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Research Article

A Novel Sulindac Derivative that Potently Suppresses ColonTumor Cell Growth by Inhibiting cGMP Phosphodiesteraseand b-Catenin Transcriptional Activity

Jason D. Whitt1, Nan Li2, Heather N. Tinsley5, Xi Chen4, Wei Zhang4, Yonghe Li4, Bernard D. Gary6,Adam B. Keeton6, Yaguang Xi6, Ashraf H. Abadi7, William E. Grizzle3, and Gary A. Piazza6

AbstractNonsteroidal anti-inflammatory drugs (NSAIDs) have beenwidely reported to inhibit tumor growth by a

COX-independent mechanism, although alternative targets have not been well defined or used to develop

improved drugs for cancer chemoprevention. Here, we characterize a novel sulindac derivative referred to as

sulindac benzylamine (SBA) that does not inhibit COX-1 or COX-2, yet potently inhibits the growth and

induces the apoptosis of human colon tumor cells. The basis for this activity appears to involve cyclic

guanosine 30,50,-monophosphate phosphodiesterase (cGMP PDE) inhibition as evident by its ability to

inhibit cGMP hydrolysis in colon tumor cell lysates and purified cGMP-specific PDE5, increase intracellular

cGMP levels, and activate cGMP-dependent protein kinase G at concentrations that suppress tumor cell

growth. PDE5 was found to be essential for colon tumor cell growth as determined by siRNA knockdown

studies, elevated in colon tumor cells as compared with normal colonocytes, and associated with the tumor

selectivity of SBA. SBA activation of PKG may suppress the oncogenic activity of b-catenin as evident by its

ability to reduce b-catenin nuclear levels, Tcf (T-cell factor) transcriptional activity, and survivin levels. These

events preceded apoptosis induction and appear to result from a rapid elevation of intracellular cGMP levels

following cGMP PDE inhibition.We conclude that PDE5 and possibly other cGMP degrading isozymes can

be targeted to develop safer andmore efficacious NSAID derivatives for colorectal cancer chemoprevention.

Cancer Prev Res; 5(6); 822–33. �2012 AACR.

IntroductionColorectal cancer (CRC) is the third most commonly

diagnosed cancer in the world that accounts for approxi-mately 600,000 deaths per year. While colonoscopy allowsfor the early detection of disease and the identification ofindividuals who are at high risk of disease progression, themortality rate from CRC has decreased only marginally inthe last 2 decades (1). In addition, certain lesions such as flatadenomas cannot be readily detected by colonoscopy (2)and surgical management of adenomas in high-risk indi-viduals, such aswith familial adenomatous polyposis (FAP)often requires complete or segmental removal of the colon(3). Given the slow progression of carcinogenesis and the

limitations of colonoscopy, much research has focused oncancer chemoprevention to reduce the development andprogression of CRC.

One class of drugs that has shown promise for chemo-prevention is the nonsteroidal anti-inflammatory drugs(NSAIDs), a chemically diverse family of drugs commonlyused for the treatment of pain, fever, and inflammation.Epidemiologic studies have shown that long-term use ofNSAIDs such as aspirin can significantly reduce the inci-dence and risk of death from CRC (4). In addition, certainprescription strength NSAIDs, such as sulindac can causethe regression and prevent recurrence of adenomas inindividuals with FAP (5). The antineoplastic activity ofNSAIDs is widely attributed to their COX inhibitory activitybecause prostaglandins are elevated in colon tumors (6)and a significant percentage of colon tumors express highlevels of the inducible COX-2 isozyme (7). However, thereis evidence that alternativemechanisms either contribute toor fully account for the CRC chemopreventive activity ofNSAIDs (8–10). For example, the non-COX inhibitorysulfone metabolite of sulindac has been reported to inhibitthe growth and induce apoptosis of colon tumor cells invitro (11, 12) and suppress colon tumorigenesis in animalmodels (13–15). Sulindac sulfone (exisulind) was alsoshown to suppress adenoma formation in individuals withFAP or sporadic adenomas (16, 17) but did not receive U.S.

Authors' Affiliations:Departments of 1Pharmacology, 2Biochemistry, and3Pathology, The University of Alabama at Birmingham; 4Drug DiscoveryDivision, Southern Research Institute, Birmingham; 5Department of Biol-ogy, Chemistry, and Mathematics, University of Montevallo, Montevallo;6Drug Discovery Research Center, Mitchell Cancer Institute, University ofSouth Alabama, Mobile, Alabama; and 7Faculty of Pharmacy and Biotech-nology, German University of Cairo, Cairo Egypt

Corresponding Author: Gary A. Piazza, Mitchell Cancer Institute, Univer-sity of South Alabama, 1660 Springhill Avenue, Suite 3029, Mobile, AL36604. Phone: 251-445-8412; Fax: 251-460-6994; E-mail:[email protected]

doi: 10.1158/1940-6207.CAPR-11-0559

�2012 American Association for Cancer Research.

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Food and Drug Administration (FDA) approval due tohepatotoxicity. Nonetheless, because the use of NSAIDs isassociated with gastrointestinal, renal, and cardiovasculartoxicities from suppressing prostaglandin synthesis (18,19), the investigation of COX-independent mechanismsmay provide insight that could lead to new drug candidatesthat are potentially safer and more efficacious for CRCchemoprevention.Previous studies have suggested that there is a close

association between the antineoplastic activity of NSAIDsand their ability to suppress Wnt/b-catenin signaling incolon tumor cells. For example, studies have shown thatcertain NSAIDs can decrease nuclear levels of b-catenin toinhibit the transcription of genes (e.g., cyclin D, survivin)that provide a survival advantage to allow for clonal expan-sion of neoplastic cells (20–22). Several groups havereported that sulindac sulfone can also induce proteosomaldegradation of oncogenic b-catenin, which suggests that theunderlying biochemical mechanism by which NSAIDs sup-press b-catenin signaling may not require COX inhibition(22–24).The mechanism responsible for the antineoplastic activ-

ity of sulindac sulfone has been previously reported toinvolve cyclic guanosine 30,50,-monophosphate (cGMP)phosphodiesterase (PDE) inhibition, although the specificisozymes involved were not identified (23, 25). Morerecently,we reported that theCOX inhibitory sulfidemetab-olite of sulindac and certain other NSAIDs also inhibitcGMP PDE and that this activity is closely associated withPDE5 inhibition and their tumor cell growth inhibitory andapoptosis-inducing properties (26–28). Cyclic nucleotidePDEs are a large superfamily of enzymes responsible forregulating second messenger signaling by hydrolyzing the30,50-phosphodiester bond in cGMP and/or cAMP. Thereare at least 11 PDE isozyme family members having differ-ent substrate specificity, regulatory properties, tissue local-ization, and inhibitor sensitivity (29). PDE1, 2, 3, 10, and11 are dual substrate-degrading isozymes, whereas PDE5, 6,and 9 are selective for cGMP and PDE4, 7, and 8 are cAMPselective. In addition, each isozyme family contains multi-ple isoforms or splice variants. Depending on the PDEisozyme content of the target cell population and inhibitorselectivity, PDE inhibitors can increase the magnitude and/or the duration of the cAMP and/or cGMP intracellularsignal(s). Increasing cyclic nucleotide levels can inducespecific signaling pathways, which, in the case of cGMP,can activate protein kinase G (PKG) to regulate cellularactivity (30).Here, we characterize the anticancer activity of a novel

benzylamine derivative of sulindac that does not inhibitCOX-1 or COX-2, yet can potently inhibit the growth ofcolon tumor cells by inhibiting proliferation and inducingapoptosis. The underlying biochemicalmechanism appearsto involve cGMP PDE inhibition as evident by its ability toselectivity inhibit cGMP hydrolysis in whole-cell lysates, aswell as purified PDE5. Moreover, treatment of colon tumorcells with sulindac benzylamine (SBA) increased intracel-lular cGMP levels and activated cGMP-dependent PKG in

colon tumor cells at concentrations that paralleled thoserequired for inhibiting cGMP PDE/PDE5 and colon tumorcell growth. PKG activation by SBA was also found to beassociated with decreased nuclear levels of b-catenin, T-cellfactor (Tcf) transcriptional activity, and the suppression ofthe apoptosis regulatory protein, survivin; all of whichpreceded apoptosis induction.

Materials and MethodsDrugs and reagents

Sulindac sulfide was purchased from Sigma-Aldrich.Recombinant PDE isozymes were purchased from BPSBiosciences. Isozyme-specific PDE antibodies were pur-chased from GeneTex, whereas vasoactive stimulatoryprotein (VASP) antibodies were purchased from BD Trans-duction Laboratories. All other antibodies were purchasedfrom Cell Signaling Technologies. Nontargeting controlsiRNA, PDE5-specific siRNAs, and the SureFECT transfec-tion reagent were purchased from SA Biosciences. All otherreagents were purchased from Sigma unless otherwisespecified.

Sulindac benzylamine synthesisSBA or [(Z)-N-benzyl-2-(5-fluoro-2-methyl-1-(4-(methyl-

sulfinyl) benzylidene)-1H-inden-3-yl)ethanamine] was syn-thesized by converting sulindac to sulindac methyl ester.Refluxing sulindac acid in methanol in the presence ofconcentrated sulfuric acid, followedby recrystallization fromethyl ether gave the methyl ester as a yellow solid in quan-titative yield. The ester was dissolved in methylene chlorideand then treatedwith diisobutylaluminumhydride (2mol/Lin toluene) at �77�C. After stirring for 4 hours, the reactionwas quenched by methanol. The temperature was slowlyraised to 0�C, and the reaction solution was washed withaqueous solution of acetic acid. The organic layer was con-centrated anddried in vacuumovernight to give the aldehydeas a yellow syrup. The aldehyde solution in ethanol wastreated with benzylamine at�77�C in for 3 hours, followedbysodiumborohydride for30minutes.Methanolwasadded,and the temperature was slowly brought up to �40�C andstirred for2hours.Acetic acidwasaddedslowly toquenchthereaction. The reaction mixture was concentrated, purifiedwith a silica gel column, and recrystallized from chloro-form/acetone/ether 3 times, which resulted in a yellowsolid. The final product was characterized by high resolutionmass, nuclear magnetic resonance, and elemental analysis.

Cell cultureThe human colon cancer cell lines, HT-29, SW480, and

HCT 116 and normal fetal human colonocytes (FHC) werepurchased from the American Type Culture Collection. HT-29, SW480, and HCT 116 cells were maintained in RPMI-1640 þ 2.0 g/L glucose pH 7.4 þ 5% FBS þ 4 mmol/Lglutamine (complete growth medium), incubated at 37�Cin 5% CO2, and passaged at subconfluent density. Thehuman fetal colon FHCcellswere cultured inDMEM:Ham’sF-12 medium supplemented with 10% FBS, cholera toxin

cGMP PDE Inhibition by a Novel Sulindac Derivative

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(10 ng/mL), 5 mg/ml transferrin, 5 mg/mL insulin, and 100ng/mL hydrocortisone. The passage number was routinelylimited to approximately 20 and morphology monitoredwith each passage, but no additional authentication of thecell lines was conducted.

COX activityCOX-1 and COX-2 activities were measured using puri-

fied ovine COX-1 and COX-2 with colorimetric assay kitsobtained from Cayman Chemical Company as previouslyreported (31). The activities of COX-1 and COX-2 weremeasured after the addition of arachidonic acid and incu-bation at 25�C for 5 minutes by absorbance at 590 nm asspecified by the manufacturer.

PGE2 assayU937 promonocytic cells (32) were differentiated into

macrophage-like adherent cells by culture in 10 nmol/Lphorbol 12-myristate 13-acetate (PMA) for 48 hours.Differentiated cells were washed with fresh media andplated at a density of 1.0 � 106 cells per well in 96-wellhalf-area plates. Cells were allowed to adhere overnightand stimulated with 10 mg/mL lipopolysaccharide (LPS).Cells were treated with sulindac sulfide or SBA for 24hours. Prostaglandin PGE2 levels in supernatants weremeasured using the HTRF PGE2 assay from Cisbio. Thisdetection method uses the binding of exogenous PGE2 todisrupt the fluorescence resonance energy transfer (FRET)between a PGE2–d2 conjugate (acceptor) and a PGE2antibody (donor).

PDE activityPDE activity was measured by the IMAP fluorescence

polarization assay (Molecular Devices) in which bindingof hydrolyzed cyclic nucleotide substrate to immobilizedmetal coordination complexes increases fluorescencepolarization as previously described (26). For studiesinvolving tumor cell lysates, human colon tumor cellswere harvested and lysed with PDE activity buffer (20mmol/L Tris-acetate, 5 mmol/L magnesium acetate, 1mmol/L EGTA, 1.0% Triton X-100, 50 mmol/L NaF, andprotease inhibitor cocktail at pH 7.4). Tetramethylrho-damine (TAMRA)-cGMP and fluorescein-cAMP were usedas substrates, each at final concentration of 50 nmol/L.Cell lysates were titrated to identify a suitable proteinconcentration that was in the mid portion of a concen-tration versus PDE activity curve. The PDE assay was doneaccording to the manufacturer’s specifications usingeither whole-cell lysates or recombinant enzymes. Fluo-rescence polarization was measured at excitation, emis-sion wavelengths of either 530,590 nm for TAMRA-cGMPor 485,530 nm for fluorescein-cAMP using a Synergy4(Biotek) microplate reader.

Growth assaysCells were plated in 96-well microtiter plates at a density

of 5,000 cells per well and allowed to adhere overnightbefore treatment. Cells were treated with a 2-log concen-

tration range of either sulindac sulfideor SBA and incubatedfor an additional 72 hours. For siRNA assays, cells weretransfected in OptiMEM media with 0.5% SureFECT trans-fection reagent and 200nmol/L of either negative control orPDE5 siRNA and incubated at 37�C for 24 hours beforetreatment. At the end of the incubation period, relative cellviability was compared with vehicle controls using theCellTiter-Glo Assay (Promega), which measures viable cellsbased on ATP content, according to the manufacturer’sspecifications.

Proliferation assaysThe antiproliferative activity of sulindac sulfide and SBA

was determined by measuring EdU (5-ethynyl-20-deoxyur-idine) incorporation during DNA synthesis. Cells wereseeded at a density of 1.5 � 106 cells per 10-cm tissueculture dish and incubated overnight at 37�C in 5% CO2.After growing the cells in serum-free medium for 24 hours,the cultures were treated with sulindac sulfide, SBA, orvehicle [0.1% dimethyl sulfoxide (DMSO)] in RPMI-1640 media with 5% serum for 4 hours. A final concentra-tion of 16 mmol/L EdU was added to each dish and incu-bated for an additional 18 hours. Cells were harvested andanalyzed using the Click-iT EdU Alexa Fluor 488 flowcytometry assay kit (Invitrogen) according to the manufac-turer’s specifications. Proliferating cells were quantifiedusing a Guava EasyCyte Plus flow cytometer. A minimumof 5,000 events were collected in triplicate for each treat-ment group.

Apoptosis assaysHCT 116 cells were seeded at a density of 1� 106 cells per

10-cm tissue culture dish, incubated for 48 hours, andtreated with the specified compound or vehicle control.After 24 hours of treatment, cells were harvested from thetreatment media and dish and fixed with 10% neutralbuffered formalin (4% formaldehyde) on ice for 15 min-utes. Samples were stained for DNA strand breaks using theAPO-BrdU terminal deoxynucleotidyl transferase–mediat-ed dUTP nick end labeling (TUNEL) assay (Invitrogen),which labels bromodeoxyuridine (BrdUrd) incorporationinto DNA strand breaks with AlexaFluor-488. The assay wasconducted according to the manufacturer’s specifications.The percentage of TUNEL-positive cells was quantifiedusing a Guava EasyCyte Plus flow cytometer. A minimumof 5,000 events were collected in triplicate for each treat-ment group with minimal electronic compensation. Datawere analyzed with CytoSoft 5.0 Software (GuavaTechnologies).

Intracellular cGMP levelsIntracellular cGMP levels were measured using the Pro-

mega GloSensor cAMP Assay modified to use a fireflyluciferase fused to the human PDE5 GAF-A cGMP bindingdomain (GloSensor cGMP-40F plasmid kindly provided byPromega Corporation). HEK293 cells were plated in 96-well white, clear bottom plates at a density of 25,000 cellsper well and incubated overnight at 37�C, 5% CO2, 95%

Whitt et al.

Cancer Prev Res; 5(6) June 2012 Cancer Prevention Research824




relativehumidity. For eachwell, a final concentrationof 100ng of DNA and 0.25 mL PLUS reagent were added to 19.75mL of Opti-MEMmedia and incubated at room temperaturefor 5 minutes; then 0.35 mL of Lipofectamine LTX reagentwas added to the solution, mixed gently, and incubated for30 minutes at room temperature. To each well 20 mL ofDNA-Lipofectamine LTX reagent was added and incubatedovernight under standard cell culture conditions. Afterincubation, the media was replaced with 100 mL of equil-ibration solution (88%CO2-independent media, 10% FBS,2% GloSensor cAMP reagent) and incubated for 2 hours atroom temperature protected from light. Background mea-surements were obtained 15 minutes before the end of theequilibration incubation by reading on a PerkinElmer Vic-tor 3 luminometer. After the background measurements,sodium nitroprusside (SNP) or SNP plus SBA was added tothe wells in 10 mL of CO2-independent media. The finalconcentration of SNPwas 50 mmol/L. Plates were read every2 minutes for 1 hour on the Victor 3 luminometer.

b-Catenin–mediated Tcf transcriptional activityExperiments to determine the effects of SBA on b-cate-

nin–mediated Tcf transcriptional activity were carried outusing the TOP/FOP-flash Tcf reporter constructs asdescribed previously (33). The TOP-flash plasmid con-tains Tcf binding sites for b-catenin, whereas the FOP-flash plasmid has mutated Tcf binding sites, which servesas a control for measuring nonspecific activation of thereporter. In brief, HCT 116 cells were plated in 24-wellplates and cotransfected with 0.1 mg TOP-flash or FOP-flash plasmids and 0.1 mg b-galactosidase–expressingvector. Following treatment with SBA for 24 hours, lucif-erase activity was determined and normalized with activ-ity of b-galactosidase. These data are expressed as themean and SEM of triplicate values of the normalizedTOP-flash and FOP-flash activity.

Western blottingFor Western blotting, cells were lysed in either ice-cold

membrane lysis buffer (1.0% Triton X-100, 1.5 mmol/LMgCl2, 10mmol/L KCl, 1mmol/LDTT, 10mmol/LHEPES,50mmol/LNaF, andprotease inhibitor cocktail), or nuclearlysis buffer (20 mmol/L HEPES, 1.5 mmol/L MgCl2, 200mmol/L EDTA, 1 mmol/L DTT, 400 mmol/L NaCl, 25%glycerol, and protease inhibitor cocktail). For nuclear frac-tionation, cell lysatewasmaintained at 4�Candvortexed for30 seconds every 5 minutes for 1 hour, followed by centri-fugation for 15 minutes at 15,000 rpm. Protein concentra-tions were determined by the Lowry method. Proteins wereseparated by SDS-PAGE on 12% PAGE before transfer tonitrocellulose membranes. Membranes were blocked for 1hour in 5% bovine serum albumin, 0.05% Tween 20, andincubated with primary antibodies overnight at 4�C. Mem-branes were incubated with horseradish peroxidase–conju-gated secondary antibody for 1 hour followed by incuba-tion with Super Signal West Pico Enhanced Chemilumines-cence Reagent (Pierce). Protein bands were visualized onHyblot CL (Denville Scientific) autoradiography film.

Molecular modelingMolecular modeling was conducted using Schr€odinger

Suite 2010 (Schr€odinger, LLC). The structural model ofPDE5 catalytic domain was derived from the crystal struc-ture of the PDE5-GMP complex from the protein databank(PDB ID: 1T9S). The induced fit docking (IFD) protocol,which takes into consideration the ligand-induced receptorconformational change, was used for all docking studies.Specifically, residues within 6 e from ligands were allowedto be flexible; docking results were scored using the extra-precision (XP) mode of Glide version 5.6 (Schr€odinger,LLC). IDF docking protocol and parameters were firstvalidated by docking GMP in the PDE5 catalytic site, whichexcellently reproduced the PDE5-GMP crystal complexconformation. The sameprotocol andparameterswere thenapplied to the docking studies of SBA.

Experimental design and data analysisFor growth assays, the IC50 values were determined by

testing a range of 8 concentrations with a minimum of 4replicates per dose. COX and PDE inhibition assays used aminimumof 3 replicates. Statistical analysis was done usingthe unpaired 2-tailed Student t test. Significance wasassumed for P < 0.05. Error bars represent SD.

ResultsSBA inhibits tumor cell growthwithout COX inhibition

We previously reported that the carboxylic acid moietyof sulindac sulfide is essential for inhibiting COX-1 andCOX-2, which could be chemically modified by substi-tuting with a positively charged amide group to effectivelyblock COX binding (31). Such derivatives were unexpect-edly found to display enhanced potency to inhibit colontumor cell growth compared with sulindac sulfide. Tochemically optimize for COX-independent tumor cellgrowth inhibitory activity, we synthesized a large seriesof sulindac derivatives with various chemical modifica-tions and screened for tumor cell growth and COX inhib-itory activity. A benzylamine derivative was identified asshown in Fig. 1A that displayed high potency for inhibit-ing tumor cell growth, but did not inhibit COX-1 orCOX-2.

As shown in Fig. 1B, SBA treatment for 72 hours inhibitedthe growth of human HT-29, SW480, and HCT 116 colontumor cell lines with IC50 values ranging from 3 to 5 mmol/L, whereas sulindac sulfide was appreciably less potent withIC50 values of 90 to 120 mmol/L. SBA completely lackedinhibitory activity for COX-1 and COX-2 at concentrationsup to 200mmol/L (Fig. 1C). By comparison, sulindac sulfideinhibited COX-1 and COX-2 with IC50 values of 2 and 9mmol/L, respectively. To determine whether SBA inhibitedother enzymes involved in arachidonic acidmetabolism,wemeasured treatments effects on the production of PGE2,which is the primary product in most tissues. Although weused a highly sensitive fluorescence-based ELISA assay formeasuring PGE2 production, the levels produced by colontumor cells (e.g., HT-29) did not result in a sufficient signal






above noise to allow for inhibition studies. As an alternativemethod, we used human U937 promonocytic cells treatedwith LPS, which are commonly used for such assays. Asshown in Fig. 1D, sulindac sulfide potently suppressedPGE2 production by these cells with an IC50 value of 0.7mmol/L, whereas SBA was ineffective, which is consistentwith its lack of COX-1 or COX-2 inhibitory activity. We alsodetermined whether the sensitivity of the colon tumor celllines to sulindac sulfide and SBA was related to the expres-sion of COX-2. As shown in Fig. 1E, HT-29 cells expressedhigh levels of COX-2 as determinedbyWestern blotting, butCOX-2 was expressed to a lesser extent in SW480 or HCT116 cells, despite no difference in their sensitivity to sulin-dac sulfide and SBA.

SBA inhibits colon tumor cell growth by inhibitingproliferating and inducing apoptosis

To determine the cellular basis for the tumor cellgrowth inhibitory activity of SBA, treatment effects onproliferation and apoptosis were measured and comparedwith sulindac sulfide. As shown in Fig. 2A, treatment ofHCT 116 colon tumor cells with sulindac sulfide for 24hours reduced the number of proliferating cells byapproximately 12% relative to vehicle-treated cells. Bycomparison, treatment with SBA inhibited cell prolifera-tion by 86% after the same treatment period. In addition,treatment with sulindac sulfide increased the number of

apoptotic cells from 3% in the vehicle group to 12%,whereas SBA increased apoptotic cells to 75%, respective-ly (Fig. 2B). These results show that the enhanced potencyof SBA compared with sulindac sulfide is associated withincreased effectiveness to inhibit proliferation and induceapoptosis.

SBA selectively inhibits cGMP PDEOn the basis of previous studies that showed a close

association between tumor cell growth inhibitory activityof sulindac sulfide and its ability to inhibit PDE5 (26–28, 34), we determined whether SBA has PDE inhibitoryactivity. For the initial studies we measured total cGMPand cAMP hydrolysis in whole-cell lysates from HT29cells using a dual substrate assay of PDE activity aspreviously described (26). As shown in Fig. 3A, sulindacsulfide inhibited total cGMP hydrolysis with an IC50 valueof 49 mmol/L, but appreciably higher concentrations wererequired to inhibit cAMP hydrolysis with an IC50 value of133 mmol/L. Corresponding with its improved potency toinhibit tumor cell growth, SBA more potently inhibitedcGMP hydrolysis with an IC50 value of 8 mmol/L as shownin Fig. 3B. In contrast with sulindac sulfide, SBA did notinhibit cAMP hydrolysis at concentrations as high as 200mmol/L, which suggests the potential for improved iso-zyme selectivity compared with sulindac sulfide. ThePDE5 inhibitory activity of SBA was next determined

Figure 1. A, chemical structures ofsulindac sulfide (SS) and SBA. B,tumor cell growth inhibitory activityof SS and SBA as measured byluciferase-based ATP assay after72 hours of treatment. C, COX-1andCOX-2 inhibitory activity of SS,but not SBA. D, inhibition of PGE2

production by SS, but not SBA inLPS-stimulated U937promonocytic cells. E, COX-2protein expression in human HT-29, HCT 116, and SW480 coloncancer cells. GAPDH,glyceraldehyde-3-phosphatedehydrogenase.

Whitt et al.





by conducting similar assays except using purified recom-binant PDE5. As shown in Fig. 3C, sulindac sulfideinhibited PDE5 with an IC50 value of 38 mmol/L. SBAwas appreciably more potent with an IC50 value of 9mmol/L as shown in Fig. 3D. The potency values as

determined using purified PDE5 were comparable withvalues as measured using whole tumor cell lysates,although less inhibition was observed in the latter, whichis likely attributed to the presence of insensitive cGMPPDE degrading isozymes as described later.

Figure 2. A, inhibition of HCT 116 colon tumor cell proliferation after 24-hour treatment with 100 mmol/L SS (middle) or 50 mmol/L SBA (right) as determined byEdU incorporation and flow cytometry. B, apoptosis induction of HCT 116 colon tumor cells after 24-hour treatment with 100 mmol/L SS (middle) or 50 mmol/LSBA (right) as determined by TUNEL and flow cytometry.

Figure 3. A, inhibition of cAMP andcGMP PDE activity by SS in HT-29tumor cell lysate. B, inhibition ofcAMP and cGMP PDE activity bySBA in HT-29 cell lysate. C, PDE5inhibition by SS. D, PDE5 inhibitionby SBA.






The PDE isozyme selectivity of sulindac sulfide and SBAwas next measured using a panel of recombinant PDEisozymes. As summarized in Table 1, SBA was highly selec-tive for PDE5. All other PDE isozymes were either insensi-tive or resulted in IC50 values that appreciably exceeded theconcentration range required to inhibit colon tumor cellgrowth. PDE5 was also the most sensitive isozyme tosulindac sulfide, although sulindac sulfide also inhibitedPDE2, PDE3, and PDE10 within the same concentrationrange as required for tumor cell growth inhibition, whichsuggests that the increased potency of SBA to inhibit tumorcell growth is associated with increased potency and selec-tivity to inhibit PDE5. However, because these studies arelimited to a group of PDE isoforms that were commerciallyavailable, we cannot rule out the potential involvement ofadditional isozymes that may be expressed in colon tumorcells and sensitive to SBA.

Elevation of intracellular cGMP by SBAWe previously reported that sulindac sulfide can increase

intracellular cGMP levels as measured by a standard immu-noassay method (26). To determine whether SBA canincrease intracellular cGMP levels, we used HEK293 cellstransfected with a construct of cGMP binding (GAF-A)protein fused to firefly luciferase. This luminescence assayin live cells allowed for kinetic measurements of intracel-lular cGMP levels in response to treatment. As shown inFig. 4A, SBA treatment caused a rapid and sustained increasein luminescence that occurred within the same concentra-tion range as required for cGMP PDE inhibition in celllysates. Others studies confirmed the expression of PDE5 inHEK293 cells (data not shown), which suggest that cGMPelevation by SBA in this cell model results from PDE5inhibition.

PDE5 suppression by siRNA inhibits colon tumor cellgrowth and increases sensitivity to SBA

To assess the possibility that PDE5 is necessary for colontumor cell growth, HCT 116 and HT-29 colon tumor cellswere transfected with PDE5 siRNA to selectively suppressthe expression of the enzyme. Western blot analysis asshown in Fig. 4B confirmed that PDE5 siRNA reduced PDE5protein levels. PDE5 knockdown by siRNA inhibited thegrowth of HCT 116 and HT-29 cells by 60% and 30%,respectively, compared with cells transfected with nontar-geted siRNA. In addition, PDE5 siRNA knockdown HCT116 cells displayed increased sensitivity to SBA treatment asevident by a 2-fold decrease in its IC50 value compared withcontrol cells as shown in Fig. 4C.

Tumor cell growth inhibition by SBA is associated withPDE5 expression

To further study the possibility that PDE5 expression caninfluence the sensitivity of colon tumor cells to SBA, PDE5levels were measured in normal human colonocytes (FHC)andHT-29 andHCT116 colon tumor cells. As shown in Fig.4D by Western blotting, PDE5 was not be detected in FHC,whereas both colon tumor cell lines expressed relativelyhigh levels of the enzyme. Consistent with these observa-tions, SBA inhibited the growthof both tumor cell lineswithan IC50 value of 5 mmol/L, whereas FHC were appreciablyless sensitive with greater than a 6-fold higher IC50 value of33 mmol/L (Fig. 4D). In contrast, sulindac sulfide did notshow evidence of tumor selectivity, whichmaybe attributedto its nonselective cGMP PDE inhibitory activity.

Mechanism of PDE5 inhibitionMolecular modeling studies were conducted to deter-

mine the mechanism by which SBA inhibits PDE5. For

Table 1. Potency of SS and SBA to inhibit cAMP and cGMP hydrolysis by PDE isozymes

SBA sensitivity SS sensitivity

PDE family Substrate cGMP IC50, mmol/L cAMP IC50, mmol/L cGMP IC50, mmol/L cAMP IC50, mmol/L

1A cAMP/cGMP >100 >100 >100 >1001B cAMP/cGMP >100 >100 >100 >1001C cAMP/cGMP >100 >100 >100 >1002A cAMP/cGMP >100 >100 97 >1003A cAMP/cGMP >100 >100 84 >1003B cAMP/cGMP >100 >100 >100 >1004B cAMP ND 75 ND >1005A cGMP 9 ND 38 ND6C cGMP >100 ND >100 ND7A cAMP ND >100 ND >1008A cAMP ND >100 ND >1009A cGMP >100 ND >100 ND10A cAMP/cGMP 62 77 70 >10011A cAMP/cGMP >100 >100 >100 >100

Abbreviation: ND, not determined.

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these studies we used a crystal structure of PDE5 com-plexed with the reaction product, GMP. Docking results asshown in Fig. 4F indicate that the heterocyclic ring scaf-fold of SBA effectively overlays with the guanine fragmentof GMP that occupies the same central hydrophobic areawithin the catalytic site of PDE5 located between Phe829and Phe786. Multiple hydrogen bonds were formed inthe PDE5-GMP structure between the guanine of GMPand Gln817, which is an important amino acid residuethat is necessary for substrate and inhibitor binding (33).Hydrogen bond interactions were also observed betweenGln817 and the amine group of SBA as evident by thedocked SBA-PDE5 complex structure. In addition, thesulfoxide group of SBA occupied the polar area near the2 metal ions, similar to the phosphate group of GMP.Likely important for enzyme inhibition, SBA occupied ahydrophobic region through its phenyl group, which isclose to the surface of PDE5 that is not occupied by theGMP molecule in the GMP-PDE5 complex structure.These results indicate that SBA directly binds the catalyticdomain of PDE5.

PKG activation by SBATo study the downstream events that may occur in

response to cGMP PDE inhibition and cGMP elevation, weinitially determined whether SBA could activate PKG incolon tumor cells. For these experiments, HT-29 cells weretreated with SBA and the phosphorylation of the knownPKG substrate, VASP, was measured using a phospho-spe-cific VASP antibody that is selective for cGMP-stimulatedphosphorylation at the Ser239 residue. As shown in Fig. 5A,the levels of phospho-VASP increased within 1 hour of SBAtreatment at a concentration of 5 mmol/L and remainedelevated for the duration of the experiment (5 hours). Theseexperiments provide evidence that PKG activation occurs inresponse to SBA treatment at concentrations and times thatparalleled those required for cGMP PDE inhibition andincreased intracellular cGMP levels, respectively.

Suppression of nuclear b-catenin levels andtranscriptional activity by SBA

Previous studies have shown that PKG can phosphorylateb-catenin to reduce cytoplasmic and nuclear levels of

Figure 4. A, luciferase-based cGMPbiosensor assay in HEK293 cellstreated with 50 mmol/L SNP in thepresence of 1, 3, or 10 mmol/L SBA.B, siRNA suppression of PDE5 andgrowth in humanHCT116 andHT-29colon tumor cells following 72-hourtransfection. C, growth inhibition bySBAofPDE5siRNAknockdownHCT116 cells compared with controls. D,PDE5 expression in human HCT 116and HT-29 colon tumor cells andFHC. E, growth inhibitory activity ofSBA and SS in human HCT 116 andHT-29 colon tumor cells comparedwith FHC. F, structuralrepresentation of GMP and SBAbinding to PDE5. The panels showGMP-bound PDE5 (left), dockedPDE5-SBA complex structure(middle), and the aligned GMP andSBA (right). Residueswithin 4Åof thebound/docked molecules are shownby lines. GMP and SBA moleculesare represented in solid sticks andtheir carbon atoms are colored ingray and green, respectively.Hydrogen-bonds are marked inyellow dashed lines.






b-catenin by a mechanism that appears to involve theactivation of ubiquitin-mediated proteosomal degradation(23, 35). To determine whether SBA can reduce b-cateninlevels within the same time period as PKG activation,nuclear b-catenin levels were measured in the same HT-29 cell lysates as used for experiments to measure PKGactivation. As shown in Fig. 5A, SBA reduced nuclear levelsof b-catenin within 1 hour of treatment, which paralleledthe time required for activating PKG. The expression of theapoptosis regulatory protein, survivin, inwhich its synthesisis under the control of the b-catenin/Tcf-Lef transcriptionfactor (36), was decreased after a slightly longer duration oftreatment (Fig. 5B). Levels of cleaved caspase-3 were alsomeasured as a biochemical marker of apoptosis and foundto be increased by SBA treatment at time points thatmatched those where survivin levels were suppressed andoccurred after the levels of nuclear b-catenin were decreased(Fig. 5C). There were no changes in PDE5 levels during thetreatment period indicating that this enzyme remainedstable during apoptosis.

To determine whether SBA can inhibit b-catenin–medi-ated Tcf transcriptional activity, we used the TOP/FOP-flashTcf luciferase assay. As shown in Fig. 5D, SBA caused asignificant decrease in b-catenin–mediated Tcf transcrip-tional activity. Finally, we confirmed that the PKG activator,8-bromo-cGMP can also suppress b-catenin levels, whichprovides additional evidence that the cGMP and b-cateninpathways are interconnected (Fig. 5E).

DiscussionThese results characterize the activity of a novel ben-

zylamine derivative of sulindac that lack COX inhibitory

activity but displays high potency to suppress the growthof colon tumor cells in vitro. The improved growthinhibitory potency of SBA was associated with increasedeffectiveness to inhibit proliferation and induce apopto-sis of colon tumor cells. Similar to observations wepreviously reported for sulindac and other NSAIDs, theunderlying biochemical mechanism responsible for thetumor cell growth inhibitory activity of SBA appears toinvolve cGMP PDE inhibition, although these results arenovel as they show that derivatives can be synthesizedthat lack COX inhibitory activity, which display higherpotency to inhibit colon tumor cell growth. The cGMPPDE inhibitory activity of SBA was evident by its abilityto selectively inhibit cGMP hydrolysis by lysates fromcolon tumor cells as well as by recombinant PDE5 withinthe same concentration range as required for inhibitionof colon tumor cell growth. Moreover, SBA increasedintracellular cGMP levels and activated PKG within thesame concentration range as required for cGMP PDE andtumor cell growth inhibition. Consistent with studiesshowing the ability of PKG to phosphorylate b-catenin toinduce degradation, SBA reduced nuclear levels of b-cate-nin, its transcriptional activity, and survivin levels withina treatment period that preceded the induction of apo-ptosis. PDE5 siRNA knockdown experiments show thatPDE5 is essential for growth of colon tumor cells that canimpact the sensitivity to SBA. These observations providestrong evidence that PDE5 inhibition represents animportant COX-independent mechanism for the antican-cer properties of NSAIDs that can be targeted to developpotentially safer and more efficacious derivatives for CRCchemoprevention.

Figure 5. A, increased VASPphosphorylation and time-dependent decrease in nuclearb-catenin levels in HT-29 cellstreated with SBA. B, decreasedb-catenin–regulated protein,survivin after 10 mmol/L treatmentwith SBA. C, caspase-3 cleavageand PDE5 expression in HT-29cells treated with 10 mmol/L SBA.D,Wnt/b-catenin signaling reporteractivity in HCT 116 cells treatedwith SBA for 24 hours. E,suppression of b-catenin by 8-Br-cGMP (100 mmol/L) in HCT 116cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Whitt et al.





Our observations are consistent with studies by otherswho have also concluded that a COX-independent mech-anism is fully responsible for or can contribute to thecancer chemopreventive activity of NSAIDs (10, 11, 37).For example, NSAIDs have been shown to suppress thegrowth of malignant cell lines that do not express COX-2(37) and supplementation with prostaglandins do notreverse the inhibitory activity on cellular growth (38). Inaddition, the rank order potency among NSAIDs toinhibit prostaglandin synthesis and growth of tumor cellsdoes not correlate (39) as higher doses of NSAIDs aregenerally required to inhibit tumor cell growth (40).Although the mechanism by which NSAIDs suppresstumorigenesis in vivo may involve both COX-dependentand independent effects, these results suggest that theirintrinsic tumor cell growth inhibitory activity onlyinvolves a COX-independent mechanism.Here, we show that SBA potently and selectively inhi-

bits cGMP hydrolysis by colon tumor cell lysates withoutaffecting cAMP hydrolysis. This was confirmed by studiesusing recombinant PDE isozymes where SBA selectivelyinhibited the cGMP-specific PDE5 isozyme. On the otherhand, sulindac sulfide inhibited multiple cGMP PDEisozymes, including PDE2, 3, 5, and 10. Consistent withthe importance of PDE5 as a target, we showed that thisisozyme is essential for colon tumor cell growth by siRNAknockdown studies. PDE5 knockdown by siRNA alsoincreased the sensitivity of colon tumor cells to SBAtreatment. In addition, PDE5 was found to be elevatedin human colon tumor cells compared with normalcolonocytes and that the expression level was associatedwith the tumor selectivity of SBA. These observations areconsistent with previous immunohistochemistry studiesshowing that PDE5 is overexpressed in human colonadenomas and adenocarcinomas as well as in bladder,lung, and breast tumors (25, 28, 41, 42). However, wecannot rule out the potential involvement of additionalcGMP PDE isozymes that might be expressed in tumorcells and sensitive to this class of compounds. In supportof this possibility, FDA-approved PDE5 inhibitors such astadalafil used for the treatment of erectile dysfunctionrequire high concentrations to inhibit tumor cell growth(micromolar levels) compared with concentrationsrequired to inhibit PDE5 in cell-free assays (nanomolarlevels; ref. 28), whereas others such as sildenafil arecompletely inactive.Of relevance to our findings that cGMP elevation can

inhibit growth and induce apoptosis of colon tumor cells,other investigators have shown an association betweencGMP elevation and inhibition of colorectal tumorigen-esis. Most notably, Shailubhai and colleagues showedthat oral administration of the enteric peptide hormone,uroguanylin, which binds a membrane-associated guany-lyl cyclase–coupled receptor to increase intracellularcGMP levels, inhibited tumor formation in the Apcmin

mouse model, and increased rates of apoptosis within thetumors (43). These experiments also reported that uro-guanylin levels were reduced in both colon adenomas

and adenocarcinomas, suggesting that cGMP levels maybe aberrantly low in colon tumors than in normal muco-sa. Other studies have shown that human colon tumorcell lines transfected with constitutively activated mutantsof PKG can undergo apoptosis and are unable to formcolonies (44). PKG is also downregulated in many cancertypes, including CRC (45), which suggests that suppres-sion of the cGMP pathway may provide a growth orsurvival advantage to tumor cells.

Given that b-catenin is an important oncogenic proteininvolved in CRC, the observations that SBA can suppressb-catenin and its transcriptional activity support the pos-sibility that this non-COX inhibitory derivative of sulin-dac (or related analogues) will be effective for CRCchemoprevention as has been established for otherNSAIDs that inhibit COX-1 and/or COX-2. We foundthat SBA treatment can induce b-catenin degradation,which is consistent with previous studies showing thatPKG can phosphorylate b-catenin to induce degradation(23, 46). The time-dependent activation of PKG by SBAparalleled the time required for cGMP elevation that isfollowed by the suppression of b-catenin nuclear andsurvivin levels. These events preceded caspase activationand suggest that the cGMP/PKG and Wnt/b-catenin path-ways are interconnected to trigger conditions that lead toapoptosis induction.

In conclusion, these results support further studies toevaluate the efficacy and toxicity of SBA or other non-COXinhibitory derivatives of sulindac for cancer chemopreven-tion in animal models. Additional studies are also war-ranted to better define the role of cGMP and cGMP-degrad-ing PDE isozymes in colorectal tumorigenesis.

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

Authors' ContributionsConception and design: N. Li, A.H. Abadi, W.E. Grizzle, G. PiazzaDevelopmentofmethodology:H.N. Tinsley, A.B. Keeton, A.H. Abadi,W.E.Grizzle, G. PiazzaAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): J.D. Whitt, N. Li, H.N. Tinsley, Y. Li, B. Gary, W.E.Grizzle, G. PiazzaAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): J.D. Whitt, N. Li, H.N. Tinsley, X. Chen,W. Zhang, A.B. Keeton, Y. Xi, A.H. Abadi, G. PiazzaWriting, review, and/or revision of the manuscript: J.D. Whitt, N. Li, H.N. Tinsley, X. Chen, W. Zhang, B. Gary, A.B. Keeton, Y. Xi, A.H. Abadi, W.E.Grizzle, G. PiazzaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): H.N. Tinsley, X. Chen, G. PiazzaStudy supervision: G. Piazza

Grant SupportThis research was supported by NIH grants, NCI 1R01CA131378 and

1R01CA148817-01A1.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 12, 2011; revised March 26, 2012; accepted April 16,2012; published OnlineFirst May 3, 2012.






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Correction

Correction: A Novel Sulindac Derivative thatPotently Suppresses Colon Tumor Cell Growthby Inhibiting cGMP Phosphodiesterase andb-Catenin Transcriptional Activity

In this article (Cancer Prev Res 2012;6:822–33), which was published in theJune 2012 issue of Cancer Prevention Research (1), the authors neglected toinclude an acknowledgment of individuals who conducted the synthesis of thecompound used in the study. The authors apologize for the omission. Theacknowledgment is as follows:

AcknowledgmentsThe authors thank Drs. Robert Reynolds, Bini Mathew, and Kocharani Jacob from Southern Researchfor the synthesis of sulindac benzylamine and other technical support.

Reference1. Whitt JD, Li N, Tinsley HN, Chen X, Zhang W, Li Y, et al. A novel sulindac derivative that potently

suppresses colon tumor cell growth by inhibiting cGMP phosphodiesterase and b-catenintranscriptional activity. Cancer Prev Res 2012;5:822–33.

Published online January 2, 2013.doi: 10.1158/1940-6207.CAPR-12-0477�2012 American Association for Cancer Research.

CancerPreventionResearch

Cancer Prev Res; 6(1) January 201370

2012;5:822-833. Published OnlineFirst May 3, 2012.Cancer Prev Res Jason D. Whitt, Nan Li, Heather N. Tinsley, et al. Transcriptional Activity

-CateninβCell Growth by Inhibiting cGMP Phosphodiesterase and A Novel Sulindac Derivative that Potently Suppresses Colon Tumor

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