS and decreas-ing ψm, which subsequently triggers apoptotic cascades (Ling et al.,2003b; Cao et al., 2010; Deeb et al., 2010). The H2DCFDA dye freelydiffuses into cells and is hydrolyzed by intracellular esterase to DCFH,which is subsequently oxidized by ROS to generate highly fluorescentDCF. Accordingly, fluorescence intensity is relative to the amount ofperoxide produced by cells.

Production of ROS activates various transcription factors (e.g., nucle-ar factor kappa-light-chain-enhancer of activated B cells [NF-κB], activa-tor protein-1, hypoxia-inducible factor-1α, and signal transducer and

Fig. 5. (a) Effect of celecoxib and/or AEE788 on inhibition of MMP-2 and -9 in HCT 15 cells. Cells were treated with varying concentrations of celecoxib and/or AEE788 for 24 h.Gelatinolytic activities of MMP-2 and -9 in conditioned media were detected by electrophoresis on 10% polyacrylamide gel. (b) Relative intensities of gelatin digested bands byMMP-2 and -9 were quantified using densitometry and expressed as relative MMP-2 and -9 activities compared to control. Means with each treatment are significantly different(**pb0.01, ***pb0.001) by Duncan's multiple range test. (c) HCT 15 cells were treated with celecoxib (IC50), AEE788 (IC50) and celecoxib (IC50)+AEE788 (3.8 μM) for 48 h andextracted DNA was electrophoresed in agarose gel to detect DNA fragmentation. A representative result from three independent experiments is shown. (d and e) Western blottingof HCT 15 cells treated with celecoxib (IC50) or AEE788 (IC50).

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activator of transcription 3). This promotes expression of proteins thatcontrol inflammation (Cox-2), tumor cell survival (Bcl-2, Bcl-xL, Aktand MAPK), tumor cell proliferation and (MMP-9), angiogenesis(VEGF) and metastasis (Gupta et al., 2012). Our results indicate thatincreased generation of ROS by a combination of celecoxib and AEE788might inhibit the transcription factor NF-κB. Consequently suppressionof COX-2, Akt and MAPK proteins leads to apoptosis in colon cancercell. AEE788 induces toxicity in HCT 15 cells through increased genera-tion of intracellular ROS. Combination treatment resulted in a furtherincrease in ROS generation, which synergistically triggered apoptoticeffects in HCT 15 cells.

Measurement of ψm provides evidence for mitochondrial involve-ment in generation of ROS that is mediated by celecoxib and/orAEE788. There is a close relationship between signaling and the cellu-lar redox state (Cao et al., 2010; Deeb et al., 2010). Previous studieshave indicated that anticancer agents induce apoptosis in partthrough generation of ROS and the disruption of redox homeostasis(ψm) (Wang et al., 2008a). Rh 123 penetrates into mitochondria asa function of membrane potential and is released during membranedepolarization due to apoptosis. Interruption of ψm is one of theearly intracellular effects that induce apoptosis (Wang et al., 2008a;Cao et al., 2010). Our experiments provide further confirmationthat AEE788 potentiation of celecoxib-mediated apoptosis occurs byreducing ψm.

The migratory ability of cancer cells is closely associated with theirmetastatic potential (Gaikwad and Prchal, 2007; Emdad et al., 2009;Makrilia et al., 2009; Xu et al., 2010; Yee et al., 2010). In this context,AEE788 (IC50) alone and in combination (celecoxib (IC50)+AEE788(3.8 μM)) resulted in a more prominent inhibition of cell migration.For this reason, celecoxib and/or AEE788 may have therapeuticpotential by inhibiting invasion and metastasis of HCT 15 cells. Addi-tionally, CAM assays suggest that celecoxib and/or AEE788 mightbe valuable as angiogenic inhibitors. ROS generation also enhancesthe production of matrix metalloproteinases (MMP-2, 72 kDa andMMP-9, 92 kDa), which are associated with reduced survival andunfavorable prognosis in various malignant tumors (Kupferman etal., 2007; Cho et al., 2010). Decreased expression and inactivation ofMMPs result in a significant decrease in invasion and metastasis ofcancer cells (Normanno and Gullick, 2006). MMP-2 and -9 are keymolecules in proteolytic digestion of the extracellular matrix (ECM),which is essential in cell growth, migration and invasion, metastasisand angiogenesis (Wang et al., 2002; Yun et al., 2010). Zymographicanalysis previously demonstrated antiangiogenic activity (inhibitionof MMPs) of celecoxib and AEE788 in salivary adenoid cystic carcino-mas and oral carcinomas, respectively (Wang et al., 2002; Thakeret al., 2005).

Western blot analysis of apoptosis signaling events supports a roleof effector caspase-3 in DNA fragmentation and apoptosis induction.

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Inhibition of the VEGFR signaling pathway by celecoxib and/orAEE788 resulted in inhibition of angiogenesis. Active Erk1/2 protectscells from multiple stimulus-induced cell death mechanisms, includingROS generation. Celecoxib- and AEE788-mediated HCT 15 cell growthinhibition and apoptosis induction might be due to increased genera-tion of ROS and blockade of cytoprotective responses in HTC 15 cells.Moreover, overexpression of the pro-apoptotic Bcl-2 family member,Bax, results from enhancement of ROS generation (Ling et al., 2003a).Bax is known to activate the caspase-3 enzyme thatmediates executionof the apoptotic program.Mitochondria play a key role in the regulationof apoptosis (Cao et al., 2010). Increased ROS also induces the collapseof ψm (an early and necessary event in apoptosis), resulting in aseries of mitochondrial-associated events that include inhibition ofantiapoptotic and MAPK (cell survival) pathways (Yu et al., 2007;Deeb et al., 2010). In addition, Gupta et al. (2012) demonstrated thatinhibition of NF-κB by anticancer agents results in down-regulation ofthe cell survival protein AKT. From our results, it is proposed thatinhibition of AKT by AEE788 might potentiate celecoxib-inducedgrowth inhibition and apoptosis. The AKT inhibition might occurthrough suppression of the transcription factor NF-κB. Further de-tailed studies are required to elucidate the mechanism of inhibition.These results, however, suggest a model in which inhibition ofmultiple survival pathways (VEGFR, apoptosis protection and COX-2pathways) might shift the balance of intracellular events towards ap-optosis. Further experiments are required to confirm this hypothesis.These findings support a potential mechanism of action of celecoxiband AEE788 in HCT 15 cells that involves inhibition of multiple signal-ing and survival pathways and provides a basis for therapeutic use ofthese compounds, particularly in combination, in colon cancer.

Conclusions

The present study demonstrates that the combination of celecoxiband AEE788 mediates cell growth inhibition, morphological changes,and induces apoptosis, ROS generation, DNA fragmentation anddiminishes ψm. Celecoxib and/or AEE788 induced apoptosis throughROS/mitochondrial-dependent pathways. Furthermore, inhibitionof multiple signaling pathways (VEGFR, apoptosis protection andCOX-2 pathways) by celecoxib and AEE788 significantly inhibitedcell proliferation and induced apoptosis. Additionally, the combina-tion treatment also significantly inhibited invasion and migration,and decreased angiogenesis. The present study provides new insightsnto the mechanism by which celecoxib and AEE788 alone or in com-bination, impact on colon cancer, as reflected by effects on HCT 15icells. Detailed in vitro and in vivo studies are necessary to validatethe synergistic growth inhibition/apoptosis and antiangiogenic activityof the combination of celecoxib and AEE788 on HCT 15 in other coloncancer cells.

Conflict of interest statement

Authors disclose that there is no conflict of interest.

Acknowledgments

We are grateful to Aarthi Drug Ltd. for generously providingcelecoxib. We wish to thank Mr. Debashis Gayen for his skilledtechnical assistance in confocal laser fluorescence microscopy. Thiswork was supported by funds from the School of Medical Scienceand Technology, Indian Institute of Technology, Kharagpur, India.Dr. Devanand Sarkar is a Harrison Scholar and Dr. Paul B. Fisherholds the Thelma Newmeyer Corman Chair in Cancer Researchin the VCU Massey Cancer Center and VCU Institute of MolecularMedicine, Virginia Commonwealth University, School of Medicine.MM is grateful to Professor Paul B. Fisher, Department of Human

and Molecular Genetics and VCU Institute of Molecular Medicinefor his kind hospitality during his visit.

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