The CXCR2 Antagonist, SCH-527123, Shows Antitumor ......SCH-527123, inhibits colorectal cancer proliferation and if it can sensitize colorectal cancer cells to oxaliplatin both in

Preclinical Development

The CXCR2 Antagonist, SCH-527123, Shows AntitumorActivity andSensitizesCells toOxaliplatin inPreclinical ColonCancer Models

Yan Ning1, Melissa J. Labonte1, Wu Zhang1, Pierre O. Bohanes1, Armin Gerger1, Dongyun Yang2,Leonor Benhaim1, David Paez1, David O. Rosenberg3, Kalyan C. Nagulapalli Venkata3, Stan G. Louie4,Nicos A. Petasis3,5, Robert D. Ladner6, and Heinz-Josef Lenz1,5

AbstractColorectal cancer is the second most common cause of cancer-related death in the United States. Recent

studies showed that interleukin-8 (IL-8) and its receptors (CXCR1 and CXCR2) are significantly upregulated in

both the tumorand itsmicroenvironment, andact askey regulatorsofproliferation, angiogenesis, andmetastasis.

Our previous study showed that IL-8 overexpression in colorectal cancer cells triggers the upregulation of the

CXCR2-mediatedproliferative pathway. The aimof this studywas to investigatewhether theCXCR2 antagonist,

SCH-527123, inhibits colorectal cancer proliferation and if it can sensitize colorectal cancer cells to oxaliplatin

both in vitro and in vivo. SCH-527123 showed concentration-dependent antiproliferative effects in HCT116,

Caco2, and their respective IL-8–overexpressing variants colorectal cancer cell lines.Moreover, SCH-527123was

able to suppress CXCR2-mediated signal transduction as shown through decreased phosphorylation of the

NF-kB/mitogen-activated protein kinase (MAPK)/AKTpathway. These findings correspondedwith decreased

cell migration and invasion, while increased apoptosis in colorectal cancer cell lines. In vivo results verified that

SCH-527123 treatment decreased tumor growth and microvessel density when compared with vehicle-treated

tumors. Importantly, these preclinical studies showed that the combination of SCH-527123 and oxaliplatin

resulted inagreaterdecrease incellproliferation, tumorgrowth, apoptosis, andangiogenesis thatwas superior to

single-agent treatment. Taken together, these findings suggest that targeting CXCR2 may block tumor prolif-

eration, migration, invasion, and angiogenesis. In addition, CXCR2 blockade may further sensitize colorectal

cancer to oxaliplatin treatment. Mol Cancer Ther; 11(6); 1353–64. �2012 AACR.

IntroductionColorectal cancer is the leading cause of death from

gastrointestinal malignancies, resulting in approximately51,690 deaths in 2012 (1). Since 2005, no new chemother-apeutic agents have been approved by the Food andDrugAdministration for treatment of patients with metastaticcolorectal cancer, resulting in a significant need todevelopmore effective targeted drugs aiming at both the tumorand its microenvironment. Recent studies have identified

that certain chemokines and their receptors act as keyregulators of colorectal cancer progression and may beimportant targets for novel drug development strategies(2).

Interleukin-8 (IL-8), a member of the neutrophil-specificC-X-C subfamily of chemokines, acts on endothelial cellsvia binding onto either CXCR1 or CXCR2 to promoteinvasion,proliferation, andangiogenesis (2–5). IL-8 expres-sion is upregulated by hypoxia, cytokines, and other envi-ronmental stresses, which are mediated by transcriptionfactors, includingNF-kB andAP-1 (6, 7). The upregulationof CXCR2 has also been correlated with promotion oftumorigenesis and angiogenesis in lung, melanoma, andovarian cancers (8–10). Previous studies by our group andothers have shown that IL-8 and its receptor CXCR2 aresignificantly upregulated in the tumor and its microenvi-ronment in colorectal cancer (11–13). These studies showedthat expression levels of IL-8 and CXCR2 were associatedwith tumor proliferation, progression, and sensitivity ofoxaliplatin-based therapy in colorectal cancer cell linemodels and genetic variants in IL-8 and CXCR2 bothpredict tumor recurrence and oxaliplatin efficacy inpatients (11, 14, 15). It is well known that oxaliplatin-basedchemotherapy is a standard of care agent used most

Authors' Affiliations: 1Division of Medical Oncology, Sharon A. CarpenterLaboratory; 2Department of Preventive Medicine; 3Department of Chem-istry; 4School of Pharmacy; 5Norris Comprehensive Cancer Center; and6Department of Pathology, Keck School of Medicine, University of South-ern California, Los Angeles, California

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Y. Ning and M.J. Labonte contributed equally to this work.

Corresponding Author: Heinz-Josef Lenz, Norris Comprehensive CancerCenter, KeckSchool ofMedicine, 1441EastlakeAve, Suite 3456,Universityof SouthernCalifornia, LosAngeles, CA 90089. Phone: 323-865-3955; Fax:323-865-0061; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0915

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

www.aacrjournals.org 1353

on September 5, 2021. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst March 5, 2012; DOI: 10.1158/1535-7163.MCT-11-0915

http://mct.aacrjournals.org/

commonly in combination with 5-fluorouracil in patientswith colorectal cancer (16). Overexpression of IL-8 level incolorectal cancer cells decreased sensitivity to the cytotoxiceffects of oxaliplatin and contributed to oxaliplatin che-moresistance (11). Therefore, targeting IL-8 or CXCR2, inaddition to having a direct antiangiogenic and antitumoreffect, may also increase chemosensitivity to oxaliplatin.

Recently, our group focused on inhibitors of the IL-8/CXCR2 pathway, which will have the potential not onlyto have antitumor activity, but to increase the efficacy ofalready available cytotoxic and targeted drugs, such asoxaliplatin, for patients with colorectal cancer by target-ing the tumor and its microenvironment. SCH-527123,as a novel and selective antagonist of the CXCR2, hasshown efficacy in the treatment of inflammatory diseases(17). Moreover, Singh and colleagues showed that SCH-527123 treatment inhibited human melanoma cancergrowth and colorectal cancer liver metastases by decreas-ing tumor cell proliferation, angiogenesis, and enhancingthe apoptosis of malignant cells (18, 19).

In this study, we show that treatment with SCH-527123alone and in combination with oxaliplatin is effective insynergistically inhibiting proliferation, angiogenesis, andenhancing chemosensitivity in colorectal cancer cells andxenografts. Taken together, these findings suggest thatCXCR2 antagonists, such as SCH-527123, may be impor-tant therapeutic candidates in treating colorectal cancerthrough attenuating the IL-8/CXCR2 signaling cascade,which influences disease progression and modulates theresponse to oxaliplatin sensitivity.

Materials and MethodsCompounds and reagents

SCH-527123 was obtained from Schering Plough andsynthesized in Petasis group. Compound purity was veri-fied by nuclear magnetic resonance and liquid chromatog-raphy/mass spectrometry.Oxaliplatinwaspurchased fromSigma-Aldrich. CellTiter96AqueousOne Solutionwaspur-chased from Promega. Recombinant human IL-8 (rhIL-8)was purchased fromR&D systems. Protease inhibitor cock-tail was purchased from Roche Molecular Biochemical.

Cell linesThe human HCT116 and Caco2 colorectal cancer cells

were purchased from American Type Culture Collectionin August 2008 (no authentication was done by theauthors). HCT116 andCaco2 IL-8–overexpressing isogen-ic cell lines (E2 and IIIe) were generated as previouslydescribed (11). HCT116 and Caco2 cell lines were main-tained in McCoy’s 5A and minimum essential medium,respectively, and supplemented with 10% fetal bovineserum, 5% penicillin/streptomycin, sodium pyruvateand L-glutamine (Mediatech). IL-8 overexpression cellswere maintained as described above with the addition of5 mg/mL blasticidin (Invitrogen). Cells were maintainedin an incubator at 37�C with 5% CO2. The cell lines wereroutinely tested to confirm that theywereMycoplasma freeusing Mycoalert Mycoplasma Detection Kit (Lonza).

ELISAThequantificationof IL-8proteinwasdeterminedusing

the Quantikine IL-8 ELISA Kit (R&D systems) accordingto manufactures’ instructions. More information isdescribed in Supplementary Methods and Materials.

Terminal deoxynucleotidyl transferase–mediateddUTP nick end labeling assay

Cellswereplated in adherent conditions in 6-well platesat 50,000 cells per well. After treatment, cells were fixedand stained for the presence of apoptotic cells by the inSitu apoptosis detection kit (Trevigen). Nuclei were coun-terstained with DAPI in red.

Growth inhibition assay and drug combinationanalysis

Growth inhibition was measured as previouslydescribed (11, 20, 21). The combination effect was deter-mined by the combination index (CI) analysis methods ofChou and Talalay (22) using Calcusyn software (Biosoft),which quantifies the degree of synergy between 2 agentsthat both induce a linear pharmacologic response. Frac-tion affected (FA) was calculated from the percentagegrowth inhibition: FA ¼ (100% growth inhibition)/100.CI values: less than 1, synergism; 1 to 1.2, additive, andmore than 1.2, antagonism. Results are representative of 3independent experiments conducted in duplicate.

Clonogenicity assayThe clonogenicity assay were conducted as previously

described (20). Cellswere subsequently treatedwith SCH-527123 and oxaliplatin either alone or in combination for aperiod of 72 hours. Cells were washed and incubated indrug-free media for 3 weeks to allow colony formation.All experiments were carried out in triplicate. Drug-trea-ted samples were compared directly with untreated con-trols set at 100%.

siRNA analysissiRNA analysis was conducted as previously described

(11). siRNAs against CXCR2 were purchased fromAmbion. Two different siRNA oligonucleotides [#14777/siRNA#1 and #4067/siRNA#2] were tested, and siRNA#2wasusedin thedescribedknockdownexperiments.Knock-down was validated by mRNA analysis at 72-hourposttransfection. siRNA-treated cells were normalized tonegative control siRNA (NC siRNA) for quantitative PCR(qPCR) and growth inhibition analyses.

Quantitative reverse transcriptase PCRTotal RNAwas extracted with QIAGENKit (QIAGEN)

according to the manufacturer’s instructions. IL-8/CXCR2/ERCC1 primers were used for reverse transcrip-tase PCR (RT-PCR), and target genes were normalized to

b-actin and quantified using the 2�DDCt method (23).

Western blotCells were solubilized in cell lysis buffer containing a

protease inhibitor cocktail. For tissue samples, Tissue

Ning et al.

Mol Cancer Ther; 11(6) June 2012 Molecular Cancer Therapeutics1354




Protein Extraction Reagent was used for tumor tissueprotein extraction. Primary rabbit polyclonal antibodieswere purchased from Cell Signaling Technology, andrabbit anti–b-tubulin antibodies were purchased fromSanta Cruz Biotechnology. Quantitative analysis of West-ern blots was conducted by Image J.

In vivo studiesXenograft experiments were conducted in male

C57Bl/6 Balb/cmice (Taconic Labs that were 6- to 8-weekold. SubcutaneousHCT116 and E2 xenografts were estab-lished and allowed to grow until they reached approxi-mately 100 mm3 (day 0). Animals were randomized totreatment groups: vehicle, SCH-527123, oxaliplatin, andcombination of SCH-527123 and oxaliplatin (n ¼ 6 pergroup). SCH-527123 was administered at 50 mg/kg byoral gavage once daily. Oxaliplatin was administered at7.5 mg/kg by i.p. injection every 4 days.

Immunohistochemistry and quantitation ofmicrovascular densityImmunohistochemistry (IHC) was carried out as pre-

viously described (11). Formalin-fixed and snap-frozenfragments of tumor specimens were paraffin embeddedand sectioned to 4 mm thickness. Primary antibody rat-anti-mouse-CD31 (BD Pharmingen) was added. Micro-vascular density (MVD) was quantified from 4 differenttumor samples by counting the total number of CD31-positive vessels across the whole section of tumors foreach experimental condition.

Statistical analysisFor all analyses, the difference between each cell lines

compared and/or treatment groups were evaluatedwith a 2-tailed t test or ANOVA fromGraphPad software.P < 0.05 was considered statistically significant.

ResultsIL-8 overexpression modulated CXCR2 mRNA levelin colorectal cancer xenograftsTo investigate the gene expression of IL-8 and CXCR2

fromxenograft samples of theHCT116andHCT116–IL-8–overexpressing cells (E2), which were established from s.c. injections in nude mice, qRT-PCR was used and datarevealed that IL-8 and CXCR2 mRNA expression weresignificantly increased at 26-fold (P < 0.005) and 2.5-fold(P < 0.05), respectively, in E2 xenografts when comparedwithHCT116 xenografts (Fig. 1A). CXCR1mRNAexpres-sion was not significantly different between the HCT116and E2 xenografts.

Knockdown of CXCR2 increased sensitivity tooxaliplatin in colorectal cancer cellsTo examine the role of CXCR2 in colorectal cancer cell

proliferation,CXCR2 siRNAwasused to suppressCXCR2expression in colorectal cancer cell lines. Two differentCXCR2 siRNA oligonucleotides were tested. After 72

hoursposttransfection, CXCR2knockdownwasvalidatedby RT-PCR and qRT-PCR, and siRNA#2 was used insubsequent CXCR2 siRNA knockdown experiments (Fig.1B). qRT-PCR analysis revealed that transfection ofCXCR2 siRNA#2 reduced the mRNA expression ofCXCR2 by 45% � 0.039 and 59% � 0.049 (siRNA#1 andsiRNA#2) in HCT116 cells compared with mock-trans-fected cells (P < 0.005, Fig. 1B). E2 cells transfected withcontrol mock and negative control siRNA had no evi-dence of CXCR2 knockdown (Fig. 1B). Our previouslystudy showed that IL-8 overexpression in HCT116 cellsdecreased sensitivity to the cytotoxic effects of oxalipla-tin (11). Having confirmed CXCR2 mRNA knockdown,we examined whether decreased CXCR2 mRNA expres-sion in colorectal cancer cell lines can reduce the IL-8–induced chemoresistance to oxaliplatin. The growthprofiles of HCT116, E2, Caco2, and Caco2-IIIe (IIIe) wereanalyzed after 24 hours treatment with CXCR2 siRNA,and then subsequently treated for 72 hours with increas-ing concentrations of oxaliplatin by growth inhibitionassay (Fig. 1C and D). There was a decrease in theIC50(72 h) of the CXCR2 siRNA/oxaliplatin-treatedcells when compared with mock and b-actin controlsiRNA/oxaliplatin-treated cells. Importantly, IC50(72 h)

of CXCR2 siRNA/oxaliplatin-treated cells that overex-pressed IL-8 (E2 and IIIe) had significant differencesthan parental siRNA/oxaliplatin-treated cells. Theseresults suggest that the knockdown of CXCR2 increasessensitivity to oxaliplatin in colorectal cancer cells, espe-cially in IL-8–overexpressing colorectal cancer cells.

SCH-527123 inhibited colorectal cancerproliferation, migration, and invasion

To investigate the impact ofCXCR2 inhibitors, the effectof SCH-527123 was evaluated in colorectal cancer cells.Single-agent efficacy of SCH-527123 was evaluated by thegrowth inhibition assays in the HCT116, E2, Caco2, andIIIe cells. Cells were treated with increasing concentra-tions of SCH-527123 for 72 hours and showed dose-dependent growth inhibitory activitywith IC50(72 h) valuesranging from 18 to 40 mmol/L (Fig. 2A). Importantly theIL-8–overexpressing cells showed a higher IC50(72 h) con-centration of SCH-527123 thanparental cells [HCT116 andE2 (P < 0.005): 28.9� 0.02 mmol/L and 39.5� 0.01 mmol/L,respectively; Caco2 and IIIe (P< 0.005): 18.8� 0.03mmol/Land 25.5� 0.02 mmol/L, respectively (Fig. 2A)]. Therefore,we concluded that SCH-527123 decreased growth inhib-itory activity in colorectal cancer cell lines. Next, the effectof SCH-527123 on inducing apoptosis was evaluatedwitha terminal deoxynucleotidyl transferase–mediated dUTPnick end labeling (TUNEL) assay in HCT116 and E2 cells.Data showed a significant increase in apoptosis in cellstreated with SCH-527123, with a 10% � 0.5 (P < 0.005;HCT116) and 25% � 0.8 (P < 0.005; E2) increase in apo-ptotic cell after 4-day treatment compared with untreatedcontrol cells (Fig. 2B). This result suggests that SCH-527123 resulted in a decrease of cell growth followed byinduction of significant apoptosis in colorectal cancer

SCH-527123 Increases Antitumor Activity

www.aacrjournals.org Mol Cancer Ther; 11(6) June 2012 1355




A B

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–1.0 –0.5 0.0 0.5 1.0

40

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HCT116 mock transfected IC50 = 0.998 ± 0.04

HCT116 negative control IC50 = 1.132 ± 0.02

HCT116 CXCR2 siRNA IC50 = 0.6321 ± 0.08

HCT116 - parental

Log [μmol/L] oxaliplatin

Log [μmol/L] oxaliplatin Log [μmol/L] oxaliplatin

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HCT116-E2 mock transfected IC50 = 1.557 ± 0.05

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–1.0 –0.5 0.0 0.5 1.020

40

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Caco2 mock transfected IC50 = 0.67 ± 0.09

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Caco2-IIIe mock transfected IC50 = 0.88 ± 0.06

Caco2-IIIe negative control IC50 = 0.82 ± 0.08

Caco2-IIIe CXCR2 siRNA IC50 = 0.384 ± 0.14

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Figure 1. CXCR2 mRNA expression in colorectal cancer HCT116 and E2 xenografts and the influence of CXCR2 knockdown in colorectal cancer ongrowth of cells treated with oxaliplatin. A, qRT-PCR determination of IL-8/CXCR2 mRNA expression in HCT116 and E2 xenografts. B, HCT116 cells weretransfected with mock, b-actin siRNA, and CXCR2 siRNA. Top, qPCR results are representative at 72-hour posttransfection. Bottom, qRT-PCR resultsare representative ofmean�SD from triplicate samples and presented asmRNA fold change relative tomock-transfected control. C and D, all cell lines weretransfected with mock, b-actin siRNA, and CXCR2 siRNA, and then treated with increasing concentrations of oxaliplatin for 72 hours.

Ning et al.





cells. Moreover, we found that 25 mmol/L SCH-527123was sufficient to block IL-8–mediatedCXCR2activation inall cell lines analyzed, inwhich phosphorylation of down-stream kinases [mitogen-activated protein kinase(MAPK)/NF-kB)] of CXCR2 was reduced in a concentra-tion-dependent manner (Supplementary Fig. S1).Because IL-8 functions to promote cellular migration

and invasion, we evaluated the effect of SCH-527123 onmigration and invasion of colorectal cancer cells. Treat-ment with SCH-527123 (24 hours) significantly inhibitedmigration and invasion of HCT116 (24% � 0.084%, P <0.05; 60%� 0.003, P < 0.005) and E2 (39%� 0.01, P < 0.005;49%� 0.004, P < 0.005) cells when compared with controltreated cells (Fig. 2C). Similar results were observed after

the additionof 10ng/mLRhIL-8 toHCT116 cells, inwhichfollowing treatment with 25 mmol/L SCH-527123 therewas a significant inhibition of migration (59%� 0.015, P <0.005) and invasion (57% � 0.004, P < 0.005; Fig. 2C,Supplementary Fig. S2).

SCH-527123 in combination with oxaliplatinsynergistically suppressed cell growth and survival

We next investigated the antiproliferative effects ofcombining SCH-527123 and oxaliplatin. All 4 cell lineswere treated with increasing concentrations of SCH-527123 and oxaliplatin alone and in combination for 72hours, then growth inhibition was measured by anMTS assay. The median effect analysis method (22)

Figure 2. SCH-527123 decreasescell proliferation, migration, andinvasion and increases apoptosis incolorectal cancer cells. A, growthinhibition assay. IC50(72 h) of SCH-527123 was determined withGraphPad Prism software. B, after72 hours treatment, the number ofapoptotic cells was evaluated, andapoptotic cells (blue) in HCT116 andE2 were detected in SCH-527123–treated cells compared withcontrols, inwhichmostly viable cells(red) were present. C, quantitation ofmigration and invasion after SCH-527123 treatment. Histogramshows fold change over the numberof HCT116 and HCT116-rhIL-8 andE2 cells that migrated or invadedcompared with no treatment.

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Caco2-IIIe IC50 25.45 ± 0.02

HCT116 IC50 28.95 ± 0.02

HCT116-E2 IC50 39.45 ± 0.01

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was used in the evaluation of the combination drugeffect. The effects of simultaneous treatment with bothagents in HCT116 and Caco2 cell lines produced syn-ergistic growth inhibition resulting in synergistic CIvalue less than 1 for the majority of concentrationstested at 0.5 FA (Fig. 3A and C, left). The combinationof SCH-527123 (10–18 mmol/L) and oxaliplatin (0.25–1.5 mmol/L) in E2 and IIIe cell lines also producedsynergistic growth inhibition resulting in FA range of0.6 to 0.8 and synergistic CI value less than 1 (Fig. 3Band D, left). These results displayed that SCH-527123synergistically increased sensitivity to oxaliplatin inHCT116 and Caco2 and IL-8–overexpressing cells,which was consistent with CXCR2 siRNA resultsdescribed above.

To determine whether alterations in sensitivity to SCH-527123 and oxaliplatin observed by growth inhibitionassay translated to changes in the ability of cells to recoverfrom drug treatment, a clonogenicity assay was con-ducted. All 4 cell lines were treated with SCH-527123 andoxaliplatin alone and selected combinations for 72 hoursfollowed by outgrow in drug-free medium for 21 days.Combined drug analysis was conducted with increasingconcentrations of both agents. As shown in Fig. 3A–D(right) and Supplementary Fig. S2, in all cell lines, SCH-527123 andoxaliplatin alone resulted in adose-dependentsuppression of colony formation. Importantly, the com-binations of SCH-527123 and oxaliplatin synergisticallysuppressed colony formation at all combinations tested inall cell lines (HCT116: 60%, E2: 64%, Caco2: 72%, IIIe:60%). Therefore, these findings suggest that targetingCXCR2 increases drug sensitivity of colorectal cancer cellsto the one of the current colorectal cancer chemotherapies,oxaliplatin.

SCH-527123 alone and in combination withoxaliplatin suppressed downstream signaling andmodulatedapoptoticmarkers in colorectal cancer celllines

To evaluate the expression of CXCR2 and IL-8 in ourcolorectal cancer cell lines treated with SCH-527123 andoxaliplatin alone and combination, qRT-PCR analysis andELISA assay were conducted. Although there were nodifferences in the level of IL-8mRNAexpression in all celllineswith treatment (datanot shown), IL-8protein expres-sion was significantly downregulated in HCT116, E2, andIIIe cells with both agents alone and in combination (Fig.4A). These data suggest that SCH-527123–mediatedantagonism may decrease IL-8 protein expression at theposttranslational level.

To evaluate the effect of combination treatment onIL-8/CXCR2 downstream signaling, the activation ofNF-kB/AKT/MAPK pathway was tested by Westernblot. All 4 cell lines were treated with SCH-527123 andoxaliplatin alone and combination for 24 hours. Phospho–NF-kB/phospho-MAPK/phospho-AKT levels were sig-nificantly suppressed in all cell lines with combinationtreatment compared with untreated and single agents

alone (Fig. 4B). These results suggest that the effects ofSCH-527123 alone and in combination with oxaliplatinmay be mediated by NF-kB/AKT/MAPK signaling cas-cade. Furthermore, using immunoblotting assay, weobserved NF-kB–targeted antiapoptotic gene BCL-2expression, proapoptotic protein BAX expression, whichacts as an antagonist of BCL-2 and promotes apoptosis byforming a heterodimer (BCL-2/BAX) and therefore losingthe proapoptotic effect of BCL-2, and apoptotic proteinPARP activation, which is involved in DNA repair (Fig.4C). After exposure of all cells to SCH-527123 and oxali-platin alone and combination, levels of BCL-2 proteinexpression decreased and BAX protein expressionincreased. HCT116 and E2 cells showed enhanced cleav-age of PARP into 116/89kDa fragments by combination ofboth agents compared with oxaliplatin alone (Fig. 4C).These findings suggest that SCH-527123 alone and incombination with oxaliplatin increased apoptosis in colo-rectal cancer cells.

SCH-527123 alone or in combination withoxaliplatin enhanced antiproliferative activity intumor xenografts

To explore the enhanced antiproliferative effects ofSCH-527123 alone or in combination with oxaliplatin ontumor growth in vivo, and to assess the potential for SCH-527123 to compromise oxaliplatin activity, nude miceimplanted with HCT116 or E2 cells were exposed to bothsingle agents and their combination. SCH-527123 wasadministered at 50 mg/kg/d, in which the dose wasdetermined by the in vitro IC50(72 h), and previously pub-lished studies. Oxaliplatinwas administered at 7.5mg/kgoxaliplatin every 4 days. Following 21 days of treatment,average tumor volume (TV) for the vehicle treated control,oxaliplatin, and SCH-527123 single-agent treatmentgroups were 831 � 133 mm3, 382 � 69 mm3, and 386 �64 mm3, respectively, in the HCT116 xenografts. Interest-ingly, in the E2 xenografts, SCH-527123 alone decreasedthe TV with an average of 229 � 56 mm3 and was morepotent than oxaliplatin alonewith a TV of 295� 61mm3 inwhich vehicle-treated control tumors had TV of 391� 121mm3. Importantly, the combination displayed a signifi-cant decrease in TV [HCT116: 340� 63mm3 (P < 0.05) andE2: 123� 26 mm3 (P < 0.05)], compared with either singleagent alone fromday 13 through the end of the study (Fig.5A). Moreover, combination treatment did not cause anysignificant difference in body weight compared withvehicle-treated control (P ¼ 0.67, Fig. 5B).

On day 21, the tumors were excised and evaluated forthe mRNA expression of CXCR2 and IL-8 followingtreatmentwith either single agent alone or in combinationby qRT-PCR. qRT-PCR analysis showed that the combi-nation of SCH-527123 and oxaliplatin resulted in a signif-icant reduction of CXCR2 and IL-8mRNA inHCT116 (P <0.05) and E2 (P < 0.005) xenografts when compared withvehicle-treated controls (Fig. 5C). Measurement of circu-lating IL-8 levels in serum which were collected at thetime of necropsy showed a dramatic decrease in both

Ning et al.





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0.125 0.25 0.5 1 1.5 2 3.25 6.5 12.5 μmol/L Oxaliplatin

5 10 15 18 22.5 25 30 40 50 μmol/L SCH-5271230.92 0.85 0.59 0.45 0.36 0.32 0.3 0.3 0.26 FA1.33 0.39 0.83 0.78 0.82 0.85 1.1 1.77 2.43 CI

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5 10 15 18 22.5 25 30 40 50 μmol/L SCh-527123

0.93 0.85 0.74 0.63 0.55 0.47 0.39 0.30 0.23 FA1.03 0.99 0.97 0.96 1.03 0.94 1 1.22 1.47 CI

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0.89 0.81 0.66 0.52 0.42 0.31 0.26 0.24 0.20 FA0.79 0.97 0.89 0.85 0.85 0.72 0.84 1.33 1.86 CI

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0.89 0.77 0.62 0.56 0.48 0.44 0.37 0.30 0.25 FA0.76 0.85 0.88 1.05 1.13 1.19 1.13 1.67 2.17 CI

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μmol/L SCH

.. .. 0.3

.. .. 0.835

0 1 1

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.. .. 0.4

.. .. 0.625

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Figure 3. SCH-527123 combined with oxaliplatin synergistically suppresses colorectal cancer cell proliferation and survival. A–D, left, growth inhibition assay.A–D, right, clonogenicity assay. All cell lineswere treatedwith increasing concentrations of SCH-527123 andoxaliplatin aloneand in combination for 72 hours.Data are presented as histograms of the mean percentage of colony formation compared with untreated controls (100%) � SD. The combined drug effectswere analyzed with the CI with FA values for combinations.






HCT116 and E2 tumor-bearing mice with agent aloneand combination compared with vehicle-treated controls(Fig. 5D).

Excision repair cross-complement group1 (ERCC1) is akey element in the nucleotide excision repair pathwaywhich has previously been associated with oxaliplatinresistance (24–26). Therefore, the effect of treatment onERCC1 mRNA expression in the xenografts followingSCH-527123, oxaliplatin alone, and their combinationwasmeasured by qRT-PCR. The analysis confirmed that boththe HCT116 and E2 xenografts displayed a significantdecrease in ERCC1 mRNA expression following treat-ment with single agents alone and their combination incomparison with vehicle controls (Fig. 5C).

The combination of SCH-527123 and oxaliplatinsynergistically suppresses downstream signalingand angiogenic activity in xenograft model

We examined the activity of NF-kB/AKT/MAPK byWestern blot in each of the xenografts after SCH-527123and oxaliplatin treatments at the end of the study. InHCT116 and E2 xenografts, SCH-527123 alone and incombination with oxaliplatin resulted in a significantdecrease in the protein activation of phospho–NF-kB/phospho-AKT/phospho-MAPK (Fig. 6A). These in vivodata are consistent with the in vitro data and confirm thatSCH-527123 inhibits the NF-kB/AKT/MAPK signalingcascades in both models.

To establish whether the decreased tumorigenicity andgrowth of tumors was associated with decreased angio-genesis, IHCwas used tomeasure the expression of CD31(mouse endothelial cell specific) to evaluate MVD in thetumor specimens. MVD in the HCT116 xenografts wasdecreased following treatment with SCH-527123 [28% �0.04 (P ¼ 0.02)] and oxaliplatin [10% � 0.05 (P ¼ 0.06)]when compared with vehicle-treated xenografts. Impor-tantly, when used in combination, MVDwere significant-ly decreased by 41% � 0.05 (P ¼ 0.006) in HCT116 xeno-grafts. Similarly, in the E2 xenografts, MVD wasdecreased by SCH-527123 by 44% � 1.08 (P < 0.05),oxaliplatin by 32% � 0.58 (P < 0.005), and their combina-tion by 67%� 0.6 (P < 0.005)when comparedwith vehicle-treated xenografts (Fig. 6B andC). These results show thatSCH-527123 in combination with oxaliplatin resulted in asignificant inhibition of angiogenic activity as determinedby CD31 IHC expression.

DiscussionThe role of IL-8 and CXCR2 in tumor development

and progression has been well documented in a widerange of cancer types (8, 27–31). Our previous data andothers have shown that the IL-8/CXCR2 pathwayplays a key role in mediating colorectal cancer devel-opment (11, 14, 15, 32). In follow-up to our previouslypublished data in colorectal cancer, this study focusedon the impact of inhibition of CXCR2 on the prolifera-tion, survival, invasion, and migration in colorectalcancer in vitro and in vivo models. To the best of ourknowledge, this study shows for the first time that theCXCR2 antagonist, SCH-527123, has significant antitu-mor activity in colorectal cancer preclinical models andcan further sensitize colon cancer cells to oxaliplatin-based treatment. Furthermore, our key findings showedthat the antitumor activity of SCH-527123 resulted frominhibition of cancer cell growth, motility, and angiogen-esis through the NF-kB/AKT/MAPK signaling path-ways, and this signaling could be further attenuatedwhen SCH-527123 was coadministered with oxaliplatin.

Previous data provide evidence that IL-8 is constitu-tively expressed in mCRC and primarily associatedwith the proliferation, metastasis, and the induction ofangiogenesis (11, 33). Important roles of its receptors,

HCT116 HCT116-E2 Caco2 Caco2-IIIe

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Figure 4. SCH-527123 in combination with oxaliplatin modulates proteinexpression of IL-8, PARP, BCL-2/BAX, and decreasedNF-kB/Akt/MAPKsignaling activity in colorectal cancer cells. A, anELISAassaywasused tomeasure IL-8 production fromall cell lines. The resultswere normalized tototal cell numbers. Histograms represent the mean fold change � SD ofIL-8–overexpressing cells compared with parental cells. B and C,Western blot analysis. All cells were treated with 25 mmol/L SCH-527123and 0.5 mmol/L oxaliplatin alone and in combination for 24 hours.b-Tubulin was used to as a loading control and normalization protein forquantitation.

Ning et al.





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Figure 5. Antitumor activity of SCH-527123 combined with oxaliplatin in HCT116 and E2 xenografts. A, examination of tumor growth in xenografts ofcolorectal cancer cells. The graphs indicate the mean tumor growth rates � SEM of each group. Statistical significance was determined by ANOVA withGraphpad Software, P < 0.05. B, mouse body weight represented as the percentage initial body weight at day 21 compared with day 1. C, qRT-PCRwas used to determine the level of IL-8, CXCR2, and ERCC1 mRNA in HCT116 and E2 xenografts with treatment. D, serum IL-8 production wasmeasured by ELISA, and the results were normalized to whole blood volume and presented as the mean � SD. The results are representativeof a minimum of 3 independent experiments.






CXCR1 and CXCR2, have also been defined in colorectalcancer progression (34, 35). Our previous study showedthat knockdown IL-8 expression can inhibit colorectalcancer cell growth and metastasis. Conversely, IL-8overexpression can increase colorectal cancer cell met-astatic and angiogenic potential, as well as increasingchemoresistance to oxaliplatin (11). However, neutral-izing antibodies against other chemokines [such as IL-6(36), IL-5 (37)] also show similar results. Importantly,CXCR2 has been shown to interact and bind diverseligands in addition to CXCR1, therefore, resulting in thetargeting of IL-8/CXCR2 signaling showing a moreeffective and broader inhibition in colorectal cancerdevelopment and progression.

Several small-molecule inhibitors targeting IL8/CXCR2 signaling have been developed to suppressinflammatory diseases (38, 39). A recent study hasshown the potential of SCH-527123 in inhibiting human

colon liver metastases using in vivo models (18). Inour in vitro study, we showed that SCH-527123 treat-ment inhibited cell proliferation and induced apoptosisin both HCT116 and Caco2 parental and IL-8–overex-pressing colorectal cancer cells. The in vitro findingswere validated in an in vivo study in which micewere given SCH-527123 orally and exhibited a reduc-tion in TV when compared with the vehicle-treatedcontrol group, which further supported the antiprolife-rative effect of SCH-527123. SCH-527123 also showed adecrease in tumor vascular density when comparedwith the vehicle-treated control group. These findingsindicated the targeting CXCR2 with antagonists suchas SCH-527123 may be a promising therapeutic forcolorectal cancer.

In prostate cancer cells, it was reported that IL-8 sig-naling contributes to the intrinsic resistance of thecancer cells to undergo apoptosis in response to either

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Figure 6. SCH-527123 in combination with oxaliplatin significantly suppressed NF-kB/Akt/MAPK downstream signaling and angiogenic activity inHCT116 and E2 xenografts. A, cell lysates of each tissue were immunoblotted with anti–phospho-NF-kB–p65/Akt/p44/42MAPK or antitotal NF-kB–p65/Akt/p44/42MAPK antibodies; b-tubulin as a loading control and normalization protein for quantitation. B and C, immunohistochemistry and quantitationof MVD (CD31) from tumor tissue slides. In HCT116 and E2 tumor specimens, rat anti-mouse CD31 antibodies were added to tissue sections. Datashown represent the mean � SD (�, P < 0.05; ��, P < 0.005).

Ning et al.





environmental or chemical stress (40). One of the keyinvestigations in this study was to evaluate the antipro-liferative effects of combining the CXCR2 antagonist withcytotoxic chemotherapy. Oxaliplatin is a platinum-basedDNA-damaging chemotherapy that shows clinical benefitfor patients with high-risk stage II and III colorectalcancer, as well as patients with advanced disease (41,42). However, cancer cells are frequently resistant tooxaliplatin (43). Our study showed CXCR2 to be a keymediator of IL-8–mediated chemoresistance to oxaliplatinand using CXCR2siRNA to knockdown its expression incolorectal cancer cells sensitized them to oxaliplatin.These results led to the hypothesis that targeting IL-8/CXCR2 in combination with oxaliplatin may increasesensitivity to oxaliplatin, providing enhanced efficacy andeventually benefit in colorectal cancer treatment. Ournovel observation showed that the combination ofSCH-527123 and oxaliplatin resulted in synergistic sup-pression of colorectal cancer cell proliferation and surviv-al with additive to synergistic effects. These synergisticantitumor propertieswere also observed in the IL-8–over-expressing cell lines, which were more insensitive tosingle-agent SCH-527123 treatment. Our in vivo data con-firmed the in vitro findings and showed that the combi-nation of SCH-527123 and oxaliplatin significantly inhi-bits tumor growth when compared with single agentsalone. ERCC1 mRNA levels have been shown to be pre-dictive of oxaliplatin cytotoxicity in the HCT116 cell line(25) and a useful marker in predicting response to oxali-platin-based treatment for colorectal cancer patients (44).High ERCC1 mRNA expression has been shown to beassociated with resistance to oxaliplatin (26). Supplemen-tary Fig. S3 showed, in an oxaliplatin-resistant cell line(HCT116-OR, in which resistance was induced by long-term culture in the presence of low-dose oxaliplatin), or inE2 cell line, that both cell lines showed an increased levelofERCC1mRNAexpression.Our in vivodata showed thattreatment with both agents in combination dramaticallylowered ERCC1 mRNA expression, which suggests thatSCH-527123 may be associated with increased sensitivityto oxaliplatin throughmodulation of the DNA nucleotideexcision repair pathway.To elucidate the mechanism of action of SCH-527123,

we analyzed the activation of the downstreampathway ofIL-8/CXCR2. IL-8/CXCR2 has previously been shown to

signal throughAKTandMAPKpathways (45, 46).Wilsongroup showed that IL-8/CXCR2 signaling confers resis-tance to oxaliplatin through NF-kB activity, which is animportant determinant of cancer cell sensitivity to oxali-platin (40). In this study, our findings suggest that SCH-527123 decreases NF-kB activity through IL-8/CXCR2signaling, which is in turn responsible for enhancingsensitivity to oxaliplatin. Moreover, our study showedthat SCH-527123 in combination with oxaliplatin signif-icantly increased apoptotic signaling in colorectal cancercells. Therefore, we propose that inhibition of CXCR2/IL-8 signaling increases oxaliplatin sensitivity that is medi-ated partially by attenuating NF-kB activity and alsoinducing apoptosis. However, further investigations arewarranted to elucidate the mechanism of oxaliplatin sen-sitivity mediated through NF-kB activity and apoptosis.

In conclusion, our studies provide significant evidencethat the CXCR2 antagonist, SCH-527123 shows antitumoreffects and increases sensitivity to oxaliplatin therapy inboth in vitro and in vivo colorectal cancer models. Theantitumor activity of SCH-527123 observed in colorectalcancer cells lines was shown to be due to decreased cellproliferation, migration, invasion, and increased apopto-sis. In addition, it was shown that combination of SCH-527123 and oxaliplatin increased sensitivity to oxaliplatinin colorectal cancer cells in vitro and in vivo. Moreover, thecombination of SCH-527123 and oxaliplatin synergistical-ly inhibited in vivo tumor growth and vascularity. Basedon these preclinical results, CXCR2may represent a noveltherapeutic target in colorectal cancer, that when targetedin combination with the DNA-damaging agent, oxalipla-tin will increase chemosensitivity.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Grant SupportThis study was supported by the NIH grant 5 P30CA14089-27S1, the.

Kroha/Casner Family Foundation, Dhont Family, and WundergloFoundation.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 29, 2011; revised February 15, 2012; acceptedFebruary 19, 2012; published OnlineFirst March 5, 2012.

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2012;11:1353-1364. Published OnlineFirst March 5, 2012.Mol Cancer Ther Yan Ning, Melissa J. Labonte, Wu Zhang, et al. Sensitizes Cells to Oxaliplatin in Preclinical Colon Cancer ModelsThe CXCR2 Antagonist, SCH-527123, Shows Antitumor Activity and

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