AnantagonistofthechemokinereceptorCXCR4induces … · Theaimof this studywas to...

An antagonist of the chemokine receptor CXCR4 inducesmitotic catastrophe in ovarian cancer cells

Joseph Kwong,1 Hagen Kulbe,1 Donald Wong,3

Probir Chakravarty,2 and Fran Balkwill1

1Centre for Cancer and Inflammation, Institute of Cancer, Bartsand The London School of Medicine and Dentistry, Queen MaryUniversity of London; 2Bioinformatics and Biostatistics Service,Cancer Research UK, London, United Kingdom; and 3ChemokineTherapeutics Corp., Vancouver, British Columbia, Canada

AbstractThe chemokine receptor CXCR4 is expressed by malig-nant cells in ovarian cancer and is implicated in theirgrowth and spread. We report here a unique mechanismof action of a small peptide antagonist of CXCR4 on ovar-ian cancer cells: induction of cell death by mitotic catas-trophe. CTCE-9908 inhibited ovarian cancer cell migrationto CXCL12, but on longer incubation, caused cell death inCXCR4-positive cells. CTCE-9908 did not cause apopto-sis or cellular senescence, but induced multinucleation,G2-M arrest, and abnormal mitosis in ovarian cancer cells.This suggests that cell death was caused by mitotic catas-trophe. Using microarray and Western blot analysis, weshowed that CTCE-9908 deregulated DNA damagecheckpoint proteins and spindle assembly checkpoint pro-teins at G2-M phases of the cell cycle. Combination treat-ment of CTCE-9908 and the drug paclitaxel led to anadditive cytotoxicity that also involved mitotic catastro-phe. We conclude that CTCE-9908 has a unique mecha-nism of action in ovarian cancer cells that seems to beCXCR4 specific. [Mol Cancer Ther 2009;8(7):1893–905]

IntroductionChemokines are chemotactic cytokines with a molecularmass of around 8 to 17 kDa and belong to a superfamily

that is divided into four groups (CXCL, CX3CL, CCL, andXCL) according to the positioning of the first two closelypaired and highly conserved cysteine (C) residues of theiramino acid sequence. The specific effects of chemokineson their target cells are mediated by members of a familyof seven-transmembrane G-protein–coupled receptors (1).To date, 20 human chemokine receptors have been identi-fied (2). In inflammation and cancer, chemokines in thediseased tissues contribute to the rolling, tethering, andinvasion of leukocytes from blood vessels, through the en-dothelial cell basement membrane, and into the parenchy-ma (3, 4).In cancer, chemokine receptors are also expressed by ma-

lignant cells. The most commonly studied chemokine recep-tor on malignant cells is CXCR4. It is found on malignantcells of epithelial, mesenchymal, and hemopoietic origin inat least 23 different human malignancies (3). In some can-cers, there is an association between levels of CXCR4 ex-pression on malignant cells in primary human tumors andthe extent of lymph node metastasis (5), and the CXCR4 li-gand CXCL12 is frequently found at common sites of metas-tasis in these malignancies (2).However, in glioma (6), astrocytoma (7), and ovarian can-

cer (8, 9) malignant cells, tumors produce both CXCL12 andits CXCR4 receptor. The biological significance of coexpres-sion of CXCR4 and CXCL12 to cancer growth and spread isnot understood.In the human ovarian tumor microenvironment, chemo-

kine CXCL12 stimulated the growth and invasion of tumorcells by establishing a protumor cytokine network in the tu-mor microenvironment (8).4 CXCL12 was also important forperitoneal dissemination of ovarian tumor cells becausethere was a CXCL12 gradient between ovarian tumor cellsand peritoneal mesothelial cells (10). In addition, CXCR4 isexpressed on the membrane and in the cytoplasm of malig-nant cells in a majority of ovarian cancer biopsies (11).4 Tak-en together, these findings suggest that CXCR4 is a potentialtarget for ovarian cancer therapy.CXCR4 antagonists include bicyclams (AMD3100; ref.

12), peptides derived from polyphemusin II proteins (T140and TN14003; ref. 13), and peptides derived from the NH2-terminal region of CXCL12 (14). CTCE-9908 is a small pep-tide that comprises a dimerized sequence of the disorderedNH2-terminal of CXCL12 and was designed to block theCXCR4 receptor (15, 16). Treatment with CTCE-9908 signif-icantly reduced lung metastasis of osteosarcoma and mela-noma cells in animal models (17). A phase I/II clinical trialof CTCE-9908 in patients with advanced solid cancers isbeing studied (16).

Received 10/8/08; revised 3/26/09; accepted 4/9/09; publishedOnlineFirst 6/30/09.

Grant support: Association for International Cancer Research and DirectGrant for Research, Chinese University of Hong Kong (J. Kwong); CancerResearch UK (H. Kulbe and P. Chakravarty); and Higher Education FundingCouncil for England (F. Balkwill).

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 toindicate this fact.

Note: Current address for J. Kwong: Department of Obstetrics andGynaecology, The Chinese University of Hong Kong, Hong Kong, China.

Requests for reprints: Fran Balkwill, Centre for Cancer and Inflammation,Institute of Cancer, Barts and The London School of Medicine andDentistry, Charterhouse Square, London EC1M 6BQ, United Kingdom.Phone: 44-207-882-6106; Fax: 44-207-882-6110.E-mail: [email protected]

Copyright © 2009 American Association for Cancer Research.

doi:10.1158/1535-7163.MCT-08-0966 4 Kulbe et al. personal communication.

Mol Cancer Ther 2009;8(7). July 2009

1893

Research. on September 13, 2020. © 2009 American Association for Cancermct.aacrjournals.org Downloaded from

Published OnlineFirst June 30, 2009; DOI: 10.1158/1535-7163.MCT-08-0966

http://mct.aacrjournals.org/

The aim of this study was to investigate the activity ofCTCE-9908 in ovarian cancer cells in vitro. We found thatCTCE-9908 inhibited migration and induced cytotoxicityin CXCR4-expressing ovarian cancer cells in a CXCR4-specific manner. Such cytotoxicity was mediated throughmitotic catastrophe. Cotreatment of CTCE-9908 and pacli-taxel resulted in additive cytotoxicity in ovarian cancer cells.

Materials and MethodsReagents

CTCE-9908 and scrambled peptide control (SC-9908)were obtained from Chemokine Therapeutics Corp. Anti-CXCR4, α-tubulin, Cdc2, Cdc25A, Cdc25C, Aurora A,Aurora B, Mad2L1, Plk1, NUMA, CENP-F, geminin, andsurvivin antibodies were purchased from Abcam; stathminantibody was from Cell Signaling; anti–phospho-MPM-2,Chk1, and Chk2 were from Upstate; CXCR4 inhibitoryantibodies were from R&D Systems; AMD3100 octahy-drochloride, paclitaxel, staurosporine, and calcein-AM werepurchased from Sigma.Cell Cultures

The ovarian cancer cell lines TOV21G and SKOV3 (allfrom American Type Culture Collection) and IGROV-1(18) were cultured in DMEM supplemented with 10% fetalbovine serum. Two CXCR4 knockdown clones fromIGROV-1 cells (shCXCR4 a9 and e10) and IGROV-1 cellswith scrambled shRNA construct were cultured in DMEMsupplemented with 10% fetal bovine serum and puromycindihydrochloride (Sigma). IOSE20C2 and IOSE25C26 arehTERT-immortalized human ovarian surface epithelial celllines from two different donors (19) that were culturedin a modified medium (NOSE-CM; ref. 20). Two endome-trial cancer cell lines (AN3CA and HEC1A and HEC1B)were also used in the study. All the cell cultures were cul-tured under endotoxin-free conditions. For CTCE-9908and SC-9908 treatment, cells were treated with freshly dis-solved CTCE-9908 or SC-9908 at 1 to 300 μg/mL for 3, 6, or10 d. For AMD3100 treatment, cells were treated with 1 and10 μg/mL AMD3100 for 6 d. For paclitaxel treatment, cellswere treated with 10 and 100 nmol/L paclitaxel for 24 or72 h. Fresh medium and drugs were replaced every 3 d.Cell Viability Assay

After drug treatments, the cell cultures were trypsinized.Viable cells were counted by Vi-Cell XR cell viability analyz-er (Beckman Coulter).Flow Cytometry

For surface expression of CXCR4, cells were dissociatedby enzyme-free PBS-based cell dissociation buffer (Invitro-gen). Monoclonal antibodies against CXCR4 (clone 44717)or IgG2B isotype control (R&D Systems) were applied tothe cell suspension and incubated. After washing, AlexaFluor 488 secondary antibody (Invitrogen) was incubated.Cells were counterstained with propidium iodide (PI) andanalyzed on a FACScan flow cytometer using CellQuestsoftware (BD PharMingen). For apoptotic cell detection, cul-ture medium and trypsinized cells were collected by centri-fugation. Cell pellets were resuspended in DMEM

containing Annexin V-carboxyfluorescein succinimidylesterand PI for flow cytometry analysis. For 5-bromo-2′-deox-yuridine (BrdUrd) fluorescence-activated cell sorting(FACS) analysis, cells were first labeled with BrdUrd (RocheApplied Science). Cells were then harvested and fixed with70% ethanol. After fixation, the cells were treated with HCland then neutralized with sodium borate. After washing,the cells were incubated with anti-BrdUrd (Roche) and fur-ther with Alexa Fluor 488–conjugated secondary antibo-dies. Cells were counterstained with PI before FACSanalysis. For cell cycle distribution, harvested and fixedcells were treated with RNase A and stained with PI forFACS analysis. For cell-cell fusion assay, ovarian cancercells were divided equally into two groups and labeledwith DiO and DiI, separately. After cell labeling, twogroups of labeled cells were mixed together and treatedwith CTCE-9908 for 3 d. FACS analyses (FL1 versus FL2)were done after the drug treatment.Migration

Chemotaxis migration assay was done using Falcontranswells (24-well format, 8-μm pore; BD Pharmingen).Serum-starved cells were treated with CTCE-9908 (100μg/mL) or SC-9908 (100 μg/mL) for 6 h. After treatment,cells were dissociated and resuspended in serum-free me-dium. Resupended cells were added to the upper chamber(5 × 105); complete medium with 10% fetal bovine serumwas added to the lower chamber. Chambers were incubat-ed overnight. Cells on the upper surface of the filter wereremoved using a cotton wool swab. Migrated cells on thelower surface were stained with calcein-AM and trypsi-nized. Trypsinized cell suspensions were collected andfluorescence was read at excitation λ 485 nm and emissionλ 520 nm.Fluorescent and Immunofluorescent Staining

Viability staining was done with the LIVE/DEAD re-duced Biohazard viability/cytotoxicity kit (Invitrogen).The plasma membrane and vesiculation were stained witha lipophilic styryl dye FM 1-43FX (Invitrogen) and the nu-clei were counterstained by 4′,6-diamidino-2-phenylindole(DAPI; Invitrogen). For immunofluorescent staining, cellswere cultured in eight-well glass chamber slides. AfterCTCE-9908 treatment, cells were fixed with 4% paraformal-dehyde. Fixed cells were permeabilized and washed. Afterblocking with bovine serum albumin and normal goat se-rum, cell were incubated with primary antibodies dilutedin immunofluorescence buffer (PBS containing 0.2% bovineserum albumin and 1% normal goat serum). Antimouse orantirabbit IgG coupled with Alexa Fluor 488 dye (Invitogen)and Alexa Fluor 633 phalloidin were diluted in immunoflu-orescence buffer and incubated. Cells were counterstainedwith DAPI and mounted with Prolong Gold antifade re-agent (Invitrogen). Microscopic images were captured byNikon Eclipse 80i microscope and Image-Pro Plus software(Media Cybernetics).5-Bromo-4-Chloro-3-Indolyl-β-D-Galactopyranoside

Staining

Cellular senescence was detected with the SenescenceDetection Kit (Biovision, Inc.).

CTCE-9908 Induces Mitotic Catastrophe in Ovarian Cancer


1894




DNA Laddering

Intranucleosomal DNA fragmentation was detected withthe Apoptotic DNA Ladder Detection Kit (Millipore).Microarray

Total RNAwas isolated using RNeasy Kit (Qiagen). RNAwas quantified and assessed for integrity using a 2100 Bioa-nalyzer (Agilent Technologies). Expression profiles of allsamples were compared with a commercial universal refer-ence RNA (Clontech). The Affymetrix GeneChip HumanGe-nome U133 Plus 2.0 arrays (Affymetrix) were used to definegene expression profiles in each sample. Probe synthesisand microarray hybridization were done according to stan-dard Affymetrix protocols at the Institute of Cancer and

the Cancer Research UK Clinical Centre microarray corefacility.Microarray Analysis

Three Affymetrix data sets were obtained from triplicatesamples of IGROV control, IGROV cells treated with CTCE-9908 for 24 h (CTCE-9908 24 h), and IGROV cells withCTCE-9908 for 48 h (CTCE-9908 48 h). Data were analyzedusing Bioconductor 1.95 running on R2.6.0. Probe set ex-pression measures were calculated using the Affy package

5 http://bioconductor.org

Figure 1. CTCE-9908 inhibitsmigration and growth in CXCR4-expressing ovarian cancer cells.A, left, quantitative real-time re-verse transcr ipt ion-PCR forCXCR4 in ovarian cancer celll ines ( IGROV, TOV21G andSKOV3) and nontransformedIOSE cell lines (IOSE20C2 and IO-SE25C26). 18S rRNA was usedas endogenous control. TheCXCR4 expression of each sam-ple was normalized to that of IO-SE20C2 cells. Right, Westernblot for CXCR4 in ovarian cancerand IOSE cell lines. β-Actin wasused as loading control. B, flowcytometric analysis for surfaceCXCR4 in ovarian cancer celllines and IOSE cell lines. IgG2Bisotype was used as antibodybinding control. C, migration as-say of IGROV and TOV21G cellsafter treatment with CTCE-9908(100 μg/mL) or SC-9908 (100μg/mL). The experiments weredone in quadruplicate. Migrationindex is a ratio of the fluores-cence reading of samples normal-ized to that of untreated control.Bars, SE. *, P< 0.05, unpaired ttest with Welch's correction. D,left, cell viability assay in ovariancancer cells and IOSE cells in thepresence of CTCE-9908 (0–300μg/mL) for 10 d. Each point ofcell survival is calculated by theaverage of two different experi-ments in duplicate as the percentof treated over untreated cells.Right, cell viability assay in ovar-ian cancer cells and IOSE cells inthe presence of scrambled pep-tide SC-9908 (0–300 μg/mL) for10 d. Bars, SE. E, left, flow cyto-metric analysis for surface ex-pression of CXCR4 in IGROV-1parental, IGROV-1 scrambled,and IGROV-1 shCXCR4 a9 ande10 cells. Right, cell viability as-say in IGROV-scrambled andIGROV-1 shCXCR4 a9 and e10cells after 10 d for treatment withCTCE-9908 (0–300 μg/mL).Bars, SE.

Molecular Cancer Therapeutics


1895




Robust Multichip Average default method. Differential geneexpression was assessed between control (IGROV-1) andtreated groups (CTCE-9908 24 h and CTCE-9908 48 h) usingan empirical Bayes t test (limma package; ref. 21); P valueswere adjusted for multiple testing using the Benjamini-Hochberg method. Any probe sets that exhibited an adjustedP value of 0.05were called differentially expressed. In addition,anyprobe sets that exhibited an absolute fold change of >2wereused to generate a heat map. Two-dimensional hierarchicalclustering of expression data using differentially expressedgenes across control and treated groups was done. Sampleswere clustered using a 1 − Pearson correlation distance matrixand average linkage clustering. Genes were clustered using aEuclidean distance matrix and average linkage clustering.Gene Set Enrichment Analysis Using Gene Ontology

Processes

Collections of gene sets based on Gene Ontology (22)term mapping to the Affymetrix human U133plus2 arrayswere created using the GSEAbase package in Bioconductor(23). The function “geneSetTest” from the limma packagewas used to assess whether each Gene Ontology term hada tendency to be associated with up-regulation or down-regulation. The function used a Wilcoxon test to generateP values, ranking the data based on t statistics. Three collectionscomprising terms from themain branches of theGeneOntology(Biological Process, Molecular Function, and Cellular Compo-nent) were tested separately. The Benjamini-Hochberg methodwas used to control the false discovery rate (24).Quantitative Real-time Reverse Transcription-PCR

Total RNA was isolated by TRI reagent (Sigma) and thefirst-strand cDNA was synthesized by SuperScriptII RTKit (Invitrogen). Predesigned probe and primer sets of hu-man CXCR4 mRNA were obtained from TaqMan Gene Ex-pression Assay (Applied Biosystems). Probe and primer setsfor human 18S rRNA were used as endogenous controls.Real-time PCR reactions were done with the ABI PRISM7700 Sequence Detector (Applied Biosystems). Data analy-ses were done using the δδCT method.Western Blotting

For the experiments with CTCE-9908 treatment, cellswere firstly synchronized at early S phase by double thymi-dine block. After the secondary thymidine (Sigma) block,cells were released by complete medium with or withoutCTCE-9908 for 3 d. Cell extract (30 μg) was run on 6%/10%/14% SDS-PAGE gel and transferred onto nitrocellulosemembranes. Themembranes were blocked in PBSwith nonfatmilk and probedwith different primary antibodies. Horserad-ish peroxidase–conjugated secondary antibodies were de-tected using enhanced chemiluminescence Western blottingdetection reagents (GE Healthcare). Protein concentrationequivalence was confirmed after probing by β-actin antibody.CXCR4 siRNA

For transient knockdown of CXCR4 in ovarian cancercells, ON-TARGET plus SMART pool of CXCR4 gene wastransfected into the cells by Dharmafect1 tranfection reagent(all from Dharmacon). SiCONTROL Nontargeting siRNAPool (Dharmacon) served as scrambled RNAi control. Thecells were incubated for 72 h after transfection.

Statistical Analysis

Statistical analysis was evaluated unpaired t test withWelch correction (GraphPad Prism version 4 software).

ResultsCTCE-9908 Inhibits the Migration and Growth of

CXCR4-Expressing Ovarian Cancer Cells

We used three different ovarian cancer cell lines (IGROV-1,TOV21G, and SKOV3) and two hTERT immortalized ovar-ian surface epithelial (IOSE) cell lines. The ovarian cancercell lines expressed varying levels of CXCR4. The IOSEcells (IOSE20 and IOSE25) were CXCR4 negative (Fig. 1Aand B).To test the effect of CTCE-9908, we treated IGROV-1 and

TOV21G cells with 100 μg/mL CTCE-9908 and performed amigration assay. Scrambled peptide (SC-9908) was also usedto test the specificity of the peptide sequence of CTCE-9908.The migration of IGROV-1 and TOV21G cells was signifi-cantly inhibited by CTCE-9908 but not by SC-9908 (Fig. 1C).Having shown that CXCL12-CXCR4 signaling increases

survival of ovarian cancer cells (8), we hypothesized thatblockade of CXCR4 by CTCE-9908 would affect the growthof ovarian cancer cells. To test this, CXCR4-positive ovariancancer and CXCR4-negative IOSE cell lines were treatedwith CTCE-9908 for 10 days and a cell viability assay wasdone. The number of viable cells in IGROV-1, TOV21G, andSKOV3 cell lines was significantly reduced by treatmentwith CTCE-9908, whereas IOSE cells (IOSE20 and IOSE25)were not affected by CTCE-9908. This result suggests thatthe effect of CTCE-9908 acts through CXCR4. In addition,the scrambled peptide had no activity on the cell lines(Fig. 1D).To further confirm that CTCE-9908–mediated growth in-

hibition is CXCR4 dependent, two clones of IGROV-1 cellsstably expressing a CXCR4-targeted small hairpin RNAconstruct, or mock scrambled shRNA construct (IGROV-1scrambled), were used. Our FACS data showed that surfaceexpression of CXCR4 was significantly down-regulated inIGROV-1 shCXCR4 clones when compared with the paren-tal cells and the scrambled control. After CTCE-9908 treat-ment for 10 days, the number of viable cells in IGROV-1shCXCR4 clones was higher than that of the scrambled con-trol (Fig. 1E). Taken together, all the above results suggestthat the growth inhibition induced by CTCE-9908 is CXCR4specific.CTCE-9908 Induces Cytotoxicity of Ovarian Cancer

but It Is Not Due to Acute Apoptosis and Cellular

Senescence

To determine whether CTCE-9908–mediated growth inhi-bition is a result of cell death, cells were stained withSYPO10 and DEAD Red nuclei acid stains after 10-dayCTCE-9908 treatment. Dead cells were detected inCTCE-9908–treated IGROV-1, TOV21G, and SKOV3 cells(Fig. 2A). The scrambled peptide SC-9908 did not cause celldeath in any ovarian cancer and IOSE cells (data notshown).To determine the mechanism for CTCE-9908 stimulat-

ed cytotoxicity, early and late events of apoptosis were



1896




investigated at day 3 posttreatment. No increase in An-nexin V–positive cells was found in CTCE-9908–treatedIGROV-1, TOV21G, and SKOV3 cells (Fig. 2B); neither wasDNA fragmentation detected in any CTCE-9908–treatedovarian cancer cells (Fig. 2C). The results indicate that acuteapoptosis is not a mechanism for CTCE-9908 stimulatedcytotoxicity.To examine whether CTCE-9908 induces cellular senes-

cence in ovarian cancer cells, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) staining was done at day 3posttreatment. No positive X-gal staining was detected inuntreated and CTCE-9908–treated IGROV-1 and SKOV3 celllines. Endogenous β-galactosidase activity was found insome of untreated TOV21G cells; however, no inductionof X-gal–positive cells was found in CTCE-9908–treatedTOV21G cells (Fig. 2D).

CTCE-9908 Induces Multinucleation and G2-M Arrest

in Ovarian Cancer Cells

Although neither apoptosis nor cellular senescence wasfound in CTCE-9908–treated ovarian cancer cells, CTCE-9908 induced significant morphologic changes in ovariancancer cells. Giant multinucleated cells were observed inCXCR4-expressing IGROV-1, TOV21G, and SKOV3 cells(Fig. 3A) but not in CXCR4-negative IOSE cells afterCTCE-9908 treatment (data not shown).Flow cytometric analysis also revealed significant

changes in DNA content and cell cycle distribution inCTCE-9908–treated ovarian cancer cells. The percentage ofcells containing more than 4N DNA content (>4N cells, i.e.,multinucleated cells) increased significantly in IGROV-1,TOV21G, and SKOV3 cells after CTCE-9908 treatment. G2-M arrest also occurred in CTCE-9908–treated IGROV-1,

Figure 2. CTCE-9908 induces cytotoxicity of ovarian cancer cells but is not mediated through acute apoptosis or cellular senescence. A, viability andcytotoxicity assays in IGROV, TOV21G, and SKOV3 cells after treatment with CTCE-9908 (100 μg/mL) for 10 d. Green, live cells; red, dead cells. B, flowcytometric analysis for Annexin V in IGROV, TOV21G, and SKOV3 cells treated with CTCE-9908 (100 and 300 μg/mL) for 3 d. Percentage of Annexin V–positive and PI-negative cells (bottom right) was indicated. SKOV3 cells were treated with staurosporine (1 μmol/L) for 18 h and used as positive control.C, DNA laddering analysis in IGROV, TOV21G, and SKOV3 cells after 3 d of treatment with increasing concentration of CTCE-9908 (0–300 μg/mL).Treatments with 1 μmol/L staurosporine (STS) for 18 h were used as positive control. DMSO, solvent control for staurosporine. D, X-gal staining in IGROV,TOV21G, and SKOV3 cells with different concentrations of CTCE-9908 (100 and 300 μg/mL) for 3 d. Cells with positive β-galactosidase activity werestained in blue.



1897




TOV21G, and SKOV3 cells as indicated by an increase in thepercentage of cells at these phases (Fig. 3B). However, multi-nucleated cells and G2-M arrest were not detected in CXCR4-negative IOSE cells after CTCE-9908 treatment (Fig. 3B andC).CTCE-9908–induced multinucleation and G2-M arrest oc-

curred in a concentration-dependent manner (Fig. 3C). Theincrease of >4N cells also occurred in a duration-dependentmanner. When IGROV-1, TOV21G, and SKOV3 cells weretreated with 100 μg/mL CTCE-9908 in a time course exper-iment, the percentage of >4N cells increased from 4 hoursposttreatment and reached a peak at 48 hours posttreatment(Fig. 3D). The scrambled peptide SC-9908 did not inducemultinucleation and G2-M arrest in ovarian cancer andIOSE cells (data not shown).CTCE-9908 Induces DNA Re-replication and Abnormal

Mitoses in Ovarian Cancer Cells

To determine whether these multinucleated cells resultedfrom DNA re-replication of 4N cells, we monitored theBrdUrd incorporation after CTCE-9908 treatment by flowcytometry analysis. The results show that the CTCE-9908–induced >4N cells were positive for BrdUrd (Fig. 4A). Thissuggests that DNA re-replication is a mechanism by whichthe multinucleated cells are formed. To determine whether

the multinucleation results from cell-cell fusion betweentwo 4N cells, we performed a cell-cell fusion assay. Similar tomany other tumor cell lines, IGROV-1, TOV21G, and SKOV3cells are able to fuse spontaneously in tissue culture (25). How-ever, we found no significant increase of cell-cell fusionwhentreating IGROV-1 cells with 100 μg/mLCTCE-9908. The per-centage of fused cells was significantly decreasedwhen treat-ing IGROV-1 cells with 300 μg/mL CTCE-9908 (Fig. 4B).Similar observations were also evident in TOV21G andSKOV3 cells after CTCE-9908 treatment (data not shown).Therefore, our observations indicate that the CTCE-9908–induced multinucleation is not due to cell-cell fusion.Multinucleation may be caused by abnormal mitosis. In-

deed, multinucleation and abnormal mitosis are morpho-logic characteristics of mitotic catastrophe, a cell death thatoccurs during mitosis or results from abnormal mitosis(26). To test this, we examined mitosis in CTCE-9908–treatedovarian cancer cells by DAPI staining. We found abnormalmitoses with multipolar metaphase and anaphase plates inIGROV-1, TOV21G, and SKOV3 cells after CTCE-9908 treat-ment. Using quantitative analysis of normal and abnormalmitoses, we found that the percentage of the normal mitosesdecreased in IGROV-1, TOV21G, and SKOV3 cells after

Figure 3. CTCE-9908 induces multinucleation and G2-M arrest in ovarian cancer cells. A, fluorescent staining for plasma membrane, lipid vesicles, andnuclei following 100 and 300 μg/mL CTCE-9908 treatment for 3 d in IGROV, TOV21G and SKOV3 cells. Green, plasma membrane and lipid vesicles; blue,nuclei. B, flow cytometric analysis of DNA content in IGROV, TOV21G, SKOV3, and IOSE25C26 cells treated with CTCE-9908 (100 and 300 μg/mL) for3 d. A representative cell cycle profile of each treatment was shown. G1: G1 phase of cell cycle; G2/M: G2-M phase of cell cycle. >4N, cells with morethan 4N DNA content. C, quantification of cell cycle distribution (sub-G1, G1, S, and G2-M) and >4N cells in IGROV, TOV21G, SKOV3, and IOSE25C26cells after 3-d treatment with increasing concentration of CTCE-9908 (0–300 μg/mL). D, quantification of cell cycle distribution and >4N cells in IGROV,TOV21G, and SKOV3 cells treated with CTCE-9908 (100 μg/mL; top) for 4 to 72 h.



1898




Figure 4. CTCE-9908 induces DNA re-replication and abnormal mitoses in ovarian cancer cells. A, flow cytometric analysis for bromodeoxyuridine(BrdUrd) incorporation in IGROV, TOV21G, and SKOV3 cells treated with CTCE-9908 (100 and 300 μg/mL) for 3 d. The quadrant shows the limits forreplicating cells (top left; BrdUrd-positive 2N and 4N cells) and re-replicating cells (top right; BrdUrd-positive >4N cells). Percentage of the re-replicatingcells is indicated. IgG1 isotype was used as negative control. B, Cell-cell fusion was detected by flow cytometry in IGROV cells after treatment with CTCE-9908. Top, unstained IGROV cells (No label), DiO-labeled cells (DiO only), DiI-labeled cells (DiI only), and double-labeled cells (DiO & DiI) were culturedwithout CTCE-9908 for 3 d. Bottom, mixture of DiO- and DiI-labeled IGROV cells were treated with CTCE-9908 (100 and 300 μg/mL) for 3 d and sub-sequently analyzed by flow cytometry. R1 represents a region for double positive (DiO and DiI positive) cells. Percentage of double positive cells is indi-cated. C, left, fluorescent staining of DNA in IGROV, TOV21G, and SKOV3 cells after treatment of CTCE-9908 (0 or 100 μg/mL) for 3 d. White circleindicates abnormal mitoses with multipolar metaphase or anaphase. Blue, DAPI stain. Right, quantification of normal and abnormal mitoses in IGROV-1,TOV21G, and SKOV3 after treatment of CTCE-9908. Fifteen to thirty of 400× objective images of each treatment were captured. Number of normalmitoses, abnormal mitoses, and total number of nuclei were counted on each image. D, immunofluorescent staining for phosphorylated MPM2 (pMPM2)following 100 μg/mL CTCE-9908 treatment for 3 d in IGROV, TOV21G, and SKOV3 cells. pMPM2 was labeled in green, F-actin was labeled in red byphalloidin, and nuclei were counterstained in blue by DAPI. White circle indicates abnormal mitoses with multipolar metaphase or anaphase. E, immuno-fluorescent staining for α-tubulin in IGROV, TOV21G, and SKOV3 cells after CTCE-9908 treatment (100 μg/mL) for 3 d. α-Tubulin was labeled in green, F-actin was labeled in red by phalloidin, and nuclei were counterstained in blue by DAPI. White circle indicates abnormal mitoses with multipolar metaphaseand multiple spindles.



1899




treatment with CTCE-9908, whereas the percentage of ab-normal mitoses increased significantly after the CTCE-9908 treatment (Fig. 4C). To ensure that those cells withmultipolar chromosome segregation are in mitosis, phos-phorylated MPM2 (a mitosis-specific marker) immunos-taining was done. Ovarian cancer cells with multipolarchromosome segregation were positive for phosphorylatedMPM2 (Fig. 4D). Staining for α-tubulin revealed multiplespindles in the abnormal mitoses in CTCE-9908–treatedIGROV-1, TOV21G, and SKOV3 cells (Fig. 4E). Taken together,

our results show that CTCE-9908 induced abnormal mitosesin ovarian cancer cells. Thus, we suggest that CTCE-9908stimulated the cytotoxicity of ovarian cancer cells by mitoticcatastrophe in a CXCR4-dependent manner.To know whether this is a phenomenon restricted to ovar-

ian cancer cells, we examined the effect of CTCE-9908 on en-dometrial cancer cells. Two endometrial cancer cell lines(HEC1B and AN3CA) were used in this study. Real-time re-verse transcription-PCR and flow cytometric analysesshowed that HEC1B cells strongly expressed CXCR4, whereas

Table 1. Significant Gene Ontology categories after CTCE-9908 treatment in IGROV-1 cells

GO ID GO Description Down FDR

IGROV-1 CTCE-9908 vs IGROV-1, 24 hGO:0006260 DNA replication <1.00e−14GO:0007049 Cell cycle <1.00e−14GO:0007067 Mitosis <1.00e−14GO:0051301 Cell division 1.71e−13GO:0006281 DNA repair 2.37e−08GO:0007051 Spindle organization and biogenesis 0.000810711GO:0000086 G2-M transition of mitotic cell cycle 0.001946096GO:0007094 Mitotic cell cycle spindle assembly checkpoint 0.004792247GO:0000085 G2 phase of mitotic cell cycle 0.006344179GO:0006270 DNA replication initiation 0.007577555GO:0000075 Cell cycle checkpoint 0.010740641GO:0000077 DNA damage checkpoint 0.011675618GO:0007059 Chromosome segregation 0.031589917GO:0007080 Mitotic metaphase plate congression 0.038362639GO:0007096 Regulation of exit from mitosis 0.039289029GO:0006333 Chromatin assembly or disassembly 0.03940174GO:0040001 Establishment of mitotic spindle localization 0.040548923GO:0007076 Mitotic chromosome condensation 0.043845871GO:0000087 M phase of mitotic cell cycle 0.046307072

IGROV-1 CTCE-9908 vs IGROV-1, 72 hGO:0006260 DNA replication <1.00e−14GO:0051301 Cell division <1.00e−14GO:0007049 Cell cycle <1.00e−14GO:0007067 Mitosis <1.00e−14GO:0006281 DNA repair 1.30e−08GO:0006270 DNA replication initiation 3.63e−07GO:0000086 G2-M transition of mitotic cell cycle 5.18e−07GO:0007001 Chromosome organization and biogenesis 0.000108262GO:0030100 Regulation of endocytosis 0.000186794GO:0007051 Spindle organization and biogenesis 0.000352454GO:0006338 Chromatin remodeling 0.001012864GO:0007059 Chromosome segregation 0.001076443GO:0006333 Chromatin assembly or disassembly 0.004379725GO:0000075 Cell cycle checkpoint 0.007719117GO:0000085 G2 phase of mitotic cell cycle 0.015893541GO:0000070 Mitotic sister chromatid segregation 0.01838485GO:0048261 Negative regulation of receptor mediated endocytosis 0.019229847GO:0007080 Mitotic metaphase plate congression 0.023838579GO:0000077 DNA damage checkpoint 0.024125364GO:0030174 Regulation of DNA replication initiation 0.027676954GO:0007095 Mitotic cell cycle G2-M transition DNA damage checkpoint 0.03143229GO:0000076 DNA replication checkpoint 0.034131394

Abbreviations: GO, Gene Ontology; FDR, false discovery rate.



1900




AN3CA cells were CXCR4 negative (Supplementary Fig. S1Aand B).6 HEC1B and ANC3A cells were treated with CTCE-9908 for 3 days and a cell viability assay was done. The num-ber of viable HEC1A cells was significantly reduced byCTCE-9908 treatment whereas AN3CA cells were not affect-ed by CTCE-9908 (Supplementary Fig. S1C)6, and giant mul-tinucleated cells were found in CTCE-9908–treated HEC1Bcells but not in CTCE-9908–treated AN3CA cells (Supple-mentary Fig. S1D).6 G2-M arrest and induction of >4N cellpopulation were also detected in HEC1B cells but not inANC3A cells after treatment with CTCE-9908 (Supplementa-ry Fig. S1E).6 These results suggest that CTCE-9908 acts in asimilar CXCR4-dependent fashion on other malignant cells.To investigate whether other CXCR4 antagonists or inhi-

bitors also induce G2-M arrest and multinucleation, ovariancancer cells were treated with CXCR4 inhibitory antibodies,

CXCR4-targeted siRNA, or nonpeptide bicyclam CXCR4antagonist (AMD3100). None of these treatments inducedmultinucleation, although AMD3100 was growth inhibitory(Supplementary Fig. S2).6 Based on these data, we concludethat CTCE-9908 exhibits a unique cytotoxicity in ovariancancer cells that seems to be CXCR4 specific.CTCE-9908 Affects DNA Damage Checkpoint and

Spindle Assembly Checkpoint at G2-M Phases

To explore the mechanism of CTCE-9908–induced mitoticcatastrophe in ovarian cancer cells, genes found to be differ-entially expressed in IGROV-1 cells after treatment with 100μg/mL CTCE-9908 (after 24 and 72 hours) versus untreatedIGROV-1 control were analyzed with Gene Set EnrichmentAnalysis to identify the Gene Ontology categories most re-presented in down-regulated genes (Table 1; SupplementaryTable S1)6. These results show that CTCE-9908 directly orindirectly affected the expression of many genes involvedin G2 andMphases of cell cycle. In particular, genes involvedin the G2-M transition checkpoints (such as Cdc2, Cdc25A,Cdc25C, and CENP-F) were significantly down-regulated in

6 Supplementary material for this article is available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Figure 5. CTCE-9908 deregulates DNAcheckpoint kinases/phosphatases and mitotickinases at G2-M phases. A, Western blots forCdc2, Cdc25A, Cdc25C, Chk1, and Chk2 insynchronized IGROV, TOV21G, and SKOV3cells after 3-d treatment of CTCE-9908(100 μg/mL). B, Western blots for AuroraA, Aurora B, Mad2L1, and Plk1 in synchro-nized IGROV, TOV21G, and SKOV3 cells fol-lowing 100 μg/mL CTCE-9908 treatment for3 d. C, immunostaining for BubR1 in IGROVcells treated with CTCE-9908 (100 μg/mL)for 3 d. BubR1 was labeled in green. Nucleiwere counterstained with DAPI. White circleindicates abnormal mitoses with multipolarmetaphase. D, Western blots for NUMA1,CENP-F, and stathmin in synchronized IGROV,TOV21G, and SKOV3 cells treated withCTCE-9908 (100 μg/mL) for 3 d. β-Actinwas used as loading control. Arrow indicatesthe bands with predicted molecular weight.



1901




CTCE-9908–treated IGROV-1 cells. These data suggest thatCTCE-9908might affectDNAdamage checkpoint and spindleassembly checkpoint at G2-M phases of ovarian cancer cells.To evaluate the integrity of DNA damage checkpoint and

spindle assembly checkpoint in ovarian cancer cells afterCTCE-9908 treatment, we examined the expression levelof several key proteins involved in these two checkpoints.In this experiment, IGROV-1, TOV21G, and SKOV3 werefirst synchronized by double thymidine block. The synchro-nized cells were then treated with 100 μg/mL CTCE-9908for 3 days. Because Cdc2 (cyclin-dependent kinase 1),Cdc25A, Cdc25C, Chk1, and Chk2 are essential to theDNA damage checkpoint, which controls cell cycle progres-sion from G2 to M phase (27), Western blots for these DNAdamage checkpoint proteins were done. Our results showedthat Cdc2, Cdc25A, Cdc25C, and Chk1 were down-regulat-ed in CTCE-9908–treated IGROV-1, TOV21G, and SKOV3cells. Chk2 expression was also decreased in TOV21G andSKOV3 after CTCE-9908 treatment (Fig. 5A).In mitosis, spindle assembly checkpoint is governed by

severalmitotic kinases, such asAuroraA,Aurora B,Mad2L1,

Plk1, and BubR1 (26). Down-regulation of Plk1 was found inCTCE-9908–treated TOV21G and SKOV3 cells (Fig. 5B),whereas up-regulation of BubR1 was detected in CTCE-9908–treated IGROV-1 cells (Fig. 5C). Other mitotic kinases,such as Aurora A, Aurora B, and Mad2L1, were not affectedby CTCE-9908 (Fig. 5B).Besides mitotic kinases, several nuclear matrix and spindle

pole proteins are also associated with spindle assemblycheckpoint. NUMA (nuclear mitotic apparatus protein 1) isa spindle pole protein in mitosis (28). Centromeric protein F(CENP-F/mitosin) is a large human nuclear protein tran-siently associated with the outer kinetochore plate in Mphase (29). Our results show that NUMA and CENP-F weredown-regulated in CTCE-9908–treated IGROV-1 andTOV21G cells (Fig. 5D). Stathmin plays an important rolein the regulation of microtubule cytoskeleton (30), and theprotein expression of stathmin was down-regulated inCTCE-9908–treated IGROV-1, TOV21G, and SKOV3 cells(Fig. 5D). Taken together, our results show that CTCE-9908 deregulated DNA damage checkpoint and spindleassembly checkpoint in ovarian cancer cells.

Figure 6. Combination treat-ment with CTCE-9908 and pacli-t a x e l r e s u l t s i n a d d i t i v ecytotoxicity by mitotic catastro-phe. A, flow cytometric analysisof DNA content in IGROV,TOV21G, and SKOV3 cells trea-ted with 100 μg/mL CTCE-9908,10 nmol/L paclitaxel, or both for3 d. A representative cell cycleprofile of each treatment wasshown. M4 represents G2-Mphase of cell cycle. M5 repre-sents >4N cells. Percentages ofcells in G2-M phase or with >4NDNA content are indicated. B,cell viability assay in IGROV,TOV21G, and SKOV3 cells trea-ted with 100 μg/mL CTCE-9908,10 nmol/L paclitaxel, or both for3 d. Cell survival is representedby the average of two differentexperiments in duplicate as thepercent of treated over DMSO-treated cells. DMSO was usedas solvent control for paclitaxel.Bars, SE. **, P < 0.01; *, P <0.05, unpa i red t test withWelch's correction.



1902




Combination Treatment of CTCE-9908 and Paclitaxel

Results in Additive Cytotoxicity by Mitotic Catastrophe

Paclitaxel (Taxol) is widely used in the treatment of breastand ovarian cancers. A low concentration (10 nmol/L) ofpaclitaxel was found to induce mitotic arrest, multinuclea-tion, and eventually cell death in HeLa cells (31). To exam-ine whether low concentrations of paclitaxel also inducedmitotic arrest and multinucleation in ovarian cancer cells,IGROV-1, TOV21G, and SKOV3 cells were treated with 10nmol/L paclitaxel for 24 hours. DNA content FACS analysisrevealed that G2-M arrest and multinucleation were foundin IGROV-1, TOV21G, and SKOV3 cells after treatment with10 nmol/L paclitaxel (Fig. 6A). Therefore, we hypothesizedthat combining CTCE-9908 with low concentration of pacli-taxel might result in additive cytotoxicity. We analyzed thecell survival after combining 100 μg/mL CTCE-9908 with10 nmol/L paclitaxel. An additive cytotoxic effect wasfound between CTCE-9908 and paclitaxel in IGROV-1,TOV21G, and SKOV3 cells after treatment with both drugsfor 3 days (Fig. 6B).To confirm that this additive cytotoxicity is due to mitotic

catastrophe, cell cycle distribution was analyzed after com-bination treatment with CTCE-9908 and paclitaxel. Additiveaccumulation of G2-M arrest and multinucleation were de-tected in IGROV-1 cells when treated with CTCE-9908 andpaclitaxel. Similarly, additive G2-M arrest was found inTOV21G cells after cotreatment of CTCE-9908 and pacli-taxel (Fig. 6A). However, no additive G2-M arrest or multi-nucleation was observed in SKOV3 cells after combinationtreatment. This might be because SKOV3 cells are moresensitive to 10 nmol/L paclitaxel (Fig. 6A and B). Takentogether, our results show that combination treatment withCTCE-9908 and paclitaxel conferred additive cytotoxicityby mitotic catastrophe.

DiscussionBecause the signals of CXCL12 in tumor microenvironmentare important for the growth, metastasis, and survival ofmalignant cells (32), blocking the signals of CXCL12 by in-activating its receptor, CXCR4, has been considered as a po-tential antitumor therapy. Preclinical studies showed thatthe major antitumor activities of CXCR4 antagonists(AMD3100, AMD3465, T140, and TN14003) are inhibitionof CXCL12-induced tumor growth and cell migration(33–37). However, these CXCR4 antagonists seldom in-duced cell death in majority of tumors. Our preliminary re-sults also showed that bicyclams and CXCR4 inhibitoryantibodies did not induce cell death in ovarian cancer cells.There are only few articles that showed AMD3100-inducedapoptosis in glioblastoma and medulloblastoma (38, 39).Different tumor cell types may account for the dissimilarityof AMD3100-induced cell death.CTCE-9908 exhibits antitumor activity. Treatment of oste-

osarcoma cells in vitro with CTCE-9908 led to decreases ingrowth rate, adhesion, migration, and invasion of the tumorcells. Using tail vein injection of osteosarcoma cells, micethat were treated with CTCE-9908 had a significant reduc-

tion in pulmonary metastasis (17). We showed in the pres-ent study that CTCE-9908 induced cell death in ovariancancer cells by mitotic catastrophe. This is a unique cytotoxi-city induced by CTCE-9908 because other CXCR4 inhibi-tors, including AMD3100, CXCR4 inhibitory antibody, andCXCR4-targeted siRNA, did not show any mitotic catastro-phe in ovarian cancer cells.CTCE-9908 induced multinucleation, G2-M arrest, and

abnormal mitosis in ovarian cancer cells. These changesmay be due to aberrant regulations of cell cycle protein orcell death signals induced by CTCE-9908-CXCR4 signaling.There are some data to suggest that different interactionswith the CXCR4 receptors may result in different biologicalsignals. CXCL12 down-regulated the phosphorylation of Rband the transcription of E2F-1 in neuronal cells throughCXCR4, whereas the binding of HIV envelope glycoproteingp120 to CXCR4 exerted opposite effects on Rb and E2F-1activities in neurons (40). Moreover, CXCL12 induced hom-ing and development of T lymphocytes, whereas the bind-ing of gp120 to CXCR4 induced autophagy and apoptosis inT lymphocytes (41). Most recently, CXCR4-gp120IIIB inter-actions were found to induce apoptosis and inhibit tumorgrowth of prostate cancer (42).CXCR4 is also expressed on primordial germ cells, neu-

ronal precursors, and hemopoietic stem cells (43). It is pos-sible that our study has uncovered an unknown action ofCXCR4 signaling in maintaining the integrity of the mitoticspindle—an action that could be important in stem andprogenitor cells.To better understand the mechanism of the CTCE-9908–

induced mitotic catastrophe, we investigated the check-points of G2-M transition. Mitotic catastrophe occurs as aresult of DNA damage or deranged spindle formation cou-pled to the debilitation of different checkpoint mechanismsthat would normally arrest progression into mitosis andhence suppress catastrophic events until repair has beenachieved. The “DNA damage checkpoint” arrests cells atthe G2-M transition in response to unreplicated DNA orDNA damage, and the “spindle assembly checkpoint” pre-vents anaphase until all chromosomes have obtained bipo-lar attachment. The combination of checkpoint deficienciesand specific types of damage (such as DNA damage or de-ranged spindle formation) would lead to mitotic catastro-phe (26).Our results showed that several G2-M checkpoint pro-

teins (Cdc2, Cdc25A, Cdc25C, Chk1, and Chk2) and spindlecheckpoint proteins (Plk1, NUMA, CENP-F, and stathmin)were down-regulated in ovarian cancer cells after CTCE-9908 treatment. Depletion of Chk1 led to premature activa-tion of Cdc2-cyclin B and mitotic catastrophe (44). Inhibitionof Chk2 would stabilize centrosomes, maintain high cyclinB1 levels, and allow for a prolonged activation of cyclin-de-pendent kinase 1. Under these conditions, multinuclearHeLasyncytia did not arrest at the G2-M boundary and rather en-teredmitosis and subsequently died during themetaphase ofthe cell cycle (45). Silencing of Plk1 caused induction of mito-tic arrest and further apoptosis in prostate cancer cells (46).Inactivation of NUMA by immunodepletion would lead to



1903




a failure of normal spindle assembly and production (47).Silencing of CENF-P induced misaligned chromosomes,premature chromosome decondensation before anaphaseonset, and mitotic cell death (29). Inhibition of stathminexpression would lead to accumulation of cells in the G2-Mphases andwas associatedwith severemitotic spindle abnor-malities and difficulty in the exit from mitosis (30). Based onthe above evidence, we suggested that CTCE-9908weakenedthe DNA damage checkpoint and spindle assembly check-point in ovarian cancer cells.Mitotic catastrophe is a common phenomenon occurring

in tumor cells with impaired p53 function following expo-sure to various cytotoxic and genotoxic agents. It is interest-ing to know whether CTCE-9908 is genotoxic and whetherthe action of CTCE-9908 is related to inactivation of p53 orBRCA1 in ovarian cancer cells. Our results showed thatCTCE-9908 did not induce DNA damage in ovarian cancercells, as indicated by no positive H2AX signal being de-tected after treatment with CTCE-9908 (data not shown).This result suggests that CTCE-9908 is not genotoxic. More-over, the p53 and BRCA1 status of the three ovarian cancercell lines we used is as follows: mutated p53 and BRCA1were found in IGROV-1; wild-type p53 and BRCA1 inTOV21G; and null p53 and wild-type BRCA1 in SKOV3.Based on these observations, we do not believe that the ac-tion of CTCE-9908 is related to p53 or BRCA1 inactivationin ovarian cancer cells.Paclitaxel is a microtubule-stabilizing agent that causes

deranged spindle formation in tumor cells, a possible expla-nation for the additive cytotoxicity between CTCE-9908and paclitaxel relating to the combination of weakenedspindle assembly checkpoints and deranged spindle forma-tion in ovarian cancer cells. This hypothesis is supported bya report showing that silencing of Plk1 in breast cancer cellsimproved sensitivity toward paclitaxel in a synergisticmanner (48).HER2 enhances the expression of CXCR4 in breast cancer,

which is required for HER2-mediated invasion and metasta-sis. HER2 also inhibits CXCL12-inducedCXCR4 degradation(49). It will be interesting to investigate the action of CTCE-9908 on CXCR4-positive breast cancer cells in the future.Although the prognostic influence of HER2/neu is not

dependent on the CXCR4/CXCL12 signaling pathway inovarian cancer (50), it would be interesting in the futureto study the action of CTCE-9908 on HER2 activity in ovar-ian cancer cells.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Prof. Iain McNeish for critical comments.

References

1. Murdoch C, Muthana M, Coffelt SB, Lewis CE. The role of myeloidcells in the promotion of tumour angiogenesis. Nat Rev Cancer 2008;8:618–31.

2. Balkwill F. Cancer and the chemokine network. Nat Rev Cancer 2004;4:540–50.

3. Balkwill F. The significance of cancer cell expression of the chemokinereceptor CXCR4. Semin Cancer Biol 2004;14:171–9.4. Sallusto F, Baggiolini M. Chemokines and leukocyte traffic. Nat Immu-nol 2008;9:949–52.5. Kaifi JT, Yekebas EF, Schurr P, et al. Tumor-cell homing to lymph nodesand bone marrow and CXCR4 expression in esophageal cancer. J NatlCancer Inst 2005;97:1840–7.6. Rempel SA, Dudas S, Ge S, Gutierrez JA. Identification and localizationof the cytokine SDF1 and its receptor, CXC chemokine receptor 4, to re-gions of necrosis and angiogenesis in human glioblastoma. Clin CancerRes 2000;6:102–11.7. Woerner BM, Warrington NM, Kung AL, Perry A, Rubin JB. WidespreadCXCR4 activation in astrocytomas revealed by phospho-CXCR4-specificantibodies. Cancer Res 2005;65:11392–9.8. Scotton CJ, Wilson JL, Scott K, et al. Multiple actions of the chemokineCXCL12 on epithelial tumor cells in human ovarian cancer. Cancer Res2002;62:5930–8.

9. Kulbe H, Hagemann T, Szlosarek PW, Balkwill FR, Wilson JL. The in-flammatory cytokine tumor necrosis factor-α regulates chemokine recep-tor expression on ovarian cancer cells. Cancer Res 2005;65:10355–62.

10. Kajiyama H, Shibata K, Terauchi M, Ino K, Nawa A, Kikkawa F. In-volvement of SDF-1α/CXCR4 axis in the enhanced peritoneal metastasisof epithelial ovarian carcinoma. Int J Cancer 2008;122:91–9.

11. Scotton CJ, Wilson JL, Milliken D, Stamp G, Balkwill FR. Epithelialcancer cell migration: a role for chemokine receptors? Cancer Res 2001;61:4961–5.

12. De Clercq E. The bicyclam AMD3100 story. Nat Rev Drug Discov2003;2:581–7.

13. Tamamura H, Xu Y, Hattori T, et al. A low-molecular-weight inhibitoragainst the chemokine receptor CXCR4: a strong anti-HIV peptide T140.Biochem Biophys Res Commun 1998;253:877–82.

14. Loetscher P, Gong JH, Dewald B, Baggiolini M, Clark-Lewis I. N-termi-nal peptides of stromal cell-derived factor-1 with CXC chemokine receptor4 agonist and antagonist activities. J Biol Chem 1998;273:22279–83.

15. Faber A, Roderburg C, Wein F, et al. The many facets of SDF-1α,CXCR4 agonists and antagonists on hematopoietic progenitor cells.J Biomed Biotechnol 2007;26065.

16. Wong D, Korz W. Translating an antagonist of chemokine receptorCXCR4: from bench to bedside. Clin Cancer Res 2008;14:7975–80.

17. Kim SY, Lee CH, Midura BV, et al. Inhibition of the CXCR4/CXCL12chemokine pathway reduces the development of murine pulmonary me-tastases. Clin Exp Metastasis 2008;25:201–11.

18. Benard J, Da Silva J, De Blois MC, et al. Characterization of a humanovarian adenocarcinoma line, IGROV1, in tissue culture and in nude mice.Cancer Res 1985;45:4970–9.

19. Li NF, Broad S, Lu YJ, et al. Human ovarian surface epithelial cellsimmortalized with hTERT maintain functional pRb and p53 expression.Cell Prolif 2007;40:780–94.

20. Li NF, Wilbanks G, Balkwill F, Jacobs IJ, Dafou D, Gayther SA. Amodified medium that significantly improves the growth of human nor-mal ovarian surface epithelial (OSE) cells in vitro. Lab Invest 2004;84:923–31.

21. Smyth GK, Michaud J, Scott HS. Use of within-array replicate spotsfor assessing differential expression in microarray experiments. Bioinfor-matics 2005;21:2067–75.

22. Ashburner M, Ball CA, Blake JA, et al. Gene ontology: tool for theunification of biology. The Gene Ontology Consortium. Nat Genet 2000;25:25–9.

23. Mootha VK, Lepage P, Miller K, et al. Identification of a gene causinghuman cytochrome c oxidase deficiency by integrative genomics. ProcNatl Acad Sci U S A 2003;100:605–10.

24. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a prac-tical and powerful approach to multiple testing. J R Statist Soc B 1995;57:289–300.

25. Duelli D, Lazebnik Y. Cell fusion: a hidden enemy? Cancer Cell 2003;3:445–8.

26. Castedo M, Perfettini JL, Roumier T, Andreau K, Medema R, KroemerG. Cell death by mitotic catastrophe: a molecular definition. Oncogene2004;23:2825–37.



1904




27. DiPaola RS. To arrest or not to G2-M cell-cycle arrest: commentary re:A.K. Tyagi et al., Silibinin strongly synergizes human prostate carcinomaDU145 cells to doxorubicin-induced growth inhibition, G(2)-M arrest, andapoptosis. Clin Cancer Res 8: 3512–3519, 2002. Clin Cancer Res 2002;8:3311–4.

28. Haren L, Merdes A. Direct binding of NuMA to tubulin is mediated by anovel sequence motif in the tail domain that bundles and stabilizes micro-tubules. J Cell Sci 2002;115:1815–24.

29. Yang Z, Guo J, Chen Q, Ding C, Du J, Zhu X. Silencing mitosin in-duces misaligned chromosomes, premature chromosome decondensationbefore anaphase onset, and mitotic cell death. Mol Cell Biol 2005;25:4062–74.

30. Rubin CI, Atweh GF. The role of stathmin in the regulation of the cellcycle. J Cell Biochem 2004;93:242–50.

31. Jordan MA, Wendell K, Gardiner S, Derry WB, Copp H, Wilson L. Mi-totic block induced in HeLa cells by low concentrations of paclitaxel (Tax-ol) results in abnormal mitotic exit and apoptotic cell death. Cancer Res1996;56:816–25.

32. Kryczek I, Wei S, Keller E, Liu R, Zou W. Stroma-derived factor (SDF-1/CXCL12) and human tumor pathogenesis. Am J Physiol Cell Physiol2007;292:C987–95.

33. De Falco V, Guarino V, Avilla E, et al. Biological role and potential ther-apeutic targeting of the chemokine receptor CXCR4 in undifferentiatedthyroid cancer. Cancer Res 2007;67:11821–9.

34. Juarez J, Bradstock KF, Gottlieb DJ, Bendall LJ. Effects of inhibitorsof the chemokine receptor CXCR4 on acute lymphoblastic leukemia cellsin vitro. Leukemia 2003;17:1294–300.

35. Yang L, Jackson E, Woerner BM, Perry A, Piwnica-Worms D, RubinJB. Blocking CXCR4-mediated cyclic AMP suppression inhibits brain tu-mor growth in vivo. Cancer Res 2007;67:651–8.

36. Liang Z, Wu T, Lou H, et al. Inhibition of breast cancer metastasis byselective synthetic polypeptide against CXCR4. Cancer Res 2004;64:4302–8.

37. Yoon Y, Liang Z, Zhang X, et al. CXC chemokine receptor-4 an-tagonist blocks both growth of primary tumor and metastasis of headand neck cancer in xenograft mouse models. Cancer Res 2007;67:7518–24.

38. Rubin JB, Kung AL, Klein RS, et al. A small-molecule antagonist of

CXCR4 inhibits intracranial growth of primary brain tumors. Proc NatlAcad Sci U S A 2003;100:13513–8.

39. Redjal N, Chan JA, Segal RA, Kung AL. CXCR4 inhibition synergizeswith cytotoxic chemotherapy in gliomas. Clin Cancer Res 2006;12:6765–71.40. Khan MZ, Brandimarti R, Musser BJ, Resue DM, Fatatis A, Meucci O.The chemokine receptor CXCR4 regulates cell-cycle proteins in neurons.J Neurovirol 2003;9:300–14.41. Espert L, Denizot M, Grimaldi M, et al. Autophagy is involved in T celldeath after binding of HIV-1 envelope proteins to CXCR4. J Clin Invest2006;116:2161–72.42. Singh S, Bond VC, Powell M, et al. CXCR4-120-IIIB interactions in-duce caspase-mediated apoptosis of prostate cancer cells and inhibit tu-mor growth. Mol Cancer Ther 2009;8:178–84.

43. Kucia M, Reca R, Miekus K, et al. Trafficking of normal stem cells andmetastasis of cancer stem cells involve similar mechanisms: pivotal role ofthe SDF-1-CXCR4 axis. Stem Cells 2005;23:879–94.

44. Niida H, Tsuge S, Katsuno Y, Konishi A, Takeda N, Nakanishi M. De-pletion of Chk1 leads to premature activation of Cdc2-cyclin B and mitoticcatastrophe. J Biol Chem 2005;280:39246–52.

45. Castedo M, Perfettini JL, Roumier T, et al. The cell cycle checkpointkinase Chk2 is a negative regulator of mitotic catastrophe. Oncogene2004;23:4353–61.

46. Reagan-Shaw S, Ahmad N. Silencing of polo-like kinase (Plk) 1 viasiRNA causes induction of apoptosis and impairment of mitosis machineryin human prostate cancer cells: implications for the treatment of prostatecancer. FASEB J 2005;19:611–3.

47. Merdes A, Ramyar K, Vechio JD, Cleveland DW. A complex of NuMAand cytoplasmic dynein is essential for mitotic spindle assembly. Cell1996;87:447–58.

48. Spankuch B, Kurunci-Csacsko E, Kaufmann M, Strebhardt K. Rationalcombinations of siRNAs targeting Plk1 with breast cancer drugs. Onco-gene 2007;26:5793–807.

49. Li YM, Pan Y, Wei Y, et al. Upregulation of CXCR4 is essential forHER2-mediated tumor metastasis. Cancer Cell 2004;6:459–69.

50. Pils D, Pinter A, Reibenwein J, et al. In ovarian cancer the prognosticinfluence of HER2/neu is not dependent on the CXCR4/SDF-1 signallingpathway. Br J Cancer 2007;96:485–91.



1905




2009;8:1893-1905. Published OnlineFirst June 30, 2009.Mol Cancer Ther Joseph Kwong, Hagen Kulbe, Donald Wong, et al. mitotic catastrophe in ovarian cancer cellsAn antagonist of the chemokine receptor CXCR4 induces

Updated version

10.1158/1535-7163.MCT-08-0966doi:

Access the most recent version of this article at:

Material

Supplementary

http://mct.aacrjournals.org/content/suppl/2009/07/13/1535-7163.MCT-08-0966.DC1

Access the most recent supplemental material at:

Cited articles

http://mct.aacrjournals.org/content/8/7/1893.full#ref-list-1

This article cites 49 articles, 21 of which you can access for free at:

Citing articles

http://mct.aacrjournals.org/content/8/7/1893.full#related-urls

This article has been cited by 4 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. (CCC)Click on "Request Permissions" which will take you to the Copyright Clearance Center's

.http://mct.aacrjournals.org/content/8/7/1893To request permission to re-use all or part of this article, use this link



http://mct.aacrjournals.org/lookup/doi/10.1158/1535-7163.MCT-08-0966

http://mct.aacrjournals.org/content/suppl/2009/07/13/1535-7163.MCT-08-0966.DC1

http://mct.aacrjournals.org/content/8/7/1893.full#ref-list-1

http://mct.aacrjournals.org/content/8/7/1893.full#related-urls

http://mct.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://mct.aacrjournals.org/content/8/7/1893


AnantagonistofthechemokinereceptorCXCR4induces … · Theaimof this studywas to...

Documents

Transcript of AnantagonistofthechemokinereceptorCXCR4induces … · Theaimof this studywas to...