The Prostate 70:1359 ^1370 (2010)

TheDisintegrinContortrostatin inCombinationWithDocetaxel Is a Potent Inhibitorof ProstateCancer

InVitro and InVivo

Edwin Lin,1 Qingcai Wang,1 Stephen Swenson,2 Hossein Jadvar,3 SusanGroshen,4 Wei Ye,4 Francis S. Markland,2 and Jacek Pinski1*

1Departmentsof InternalMedicine,Norris Cancer Center,Universityof SouthernCaliforniaKeck SchoolofMedicine,Los Angeles,California

2Departmentof BiochemistryandMolecular Biology,Norris Cancer Center,Universityof SouthernCaliforniaKeck SchoolofMedicine, Los Angeles,California

3Departmentof Radiology,Norris CancerCenter,Universityof SouthernCaliforniaKeck SchoolofMedicine,Los Angeles,California

4Departmentof Biostatistics Core Resource,Norris Cancer Center,Universityof SouthernCaliforniaKeck SchoolofMedicine, Los Angeles,California

BACKGROUND. There are few available treatments for hormone refractory prostate cancer.Through the inhibition of integrins, contortrostatin (CN) effects tumor cell growth directly aswell as through the inhibition of angiogenesis. The effect of CN in combination with docetaxelon prostate cancer cell lines in vitro and in vivo is evaluated in the present study.METHODS. FACS analysis of integrin expression, assessment of CN and docetaxel exposureon viability of plated cancer cells, and scratch test migration analysis were performed on PC-3prostate cancer cells. CN and docetaxel inhibition of both PC-3 and CWR-22 prostate cancer celllines were evaluated in a mouse xenograft bone model. Angiogenic activity in tumors wereassessed using IHC with antibodies to CD31.RESULTS. Cell culture experiments indicate that the combination of docetaxel and CN inhibitsgrowth in an additive fashion. FACS analysis of PC-3 cells shows expression of a5b1 and avb5integrins, but little expression of the avb3. CN showed complete inhibition of PC-3 migration incultures grown on matrigel plates. In mice xenograft bone models, CN with docetaxel showedincreased inhibition of both PC-3 and CWR-22 derived tumors. Analysis of treated xenografttumors showed significantly decreased expression of CD31 indicating suppression of angio-genesis. Prostate � 2010 Wiley-Liss, Inc. Prostate 70: 1359–1370, 2010. # 2010 Wiley-Liss, Inc.

KEY WORDS: prostate cancer; contortrostatin; integrin; docetaxel; angiogenesis; bonemetastasis

INTRODUCTION

Prostate cancer is currently the most common cancerin males in the US with more than 220,000 new casesand approximately 27,000 deaths in 2007 [1]. Althoughlocal therapy and hormone ablation offer an initialdurable response in early stages, development ofhormone refractory prostate cancer (HRPC) andresistance to chemotherapy remains a significantchallenge in advanced disease. Currently, HRPCresponse to cytotoxic therapies is short-lived with amedian survival for patients with metastatic prostate

There is no conflict of interest with any of the material presented inthis manuscript for any of the authors.

Grant sponsor: NIH; Grant numbers: 1R41 CA126001, R01-CA111613; Grant sponsor: US Army Prostate Cancer Program;Grant number: PC030704; Grant sponsor: Norris Cancer Center;Grant number: P30-CA14089.

*Correspondence to: Jacek Pinski, MD, PhD, Division of MedicalOncology, Norris Comprehensive Cancer Center, 1441 Eastlake Ave,Suite 3449, Los Angeles, CA 90089.E-mail: pinski_j@ccnt.hsc.usc.eduReceived 16 October 2009; Accepted 3 March 2010DOI 10.1002/pros.21173Published online 26 May 2010 in Wiley InterScience(www.interscience.wiley.com).

) 2010 Wiley-Liss, Inc.

cancer requiring chemotherapy of approximately18 months. Therefore, novel approaches to treatmentof prostate cancer are needed.

With recent clinical success of therapy targetingangiogenesis in the treatment of colorectal and lungcancer, significant efforts have been placed on furtheradvancing this approach to treatment of other cancers[2]. The tumor vasculature provides an attractive targetfor cancer therapy because of the reliance of most tumorcells on an intact vascular supply for their growth andsurvival [3,4]. Angiogenesis has been shown to be notonly a requirement for primary tumor expansion, butalso a critical component of tumor metastasis [5].Antivascular agents diminish the effective bloodsupply to the tumor, killing malignant cells bydepriving them of oxygen and nutrients. A potentialadvantage of this approach is that by targeting normalendothelium as opposed to the malignant cells them-selves, there may be a decreased likelihood for thedevelopment of drug resistance [6].

Integrins have been shown to be vital to not onlyangiogenesis, but other aspects of tumor growth andmetastasis [7–9]. Cell migration, invasion, matrixdegradation, proliferation, and angiogenesis are allmediated by integrins and integrin signaling [10].Several studies have indicated that different membersof the integrin family are expressed in various prostatecancer lines, as well as human cancer and benignprostatic tissue [11,12]. Similar to normal cells, theseintegrins can be presumed to be of importance in themotility of cancer cells as well as the interaction of thesecells with the local milieu. Of interest is the finding thatdifferent prostate cancer cell lines express differentprofiles of integrins, correlating with the ability ofthese prostate cancer cells to progress and metastasize[12–15].

Contortrostatin (CN), a disintegrin isolated from thevenom of the southern copperhead snake, is a novelagent that has shown antitumor activity in a variety oftumors including melanoma and breast cancer [16–18].CN is a non-toxic component of the venom thatfunctions as a platelet-integrin antagonist, inhibitingaggregation, allowing for the efficient spread of theother toxic venom components. Previous studies haveshown that CN exposure does not display a toxic effecton cells in vitro nor animals treated with CN. Addi-tionally, through antagonism of integrins on a numberof different cell types, CN blocks adhesion, migrationand invasion of many forms of cancer and vascularendothelial cells, thereby exerting antimetastatic andantiangiogenic activities. Previous studies from theMarkland laboratory have shown that CN binds to andaffects the function of several integrins expressed oncancer, platelet, and endothelial cell surfaces, includingavb3, a5b1, avb5, and aIIbb3 [16,18–22]. By targeting

various integrins expressed on both cancer cells andnormal endothelial cells, CN is unique in havingthe potential to have a significant effect directly on thetumor cells themselves as well as on angiogenicvascular endothelial cells. Additionally, due to itsunique structure in comparison to other disintegrins,contortrostatin has shown a unique ability to effect abroad array of integrins as opposed to specificsubtypes.

In view of prior studies establishing antitumoractivity of CN and the potential benefits of using adrug with a known mechanism of action, we examinedthe effect of CN on the PC-3 prostate cancer cell linein vitro and both PC-3 and CWR-22 prostate cancer celllines in vivo. In this publication, we report that CNshows an additive inhibitory effect with docetaxel onthe growth of both the PC-3 and CWR-22 cancer linecells.

MATERIALSANDMETHODS

PC-3 and CWR-22 cells were obtained from theAmerican Type Culture Collection (ATCC, Manassas,VA). CN was purified from crude lyophilized venomand purity confirmed as previously described [18].Venom was obtained from Miami SerpentariumLaboratories (Punta Gorda, FL). 7E3 is a generous giftfrom Dr Mohit Trikha, Centocor, Inc. (Malvern, PA).Primary anti-integrin and secondary detection anti-bodies were obtained from Santa Cruz Biotechnology(Santa Cruz, CA). Docetaxel was obtained from LCLaboratories (Woburn, MA), prepared in ethanol, anddiluted prior to use in PBS.

InVitro Experiments

Effect of CN and docetaxel on cell survival. Twelve-well plates were coated with 1 ml of matrigel. Each wellwas seeded with approximately 2.5� 105 prostatecancer cells and cells were allowed to adhere overnight.Wells were washed to remove non-adherent cells andthen treatment was initiated as follows: (i) Untreated(negative control); (ii) 20 mg/ml actinomycin-D (pos-itive control); (iii) 100 nM CN; (iv) 50 nM docetaxel;(v) 100 nM CNþ 50 nM docetaxel. The decision toutilize 50 nM of docetaxel was made following multipletrials using different concentrations showing 50 nM ofdocetaxel yielded an optimal percentage of cell death asa single agent. After 48 hr of treatment, the cells wereharvested, resuspended and adjusted to 300,000 cells/ml. Cells were then stained with trypan blue andobserved under a light microscope. The number of non-viable, blue staining cells were counted and thepercentage of cell viability determined through com-parison to a UV exposed control (in which all cells are

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non-viable following exposure). UV exposure wascarried out through placement of the flask of cells ona UV Trans-Illuminator for 15 min (ChromaVue T-40,UVP San Gabriel, CA). Analysis of variance (ANOVA)was used to test the effects of various treatments on celldeath of PC-3 cells. Prior to analysis, the squared roottransformation was performed for the percent celldeaths in order to render the assumption of normaldistribution and homoscadesticity. Pair-wise compar-isons between the treatments were performed using theleast significant difference procedure once the overallP-value was significant. The means of their 95% CIswere calculated and the data was transformed back tothe original scale.

Evaluation of integrin display. The integrins dis-played on the surface of PC-3 prostate cancer cells wasdetermined by flow cytometric (FACS) analysis. PC-3cells were harvested, washed with 1% bovine serumalbumin (BSA) in phosphate-buffered saline (PBS), andincubated with monoclonal antibody 7E3 (anti-avb3,2.5 mg/ml), P1F6 (anti-avb5, 1 mg/ml), or JBS5 (anti-a5b1, 1:2,000) for 30 min on ice. After washing of thecells with 1% BSA in PBS, FITC-conjugated secondaryantibody was added to the cells and incubated foranother 30 min. The fluorescent intensity of the washedcells was analyzed with a FACScan flow cytometer inthe USC/Norris core laboratory.

Effect of CN on PC-3 cell adhesion to various ECMproteins and migration of PC-3 cells on matrigel. Theadhesion assay using immobilized ECM proteinswas performed as described previously [23]. In brief,individual wells were coated with vitronectin (0.5 mg/well), fibronectin (0.5 mg/well), type IV collagen(2.5 mg/well) or laminin (2 mg/well) overnight. Prostatecancer cells, starved in serum-free medium for 2 hr,were briefly detached by trypsin and resuspended inserum-free medium containing soybean trypsin inhib-itor. The cells were then held in solution for >2 hr priorto the exposure to CN and adherence to the differentECM proteins. To carry this out, the cells (5� 105 cells/ml) were exposed to CN (100 nM) and then allowed toadhere to immobilized ECM proteins for 30 min at378C. The number of adherent cells �CN was obtainedby use of Cell Titer 96 Aqueous Proliferation AssayKit and reading the absorbance at 490 nm on a platereader.

The effect of CN on migration on matrigel wasmeasured in the in vitro wound closure assay aspreviously described [24]. Briefly, cells are seeded on asix-well matrigel (125 mg/ml) coated plastic plate andallowed to grow to confluency. A scratch with a pipettip is made in the cell monolayer followed by extensivewashing with serum-free medium to remove cell

debris. The cells were then incubated in serum-freemedium containing mitomycin C (10 mg/ml). Mitomy-cin C was used in this assay to assess the relativecontribution of cell migration to in vitro wound closurein the absence of cell proliferation. After 2 hr incubationat 378C, the medium was removed and fresh mediumcontaining 10% FBS and CN at 100 nM was added to thecells. After incubation for 12 hr at 378C, microscopicfields were photographed under phase contrast.

InVivo Experiments

Inhibition of tumor growth and progression inxenograft models of human prostate cancer. MaleNu/Nu mice, approximately 5 weeks old, wereobtained from Charles River Laboratories, Inc. andhoused in a laminar airflow cabin under pathogen-freeconditions with a 12-hr light/12-hr dark schedule andfed autoclaved standard chow and water ad libitumthroughout the experiments. The care of all animalswas in accord with institutional guidelines.

A mixture of 100 ml of PC-3 cell suspension (1.0� 106

cells) and 100 ml Matrigel basement membrane matrix(BD Biosciences, Bedford, MA) were injected subcuta-neously into the flank region of athymic male nudemice. After 4 weeks, 40 mice were randomized into fourgroups, which received the following treatments: (i)Control group—These mice received PBS intraperito-neally; (ii) Docetaxel only group—These mice weretreated with docetaxel injected intraperitoneally, 6 mg/kg, twice a week for 2 weeks, then 4 mg/kg for 2 weeks(for a total of 4 weeks of treatment); (iii) CN onlygroup—These mice had Alzet mini-osmotic pumpssubcutaneously placed near the growing tumor. Thesepumps released 60 mg of CN per day for 28 days; (iv) CNplus docetaxel group—These mice had the combina-tion treatment of CN infused with subcutaneouslyplaced osmotic pumps and docetaxel injection asdescribed above for the second and third groups.Dosing of contortrostatin and docetaxel was based onprior experiments in mice models. Tumor volumeswere measured once a week for 4 weeks and thevolumes were calculated according to the formula:volume¼ length�width2� 0.52. Statistical analysis ofthe tumor growth data was performed via an unpairedt-test, and P values <0.05 were deemed to besignificant. Statistical significance between the controland the treatment groups was evaluated as well asbetween the individual treatment groups.

For the androgen dependent CWR-22 prostatecancer cell line, each of 40 nude mice was implantedwith one testosterone pellet (12.5 mg) for 3 days. Amixture of 100 ml of CWR-22 cell suspension (1.0� 106

cells) and 100 ml Matrigel basement membrane matrix(BD Biosciences) were injected subcutaneously into the

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Contortrostatin Inhibits Prostate Cancer 1361

flank region of athymic male nude mice. After 4 weeks,mice were randomized into four groups, whichreceived the following treatment: (i) Control group—These mice received PBS intraperitoneally; (ii) Doce-taxel only group—These mice were treated withdocetaxel injected intraperitoneally, 4 mg/kg twiceper week for 4 weeks; (iii) CN only group—These micehad Alzet mini-osmotic pumps subcutaneously placednear the growing tumor. These pumps released 60 mg ofcontortrostatin per day for 28 days; (iv) CN plusdocetaxel group—These mice had the combinationtreatment of contortrostatin infused with subcutane-ously placed osmotic pumps and docetaxel injection asdescribed above for the second and third groups.Tumor volumes were determined as described pre-viously and statistical significance was determined asdescribed previously.

Evaluation of the effect of CN on tumor inducedangiogenesis. PC-3 tumors from treated anduntreated mice in the therapeutic efficacy studies werefixed in 4% normal buffered formalin and embedded inparaffin blocks as described previously [17,25]. Theparaffin blocks were cut into 5 mm sections and placedon glass slides. Tissue sections underwent deparaffini-zation, rehydration, and antigen retrieval following anestablished protocol. Endogenous peroxidase activitywas blocked by exposure of the sections to 3% H2O2.Specimens were blocked with normal goat serum (1:20)for 30 min, followed by incubation with the primaryantibody for 1 hr. Rabbit monoclonal antibody toCD31 (Sigma) was used to detect small vessels.The secondary (detection) goat anti-rabbit antibodyconjugated with peroxidase (Zymed) was then appliedto the samples and incubated for 10 min at roomtemperature followed by removal of unbound anti-body by multiple washes with PBS. Detection ofthe secondary antibody using 3,30-diaminobenzidine(DAB) as the chromogen was performed following themanufacturers instructions (Zymed HistoMouse Max).Slides were counterstained with hematoxylin. Quanti-tation of the stained vessels was performed using ‘‘hotspot’’ analysis [26], with ‘‘hot spots’’ being defined asareas of high vessel density. With 100-fold magnifica-tion, areas showing positive staining were quantitatedin terms of pixels within a given hot spot usingSimplePCI advanced imaging software (C-ImagingSystems, Cranberry Township, PA). This programallows the quantification of a specific color even in thepresence of multiple other colors. The same numberof sections and visual fields from each of the control andtreatment groups were evaluated to eliminate quanti-tation bias based on tumor size. Anti-CD31 (PECAM-1)monoclonal antibody was used for immunohistochem-istry because it has been reported to be highly

expressed on angiogenic vasculature (�1 millioncopies of PECAM-1 reported on the surface of anendothelial cell [27]. Analysis of variance (ANOVA)was used to test the effects of various treatments andpixels of microvessel CD31 staining. Prior to analysis,the squared root transformation was performed for thepixels of microvessel CD31 staining in order to renderthe assumption of normal distribution and homosca-desticity. Pair-wise comparisons between the treat-ments were performed using the least significantdifference procedure once the overall P-value wassignificant. The means of their 95% CIs were calculatedand the data was transformed back to the original scale.

Evaluation of the effect of CN on bone tumorsites. Nude mice were injected with 50,000luciferase transfected PC-3 cells (PC-3-Luc) withMatrigel into the tibia. The mice were randomizedinto the following groups (eight animals per group):(i) Control group; (ii) CN group—Each mousewas implanted under the skin with an Alzet osmoticpump releasing about 43 mg of CN daily directedtoward the tumor site; (iii) Docetaxel group—Docetaxelwas injected into mice via tail vein at dose of4 mg/kg weekly; (iv) Combination group of CN anddocetaxel. Tumor growth in the bone was monitoredwith Xenogeny (Xenogen, IVIS200, Caliper, San Diego,CA) at the USC Molecular Imaging Center at the timepoint 0 and after 7 and 14 days post PC-3-Luc injection.

For the Xenogen measurements of the prostatictumors, mice were given a single intraperitonealinjection of ketamine (50 mg/kg) and xylazine(10 mg/kg) followed by intravenous injection of luci-ferin (100 mg/kg). After waiting for 10 min to allowproper distribution of luciferin, the mice were placed inthe chamber of an IVIS 200 optical imaging system.Photons were collected for a period of 3 min. Signalintensity was quantified for defined regions of interestby the detector (units of photons/sec/cm2/steradian).Images simulating the three-dimensional location ofbioluminescent sources within tibia were generatedusing the single-view diffuse tomography capability ofthe IVIS 200 and LIVING IMAGE 3D v. 2.50. Imageswere analyzed using LIVING IMAGE software v. 2.50(Xenogen).

RESULTS

Contortrostatin InhibitsthePC-3CellLineViabilityinan Additive FashionWith Docetaxel InVitro

We examined the effect of treatment by contortros-tatin alone and in combination with docetaxel on PC-3cell proliferation and viability. After 48 hr of treatment,we observed through the use of trypan blue stainingthat in comparison to the untreated control group, both

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the CN treated group and the CNþdocetaxel treatedgroup showed decreased viability (Fig. 1). The slightlimitation in viability with the CN treatment can beattributed to inhibition of cell adhesion to the tissueculture plastic. As expected, untreated samples dis-

played very little staining (4� 2% of the cells staining)indicating a high level of viability. The actinomycin-Dpositive control indicated 87� 5% cell death. Treat-ment of the cells with CN alone resulted in 10� 1% celldeath while the docetaxel and CNþdocetaxel treat-ment groups showed a 48� 3% and 66� 6% cell deathrespectively. Overall, the effects of the treatments oncell deaths were significantly different from oneanother (P-value< 0.001). All pair-wise comparisonsamong the treatments were also significant. However,there was no significant synergistic effect between CNand docetaxel on cell deaths (P-value¼ 0.42). Theseresults indicate that the combination of docetaxel andCN appears to work in an additive fashion.

FACSAnalysis of Integrin Expression in thePC-3 Cell Line

PC-3 is a human prostate cancer cell line derivedfrom bone metastases and is hormone independent.FACS analysis of PC-3 cells displays significant shifts inthe fluorescent signal when probed with antibodiesagainst a5b1 (FN receptor) and avb5 (VN receptor)integrins. There is also expression of the b1 subunit.There appears to be little expression of the avb3 (VNreceptor) integrin. These integrin evaluations wererepeated at three intervals using cells passagedindependently (Fig. 2).

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Fig. 1. Contortrostatinappliedincombinationwithdocetaxeldis-plays an enhanced cytotoxic effect on PC-3 cells. PC-3 cells platedandgrownin thepresence ofActinomycin-D (positive control),UVlight (positive control),CN (100nm), docetaxel (Doc, 50nm) and acombination of CNanddocetaxel.While CNalone displays limitedtoxicity itenhances the cytotoxic activityof docetaxel.Overall, theeffectof thetreatmentsoncelldeathwas significantlydifferentfromoneanother (P-value<0.001).

Fig. 2. IntegrinstatusofPC-3cellsevaluatedbyFACSanalysis.TheandrogenindependentPC-3cellswereevaluatedforintegrindisplay(avb3,a5b1, andavb5)byFACSanalysis.Cellswereincubatedwith theantibodies7E3 (antiavb3),P1F6 (antiavb5), and JBS5 (antia5)andsubjected toFACS.As seenfromthegraphsof theFACSanalysis,positive shiftsareobservedfora5b1andavb5whileno shiftis observedforavb3.

Contortrostatin Inhibits Prostate Cancer 1363

CNInhibits Adhesion and Migration ofPC-3 Prostate Cancer Cells

Following determination of the integrin displayprofile of the PC-3 cells, we determined the effect oncellular adhesion to various extracellular matricesfollowing treatment of this cell line with CN. Thesestudies showed that there was extensive inhibition ofPC-3 cell adhesion to fibronectin but not as extensiveinhibition to vitronectin (data not shown). Theseobservations support the integrin profile data. For themigration assay, PC-3 cells were grown in a 12-well cellculture plate until confluent. A scratch of equal widthwas made in the middle of each well. After washing, thecells were incubated with or without CN (Fig. 3). After12 hr, microscopic examination demonstrated that thescratch in the control well was filled in by migratingcells, whereas the scratch remains the original widthwith clear-cut edges in 100 nM CN-treated cells. Thus,

migration of PC-3 cells on Matrigel was completelyinhibited by CN.

Contortrostatin Inhibits the Growthof AndrogenIndependent PC-3 Tumors Implanted in Mice

Having shown in vitro the effectiveness of thecombination of docetaxelþCN, we evaluated the invivo efficacy of this combination using the PC-3 cell linein a xenograft nude mouse model. We used a subcuta-neous tumor model to evaluate the efficacy of CN ontumor growth and angiogenesis. A PC-3 cell suspen-sion (1.0� 106 cells, 100 ml) and 100 ml Matrigel base-ment membrane matrix were injected subcutaneouslyinto the flank region of athymic male nude mice.Following tumor cell implantation, mice were treatedfor 4 weeks as described in the Materials and MethodsSection. Treatment results are shown in Figure 4.Tumor volumes were measured once a week andcalculated via the formula: v¼ length�width2� 0.52(where v is tumor volume). During this experiment,there were no significant differences in body weightsbetween groups. Both docetaxel and CN significantlysuppressed growth of PC-3 tumors. After 4 weeks,the mean tumor volume was significantly reduced ingroups receiving CN to 195� 40 mm3 (reduction of

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Fig. 3. CN inhibits migration of PC-3 tells: Scratches of equalwidthsweremade in themiddle of wells containing confluent PC-3cells. The cells were incubated with or without CN. After 12hr,microscopicexaminationdemonstrated that the scratchin thecon-trol well (upper panel) was almost covered by migrating cells,whereas the scratchremains theoriginalwidthwithclear-cutedgesinCN-treatedcells (lowerpanel).Theexperimentwasrepeatedintriplicatewith theabove figurerepresentativeofresults.

Fig. 4. PC-3xenografts treatedwithCN.Tumorvolumesinathy-micnudemicebearingsubcutaneousxenograftsof theprostatecan-cer cell line PC-3 during treatment with Contortrostatin (CN),administered either by itself or together with docetaxel (DT).CNwas administered by continuous release from Alzet mini-osmoticpumps at a dose of 60mg/day.Docetaxel was injected intraperito-neally at a dose of 6mg/kg twice a week for 2 weeks and thenswitched to 4mg/kg twice a week. Control animals were infusedwith PBS. Treatment commenced 4 weeks after implantation oftumorcells andlasted for4weeks.Verticalbars indicateSEM.Thereis a significant improvement in outcome when the combination ofDTþCNiscomparedto eitherCNorDTalone.

1364 Lin et al.

48%), and in the group receiving docetaxel to259� 38 mm3 (reduction of 31%), as compared withthat of the control group (378� 49 mm3). Therapywith the combination of CN plus docetaxel was themost effective and resulted in a tumor volume of95� 38 mm3 (a reduction of 75% as compared tocontrol, Fig. 4). As indicated in the figure legend,there is statistical significance between all treatmentgroups and the control (P< 0.05) in all cases. The resultof the combination treatment versus either CN orDoc alone also shows that the combination treatmentis statistically significant in the limitation of tumorgrowth.

Contortrostatin Inhibits the Growthof AndrogenDependent CWR-22 Tumors Implanted in Mice

In order to demonstrate the in vivo effect ofcontortrostatin in other prostate cancer cell lines, weevaluated the androgen dependent prostate cancer cellline CWR-22 in a similar fashion as performed with thePC-3 cancer cell line. Subcutaneous tumors weredeveloped in mice pretreated with testosterone byinjecting a suspension of CWR-22 cells. After allowingthese tumors to grow for 4 weeks, each group of micewas treated as described above. Treatment results areshown in Figure 5. Tumor volumes were measuredonce a week and calculated via the formula:v¼ length�width2� 0.52 (where v is tumor volume).Both docetaxel and CN significantly suppressedgrowth of CWR-22 tumors. After 4 weeks, the meantumor volume was significantly reduced in groupsreceiving CN to 4,362� 1,029 mm3 (reduction of 39%),and in the group receiving docetaxel to 4,053�595 mm3 (reduction of 44%), as compared with that ofthe control group (7,244� 1,896 mm3). Similar to thePC-3 cancer cell line results, therapy with the combi-nation of CN plus docetaxel was the most effectiveand resulted in a tumor volume of 2,946� 479 mm3

(a reduction of 59% as compared to control) (Fig. 5).In these studies the combination (CNþDoc) treatmentresults show a statistically significant advantage(P< 0.02) as compared to the control. Furthermore,the results show significance when evaluating thecombination arm versus Doc alone (P< 0.04) andapproaches significance when compared to CN alone(P< .07).

CNDemonstrates the Ability to InhibitAngiogenesisin PC-3 Xenographic MiceTumor Models

In order to evaluate the antiangiogenic propertiesof CN, we utilized immunostaining of the moleculePECAM-1 to show the downregulation of newlyformed blood vessels. Anti-CD31 (PECAM-1) mono-

clonal antibody was used for immunohistochemistrybecause it has been reported to be highly expressed onangiogenic vasculature (�1 million copies of PECAM-1reported on the surface of an endothelial cell). CD31 hasbeen reported to be involved in the initial formation ofcell–cell contacts at junctions of endothelial cells, themaintenance of the vascular permeability barrier, theregulation of cell migration, and the formation of newblood vessels during angiogenesis [28–31]. Theseproperties of CD31 make it an optimal reportermolecule for determination of angiogenic growth.

In comparison to controls, treatment with either CNor docetaxel resulted in decreased immunohistochem-ical staining of CD31. This effect was further enhancedwhen CN and docetaxel were used in combination(Fig. 6a). When examined quantitatively using C-ImageSimple PCI software. Docetaxel treated PC-3 tumorsshowed �78% decrease, CN showed �82% decrease,while a combination therapy (CNþdocetaxel) showeda �85% decrease in CD31 staining as compared to PBStreated tumors (Fig. 6b). In comparison to controls,either CN or docetaxel alone resulted in significantly

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Fig. 5. CWR22 tumors xenografts treated with CN. Tumorvolumes in athymic nudemice bearing subcutaneous xenografts ofthe prostate cancer cell line CWR-22 during treatmentwith Con-tortrostatin (CN), administered either by itself or together withdocetaxel.CNwas administered by continuous release fromAlzetmini-osmotic pumps at a dose of 60mg/day.Docetaxelwas injectedintraperitoneallyatadoseof4mg/kg twiceaweek.Controlanimalswere infusedwith PBS.Treatmentlasted for 4weeks.The combina-tion (CNþDoc) treatment results show a statistically significantadvantage (P< 0.02) as compared to the control. In addition, theresults show significance when evaluating the combination versusDoc alone (P< 0.04) and approach significancewhen compared toCNalone (P< 0.07).

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decreased staining of CD31, both with a P-value<0.001. Interestingly, the synergistic effect of CN anddocetaxel on microvessel CD31 staining was significant(P-value <0.001 for the interaction between CN anddocetaxel), indicating that the combination of CN anddocetaxel enhanced the effect of each individual agent.This is in contrast to the in vitro and other in vivostudies showing only an additive effect.

Contortrostatin Inhibits the Growthof PC-3 TumorsImplanted Directly Into the Bones of Mice

After 2 weeks of therapy there was no significantdifference in body weights between groups. Also,treatment with CN or the combination of CN withdocetaxel was not associated with other signs oftoxicity, such as animal death, weakness or changesin animal behavior. Figure 7 shows typical xenogenpictures of orthotopic prostate cancer bone lesions in acontrol animal and a mouse treated for 14 days withCN.

After 7 days of treatment, the xenogen signal wasreduced in groups receiving docetaxel by weeklyintravenous injections or CN released by continuousinfusion from Alzet mini-osmotic pumps by 27% and43%, respectively, compared to the control group(Fig. 8). The combination group of CN and docetaxelshowed the most marked decrease in signal ascompared to control animals (65%). After 2 weeks oftreatment, the signal in the docetaxel and CN groupswas decreased by 60% and 62%, respectively, com-pared to control. At 14 days, the combination groupdemonstrated a statistically significant (P< 0.05) 87%decrease in signal compared to controls (Fig. 8).

DISCUSSION

Tumor growth and metastasis are complex proc-esses that require myriad interactions with environ-mental proteins, cytokines, and other cells. Metastases,in particular, requires several distinct actions includingthe following: (1) migration of the tumor cells through

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Fig. 6. a: Immunohistochemical staining of microvessels withCD31.PC-3tumorsgrowninmiceandtreatedwithdocetaxel�CNwereassessedforangiogenesiswithCD31mAbusingDABforvisual-ization.CN (B), docetaxel (C), and combination therapy of CNþdocetaxel (D) showeddecreasedangiogenesis, as compared toPBStreated tumors (A). In panel A there appears to be excess diffusestaining which is an artifact of the chromagen used in staining.Forquantitation of staining, the color evaluatedwas thebrown as seeninpanels B^D. At least15 areas of each tumorwere evaluatedwiththe above results representative. b: Quantitation of MicrovesselCD31 staining.CD31 stained slides were visualized at 40� with anOlympusVanox-SandcapturedwithanOlympusC-5050Zoomdig-ital camera.The digital imageswere thenquantitated forCD31pos-itive stainingwithC-ImageSimplePCI software.Docetaxel treatedPC3 tumors showed�78% decrease,CN showed�82% decrease,while a combination therapy (CNþDocetaxel) showed a �85%decrease in CD31 staining as compared to PBS treated tumors(P-value for each treatment as compared to control is <0.001). Toeliminate bias from the varying tumor sizes between the controlandtreatmentgroups,thesamenumberofsectionsandopticalfieldswerequantitated foreach tumor sample.

Fig. 7. Xenogen pictures of xenograft bone tumors. Nude micewere injected with PC-3-Luc cells directly into the tibia. Figureshowsxenogenpicturesoforthotopicprostate cancerbonelesionsin a control animal (left) and a mouse treated for14 days with CN(right).Pictures arerepresentativeofresults.

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the extracellular matrix surrounding the tumor; (2)invasion of tumor cells into angiogenic blood vesselsgrowing into the tumor; (3) adhesion of the metastaticcell at a distant site where the microenvironment isreceptive to tumor growth; and (4) newly attached cellsmust proliferate and induce angiogenesis at themetastatic site. Due to the complex multistep natureof these processes, a combination of inhibitory actionsis thought to be necessary to effectively limit theirdevelopment.

As essential components in the communicationbetween cells and their milieu, integrins provide anattractive target in the inhibition of both cell migrationand angiogenesis. Integrins are an important class ofcell surface receptors that are critically involved in cell–cell and cell–matrix interactions [32]. All integrins arealpha/beta heterodimers that require metal ions fornon-covalent association of their subunits [33]. Thedifferent alpha subunits combine with distinct betasubunits resulting in a range of specificities towardvarious extracellular matrix proteins. Several classes ofintegrins recognize the RGD sequence present in ECMproteins, allowing integrins to link cytoskeletal pro-teins with the ECM and to be involved in bi-directionalsignaling that alters cellular functions. Among theseinteractions are the adhesion of both endothelial cellsand cancer cells to ECM proteins, such as fibronectin,vitronectin, type I and IV collagen, and laminin. Theseinteractions provide an ideal target for suppressionof tumor growth. Although much emphasis revolvesaround growth factors such as vascular endothelialgrowth factor (VEGF) in angiogenesis, cytokine func-tion is also intimately linked to the ability of endothelial

cells to interact with the ECM. Endothelial cell (EC)adhesion to ECM is required for EC proliferation,migration, morphogenesis, survival, and ultimatelyblood vessel stabilization, all of which are critical forneovascularization. The specific mechanisms throughwhich ECM supports EC functions are complex andinvolve both external structural support and regulationof multiple signaling pathways within the cell, includ-ing signaling pathways that control apoptosis, prolife-ration, the cytoskeleton, and cell shape.

The importance of integrins is underscored by thedifferent expression of integrins on various types ofnormal cells and cancer cell lines as described in earlierstudies. It was shown that integrin avb3 in the activeform was found only on angiogenic endothelium[34,35]. Additionally, VEGF was shown to induceactivation of various integrins involved in angio-genesis, including avb3, avb5, a5b1, and a2b1 [36].Blockage of these integrins had an inhibitory effect onendothelial function [37–40]. Prior studies have alsoelucidated the expression profile of integrins in variouscancer cell lines, including prostate cancer [12]. Theexpression of certain integrins, such as avb3, appears tocorrelate with each cell lines’ ability to adhere andmigrate on ECM proteins in vitro, as well as theaggressiveness of the cell lines in vivo [15]. Thus,inhibition of these integrins may prove to be anattractive method of preventing metastasis.

CN is a unique homodimeric disintegrin with atruncated amino-terminus in comparison to othermonomeric medium size disintegrins; the missingsequence containing two cysteine residues [41]. Allcysteines in other disintegrins are involved in disulfidebonds. Since there are two unpartnered cysteineresidues in the residual CN sequence, they are likelyinvolved in its dimerization. CN binds competitively tointegrins of the b1, b3, and b5 subclasses, includingintegrin receptors for fibronectin (a5b1), vitronectin(avb3, avb5), and fibrinogen (aIIbb3) [19–22]. Priorstudies have shown that there are different pathwaysinvolved in angiogenesis, in part regulated by thesevarious integrins [42]. The lack of integrin specificity byCN may prove to be an advantage in controlling cancergrowth and dissemination by allowing it to blockmultiple pathways [19,37]. CN disrupts angiogenesisinduced by both bFGF and VEGF, consistent with theobservation that a cyclic RGD peptide had no selectiv-ity amongav family members. The effect of CN appearsto be cytostatic as opposed to cytotoxic and preferen-tially effects the growth of new vessels as opposed topreexisting vessels [37].

Prior studies have shown that CN may be a viableoption for the treatment of cancer. Since CN is obtainedfrom snake venom, it is difficult to produce in largequantities. However, recent developments in recombi-

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Fig. 8. NudemiceinjectedwithPC-3-Luccellswererandomizedinto four groups: (1) control, (2) treated with contortrostatin(CN) released from Alzet osmotic pumps, (3) injected withdocetaxel, (4) treatedwith both CNand docetaxol.Tumor growthin the bone was monitored with Xenogeny at 7 and 14 dayspost-PC-3-Luc injection.Ten minutes after IP injection of luciferin(100mg/kg),micewereplacedin the chamberof an IVIS 200 opticalimaging system. Photons were collected for a period of 3min.Thebars show the total fluxof signal fromeachgroup.

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nant protein expression have made the production of amodified form of the drug in high yield a reality.Additionally, new formulations of CN are improvingthe pharmacokinetic profile of the drug. A recentlydescribed liposomal formulation of CN (LCN) dramat-ically increased the circulatory half-life of LCN overnative CN allowing increased number of passes of LCNthrough the tumor [17]. This leads to passive accumu-lation of LCN at the tumor due to the ‘‘leakiness’’ of theangiogenic vasculature of the tumor. As compared tonative CN, LCN has no platelet activity and is notrecognized by the immune system.

In the present study, we demonstrate for thefirst time the efficacy of CN in inhibiting progressionof two prostate cancer cell lines. CWR-22 is anandrogen-dependent prostate cancer cell line and PC-3 is a cell line derived from bone metastases and isandrogen independent. We tested CN both as a singleagent and as an adjunctive treatment with docetaxel.Docetaxel is a microtubule inhibitor that is one of thefew current primary active chemotherapy drugs usedin prostate cancer. It was therefore important todemonstrate compatibility between the two drugs intheir effects on cancer cell inhibition.

We analyzed the PC-3 cell line integrin profile indetail in order to correlate with CN’s ability to inhibitthe growth of this particular cell line. FACS analysis ofPC-3 cells indicates the presence of integrins a5b1 andavb5. Of note, our results did not show the presence ofthe avb3 (VN receptor) integrin which is consistentwith prior studies by Nemeth et al. [43]. Other studieshave evaluated the expression of individual integrinsubunits in the PC-3 cell line including av and b3 withsomewhat varied results [11,12]. Differences in integrinexpression may be reflective of the specific antibodiesused. There may also be some variation of integrinexpression within the same prostate cancer cell line andvariation as a result of differing cell culture conditions[11,44]. Adhesion assays revealed that PC-3 cells bindto immobilized fibronectin, vitronectin, and laminin.CN inhibited adhesion to fibronectin and vitronectindose-dependently, but did not have a significant effecton binding to laminin. This correlates with both the PC-3 integrin profile and CN’s ability to inhibit theseintegrins. These results are consistent with prior resultsusing similar methods with different cancer cell lines[18,19]. The general effect on migration was furtherillustrated by the scratch test that showed decreasedmigration on a matrigel plate in the presence of CN.The use of matrigel, which is bound by a number ofdifferent integrins based on the composition of anartificial ECM, is important for a number of reasons inthis experiment. Substituting matrigel with anotherindividual ECM protein (i.e., fibronectin or vitronectin)in the presence of CN would cause the cells to detach

and prevent analysis of migration. By using matrigel,the effect of disintegrins binding to integrins onadherent and potentially migrating cells can beobserved. Additionally, in this migration experimentthe removal of serum allows for observation of theeffect of CN binding only in the initial 2-hr period andnot subsequent binding events. When examining theability of CN to cause cell death, the use of CN aloneshowed the ability to partially inhibit PC-3 cells grownon a matrigel basement membrane matrix. Perhapsmore importantly, this effect was additive to theinhibitory effect shown by docetaxel.

The in vivo results of subcutaneously placed mousexenograft tumors showed a similar effect of CN both asa single agent and as a complementary treatment withdocetaxel. In these experiments, we were able to extendthese results to the androgen-dependent prostatecancer cell line CWR-22. This demonstrates that CN’sinhibitory effect is not isolated to only the PC-3 prostatecancer cell line. In reviewing the results, it should benoted that the in vivo inhibition of tumor growth withCN alone was comparable to docetaxel treatment alonein both the PC-3 and CWR-22 cell lines. This is incontrast to the in vitro experiments that showed modestinhibition of prostate cancer cell growth when CN wasused as a single agent. These results are consistent withother in vitro and in vivo experiments using CN inother cancer cell types [19]. These outcomes may be areflection of the in vivo inhibition of tumor angio-genesis not relevant to the in vitro setting. Our in vivodata shows CN decreases tumor expression of theangiogenic marker CD31, thus supporting this hypoth-esis. Another possibility for the increased efficacy is thein vivo effect of single agent CN integrin modulation onthe tumor milieu as opposed to the tumor itself. Also ofsignificance is that although the combined efficacy ofCN and docetaxel on tumor growth in vivo appearsto be additive, the inhibition of CD31 expression intreated PC-3 tumors did show some evidence of asynergistic effect. While this did not translate into asimilar effect on tumor growth, it will be of interest toevaluate in the future if this relationship changes withdifferent concentrations of drug, different methods ofdrug delivery, or larger sample sizes.

Of paramount importance in the treatment ofprostate cancer is its propensity to metastasize to bone.A vast proportion of hormone refractory prostatecancer will become metastatic to the bones, causing asignificant amount of patient morbidity. Estimatesshow that >80% of patients who die from prostatecancer have involvement of bone at death [45]. Giventhe predilection of prostate cancer to metastasize tobone, it is relevant to assess the ability of potentialtherapeutic agents to inhibit prostate cancer specificallyin the bone milieu. Prior experiments have implicated

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the importance of integrins avb3 and avb5 in a varietyof cancers, including prostate cancer [43][46–48].Furthermore, Nemeth et al. [43] demonstrated thatblockage of the avb3 integrin using monoclonal anti-bodies in a PC-3 tumor mouse model resulted in bothdecreased tumor growth and decreased tumor angio-genesis despite the lack of expression of avb3 on thetumor cells themselves. Given that CN has been shownto inhibit these integrins that appear vital in theproliferation of bone metastases, we examined theeffect of contortrostatin on prostate cancer cellsimplanted directly into bone in mice models. Ourresults demonstrate that CN inhibits PC-3 tumorsimbedded in bone. Similar to the other previouslydescribed experiments, this effect appeared to beadditive to docetaxel (Fig. 8). Whether this inhibitionis secondary to CNs effect on the tumor cells them-selves, blood vessel formation, integrins found in thebone milieu, or some combination of the above isunclear.

These experiments demonstrate the potential of CNas an adjunctive agent in the treatment of prostatecancer, and merit further study. Additional studies willbe required to find optimal dosing for the CN anddocetaxel combination as well as to further evaluatedifferent formulations and drug delivery. One of thedifficulties in performing dosing studies in the past hasbeen the relative lack of available CN, as CN representsonly a small proportion of snake venom. Likewise, theeffect of CN on other prostate cancer cell lines usingin vitro as well as in vivo bone models would providemore insight into some of our current findings. Finally,upcoming experiments may focus on the capability ofCN to prevent the formation of bone metastases asopposed to its effect on tumor already present in bone.

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