Anti-tumor activity of an anti-DR5monoclonal antibody,TRA-8,in combination with taxane/platinum-based chemotherapy in an ovarian cancer model ☆
Kerri S.Bevis a ,⁎,Lacey R.McNally b ,Jeffery C.Sellers b ,Deborah Della Manna b ,Angelina Londoño Joshi c ,Hope Amm d ,J.Michael Straughn Jr.a ,Donald J.Buchsbaum b
a
Division of Gynecologic Oncology,University of Alabama at Birmingham,61919th Street South,176F,Room 10250,Birmingham,AL 35249,USA b
Department of Radiation Oncology,University of Alabama at Birmingham,Birmingham,AL 35294,USA c
Department of Pathology,University of Alabama at Birmingham,Birmingham,AL 35294,USA d
Department of Pharmacology,University of Alabama at Birmingham,Birmingham,AL 35294,USA
a b s t r a c t
a r t i c l e i n f o Article history:
Received 27August 2010
制度Available online 5January 2011Keywords: Monoclonal antibody Death receptors
Tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL)Chemotherapy Ovarian cancer
Objective.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)mediates apoptosis via binding to death receptors and enhances the anti-tumor effect of conventional cancer therapies.We evaluated the ef ficacy of TRA-8,an agonistic antibody to DR5,combined with docetaxel and carboplatin in vitro in an intraperitoneal (IP)ovarian cancer model.
Methods.Luciferase positive ES2cells (ES2H)were treated in 96well plates with TRA-8,carboplatin,docetaxel,and combination therapy.Cell viability was assessed using ATP-lite assay.Apoptosis was con firmed via Western blot analysis.ES2H cells were injected IP into female athymic nude mice.Animals were sorted based on bioluminescent signal with the following treatments:1)untreated;2)TRA-8alone;3)docetaxel+carboplatin;and 4)docetaxel +carboplatin +TRA-8.女法官陈玉莹
Animals receiving TRA-8antibody were injected IP with 200μg of TRA-8twice weekly until death.Animals receiving docetaxel+carboplatin were injected IP with 5mg/kg and 15mg/kg respectively every 3weeks until death.Animals were assessed for tumor burden using bioluminescence imaging and overall survival.
Results.Combination therapy reduced viability of ES2H cells in vitro over single agent therapy.Tumor burden was lowest in the chemotherapy +TRA-8group at days 23(p b 0.001)and 30(p =0.04).Mean survival was greatest in the chemotherapy +TRA-8group (41days)compared to the chemotherapy only group (34days)and control group (27days)as determined by Kaplan –Meier analysis (p b 0.001).
Conclusion.Conventional chemotherapy combined with TRA-8reduced cell-viability via activation of apoptotic pathways,reduced tumor burden and improved survival in this ovarian cancer model.
©2010Elsevier Inc.All rights reserved.
Introduction
Epithelial ovarian cancer remains the deadliest gynecologic malignancy with approximately 21,550new cases diagnosed in 2009and 14,600related deaths [1].While 80%of patients with advanc
ed disease achieve clinical remission with surgical cytoreduc-tion and taxane/platinum-based chemotherapy,the majority of these patients recur resulting in 5-year survival rates of 35–40%for patients with stage III disease [2–5].Less than 20%of patients with recurrent disease will experience a clinical remission with second-line chemo-therapy [6].Because of poor long-term survival,the search for better
therapeutic options for the treatment of epithelial ovarian cancer continues.Recent research has focused on agents targeting speci fic cellular targets including vascular endothelial growth factor,speci fic tyrosine kinases,and speci fic cell surface receptors involved in mediating cellular death.One such agent demonstrating particular promise in the treatment of a variety of tumor types is tumor necrosis-factor related apoptosis-inducing ligand (TRAIL).
Since its discovery more than a decade ago,TRAIL has been the focus of intense anticancer research because of its ability to stimulate apoptosis in cancer cells while having minimal effect on normal cells [7,8].TRAIL belongs to the tumor necrosis factor (TNF)superfamily which includes cytokines such as TNF and FasL.It is a type II transmembrane protein that can be cleaved into soluble form by cysteine proteases [9].Soluble TRAIL is a trimer which induces apoptosis through binding to TRAIL receptors on the surface of target cells.While five TRAIL receptors have been identi
fied only two of them,death receptor 4(DR4)(TRAILR1)and death receptor 5(DR5)(TRAILR2),contain the cytoplasmic death domain required for activation of the
Gynecologic Oncology 121(2011)193–199
☆Oral Presentation at the 41st Annual Meeting of the Society of Gynecologic Oncologists San Francisco,CA,March 2010.
⁎Corresponding author.University of Alabama at Birmingham,Department of Obstetrics and Gynecology,Division of Gynecologic Oncology,61919th Street South,176F,Room 10250,Birmingham,AL 35249,USA.Fax:+12059756174.
E-mail address:ksbevis@gmail (K.S.
邻苯二甲酸酐
Bevis).0090-8258/$–see front matter ©2010Elsevier Inc.All rights reserved.doi:
10.2010.11.046
Contents lists available at ScienceDirect
Gynecologic Oncology
j o u r n a l h o me p a g e :w w w.e l s e v i e r.c om /l o c a t e /y g yn o
extrinsic apoptotic pathway[10–13].The remaining three receptors lack this intracellular death domain and serve as decoy receptors capable of blocking TRAIL mediated apoptosis.Because of the relative lack of specificity of TRAIL,agonistic monoclonal antibodies have been developed to selectiv
ely target the death receptors while avoiding the anti-apoptotic effects of the decoy receptors.TRA-8is an agonistic monoclonal antibody to DR5which has demonstrated anti-tumor activity,both alone and in combination with chemotherapy,in breast and ovarian cancer models[14–18].In vitro experiments using ovarian cancer cell lines indicate treatment with cytotoxic drugs including platinum based therapies results in upregulation of DR5[16]with varying degrees of cytotoxicity achieved by treatment with TRAIL or agonistic monoclonal antibodies.Based on these results,the objective of the current study is to evaluate the efficacy of TRA-8,a monoclonal antibody to DR5,in combination with docetaxel and carboplatin in an in vivo intraperitoneal ovarian cancer model.
Methods
Cell lines and reagents
ES2and OvCar3cells were obtained from the American Type Culture Collection(Manassas,VA)and HeyA8and A2780cells were provided courtesy of Gordon Mills at the University of Texas M.D.Anderson Cancer Center.All cells were maintained in RPMI medium supplemen-ted with10%FBS and1%L-glutamine(Cellgro by Mediatech,Manassas, VA).Cells were maintained in antibiotic-free medium at37°C and a5% CO2atmosphere.Purified TRA-8was provided by Dr.Tong Zh
ou (University of Alabama at Birmingham).Carboplatin(Sigma-Aldrich,St. Louis,MO)was stored as25mM stock solutions in sterilized water.The clinical formulation of docetaxel was obtained from the University of Alabama Hospital Pharmacy(Birmingham,AL)and diluted to the appropriate concentration in RPMI or PBS for in vitro and in vivo experiments,respectively.Docetaxel was selected due to the improved water solubility of this taxane compared to paclitaxel.
Construction of luciferase positive cell line
ES2cells were transduced with a non-replicating retrovirus containingfirefly luciferase gene(Stratagene,La Jolla,CA).A single cell clone,ES2H,was isolated and bioluminescent signal was confirmed using the IVIS100imaging system(Caliper,Hopkinton, MA).The growth rates of parental ES2cells and the ES2H clone were identical in vitro and in vivo.Stable luciferase expression of ES2H cells was documented across passages.
Cell viability assays
Cells were trypsinized and re-suspended in complete culture medium.Two thousand cells per well were plated in optically clear 96-well black plates(Costar#3904,Corning,NY)and incubated overnight at37°C.Drugs and antibody were diluted in culture medium immediately before use.For individual dru
g experiments,cell viability was assessed after24hour exposure to single agent TRA-8and48hour exposure to carboplatin,and docetaxel individually.For combination treatments,cells were treated with combination of carboplatin and docetaxel for24h prior to adding antibody for an additional24h prior to analysis.Cell viability was assessed via measurement of cellular ATP levels using ATPLite luminescence-based assay(Perkin Elmer,Waltham, MA).All samples were assayed in quadruplicate in three independent experiments and are reported as the mean and standard error.
DR5expression viaflow cytometry
Cells in exponential growth phase were washed twice with Dulbecco's PBS(Ca2+and Mg2+deficient)and harvested with2ml Cellstripper(Mediatech,Manassas,VA)at37°C.Cells were collected by centrifugation at4°C for5min at1000rpm and re-suspended in HBSS at4°C.Cells were incubated with10μg/ml purified TRA-8or an isotype-specific IgG1control antibody for15min at4°C,washed once with HBSS,then incubated with10μg/ml Alexa-647conjugated goat anti-mouse IgG1for15min at4°C.After antibody staining,cells were washed once with HBSS and re-suspended in400μl HBSS for immediate analysis.Samples were analyzed on an LSRII(BD Biosciences,San Jose,CA)and data were analyzed using FlowJo7.5 software(TreeStar Inc.,Ashland,OR).
Western blot analysis of ES2H cell line
Cells in the exponential growth phase were washed with Dulbecco's PBS and harvested with0.25%trypsin at37°C.Cells were plated in sterile six well plates(Corning)at a density of2.0×105 cells per well in3ml medium.Following24hour incubation at37°C and5%CO2,cells were treated with chemotherapy followed24h by TRA-8treatment under the following conditions:1)no treatment;
2)TRA-8alone at125ng/ml and1000ng/ml;3)docetaxel20nM;
4)docetaxel20nM+TRA-8at125ng/ml and1000ng/ml;5)carbo-platin100μM;6)carboplatin100μM+TRA-8125ng/ml and 1000ng/ml;7)docetaxel20nM+carboplatin100μM(combination therapy);and8)combination therapy+TRA-8at125ng/ml and 1000ng/ml.Three hours following addition of TRA-8,cells were harvested using a cell scraper and washed twice with cold PBS. Cells were homogenized on ice in50μl lysis buffer containing10mM Tris–HCl(pH7.6),1%Nonidet P40,1mM sodium orthovanadate,and Protease Inhibitor Cocktail(Roche,Indianapolis,IN).The lysates were then sonicated on ice for10s and centrifuged at14,000rpm for5min at4°C.
Equal amounts of total protein from each lysate were boiled for 5min in SDS-PAGE sample buffer.A total of15μg of protein was loaded and separated on10–15%SDS-PAGE gels,and electrophoretically transferred to PVDF(polyvinylidenefluoride)membranes.The blots were blocked with5%nonfat dry mil
k in TBST buffer(20mM Tris–HCl at pH7.4,500mM NaCl,and0.1%Tween20)and incubated with primary antibodies to caspase-3(Stressgen,Ann Arbor,MI),caspase-8,PARP(BD PharMingen,San Diego,CA),caspase-9,and Bid(Cell Signaling Technology,Beverly,MA)in blocking buffer at4°C overnight.The blots were then washed three times with TBST and probed with HRP (horseradish peroxidase)-conjugated secondary antibodies(Bio-Rad Laboratories,Hercules,CA)for1h at room temperature.After washing three times with TBST,the probed proteins were visualized using the ECL Western blotting detection system(GE Healthcare,Piscataway,NJ) according to the manufacturer's instructions.
In vivo model
Sixty athymic nude mice were injected IP with5×104ES2H cells. Nine days later all mice were imaged using the IVIS100bioluminescence imaging system and luminescence was quantified using the region of interest method in Living Image software(Caliper,Hopkinton,MA).The mice with the highest and lowest tumor burdens were discarded to provide40mice with similar tumor burden which were then sorted into groups of equal mean bioluminescent signal.The groups received the following treatments beginning9days after tumor injection: 1)untreated control;2)TRA-8;3)carboplatin+docetaxel;and 4)carboplatin+docetaxel+TRA-8.Animals receiving TRA-8antibody we
re injected IP with200μg of TRA-8twice weekly.Animals treated with docetaxel+carboplatin were injected IP with5mg/kg and 15mg/kg respectively every three weeks.Animals were treated until death.
Treatment efficacy was assessed by measuring tumor burden prior to therapy,on days23and30.Tumor burden measurements were
194K.S.Bevis et al./Gynecologic Oncology121(2011)193–199
analyzed using ANOVA and the Tukey test.Overall survival of animals was assessed using Kaplan –Meier analysis.Results
DR5expression via flow cytometry
As illustrated in Fig.1,all four ovarian cancer cell lines were DR5positive with a range of expression from strongly positive (A2780and HEYA8)to weakly positive (OVCAR3).ES2H exhibited intermediate DR5expression.
Cytotoxicity of chemotherapy and TRA-8in ovarian cancer cell lines Cell viability assays were completed for docetaxel,carboplatin,and TRA-8in ES2H cells both alone and in combination (Fig.2).
李庆善 标本Speci fically,ES2H cells were noted to be sensitive to treatment with single agent TRA-8with an IC 50of 240.6ng/ml (Fig.2A).ES2H cells were resistant to treatment with both single agent carboplatin and single agent docetaxel with IC 50values of 850μM and N 1000nM
respectively (Figs.2B and C).Combination treatment with docetaxel,carboplatin and TRA-8demonstrated enhanced cytotoxicity over each single agent therapy (Fig.2D).The ES2H cells were selected for use in further experiments because the in vitro data indicated a more robust inhibitory effect with combination therapy.Furthermore,these cells demonstrated both robust expression of luciferase and growth in vivo .Western blot analysis
Western blot analysis of ES2H cell lysates following treatment with chemotherapy and TRA-8was analyzed for caspase activation and PARP cleavage.Treatment with TRA-8,docetaxel,or carboplatin alone resulted in no activation of apoptosis as evidenced by lack of cleavage of caspase-3,no cleavage of PARP or reduction in Bid.When treated with docetaxel+TRA-8,caspases-8,-9,-3and PARP cleavage,as well as a reduction in the pro form of Bid occurred.Caspase-3and PARP cleavage were seen in cells treated with carboplatin +TRA-8(1000ng/ml).Notably,combination therapy with carboplatin,docetaxel,and TRA-8resulted in the highest levels of caspase-3and PARP cleavage in ES2H cells (Fig.3
).
Fig.1.Flow cytometry analysis of DR5cell surface expression in a panel of human ovarian cancer cell lines.Ovarian cancer cells were harvested using Cellstripper and stained with 10μg/ml TRA-8mAb for 15min at 4°C followed by Alexa-647-conjugated goat antimouse IgG1,then analyzed using LSRII and FlowJo 7.5software.Gray-filled histograms ,TRA-8staining;Black histograms ,incubation with mouse IgG1isotype control
antibody.
1
101001000% c o n t r o l (c o u n t s /s )
% c o n t r o l (c o u n t s /s )
% c o n t r o l (c o u n t s /s )
% c o n t r o l (c o u n t s /s )
TRA-8 dos e (ng/ml)TRA-8
TRA-8
Docetaxel dose (nM)
Docetaxel
Carboplatin dos e (µM)Carboplatin
TRA-8 dos e (ng/ml)
ES2-H
A
B
C
D
Fig.2.ES2H cell viability after treatment with TRA-8(A),carboplatin (B),docetaxel (C),and TRA-8in combination with docetaxel and carboplatin (D).Cells were trypsinized and plated in 96-well plates at a density of 2000cells/well and incubated overnight.Cells were then treated for 24h with docetaxel and carboplatin and TRA-8was added 24h later.Cell viability was measured 24h following the addition of TRA-8and thus 48h following the addition of combination chemotherapy using ATPLite assay.ATP levels are reported relative to untreated control cells as the mean and standard error from quadruplicate determinations in three independent experiments.
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In vivo ef ficacy of TRA-8and chemotherapy in ovarian cancer xenografts The mouse with the median bioluminescence signal from each treatment group illustrates the effect of each therapy on tu
mor growth in the ES2H human ovarian cancer xenograft model (Fig.4).Therapy with TRA-8alone produced limited tumor growth inhibition compared to the untreated group as measured on day 23.Both groups demonstrated an overall 100-fold increase in tumor burden compared to baseline values.However,when comparing the TRA-8group and the untreated control group at day 23,the TRA-8mice demonstrated 20.4%less tumor burden relative to controls (p N 0.05)(Fig.5).Comparison at day 30was not performed due to lack of surviving animals in the control group.As anticipated based on tumor burden measurements,median survival was similar between the TRA-8(30days)and untreated groups (27days;p N 0.05)(Fig.6).
The chemotherapy treatment group showed an 82.1%reduction in tumor burden relative to untreated controls (p b 0.001),and a 77.8%reduction compared to the TRA-8group (p b 0.001)as measured on day 23.At day 30,tumor burden in the chemotherapy group was 14.8%less than the TRA-8group (p=0.14)and no untreated animals remained for comparison (Fig.5).As shown in Fig.6,median survival was signi ficantly increased in the chemotherapy treatment group compared to the untreated controls (34vs.27days;p b 0.001)and to the TRA-8treated group (34vs.30days;p=0.012).
Animals treated with combination of carboplatin,docetaxel,and TRA-8demonstrated better outcomes than all other treatment groups (Fig.4).When compared to untreated controls,the combination therap
y group demonstrated 96.0%less tumor burden (p b 0.001)at day 23(Fig.5)and exhibited signi ficantly prolonged survival (41vs.27days;p b 0.001)as shown in Fig.6.When compared to the TRA-8group,the combination treatment group showed 95.0%less tumor burden at day 23(p b 0.001)and 49.6%less tumor burden at day 30(p=0.04).Additionally,mice treated with combination therapy lived longer than the TRA-8treated mice (41vs.30days;
p b 0.001).Finally,compared to the chemotherapy group,animals treated with combination therapy showed 77.7%less tumor burden (p b 0.001)at day 23and 40.9%less at day 30(p=0.04)(Fig.5).Overall survival was also longer in the combination group when compared directly to the chemotherapy group (41vs.34days;p b 0.001).Median survival was greatest in the combination group (41days)compared to the chemotherapy group (34days),TRA-8group (30days)and control group (27days)as determined by Kaplan –Meier analysis (p b 0.001)(Fig.6).Discussion
Despite success with surgical cytoreduction and adjuvant taxane/platinum-based chemotherapy,prognosis for patients with advanced stage epithelial ovarian cancer remains poor due to high recurrence rates.Multiple clinical trials have investigated the addition of other cytotoxic chemotherapeutic agents to the standard regimen with results indicating limited ef ficacy and increased toxicity [19].More recently,there was a resurgence of interest in intraperitoneally administe
red chemotherapy as a means of improving patient out-comes.Armstrong ported a 16month survival advantage in patients with stage III,optimally cytoreduced ovarian carcinoma receiving intraperitoneal chemotherapy compared to the standard intravenous regimen [20].However,only 40%of patients in the intraperitoneal arm were able to complete the prescribed treatment due to the marked toxicity of the regimen.Despite the promise of this treatment strategy,the shortcomings of cytotoxic chemotherapeutic agents remain.Recent data from a large phase III trial evaluating the ef ficacy of bevacizumab in combination with taxane-platinum based chemotherapy in advanced ovarian cancer demonstrated an improve-ment in progression free survival [21].It is for these reasons that targeted agents including VEGF inhibitors and TRAIL analogues are being further evaluated in combination with taxane/platinum-based therapies in patients with advanced ovarian cancer.TRAIL and
its
Fig.3.Caspase-8,-9,-3and PARP cleavage and decreased Bid levels were induced by combination tr
彩虹原则
eatment in ES2H ovarian cancer cells.Cells were pretreated for 24h with docetaxel or carboplatin before the addition of TRA-8(ng/ml)for 3h.Whole cell lysates were analyzed by Western blotting.β-Actin was used as a loading control.
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agonistic anti-death receptor antibodies provide an attractive molec-ular approach to cancer therapy due to induction of apoptosis in malignant cells combined with an overall lack of toxicity in normal cells.Recombinant forms of TNF-αand soluble TRAIL developed in earlier studies produced signi ficant cytotoxicity against cancer cells but also exhibited substantial hepatotoxicity and had short plasma half-lives [22,23].As a result,monoclonal antibodies targeting either DR4or DR5are being developed as alternative agents with higher speci ficity,no hepatotoxicity,and longer serum half-lives [24–28].This would allow for greater ef ficacy through avoidance of decoy
Day
C o u n t s /s
Fig.5.Tumor burden measurements of ES2H human ovarian cancer xenografts.Based on imaging data obtained 9days following injection,mice were sorted into groups to achieve equal mean tumor burden between groups.Twenty-three days
following injection,tumor burden in the untreated controls,TRA-8,carboplatin and docetaxel,and combination groups were 5.2×107,4.1×107,9.3×106,and 2.1×106photons/s,respectively.By day 30,all untreated control animals were dead and mean tumor burden in the TRA-8,chemotherapy,and combination treatment groups were 4.4×107,3.77×107,and 2.2×107,respectively.
Fig.4.Representative tumor burden of ES2H intraperitoneal xenografts.Tumor burden was measured by bioluminescence imaging in mice 9days post implant,on day 23,and day 30.Using Living Image software,images were equalized for time and binning to allow direct comparison between groups and time points.The mouse with the median bioluminescence signal is shown at each time point.There were no surviving mice in the control group at day 30.
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