identificationofasmallmoleculeclasstoenhance cell...

Identification of a small molecule class to enhancecell-cell adhesion and attenuate prostate tumorgrowth and metastasis

Girish V. Shah, Anbalagan Muralidharan,Shibu Thomas, Mitan Gokulgandhi, Mudit Mudit,Mohammad Khanfar, and Khalid El Sayed

Department of Pharmaceutical Sciences, University of LouisianaCollege of Pharmacy, Monroe, Louisiana

AbstractExpression of calcitonin (CT) and its receptor (CTR) is elev-ated in advanced prostate cancer, and activated CT-CTRautocrine axis plays a pivotal role in tumorigenicity andmetastatic potential of multiple prostate cancer cell lines.Recent studies suggest that CT promotes prostate cancermetastasis by reducing cell-cell adhesion through the dis-assembly of tight and adherens junctions and activationof B-catenin signaling. We attempted to identify a class ofmolecules that enhances cell-cell adhesion of prostate cellsand reverses the disruptive actions of CT on tight andadherens junctions. Screening several compounds led tothe emergence of phenyl-methylene hydantoin (PMH) as alead candidate that can augment cell-cell adhesion andabolish disruptive actions of CT on junctional complexes.PMH reduced invasiveness of PC-3M cells and abolishedproinvasive actions of CT. Importantly, PMH did not displaysignificant cytotoxicity on PC-3M cells at the tested doses.I.p. administered PMH and its S-ethyl derivative remarkablydecreased orthotopic tumor growth and inhibited theformation of tumor micrometastases in distant organs ofnude mice. PMH treatment also reduced the growth ofspontaneous tumors in LPB-Tag mice to a significant extentwithout any obvious cytotoxic effects. By virtue of itsability to stabilize cell junctions, PMH could reverse theeffect of CT on junctional disruption and metastasis, whichstrengthens the possibility of using PMH as a potentialdrug candidate for CT-positive androgen-independent pros-tate cancers. [Mol Cancer Ther 2009;8(3):509–20]

IntroductionProstate cancer is the most commonly diagnosed cancerand the second leading cause of cancer deaths in men inAmerica (1, 2). Although androgen ablation therapy iseffective in prostate cancer patients for some time, thedisease progresses to an androgen-independent stage (3)when the tumor becomes metastatic, chemoresistant, andlife threatening (4, 5). The population of prostate cellsexpressing neuropeptides, such as calcitonin (CT) and itsreceptor (CTR), also increases during this progression (6, 7).Earlier results from this laboratory have shown that CT andCTR are exclusively localized in basal, but not secretory,compartment of normal prostate epithelium. This spatialspecificity is lost during tumor progression as evidenced bya large increase in CT/CTR mRNA abundance in secretoryepithelia of advanced prostate cancers (8). Moreover,exogenous CT or enforced activation of CT-CTR autocrineaxis remarkably raises tumorigenicity, metastatic potential,and apoptosis resistance of several prostate cancer cell lines(8–14). Our recent studies suggest that the disruption ofcell-cell adhesion may be a key mechanism associated withCT-stimulated prostate cancer progression and metastasis(15, 16).Because metastasis is the primary cause of morbidity and

mortality associated with prostate cancer, we focused toidentify a class of molecule(s) that augments cell-celladhesion complexes and attenuates CT-stimulated prostatecancer growth and metastasis. We began by screeningseveral structurally diverse marine natural products isolat-ed from Red Sea sponges and identified phenyl-methylenehydantoins (PMH) as a lead class that strengthens cell-celladhesion complexes of prostate cancer cell lines andattenuates CT-stimulated tumor growth and metastasis ofprostate cancer cell lines. Moreover, PMH molecules can beeasily, cost effectively, and regioselectively synthesized anddisplay minimal side effects at the therapeutic doses inxenograft and transgenic mouse models.

Materials andMethodsAnimalsMale BALB/c nu/nu mice (6–8 wk old) were purchased

from Harlan and housed two per cage in microisolator unitsunder controlled temperature/humidity. The animals werefed ad libitum on a standard sterilizable laboratory chow(Harlan Teklad) and quarantined for 1 wk before their use.LPB-Tag transgenic mice (12T-7fast) were provided by

Dr. Robert J. Matusik. The colony of these mice wasestablished in our facility and newborn mice wereidentified by genotyping as previously described (17).Positive adult mice were used in the present study.

Received 7/23/08; revised 11/19/08; accepted 12/9/08; publishedOnlineFirst 3/10/09.

Grant support: NIH grant R01CA96534 (G.V. Shah) and LBRNP20RR16456 (K.E. Sayed).

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.

Requests for reprints: Girish V. Shah, University of Louisiana College ofPharmacy, 1800 Bienville Drive, Monroe, LA 71209.Phone: 318-342-1693; Fax: 318-342-1737. E-mail: [email protected]

Copyright C 2009 American Association for Cancer Research.

doi:10.1158/1535-7163.MCT-08-0693

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All animal procedures were conducted in accordancewith the principles and procedures outlined by the NIHand Institutional Animal Care and Use Committee atUniversity of Louisiana-Monroe.

Surgical Orthotopic ImplantationThe surgical orthotopic implantation on nu/nu mice was

done as described before (18). Briefly, the mice were anes-thetized with ketamine (100 mg/kg)–xylazine (10 mg/kg)and placed in a supine position. A midline incision wasmade in the lower abdomen. Tumor cell suspensions(1 � 106 cells/20 AL) were injected into the dorsal prostateusing a 30-gauge needle (n = 6 per molecule or diluent) andsingle-stitch sutures closed abdomen. The mice wereperiodically fluoroimaged with Kodak 4000 MM imagingstation under anesthesia to monitor tumor growth andmetastasis. The mice were sacrificed as described in Resultsand organs were collected for further analysis.Because the goal of the present study was to identify

novel molecules that inhibit CT-stimulated disruption ofcell-cell adhesion and reduce invasive virulence of ad-vanced prostate cancer, we chose PC-3M prostate cancercells as a model cell line. This is because PC-3M cells arepoorly differentiated, androgen-refractory, coexpress CTand CTR, and are highly metastatic (8, 19). The cellswere maintained in the complete medium (RPMI 1640supplemented with 10% FCS, 100 IU/mL penicillin G,and 100 mg/mL streptomycin) under standard cultureconditions. Stable PC-3M-CT+ subline overexpresses CTand is more metastatic than PC-3M cells. This wasgenerated by stable transfection of recombinant plasmidpcDNA 3.1 containing CT cDNA and selected in G418. Thecell line was extensively characterized in recent studies (9).Initially, phenylmethylene hydantoin was extracted from

Hemimycale arabica (a Red Sea sponge) and characterized byspectral methods, including full nuclear magnetic reso-nance analysis. Subsequently, PMH as well as its mostpotent analogue S-ethyl PMH (S-PMH) were synthesized(Supplementary Fig. S1).1 Extracted PMH and syntheticPMH showed identical nuclear magnetic resonance pro-files. They were tested in multiple in vitro assays and werefound to be equipotent. Synthetic PMH was used in thepresent studies.

In vitro Assays: Cell-Cell AdhesionTransepithelial Resistance. Approximately 1 � 105 cells

were plated and grown to confluency on 12-well transwellfilters (0.4-Am pore size) in complete medium for the first12 h and then in serum-free medium. Electrodes wereplaced at the upper and lower chambers and transepithelialresistance (TER) was measured in triplicate wells atmultiple time points after CT/PMH addition with EVOMV-ohm meter (World Precision Instruments). The TERvalues were normalized to the area of the monolayer filter

and calculated by subtracting the blank values derivedfrom the filters containing only bathing medium. Theintegrity and cell density of monolayers was carefullymonitored during TER measurement studies.Paracellular Permeability. This assay measured the

diffusion of tetramethyl rhodamine– labeled dextran(TMR-dextran, 4 kDa) across a cell layer grown on amembrane of transwell insert (paracellular permeability).PC-3M cells were seeded (1 � 105 per insert) and culturedto form a monolayer for 5 d. The culture medium was thenreplaced with HBSS a few minutes before the addition ofTMR-dextran (1 mg/mL in HBSS). TMR-dextran wasadded to the upper chamber and 100 AL of the sampleswere removed from the lower chamber after 1 h.Fluorescence was measured on a Bio-Tek ELISA platereader (Ex530 nm/Em590 nm). The diffusion of TMR-dextran across the insert without cells was also measured toascertain the integrity of cell monolayers. Each data pointwas in quadruplicates.Preparation of Cell Lysates: Triton X-100–Soluble and

Triton X-100– insoluble Fractions. Confluent 100-mmplates of PC-3M cells were serum-starved overnight andtreated with diluent/CT/PMH as described in Results.Triton X-100–soluble extract (cytosolic) was obtained byincubating cells with 10 mmol/L Tris-HCl (pH 7.4;containing 150 mmol/L NaCl, 2 mmol/L CaCl2, 1 mmol/Lphenylmethylsulfonyl fluoride, 40 units/mL aprotinin,15 Ag/mL leupeptin, 1% NP40, and 1% Triton X-100)for 30 min with occasional agitation. After washing theplates with TBS (containing protease inhibitors), the TritonX-100–insoluble fraction (plasma membrane-associated)was scraped out from the plates with TBS containing0.5% SDS, 1% NP40, 40 units/mL aprotinin, and 15 Ag/mLleupeptin. The contents were homogenized and centrifugedat 14,000� g for 5 min at 4jC to obtain the Triton X-100–insoluble fraction. Protein content of lysates was deter-mined using Bio-Rad Reagent (Bio-Rad).Western Blotting. The lysate fractions (100 Ag protein per

lane) were boiled for 5 min in 2� Laemmli solutioncontaining 20 mmol/L DTT, loaded on 10% SDS-polyacryl-amide gel, and fractionated proteins were electricallytransferred to a nitrocellulose membrane. The blots wereincubated with appropriate antisera as described in Resultsfor 18 h at 4jC. Following three washes, the blots wereincubated with appropriate horse radish peroxidase–conjugated secondary antiserum (either antirabbit orantimouse IgG). The immune complexes were visualizedon chemiluminiscence radiography film using Western BlotEnhanced Chemiluminescence Detection System (Radio-chemical Center, Amersham). The blots were then washedand reprobed for a-tubulin or h-actin. The same experi-ment was repeated two more times.

In vitro Assays: Cell Growth and InvasionCell Proliferation Assay. The proliferation of prostate

cancer cells was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using the kitpurchased from American Type Culture Collection. Expo-nentially growing PC-3M cells were plated in a 96-well

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

PMH Inhibits Prostate Cancer Metastasis510





plate at a density of 8 � 103 per well and cultured for 24 h.The complete growth medium was then replaced with100 AL of basal incubation medium (RPMI 1640 containing0.1% bovine serum albumin, 10 mmol/L HEPES, 4 mmol/LL-glutamine, 100 IU/mL penicillin G, and 100 mg/mLstreptomycin) and incubation was continued for additional24 h. The cells were then treated with the MTT solutionat 37jC for 4 h and then stop solution was added (100 ALper well). Absorbance of the samples was determined at595 nm with an ELISA plate reader (Bio-Rad).In vitro Invasion Assay. Invasion experiments were

conducted in 24-well, two-compartment, Matrigel invasionchambers (Becton Dickinson). Exponentially growing PC-3M cells were serum-starved for 24 h with basal incubationmedium and then seeded at a density of 25 � 103 cells perwell in the upper insert of the Matrigel chamber. The lowerchamber received the chemoattractant medium, whichconsisted of 90% basal RPMI and 10% conditioned mediumfrom the cultures of PC-3M cells expressing constitutivelyactive Gas protein (20). The incubations were carried outfor 24 h, after which Matrigel (along with noninvadingcells) was scraped off with cotton swabs, and outer side ofthe insert was fixed and stained using Diff Quick kit (Dade-Behring Diagnostics). Invaded cells on the underside ofthe insert were counted in six or more randomly selected�100 fields. The results were expressed as mean F SEinvaded cells per �100 field. Each experiment was done intriplicates and the experiment was repeated twice.Growth Correction. Because PC-3M cells exhibit a high

proliferation rate, it is conceivable that the cells thatmigrated during the early part of the 24-h incubationperiod could proliferate during the remaining period ofincubation, causing a slight overestimation of final results.To correct this, we determined the growth rate of PC-3Mcells under identical conditions. Cells (25 � 103) wereplated at hourly intervals in six-well dishes and culturedfor 1 to 24 h. Mean percentage increase in cell number ineach well was determined. The relative CT-inducedincrease of the pooled results of all time points was foundto be 1.19 (vehicle control = 1). This correction was appliedto the results of invasion assays.Spheroid Disaggregation Assay. Current evidence has

shown that the cells of primary tumors in situ are released inclumps, which then attach to a favorable extracellularmatrix (ECM), and are released gradually to migrate in alldirections (21, 22). Based on this model, we have establisheda spheroid disaggregation assay where we first producespheroids of prostate cancer cells, then place them on anECM and monitor their disaggregation and the radialcell migration of tumor cells over a period of 48 h (23). Inour experience, this model reliably predicts the relativemetastatic potential of prostate cancer cell lines. Weexamined the effect of PMH and S-PMH on the metastaticpotential of PC-3M cells in spheroid disaggregation assay.In brief, a 100-AL suspension of 5 � 104/mL PC-3M cellsin RPMI 1640 serum-free medium was placed on 96-welllow-attachment tissue culture plates. The plates wererocked on gyrotory shaker in a CO2 incubator at 37jC for

2 d, at the end of which the spheroids measuring 150 to300 Amdiameter (f4� 104 cells per spheroid) were formed.A single spheroid was then placed in the center of each wellof ECM-coated 24-well microplate in 200 AL of serum-freemedium, was allowed to attach to the ECM for 1 h, anddigitally photographed (t = 0). The spheroids were thencultured at 37jC for 48 h, fixed, stained with Diff-Quik(Dade-Behring), and rephotographed. The diameter of thearea covered with cells was measured in a microscopecalibrated with a stage and ocular micrometer. The resultsare presented as Am migration F SE, where migration =(diameter of the area covering migrated cells � diameter ofthe spheroid) / 2 from three separate experiments, totaling5 to 10 spheroids.

In vivo Assays: Orthotopic Tumor Growth andMetastasisStable Expression of Red Fluorescence Protein in

PC-3M Cells. To detect micrometastases of implantedtumor cells in mice, we stably transfected PC-3M cells withDsRed-MCherry-Hyg-N1, a mammalian expression vectorthat encodes DsRed-MCherry, a derivative of red fluores-cent protein (Clontech). Hygromycin-resistant colonieswere selected and observed over a period of 4 wk. All celllines expressed strong red fluorescence at a steady levelover the duration of the observation period. These cellswere then used for orthotopic implantation in nu/nu mice.

Administration of PMH/S-PMHinMiceNu/nu Mice. PMH (or S-PMH) solution was prepared at

100� concentration in 10% DMSO and diluted to 1� withnormal saline. The molecules were administered i.p. to agroup of six animals per molecule or a diluent as describedin Results. The doses for PMH and S-PMH were 200 Ag/100 AL per day or 5 Ag/g body weight per day and 40 Agper day or 1 Ag/g body weight per day, respectively. Thesewere the maximal doses for each molecule withoutapparent cytotoxic effects as determined by initial studies.Tumor growth and metastases were monitored every 3 dbeginning after day 25 postimplantation by fluoroimagingwith Kodak 4000 MM image station. The scanned fluores-cent images were quantitated with Molecular ImagingSoftware version 6.05f7 (Kodak).The S-PMH solution was administered i.p. at the dose of

80 Ag/wk or 2 Ag/g of body weight per week. Thetreatment was started at the age of 30 d postnatal andcontinued until day 90. The mice were sacrificed on day 90and their male reproductive organs were isolated, weighed,and fixed for histology.At necropsy, the primary tumor and other organs were

harvested and weighed. Wet sections of organs wereexamined for the presence of red fluorescent protein.Fluorescent images of tumor cells were acquired with acharge-coupled device Retiga 2000 RT digital cameraconnected to a microscope (Nikon Optiphot 2) and acomputer. The images were then processed with IPLabImage Analysis Software (BD Biosciences). The remainingtissue portions were fixed in neutral buffered formalin andembedded in paraffin. The 5-Am-thick sections of thetumors were processed for H&E staining.

Molecular Cancer Therapeutics 511





Statistical AnalysisThe results were statistically evaluated by one-way

ANOVA or other analyses as described in Results and sig-nificance was derived from Newman-Keuls test. The diffe-rence was considered statistically significant when P < 0.05.

ResultsCTDisruptsTight andAdherensJunctions inPolarized

PC-3MCells and PMHAbolishesThis ActionA primary function of epithelia is to create a barrier that

separates distinct tissue compartments by forming gapjunctions, desmosomes, and tight and adherens junctions(TJ and AJ). TJs are located on most apical region of the cellmembrane, whereas AJs are located immediately basal toTJs. AJs maintain cell-cell adhesion, whereas TJs function asa ‘‘fence’’ by sealing the spaces between adjacent cells andmaintain apico-basal polarity and as a ‘‘barrier’’ byregulating paracellular permeability to control the diffusionof electrolytes and small molecules (24–26). We haverecently reported that CT promotes prostate cancermetastasis by disrupting cell-cell adhesion through thedisassembly of TJs and AJs (16). Therefore, we screenedseveral molecules and identified PMH as a molecule thatreverses the actions of CT on TJs and AJs.PMH and TER of Polarized PC-3M Cells. The optimal

concentrations of CT, PMH, and S-PMH were chosen byprevious experimentation. We observed that 50 Amol/LPMH was most beneficial for the enhancement of TJ/AJfunction, whereas 50 nmol/L CT had the most disruptiveeffect. These concentrations were then used in all in vitrostudies. We first examined the effect of PMH on TER ofdiluent- and CT-treated polarized PC-3M cells. TER of PC-3M cultures steadily increased up to 48 hours and then itstabilized (Fig. 1A). CT significantly reduced TER. Incontrast, PMH significantly increased TER of diluent- andCT-treated PC-3M cultures, suggesting that PMH hasbeneficial effects on TJs and it completely reverses theactions of CT on TER.PMH and Paracellular Permeability of Polarized PC-3M

Cells. Barrier function of TJs of PC-3M cultures can beexamined by the ability of TMR-dextran to penetratethrough the cell layer. Because PMH promoted TJformation and reversed the action of CT on TER, weexamined its effect on paracellular permeability of diluent-and CT-treated PC-3M cell layers. As expected, CT causedan almost 2-fold increase in paracellular permeability(Fig. 1B). In contrast, PMH decreased baseline paracellularpermeability and abolished CT-induced increase.PMH and Translocation of Key Junction Proteins from

Triton X-100–Insoluble to Triton X-100–Soluble Compart-ment. Cell-cell adhesion is controlled by key AJ and TJproteins that are located either on the membrane (such asclaudins, occludins, or E-cadherin) or on the intracellularside of the membrane (such as zonula occludens-1 or h-catenin). Disruption of these junctions causes internaliza-tion of integral membrane proteins into the cytoplasm,which can be identified by the change in their solubility

in Triton X-100 (membrane proteins are Triton X-100insoluble and cytoplasmic ones are soluble; ref. 27).Because the results of Fig. 1A and B suggested that PMHattenuated CT-stimulated disassembly of junctions, weverified this by testing the effect of PMH on CT-stimulatedchanges in the solubility of key TJ proteins, such as zonulaoccludens-1 and occludin, and AJ proteins, such asE-cadherin and h-catenin. Equal amounts of cell lysateproteins of Triton X-100–insoluble or Triton X-100–solublefractions were loaded onto a SDS-PAGE and immunoblot-ted for zonula occludens-1, occludin, E-cadherin, andh-catenin. Occludin and E-cadherin were predominantlypresent in the Triton X-100–insoluble fraction (Fig. 1C andD, lanes 1), whereas zonula occludens-1 and h-cateninwere present in Triton X-100–insoluble as well as TritonX-100–soluble fractions (Fig. 1C and D, lanes 1). CTincreased the solubility of all four proteins in Triton X-100 (Fig. 1C and D, lanes 2). Interestingly, PMH decreasedthe solubility of all four proteins in Triton X-100, underbaseline as well as CT-stimulated conditions (Fig. 1Cand D, lanes 3 and 4). These results confirm the results ofFig. 1A and B and show that PMH augments cell-celladhesion by enhancing TJs and AJs and by abolishing thedestabilizing actions of CT on these complexes.

Effect of PMH on Prostate Cancer Tumor Growth/MetastasisOur recent studies have shown that CT significantly

increases tumor growth and metastasis of prostate cancercell lines and these effects may be mediated by thedisruption of cell-cell adhesion (15, 16). Because PMHreversed the disruptive actions of CT on cell-cell adhe-sion, we tested the hypothesis that PMH attenuates CT-stimulated tumor growth/metastasis.PMH and PC-3M Cell Proliferation. PMH caused a

small but significant decline in the rate of baseline PC-3Mcell proliferation (Fig. 2A). CT (50 nmol/L, an optimal dosefor stimulation of cell proliferation/invasion as determinedin ref. 11, 28) caused a modest but significant increase inPC-3M proliferation, and PMH remarkably reduced theCT-stimulated increase. Moreover, PMH (50 Amol/L)displayed a minimal cytotoxic effect on PC-3M cells assuggested by no decrease in cell viability in its presence.PMH and PC-3M Cell Invasion. PC-3M cells displayed a

high rate of invasion, and CT (50 nmol/L) caused almost a2-fold increase in invaded cells (Fig. 2B). PMH (50 Amol/L)had minimal effect on baseline invasion but almostattenuated the CT-stimulated increase.Effect of PMH on Metastatic Potential: Spheroid

Disaggregation Assay. To avoid repeated addition ofexogenous CT during the long incubation period of 48hours in a spheroid disaggregation assay, we used PC-3M-CT+ cells, which secrete large amounts of CT and have CTR(9). We first examined the migration of cells from diluent/PMH/S-PMH–treated PC-3M-CT+ spheroids on vitro-nectin, fibronectin, collagen, and laminin (Fig. 2C). This isbecause the migration of cancer cells depends on surfaceintegrins and integrins have preferential affinity fordifferent ECM proteins (29). PC-3M-CT+ cells displayed






maximal migration on vitronectin and minimal migrationon laminin. These results are consistent with our earlierobservation that CT increases surface activity avh3 integ-rins, which serve as vitronectin receptors (23). Consideringthe involvement of avh3 in bone metastasis (30, 31), wethought it was important to examine whether PMH andS-PMH affect CT-stimulated spheroid disaggregation onvitronectin. Interestingly, PMH, as well as S-PMH, main-tained integrity of spheroids (Fig. 2E) and blocked spheroiddisaggregation and attachment of the cells to vitronectinand, to a smaller extent, to other various ECM proteins(Fig. 2C). Therefore, all subsequent assays were doneon vitronectin.The experiment was then repeated at multiple doses of

PMH and S-PMH. Both molecules inhibited spheroiddisaggregation/cell migration in a dose-dependent mannerwith EC50 concentration of 150 Amol/L for PMH (Fig. 2D).S-PMH was approximately three times more potent thanPMH in inhibiting PC-3M-CT+ spheroid dispersal/cellmigration on vitronectin, suggesting that both drugsblocked CT-stimulated avh3 activity, a key factor in bonemetastasis (23, 32).Figure 2E depicts a typical example of untreated/

S-PMH–treated PC-3M spheroids after 48 hours. The

untreated spheroid is disaggregated as shown by the largecircle of migrated cells around the spheroid. However, thepresence of S-PMH (50 Amol/L) completely prevented thespheroidal disaggregation. Figure 2F provides the quanti-tative data of Fig. 2E.PMH and In vivo Tumor Growth and Metastasis:

Orthotopic PC-3M Xenografts in Nude Mice. The ortho-topic microenvironment of mouse prostate is more suitableto study tumor growth/metastatic potential of humanprostate cancer cells than the ectopic microenvironment(19). We used orthotopic xenograft model to test the effectof PMH/S-PMH on tumor growth/metastasis of PC-3M-CT+ cells. PC-3M-CT+ tumors grew fairly quickly andmetastasized to several host organs as suggested by thefluorograms and growth curves generated from tumor cellfluorescence in diluent-treated mice (Fig. 3A and B).However, fluorograms as well as growth curves suggestthat tumors of PMH- and S-PMH–treated mice wereremarkably smaller than diluent-treated mice andremained localized within the abdominal cavity. Tumorburden of diluent-treated mice became very large within 60days after implantation and the mice displayed signs ofmorbidity, such as immobility and remarkably decreasedfood intake. At necropsy, tumors of diluent-treated mice

Figure 1. PMH and cell-cell adhesion. A, TER. Polarized monolayer cultures of PC-3M cells were exposed to diluent, 50 nmol/L CT, 50 Amol/L PMH,and 50 Amol/L PMH with 50 nmol/L CT 24 h after plating. TER values were measured from 5 min to 48 h after CT treatments. Results are mean TER F SE(n = 3) normalized to control monolayers of PC-3M cells at each time point. Data were compiled from three separate experiments. B, paracellularpermeability. Polarized monolayer cultures of PC-3M cells treated with 50 nmol/L CT for 1 h. The cells were then treated with TMR-dextran (500 AL of1 mg/mL solution) in the upper chamber. After 1 h, 100 AL of the medium were drawn from the lower chamber and assayed for fluorescence using aspectrophotometer at 530-nm excitation and 590-nm emission. *, P < 0.05, significantly different from their respective controls (one-way ANOVA andNewman-Keul’s test). ^, P <0.01, significantly different from CT treated (one-way ANOVA and Newman-Keul’s test). C-D, localization of junctionalproteins: immunoblotting. Confluent monolayers of PC-3M cells were treated with cytosolic lysis buffer for 30 min to collect cytosolic fraction. Then, thecells were lysed in membrane lysis buffer. Western blot analysis for zonula occludens-1, occludin, h-catenin, and E-cadherin was carried out after loading40 Ag of cytosolic proteins and 20 Ag of membrane proteins. a-Tubulin and h-actin were used as loading controls for membrane and cytoplasmic fractions,respectively. The experiment was repeated three separate times.






Figure 2. Effect of PMH on the growth and invasion of PC cells. A, PC-3M cells were cultured for 3 d. After 2 d, complete growth medium was replacedwith serum-free basal medium. Cells were then treated with diluent, 50 nmol/L CT, 50 Amol/L PMH, and 50 Amol/L PMH with 50 nmol/L CT for 24 h.Cell proliferation was assessed by the MTT assay. Absorbance at 595 nm was measured and error bars are mean F SE of four independent experiments(n = 8). *, P < 0.05, significantly increased over the untreated PC-3M values (one-way ANOVA and Newman-Keul’s test). B, invasion of PC-3M cellstreated with diluent, 50 nmol/L CT, 50 Amol/L PMH, and 50 Amol/L PMH with 50 nmol/L CT was examined as described in Materials and Methods.Triplicate experiments were done and six �100 fields were counted for each data point. Results are mean F SE for cells per �100 field (n = 3 independentexperiments �6 fields). *, P < 0.01, significantly different from the control (diluent treated; one-way ANOVA and Newman-Keul’s test). C,disaggregation/migration of PC-3M-CT+ spheroids on different ECMs in the presence or absence of PMH/S-PMH. Radial migration of spheroids under eachcondition [with and without the treatment of PMH or S-PMH on vitronectin (VN ), fibrinogen, collagen, and laminin] was quantitated as described inMaterials and Methods and graphed. The cell outgrowth was measured after 48 h of incubation. The results are presented as mean Am radial migration FSE for n = 4. *, P <0.05, significantly different from their respective controls (one-way ANOVA and Newman-Keul’s test). D, effect of differentconcentrations of PMH/S-PMH on disaggregation/migration of PC-3M-CT+ spheroids on vitronectin. Spheroids prepared from PC-3M-CT+ were treatedwith different concentrations of PMH and S-PMH and their disaggregation/migration was examined on vitronectin. The cell outgrowth was measured after48 h of incubation. The results are presented as mean Am radial migration F SE for n = 4. *, P <0.05, significantly different from their respective controls(one-way ANOVA and Newman-Keul’s test). E, typical photomicrographs of PC-3M-CT+ spheroids at the end of the experiment. Cell migration ofspheroids prepared with PC-3M-CT+ cells was examined in the absence or presence of 50 Amol/L S-PMH on vitronectin as described in Materials andMethods. F, the cell outgrowth was measured and photographed after 48 h of incubation. *, P <0.05, significantly different from vehicle-treated controls(one-way ANOVA and Newman-Keul’s test).






weighed 2.673 g F 0.268 (n = 6). The corresponding tumormasses of PMH-treated and S-PMH–treated mice weresignificantly lower and were 1.597 g F 0.138 (n = 12) and1.405 g F 0.305 (n = 8), respectively (Fig. 3C). AlthoughPMH and S-PMH displayed a similar antitumor activity,that of S-PMH was more robust as it was given only at20% of PMH dose. Survival curves of these mice suggestthat the treatment with PMH extended the life span ofmice from 50–57 to 56–65 and that with S-PMH to 65–70(Fig. 3D).PMH and S-PMH Reduce the Formation of Distant

Micrometastases in Nu/Nu Mice. Because we implantedPC-3M-CT+ cells expressing red fluorescence protein, wecould detect the presence of fluorescent micrometastases inwet sections of organs. We examined several organs forfluorescent tumor cell colonies and metastases were gradedfrom � (no cells) to ++++ (large tumor mass) as depictedin Table 1A. Both PMH and S-PMH significantly reducedincidents of metastases in several organs (Table 1B).Figure 4 (A and B) provides representative fluromicro-

graphs of different organs of diluent/PMH/S-PMH–treated mice. Diluent-treated mice showed large tumor cellcolonies in lymph node, seminal vesicles, bone, lungs, andsmaller tumor cell numbers in other organs including brain.Both PMH and S-PMH remarkably decreased metastasis ofPC-3M-CT+ cells in most organs and reduced the size oftumor cell colonies in organs wheremetastases were present.Comparatively, S-PMH seemed more antimetastatic thanPMH. H&E staining of these organs confirmed the presenceof micrometastases in these organs (Supplementary Fig. S2).1

S-PMH and Tumor Growth/Metastasis in LPB-TagMice. Because S-PMH displayed potent antimetastaticeffects in orthotopic xenograft model, we tested thismolecule in LPB-Tag mice (33). This line of transgenicmice was established as a model for prostate cancer. Themice develop spontaneous prostate tumors around day 30postnatal. The mice were given 2 Ag S-PMH/g body weightper week i.p. in three equally divided doses per weekbeginning at day 30. The treatment was continued for theperiod of the next 60 days and was terminated on day 90.

Figure 3. Growth of PC-3M-CT+ xenografts in nu/nu mice. PC-3M-CT+ red fluorescent protein cells (1 � 106) were orthotopically implanted in theprostate. Periodically, tumor growth was monitored by fluoroimaging. The experiment was terminated 60 d after cell implantation and the tumors wereremoved, cleaned, and weighed. A, fluorographs of diluent-, PMH-, and S-PMH–treated mice on day 41 postimplantation. B, growth curves of tumorscomputed from quantitation of fluorographs through the course of the experiment (mean F SE of n = 6 for each group). C, the bar graphs present finalweights of tumors at the termination of experiments (mean g tumor weightF SE for n =6). *, P <0.05, significantly different from diluent-treated tumors(one-way ANOVA and Newman-Keul’s test). D, Kaplan-Meier survival curves of diluent- and drug-treated mice.






The reproductive organs of the mice were harvested andweighed. All LPB-Tag mice spontaneously producedprostate tumors as depicted in Fig. 5A. However, S-PMHtreatment reduced the organ growth by 65% (Fig. 5B). H&Estaining of prostate sections suggest the presence of largetumor mass in the prostate of diluent-treated mice. Incontrast, S-PMH–treated mice displayed relatively normalprostate epithelium (Fig. 5C and D).

DiscussionExpression of CT and CTR is elevated in advanced prostatecancer, and activated CT-CTR autocrine axis increasestumorigenicity and metastatic potential of multiple prostatecancer cell lines (8–10). Recent results from this laboratorysuggest that CT promotes prostate cancer metastasis byreducing cell-cell adhesion through the disassembly of TJsand AJs and activation of h-catenin signaling. The TJs areoccluding junctions, which seal cells together in anepithelial sheet, and are critical for the determination ofepithelial cell polarity. Either disassembly or remodeling ofTJs can cause a loss of cell polarity and increase in motility(34, 35). Adherens junctions are adhesive contacts that holdcells together in a fixed position within the tissue. It has

been shown that TJ along with AJ and desmosomes providetissue integrity and promote cellular polarity, and AJ playsa pivotal role in regulating the entire activity of junctionalcomplex (36–38). There is increasing evidence for theassociation between the loss cell-cell adhesion structuresand metastasis of several other epithelial cancers (39, 40).Therefore, the primary objective of the present study was toidentify a class of molecules that enhances cell-celladhesion of prostate cells and abolishes disruptive actionsof CT on TJs and AJs. PMH increased TER, decreasedparacellular permeability, and prevented the translocationof key integral TJ and AJ membrane proteins into thecytoplasm, suggesting that PMH enhanced TJs and AJs.More importantly, PMH attenuated the disruptive effects ofCT on TER, paracellular permeability, and internalizationof key AJ and TJ proteins, such as zonula occludens-1,occludin, E-cadherin, and h-catenin.Cell adhesion, proteolytic degradation, and cell migration

are interrelated processes responsible for invasion andmetastasis of cancer (41–43). Because PMH attenuated thedisruptive actions of CT on cell-cell adhesion complexes,we tested a second hypothesis that PMH reduces invasive-ness of highly metastatic PC-3M cells. We used a combina-tion of in vitro and in vivo models to test this hypothesis.The results from cell proliferation assays revealed that PMHdid not significantly alter baseline proliferation of PC-3Mcells but abolished CT-stimulated cell proliferation. Moreimportantly, PMH did not display significant cytotoxicityon PC-3M cells at the tested doses but reduced invasivenessof PC-3M cells and attenuated proinvasive actions of CT asassessed by Matrigel invasion assays and spheroid disag-gregation assays. If cell-cell adhesion plays a key role intumor metastasis, PMH should attenuate the metastaticpotential of PC-3M sublines. We tested this possibility intwo in vivo models. It has been shown that in vitro invasionassays are useful for studying early events of metastasis.However, the formation of distant metastases requires notonly invasiveness of tumor cells but also additional char-acteristics like the capacity to survive during the process ofintravasation into blood and lymphatic vasculature, extrav-asation into parenchyma of distant tissues, and ability togrow in ectopic environment (11, 44, 45). These character-istics can only be examined in animal models. In addition,in vivo assays provide other key information, such as thestability of the therapeutic molecule in the body and itsability to reach the target organ and produce intendedbeneficial effects. First, we tested the effect of PMH andS-PMH on tumor metastasis in an orthotopic xenograftmodel. As expected, PC-3M-CT+ cells formed large ortho-topic tumors and distant metastases in multiple organsof nude mice. I.p. administered PMH and S-PMH remark-ably decreased orthotopic tumor growth and inhibitedthe formation of tumor micrometastases in distant organs.More importantly, the mice tolerated therapy welland did not display apparent cytotoxic effects of PMH andS-PMH at the tested doses. However, the mice producedremarkable beneficial responses, such as decreased morbidityand increased survival, reinforcing a possibility that the

Table 1.

A. Micrometastases of PC-3M cells in host organs

Organs Diluent PMH S-PMH

Seminal vesicles ++++ ++ +Testis ++++ +++ +Lymph nodes +++ ++ +Bone ++++ � �Lungs ++ � �Liver ++ � �Mesentary ++ � �Kidneys ++ � �Rain ++ � �

B. Frequency of micrometastases in host organs

Organ Diluent PMH S-PMH n

Seminal vesicles 6 3 2 6Testis 6 2 1 6Lymph nodes 5 2 0 6Bone 4 0 0 6Lungs 3 0 0 6Liver 2 0 0 6Liver 2 0 0 6Mesentery 2 0 0 6Kidneys 2 0 0 6Brain 1 0 0 6

NOTE: �, no tumor cells; +, isolated tumor cells; ++, few tumor cell colonies;+++, several tumor cell colonies concentrated in one area of the organ; ++++,tumor cell colonies spread throughout the organ. One-way ANOVA, level ofsignificance: P < 0.001 (PMH versus diluent). P < 0.001 (S-PMH versusdiluent). P > 0.05 (PMH versus S-PMH).






disassembly of junctional complexes may be a key eventin the progression of a localized prostate tumor to itsmetastatic form, and PMH can attenuate this process.Because S-PMH was more potent in attenuating PC-3M-

CT+ xenograft growth and metastasis, it can potentially be

useful for the treatment of patients with invasive or highCT prostate tumors. However, human tumors are nothomogenous like PC-3M-CT+ xenografts. Therefore, wetested S-PMH therapy in LPB-Tag mice, a transgenic modeldeveloped to study prostate carcinogenesis (46). Prostate

Figure 4. Metastasis of PC-3M-CT+ cells in organs of nu/nu mice. A, fluorescent micrographs of tumor micrometastases of diluent- and PMH-treatedmouse organs. Red fluorescent protein–expressing PC-3M-CT+ cells (1 � 106) were orthotopically implanted into the prostate of nude mice. Animalswere sacrificed 5 wk after implantation; fresh organs were removed from mice and were observed directly under fluorescent microscope. Mostmicrometastases could not be seen under bright-field microscopy (data not shown). The images were captured at �100 magnification. B, fluorescentmicrographs of tumor micrometastases of diluent- and S-PMH–treated mice (as described in A).






tumors of LPB-Tag mice display significant similaritieswith human tumors, including tumor heterogeneity,neuroendocrine features, and relatively slower tumorgrowth (46). Our results show that S-PMH therapy waseffective in LPB-Tag mice as assessed by a remarkablyreduced growth of prostate tumors. These results stronglysupport S-PMH as a novel candidate therapeutic moleculefor metastatic prostate cancer and a chemopreventiveagent.PMH has been tested for a possible anticonvulsant acti-

vity in experimental animals but no beneficial therapeuticeffects have been reported. In contrast, S-PMH is a novelmolecule. There have also been a few reports onpharmacologic activity of molecules that are structurallyrelated to PMH/S-PMH. For example, cyclic imidehydantoins class of compounds has been investigated foranticonvulsant activity (47). Pharmacologic activity ofhydantoins could be significantly modulated by substitu-tion of active groups to produce fungicidal, herbicidal,anti-inflammatory, anti-HIV, antihypertensive, and hypo-lipedemic activities (47). Hydantoin analogues, such assubstituted diisopropylbenzylidene hydantoins and 2-thiohydantoins, were patented for the inhibition of

tyrosine kinase and anti-allergic activities (48). However,the compounds were potent only at a superpharmacologicdose of 100 mmol/L in guinea pigs (48). Propanoic acid, 2-[4-[(5-oxo-2-thioxo-4-imidazolidinylidene)methyl]phe-noxy], and related analogues were recently patented fortheir ability to suppress cell proliferation and inhibitbinding between extracellular signal-regulated kinases andtheir substrates (49). Imidazol-2-amine, 5-chloro-N-(4-methylphenyl)-4-(phenylmethylene), was patented forin vitro anti2melanoma activity (50). However, the presentreport for the first time shows potent antimetastatic effectsof PMH and S-PMH on prostate cancer in multiple in vitroand in vivo model systems through the enhancement ofcell-cell adhesion.Although additional studies are required to identify

specific targets of PMH and precise molecular eventsthrough which PMH enhances junctional complexes, thepresent results suggest that S-PMH can potentially serve asa novel therapeutic molecule for treatment of advancedprostate cancer and cell-cell adhesion can be an importantparadigm to screen or develop new class of therapeuticmolecules for the treatment of advanced prostate cancerand possibly other epithelial tumors.

Figure 5. Treatment of LPB-Tag mice with S-PMH.Adult LPB-Tag mice between ages of 30 and 90 d wereinjected with S-PMH i.p. thrice a week as describedin Materials and Methods. The mice were sacrificed at90 d of age and tumors were removed, cleaned, andweighed. A, all LPB-Tag mice spontaneously produceprostate tumors as depicted in diluent-treated mice.S-PMH treatment remarkably reduced the organ mass.B, the bar graphs represent final weights of organs(prostate + seminal vesicles) at necropsy (mean gtumor weight F SE for n = 6). *, P < 0.05, significantchange from diluent (one-way ANOVA and Newman-Keul’s test). C, H&E sections of prostates from diluent-treated and S-PMH–treated mice.






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

Acknowledgments

We thank Dr. Isiah Fidler (M. D. Anderson Cancer Center, Houston, TX) forthe contribution of PC-3M cells and Dr. Robert Matusik (VanderbiltUniversity, Nashville, TN) for the LPB-Tag mice.

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2009;8:509-520. Published OnlineFirst March 10, 2009.Mol Cancer Ther Girish V. Shah, Anbalagan Muralidharan, Shibu Thomas, et al. metastasisadhesion and attenuate prostate tumor growth and Identification of a small molecule class to enhance cell-cell

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