ursolic acid inhibits growth and metastasis of...

Cancer Therapy: Preclinical

Ursolic Acid Inhibits Growth and Metastasis of HumanColorectal Cancer in an Orthotopic Nude Mouse Model byTargeting Multiple Cell Signaling Pathways:Chemosensitization with Capecitabine

Sahdeo Prasad, Vivek R. Yadav, Bokyung Sung, Simone Reuter, Ramaswamy Kannappan, Amit Deorukhkar,Parmeswaran Diagaradjane, Caimiao Wei, Veerabhadran Baladandayuthapani, Sunil Krishnan,Sushovan Guha, and Bharat B. Aggarwal

AbstractPurpose: Development of chemoresistance, poor prognosis, and metastasis often renders the current

treatments for colorectal cancer (CRC) ineffective. Whether ursolic acid, a component of numerous

medicinal plants, either alone or in combination with capecitabine, can inhibit the growth and metastasis

of human CRC was investigated.

Experimental design: The effect of ursolic acid on proliferation of CRC cell lines was examined by

mitochondrial dye uptake assay, apoptosis by esterase staining, NF-kB activation by DNA-binding assay,

and protein expression byWestern blot. The effect of ursolic acid on the growth and chemosensitizationwas

also examined in orthotopically implanted CRC in nude mice.

Results:We found that ursolic acid inhibited the proliferation of different colon cancer cell lines. This is

correlated with inhibition of constitutive NF-kB activation and downregulation of cell survival (Bcl-xL, Bcl-

2, cFLIP, and survivin), proliferative (cyclin D1), and metastatic (MMP-9, VEGF, and ICAM-1) proteins.

When examined in an orthotopic nude mouse model, ursolic acid significantly inhibited tumor volume,

ascites formation, and distant organ metastasis, and this effect was enhanced with capecitabine. Immu-

nohistochemistry of tumor tissue indicated that ursolic acid downregulated biomarkers of proliferation (Ki-

67) and microvessel density (CD31). This effect was accompanied by suppression of NF-kB, STAT3, andb-catenin. In addition, ursolic acid suppressed EGF receptor (EGFR) and induced p53 and p21 expression.

We also observed bioavailability of ursolic acid in the serum and tissue of animals.

Conclusion:Overall, our results show that ursolic acid can inhibit the growth andmetastasis of CRC and

further enhance the therapeutic effects of capecitabine through the suppression of multiple biomarkers

linked to inflammation, proliferation, invasion, angiogenesis, and metastasis. Clin Cancer Res; 18(18);

4942–53. �2012 AACR.

IntroductionColorectal cancer (CRC) is the second most common

cause of cancer death among men ages 40 to 79 years (1),with an annual global mortality of greater than 639,000persons. In the year 2012 in the United States alone, 73,420men and 70,480 women will be diagnosed with CRC.

Deaths from CRC are expected to be 51,690 in 2012, upfrom 49,920 in 2009. Chemotherapy, radiotherapy, andsurgery are the basic treatment methods used against CRC.However, long-term use of chemotherapy can makepatients feel very sick and can also make their conditionworse. Tumors also develop resistance to the chemotherapyover time.

Accumulating evidence suggests that the developmentand progression of many malignancies, including CRC, areassociated with constitutive activation ofmultiple signalingpathways that promote proliferation, inhibit apoptosis, andinduce metastasis (2). NF-kB and STAT3 transcription fac-tors, which primarily regulate inflammation, play a crucialrole in regulating several pathways that affect tumor cellsurvival, angiogenesis, motility, and invasiveness (3). EGFreceptor (EGFR), one of the family of 4 erbB receptors, isfound to be overexpressed in a variety of human tumors,including CRCs (4). Aberrant activation of EGFR and the

Authors' Affiliation: CytokineResearch Laboratory, Department of Exper-imental Therapeutics, The University of Texas MD Anderson CancerCenter, Houston, Texas

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Bharat B. Aggarwal, The University of Texas M DAnderson Cancer Center, 1515 Holcombe Blvd., Box 143, Houston, TX77030. Phone: 713-794-1817; Fax: 713-745-6339; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-11-2805

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(18) September 15, 20124942

on February 11, 2020. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 25, 2012; DOI: 10.1158/1078-0432.CCR-11-2805

http://clincancerres.aacrjournals.org/

EGF signal pathway is associated with neoplastic cell pro-liferation, migration, stromal invasion, resistance to apo-ptosis, and angiogenesis (5). Thus, agents that can controlthese multiple signaling pathway have potential for useagainst CRC.Although natural remedies have been claimed to have

potential for the prevention or treatment of CRC, neither anactive component nor a mechanism has been well charac-terized. Ursolic acid, a pentacyclic triterpenoid, as a com-mon active compound of numerous medicinal plantsincluding holy basil (6) has been shown to exhibit anti-cancer potential in vitro and in vivo. It suppressed prolifer-ation and induced apoptosis in cells of various cancers,including breast cancer (7), colon cancer (8), non–smallcell lung cancer (9), cervical cancer (10), multiplemyeloma(11), pancreatic cancer (12), melanoma (13), and prostatecancer (14). In animal models, ursolic acid inhibitedtumorigenesis (15–17), suppressed tumor invasion (18),and inhibited metastasis of esophageal carcinoma (19).How ursolic acid acts as an anticancer agent is not yet clear.However, several reports have suggested that it inhibitsDNA replication (20), induces Ca2þ release (21), activatescaspases (13, 22) and c-jun-NH2-kinase (JNK; ref. 14),downregulates antiapoptotic genes (23, 24), inhibitsCOX-2 and iNOS (25, 26), suppresses MMP-9 (18), andinhibits protein tyrosine kinase (27), STAT3 (11), and NF-kB activation (24). All these reports suggest that ursolic acidhas potential against CRC.In the current report, we investigatedwhether ursolic acid

inhibits the growth and metastasis of human colon cancerin vitro and in an orthotopic mouse model of CRC. Wefound that ursolic acid significantly inhibited the growthand metastasis of CRC and further enhanced the effect of

capecitabine through downregulation of multiple cell sig-naling pathways.

Materials and MethodsMaterials

Ursolic acid (Fig. 1A) was kindly supplied by King SingGuan (Haikou, China), and its purity was ascertained byhigh-performance liquid chromatography (HPLC; Fig 1A,left). Polyclonal antibodies against p65, ICAM-1, cyclinD1,MMP-9, survivin, and cIAP-1, and monoclonal antibodiesagainst VEGF, c-myc, Bcl-2, and Bcl-xL were obtained fromSanta Cruz Biotechnology. The liquid 3,3-diaminobenzi-dine þ substrate chromogen system–horseradish peroxi-dase used for immunohistochemistry was obtained fromDakoCytomation. Penicillin, streptomycin, Dulbecco’smodified Eagle’s medium (DMEM)/F12 medium, and FBSwere obtained from Invitrogen. All other chemicals wereobtained from Sigma unless otherwise stated.

Cell linesThe human colon cancer cell lines, HCT116, HT29, and

Caco2, were obtained from American Type Culture Collec-tion. HCT116 cells were cultured in DMEM, and HT29 andCaco2 cells were cultured in RPMI-1640 medium supple-mentedwith 10%FBS, 100U/mLpenicillin, and100mg/mLstreptomycin.

Proliferation assayThe effect of ursolic acid on cell proliferation was

determined by the MTT uptake method as describedpreviously (28). The absorbance was measured at 570nm using an MRX Revelation 96-well multiscanner(Dynex Technologies).

Clonogenic assayHCT116 cells (3,000 cells/well) were seeded in 12-well

plates, incubated for 12 hours, and treated with differentconcentrations of ursolic acid for 12 hours. The cells werethen reseeded in 6-well plates and allowed to form coloniesfor 14 days. Colonies were stained with 0.3% crystal violetsolution for 20 minutes. The excess crystal violet solutionwas washed with distilled water to visualize the clonogenicpotential of the cells.

Apoptosis assayTo determine whether ursolic acid can potentiate the

apoptotic effects of capecitabine in colon cancer cells, weused a Live/Dead Assay Kit (Molecular Probes). In brief,cells (5,000 cells per well) were incubated in chamber slidesand coincubated with ursolic acid and capecitabine for 24hours. Cells were then stained with assay reagents for 30minutes at ambient temperature. Cell viability was deter-mined by a fluorescence microscope by counting live(green) and dead (red) cells.

AnimalsMale athymic nu/nu mice (4 weeks old) were obtained

from the breeding section of the Department of

Translational RelevanceDespite advances in earlier detection and therapy,

colorectal cancer (CRC) is the leading cause of deathfrom gastrointestinal malignancy. Existing chemothera-peutic regimens are associated with lack of efficacy andyet are highly toxic. Long-term use of chemotherapeuticdrugs also resulted into the development of tumorresistance over time. Thus the agents, which can over-come the resistance and can enhance the effect of che-motherapeutic drugs, are urgently needed. Throughin vitro and in vivo experiments, we show for the firsttime that ursolic acid, an active compound present innumerous medicinal plants including holy basil, canpotentiate the effect of capecitabine, a standard drug,against CRC. Our preclinical findings show that ursolicacid is effective in inhibition of tumor growth andmetastasis and that this effect is further enhanced whencombined with capecitabine. This antitumor effect ofursolic acid was mediated through multiple pathwayslinked to inflammation, proliferation, invasion, angio-genesis, and metastasis.

Ursolic Acid Inhibits Growth and Metastasis of CRC

www.aacrjournals.org Clin Cancer Res; 18(18) September 15, 2012 4943




Experimental Radiation Oncology at The University ofTexasMDAnderson Cancer Center (Houston, Texas). Threemice per cage were housed in standard acrylic glass mousecages in a room maintained at constant temperature andhumidity with a 12-hour light:dark cycle; mice were fedregular sterilized chow diet with water ad libitum. Ourexperimental protocol was reviewed and approved by theInstitutional Animal Care and Use Committee at the MDAnderson Cancer Center.

Orthotopic implantation of HCT116 cellsHCT116 cells were stably transfected with luciferase as

previously described (28). Luciferase-transfected HCT116cells were harvested from subconfluent cultures after a briefexposure to 0.25% trypsin and 0.2% EDTA. Trypsinizationwas stopped with medium containing 10% FBS. The cellswere washed once in serum-free medium and resuspendedin PBS. Only suspensions consisting of single cells, with

more than 90% viability, were used for the injections. Micewere anesthetized with ketamine–xylazine solution, asmall, left abdominal flank incisionwasmade, andHCT116cells (1.5� 106 cells) in 50 mL of PBS were injected into thececum using a 30-gauge needle and a calibrated pushbutton–controlled dispensing device (Hamilton SyringeCompany). To prevent leakage, a cotton swab was heldcautiously for 1 minute over the site of injection. Theabdominal wound was closed in one layer with woundclips (Braintree Scientific, Inc).

Experimental protocolOne week after implantation, mice were randomly

assigned to the following treatment groups (6 mice pergroup): (i) corn oil (vehicle; 100 mL daily); (ii) ursolic acidalone (250 mg/kg once daily, orally), (iii) capecitabinealone (60 mg/kg, twice weekly by gavage), and (iv) ursolicacid (250 mg/kg, once daily, orally), and capecitabine

Figure 1. Ursolic acid inhibits thegrowth and proliferation of CRCandenhances the apoptotic effectsof capecitabine. A, chemicalstructure and HPLC profile ofursolic acid (left). HCT116, HT29,and Caco2 cells were pretreatedwith the indicated concentrationsof ursolic acid for 1, 2, 3, 4, and 5days. Cell viability was thenanalyzed by the MTT method asdescribed under Materials andMethods (right). B, HCT116 cellswere pretreated with indicatedconcentration of ursolic acid for 24hours. Whole-cell extracts wereprepared andsubjected toWesternblotting (left). HCT116 cells werepretreatedwith 20mmol/Lof ursolicacid for 8 hours and then exposedto capecitabine (20 mmol/L) for 24hours. Whole-cell extracts wereprepared andsubjected toWesternblotting (right). C, HCT116 cellswere treated with the indicatedconcentrations of ursolic acid for 8hours, and then nuclear extractswere prepared and assayed forNF-kBactivation by EMSA. D, HCT116cells were treated with indicatedconcentration of ursolic acid for 12hours. The cells were thenreseeded in 6-well plates andallowed to form colonies for 14days, after which theywere stainedwith crystal violet, as described inMaterials and Methods (left).HCT116, HT29, and Caco2 cellswere pretreated with 20 mmol/L ofursolic acid for 8 hours. The mediawere removed, and the cells werethen exposed to capecitabine for24 hours. Cell viability was thenanalyzed by the Live/Dead assay(right). �, significant [Bonferroni Adj(for 3 comparisons) P < 0.05] overvehicle control within each cell line.

Prasad et al.

Clin Cancer Res; 18(18) September 15, 2012 Clinical Cancer Research4944




(60mg/kg, twice weekly by gavage). In combination group,ursolic acid was given 6 to 8 hours before capecitabine.Tumor volumes were monitored weekly by the biolumi-nescence imaging system (IVIS) 200 and Living Imagesoftware (Caliper Life Sciences). Before imaging, animalswere anesthetized in an acrylic chamber with 2.5% isofluor-ane/air mixture and injected intraperitoneally with D-lucif-erin potassium salt in PBS at a dose of 150 mg/kg bodyweight. After 10 minutes of incubation with luciferin, micewere placed in a right lateral decubitus position, and adigital gray scale animal image was acquired, followed byacquisition and overlay of a pseudocolor image represent-ing the spatial distribution of detected photons emergingfrom active luciferase within the animal. Signal intensitywas quantified as the sumof all detected photonswithin theregion of interest per second per steradian. Mice wereimaged on days 0, 7, 14, 21, and 28 of treatment. Therapywas continued for 4 weeks, and the mice were sacrificed 1week later. Primary tumors in the cecum were excised, andthefinal tumor volumewasmeasured asV¼2/3pr3,where ris themean of the 3 dimensions (length, width, and depth).The number ofmetastasis colonies was counted in the liver,intestines, lungs, rectum, and spleen. Each tumor excisedfrom mice was divided into 3 parts. The first part of thetumor tissue was formalin-fixed, second part fixed in opti-mum cutting temperature (OCT) and third part was snapfrozen in liquid nitrogen and stored at�80�C.Hematoxylinand eosin staining confirmed the presence of tumors in eachcecum.

NF-kB activation in colon cancer cellsTo assess NF-kB activation, we isolated nuclei from the

colon cancer cell lines and conducted the electrophoreticmobility shift assay (EMSA) essentially as previouslydescribed (29).

Immunolocalization of NF-kB p65, b-catenin, VEGF,and MMP-9 in tumor samplesThe nuclear localization of p65 and the expressions of

MMP-9 and VEGF were examined using an immunohisto-chemical method described previously (28). Pictures werecaptured with a Photometrics CoolSNAP CF color camera(Nikon) and MetaMorph version 4.6.5 software (UniversalImaging).

Ki-67 immunohistochemistryFrozen sections (5 mm) were stained with anti-Ki-67

(rabbit monoclonal clone SP6; NeoMarkers) antibody aspreviously described (28). Results for Ki-67–positive cellsare shown as �40 magnification. A total of ten �40 fieldswere examined from 3 tumors of each of the treatmentgroups.

Microvessel densityFrozen sections (5 mm) were fixed in cold acetone and

stained with rat anti-mouse CD31 monoclonal antibody(Pharmingen) as previously described (28). The CD31-stained slides were observed under Leica DM4000B fluo-

rescence microscope (Leica Microsystems, Inc.) equippedwith a SPOT-RTKE digital camera, and the images wereacquired and stored using SPOT advanced software (Diag-nostic Instruments).

Protein extraction and Western blot analysisColorectal tumor tissues (75–100 mg) from control and

experimentalmice wereminced and incubated on ice for 30minutes in 0.5 mL of ice-cold whole-cell lysis buffer. Theminced tissue was homogenized using a Dounce homog-enizer and centrifuged at 16,000� g at 4�C for 10 minutes.The proteins were then fractionated by SDS-PAGE, electro-transferred to nitrocellulose membranes, blotted with eachantibody, and detected by enhanced chemiluminescence(Amersham Pharmacia Biotech).

HPLC analysisThe extraction of ursolic acid was conducted as described

by Chen and colleagues (30). Frozen tissue (100 mg) wasthawed, 400 mL of ethanol was added, and the mixture washomogenized for 30 seconds. Next, 500 mL of water wasadded, and the solution was homogenized for 15 seconds.Then, 500 mL of hexane was added, and the solution wasagain homogenized for 15 seconds. The samples werecentrifuged at 5,000 rpm for 5 minutes at 4�C. The organiclayer was evaporated to dryness under purified air at 40�C.The pellets were reconstituted with mobile phase and esti-mated by HPLC (Waters Inc.). A silica-based C18 columnand a mobile phase that was the mixture of acetonitrile:methanol:acetic acid (10%) in a ratio of 45:45:10were usedfor separation. Analyses were conducted at a flow rate of 0.4mL/min with the detector operating at a wavelength of 291nm. Tissues from vehicle-treated mice were set as controlsthat reflect the baseline.

Combination effects of ursolic acid and capecitabineAssessment of synergistic drug combination treatments

between ursolic acid and capecitabine were evaluated usingMTT assays on HCT116 cells. A total of 4.0� 103 cells wereplated in triplets and treated with ursolic acid alone (0.25�IC50, 0.5� IC50, 1� IC50, 2� IC50, 4� IC50), capecitabinealone (0.25� IC50, 0.5� IC50, 1� IC50, 2� IC50, 4� IC50),and ursolic acid in combination with capecitabine at fixedratio (Supplementary Table S1). After incubation, 5.0 mg/mL MTT reagent was added into each well, incubated for 2hours in the dark at 37�C and then MTT lysis buffer wasadded as described previously (28). The absorbance wasmeasured at 570 nm using an MRX Revelation 96-wellmultiscanner (Dynex Technologies).

Statistical analysisFor the effect of ursolic acid on cell proliferation over

time, wemodeled the percentage of viable cells using linearmixed effects models with fixed effects as dose, day ofmeasurement, and their interaction and (replicated) cellgrowth as random effects. In cases where the fixed effectswere statistically significant, pairwise comparisons betweendoses (within each time point) were conducted by






comparing the least square means. Multiple comparisonadjustments were made using the Bonferroni method.Similarly, we examined the effect of ursolic acid and cape-citabine on tumor growth of nude mice over time using alinear mixed model with fixed effects as ursolic acid orcapecitabine, day of measurement, and interactionsbetween treatment and day (other 2-way and 3-way inter-actions were not significant), along with random effect ofmice nested within treatment group. Repeated measure-ments over time were modeled using an (first order) auto-regressive covariance structure.

To model the effects of ursolic acid and capecitabine onapoptosis,metastatic score, and tumorweight of nudemice,we first modeled the outcomes using linear models withfixed effects as ursolic acid and capecitabine, and theirinteraction. Pairwise comparisons were conducted as abovewith multiple comparison adjustments made using theBonferroni’s method. When multiple cell lines were tested,we examined each cell line separately.

For all linear models, we examined the normalityassumption of the residuals using Q–Q plots and usedtransformation to approximate normality where thisassumption was violated (e.g., for tumor weight we usedsquare-root transformation). We computed the incidencerate of ascites with exact 95% confidence interval. Thedifferences in ascites incidence rate among treatment groupswere compared using Fisher exact test.

Assessment of the type of drug interactions wasdone using Chou and Talalay method. Assuming that adose-effect curve follows Chou and Talalay median effect

equation, E ¼�

dDm

�m

1þ�

dDm

�m, where d is the dose of a drug, Dm is

the median effective dose of the drug, and m is a slopeparameter. The above equation can be rewritten as

log E1�E ¼ m log d� logDm

� � ¼ b0 þ bl log d. We regress

log E1�E on logd to get a marginal dose-effect curve. Then

the dose level to achieve certain effect can be estimated foreach of the 3 regimens (drug 1 alone, drug 2 alone, and thecombinationof drugs 1 and2with constant relative potencybetween them). Suppose that combination dose (d1,d2)elicits the same effect y as drug 1 alone at dose Dy,1, anddrug 2 alone at dose Dy,2, then the interaction index (II) as

defined by the Loewe additivity model (31), II ¼ d1Dy;1

þ d2Dy;2

,

which measures the magnitude of drug interaction. Thevalue of II is estimated by the above equation and thecorresponding 95% confidence interval is computed usingthe delta method (32) to calculate the variance of II. Thevalue of II < 1, II ¼ 1, and II >1 correspond to the druginteractions’ being synergistic, additive, and antagonistic,respectively. Statistical software SAS 9.1.3 (SAS) and S-Plus8.0 (TIBCO Software Inc.) were used for all the analyses.

ResultsOur goal in the current study was to determine whether

ursolic acid can affect the growth and metastasis of CRCand enhance the effect of capecitabine, which is used

routinely to treat CRC, and if so, to determine the mech-anism by which ursolic acid manifests its effects in vitroand in vivo against human CRC. We used 3 different well-characterized human colon cancer cell lines (HCT116,HT29, and Caco2) that exhibit distinct characteristics. Tofacilitate the monitoring of tumor growth in animals,HCT116 was stably transfected with the luciferase report-er gene and used in an orthotopic transplant model inmice.

Ursolic acid inhibits the proliferation of CRC cells invitro

We first determined whether ursolic acid inhibits theproliferation of human CRC cells. We used one cell linewith mutant K-ras (HCT116) and 2 cell lines with wild-type K-ras (HT29 and Caco2). We found that ursolic acidsignificantly (P < 0.001) inhibited cell proliferation in all3 cell lines in a dose- and time-dependent manner. HT29and Caco2 cells were found to be more sensitive thanHCT116 cells as indicated by their IC50 value (HT29,8.7 mmol/L; Caco2, 6.2 mmol/L, and HCT116, 9.8 mmol/L;Fig. 1A, right).

Ursolic acid alone and in combination withcapecitabine downregulates the expression of proteinslinked to survival, proliferation, andmetastasis in CRCcells

We also examined whether ursolic acid alone or incombination with capecitabine can downregulate theexpression of proteins associated with survival, prolifera-tion, invasion, and metastasis. Our results showed thatursolic acid inhibited the expression of CRC survival (c-FLIP, Bcl-2, survivin, Bcl-xL), proliferation (cyclin D1), andmetastasis (MMP-9, VEGF, ICAM-1) proteins in dose-dependent manner (Fig. 1B, left). When ursolic acid wastreated in combination with capecitabine, the effect wasprominent (Fig. 1B, right). However, induction of cFLIP,Bcl-2, VEGF, and ICAM-1 was observed by capecitabinealone treatment.

Ursolic acid inhibits the constitutive action of NF-kB inCRC cells

Because NF-kB activation has been closely associatedwith proliferation of tumor cells (33) and CRC is knownto express constitutively active NF-kB, we investigatedwhether ursolic acid inhibits this transcription factor incolon cancer cells. Therefore, we conducted the DNA-bind-ing assay to determine whether ursolic acid inhibits con-stitutive NF-kB activation. We found that ursolic acidinhibited NF-kB activation in a dose-dependent manner(Fig. 1C).

Ursolic acid suppresses colony formationNext, we determinedwhether ursolic acid could affect the

long-term colony formation assay. We found that ursolicacid treatment significantly suppressed the colony-formingability of HCT116 tumor cells in a dose-dependent manner(Fig. 1D, left).

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Ursolic acid sensitizes CRC cells to apoptosis bycapecitabineWe next investigated whether ursolic acid can enhance

the effect of capecitabine-induced apoptosis in all 3 celllines. For this, we conducted the Live/Dead apoptosis assay.Our linear models showed that there was significant inter-action between the 2 drugs for all 3 cell lines. The ursolicacid significantly enhanced capecitabine-induced apoptosison all 3 cell lines (P value for interaction term, <0.001; Fig.1D, right). These observations together suggested that urso-lic acid has potential in the treatment of CRC. Therefore wedesigned in vivo studies.

Ursolic acid synergistically enhances capecitabine-induced cytotoxicityOn the basis of MTT assay and Chou and Talalay median

effect results, it was found that ursolic acid was able to

reduce the viability of HCT116 cells to a higher extent whenused in combination with capecitabine. Drug interactionindex (II) analysis indicates synergistic effects for all com-binations where ursolic acid and capecitabine were usedequal to or higher than IC50 concentration. However, addi-tive effects were observed where ursolic acid and capecita-bine were used less than IC50 concentration (Supplemen-tary Table S1).

Ursolic acid inhibits the growth of human CRC in anorthotopic nude mouse model

On the basis of these results, we decided to determinewhether ursolic acid affects the growth of CRC, alone and incombination with capecitabine. For this, we used orthoto-pically implanted human HCT116 cancer cells in nudemice. The experimental protocol is depicted in Fig. 2A.Luciferase-transfected HCT116 cells were implanted in the

Figure 2. Ursolic acid enhances theability of capecitabine to inhibit thegrowth of orthotopically implantedCRC tumors in nude mice. A,schematic representation of theexperimental protocol described inMaterials and Methods (n ¼ 6). B,bioluminescence imaging oforthotopically implanted CRC in live,anesthetized mice (lef) every week,showing measurements (photons/s)of tumor volume at various time pointusing live bioluminescence imaging(BLI) at the indicated times (right) �,significant over control at each timepoints [Bonferroni Adj (for a total of 15comparisons) P < 0.05]. C, tumorvolumes measured on the last day ofthe experiment at autopsy usingVernier calipers and calculated usingthe formula V ¼ 2/3 pr3 (n ¼ 6). �,significant [Bonferroni Adj (for 3comparisons) P < 0.05) over vehiclecontrol. D, average tumor weight ofeach group weighed on the last dayof the experiment. �, significant[Bonferroni Adj (for 3 comparisons)P < 0.05] over vehicle control.






colorectal part of nude mice. After 1 week, on the basis ofthe initial IVIS image, we randomly assigned the mice to 4groups (6 mice per group) and began the treatment as perthe experimental protocol. The treatment was continuedfor 4 weeks, and the mice were sacrificed 6 weeks aftertumor cell injection. The IVIS imaging was conductedweekly after tumor implantation (Fig. 2B, left). Figure 2B(right) shows the bioluminescence imaging of tumorvolume over time. Results showed a gradual increase intumor volume in the control group (Fig. 2B, right) com-pared with the remaining groups. Overall, there wassignificant day and treatment-by-day effect (type III test,P < 0.0001). However, the differences in IVIS imagingwere not significant on day 0 and day 7. On day 14, 21,and 28, the differences between the control and thecapecitabine group and between the control and ursolicacid þ capecitabine group were significant. Figure 2Cshowed the effect of capecitabine and ursolic acid ontumor volumemeasured on the last day of the experimentat autopsy. Capecitabine significantly reduced tumor vol-ume (P ¼ 0.0335). There was a trend that ursolic acid alsoreduced tumor volume, but the effect was not statisticallysignificant (P ¼ 0.11). Tumor volume of the mice treatedwith ursolic acid plus capecitabine was significantly smal-ler than that of the vehicle control group. The linearmodel showed that capecitabine significantly decreasestumor weight (P ¼ 0.021). The effect of ursolic acid is notstatistically significant (P ¼ 0.12), possibly due to smallsample size. Compared with the vehicle control, tumorweight of the combination treatment was significantlysmaller [Bonferroni adjusted (for 3 comparisons), P ¼0.027; Fig. 2D)].

Ursolic acid inhibits distant organ metastasis andascites

We also evaluated whether ursolic acid alone or in com-bination with capecitabine can inhibit ascites formationand distant organ metastasis in nude mice. We monitoredthe animals every day for ascites tumors by observing theincrease in belly size. Vehicle-treatedmice developed ascitestumors 27 days after tumor cell implantation, and thepresence of ascites tumor was confirmed at the time ofdeath. There were no statistically significant differences inascite incidence rate among treatment groups (Fisher exacttest P¼ 0.57) due to small sample size (Fig. 3A). On day 42,mice were sacrificed and examined for the presence ofmetastases. The examination showed that colon cancermetastases developed in the liver, intestines, lungs, andspleen of vehicle-treated mice. Ursolic acid alone inhibitedmetastasis to some extent, as did capecitabine in mostorgans; however, when they were used in combination,the inhibition of metastasis was maximal in these mice(Fig. 3B).

Ursolic acid is bioavailable in serum and CRC tumortissue

Because ursolic acid inhibited tumorigenesis in nudemice, we therefore investigated whether ursolic acid is

bioavailable in the serum and the CRC tumor of themice. HPLC results showed that a significant amount ofursolic acid was available in the serum of the micetreated with ursolic acid alone (480 ng/mL) or withursolic acid plus capecitabine (477 ng/mL). The presenceof capecitabine did not affect the level of ursolic acidconcentrations in serum. Ursolic acid was also found inCRC tumors (Fig. 4A). Although the amount of ursolicacid in CRC tumor was lesser than that in the serum ofmice, it was comparable. Purified ursolic acid was used asa control.

Ursolic acid inhibits CD31 and Ki-67 expressionThe Ki-67–positive index is used as a marker for cell

proliferation, and the CD31 index is a marker for micro-vessel density. We examined whether ursolic acid andcapecitabine can modulate these biomarkers. We foundthat both ursolic acid alone and capecitabine alone

Figure 3. Ursolic acid potentiates capecitabine to inhibit ascites anddistant organ metastasis in orthotopic CRC in nude mice. A ursolic acidcombinedwith capecitabine inhibited the development of ascites inCRC.The number of mice with ascites was counted on the day of experimenttermination, and the percentages of mice with ascites tumors wereplotted. B, combined ursolic acid and capecitabine inhibited metastasisto the liver, intestines, lungs, and spleen. Mice were sacrificed, andabdomenswere opened surgically. The number ofmetastatic foci in eachorganwas then counted. �, significant [Bonferroni Adj (for 3 comparisons)P < 0.05] over vehicle control (V, vehicle; UA, ursolic acid; CAP,capecitabine).

Prasad et al.





downregulated the expression of Ki-67 in CRC tissue andthe combination of the 2 was more effective (Fig. 4B).Similarly, we found that both agents alone reduced CD31expression, compared with the control group, and thecombined ursolic acid plus capecitabine was the mosteffective (Fig. 4B). The differences in expression of Ki-67and CD31 between each of the 3 active treatments and thevehicle control were statistically significant (Bonferroniadjusted P value, <0.007).

Ursolic acid inhibits NF-kB in CRC tumorsNF-kB is known to be overexpressed in CRC tumors

(34). Therefore, we determined whether ursolic acidcould inhibit activated NF-kB in the CRC tumors. Wefound that ursolic acid inhibited activated NF-kB in thesetissues and that combined ursolic acid plus capecitabinewas more effective (Fig. 5A). The differences in expres-

sion of NF-kB between each of the 3 active treatmentsand the vehicle control were statistically significant[Bonferroni adjusted (for all 6 pairwise comparisons),P value <0.001).

Ursolic acid inhibits b-catenin expression in CRCtumors

Overexpression of nuclear b-catenin in CRC is knownand found to be strongly associated with metastasis (35).Therefore, we investigated to determine whether ursolicacid inhibits the expression of b-catenin in tumors.We observed that the treatment of ursolic acid resultedin suppression of b-catenin. The combination of ursolicacid and capecitabine treatment results in greater inhi-bition of b-catenin than each agent alone [Bonferroniadjusted (for all 6 pairwise comparisons) P value<0.01; Fig. 5B].

Figure 4. A, ursolic acid isbioavailable in the serumof nudemiceand in CRC tumors. Animals were fedwith ursolic acid 4 hours before beingsacrificed. Mice were anesthetizedand blood was collected by cardiacpuncture, and then the mice weresacrificed humanely. HPLCanalysis was conducted todetermine ursolic acid bioavailabilityin the serum and CRC tumors. B,immunohistochemical analysis of theproliferation marker Ki-67 and CD31microvessel density in tumor tissue.Percentages indicate positive stainingfor the given biomarker.






Ursolic acid suppressed the activation of transcriptionfactor STAT3

Next, we examined whether ursolic acid modulatesthe activation of STAT3 overexpression because STAT3is found to be constitutively active (36) in CRC tumors.We found that ursolic acid and capecitabine alone mod-erately suppressed the activated STAT3 by inhibitingphosphorylation at Tyr705; however, ursolic acid incombination with capecitabine markedly inhibited itsphosphorylation (Fig 5C, left). Thus, inhibition of thistranscription factor by ursolic acid could be associatedwith enhancement of capecitabine-induced CRC tumorregression.

Ursolic acid suppressed the activation of EGFRMost of the CRCs are characterized with overexpression

of EGFR and predicted with high risk of metastasis andrecurrence (37). Targeting EGFR seems to be a promisingapproach for the CRC treatment. In present experiment,moderate inhibition of EGFR activation was observed byeither ursolic acid alone or capecitabine alone or by com-bination (Fig 5C, right).

Ursolic acid inhibits the expression of variousbiomarkers

We also examined the expression of various biomarkersinCRC tumor samples byWesternblot (Fig. 5D). The results

showed that ursolic acid alone or capecitabine suppressedthe expression of cyclin D1, c-myc (involved in prolifera-tion), Bcl-2, Bcl-xL, and survivin (involved in cell survival).Ursolic acid in combination with capecitabine inhibitedproteins like cyclin D1, c-myc, and Bcl-2 more profoundlythan either drug alone (Fig. 5D). However, inhibition ofsurvivin and Bcl-xL was not prominent in combinationcompared with either ursolic acid alone or capecitabinealone.

Immunohistochemical analysis showed reductions inthe expression of MMP-9 and VEGF (involved in invasionand metastasis) in tumors from the ursolic acid alonegroup (Bonferroni adjusted P value ¼ 0.053) and thosefrom the capecitabine alone group (Bonferroni adjustedP value <0.001) compared with the control group (Fig. 6A).Results also indicated that the combination of ursolicacid and capecitabine was more effective than treatmentwith either ursolic acid alone [Bonferroni adjusted (for all6 pairwise comparisons) P value <0.001] or capecitabinealone (Bonferroni adjusted P value ¼ 0.053). Westernblotting results also confirmed that expression of proteinsinvolved in invasion and metastasis like MMP-9, VEGF,and ICAM-1 decreased by either drug compared with thecontrol treatment. However, in combination with capeci-tabine, decrease of MMP-9, VEGF, and ICAM-1 expressionby ursolic acid was more prominent compared with eitheragent in CRC tumor tissues (Fig 6B).

Figure 5. Ursolic acid enhances theability of capecitabine to inhibitthe expression of NF-kB andgenes linked to proliferation,invasion, angiogenesis, andmetastasis of CRC tumors.Immunohistochemical analysis ofnuclear p65 (A) and b-catenin (B) intumor tissues. Percentagesindicate positive staining for thegiven biomarker. C, Western blotanalysis of phospho-STAT3 (left)and EGFR phosphorylation (right).D, Western blot analysis ofproteins involved in proliferation(cyclin D1and c-myc), cell survival(Bcl-2, Bcl-xL, and survivin). Threesamples were taken from eachgroup.

Prasad et al.





Ursolic acid upregulates tumor suppressor proteinsThe activation of oncogenes and inactivation of tumor

suppressor genes have been implicated in the developmentof many human and animal malignancies. Therefore, wedetermined whether ursolic acidmodulated p53.We foundthat p53 and its downstream target p21 was upregulated byeither ursolic acid or capecitabine treatment. Combinationof ursolic acid and capecitabine enhance the level of p53and p21 (Fig 6C).

DiscussionAlthough capecitabine as a chemotherapeutic drug is

frequently used in patients with stage III CRC after surgeryto remove their tumors, this agent has limited efficacy andhas side effects (38). Therefore, in this study,we investigatedwhether ursolic acid derived from various fruits and vege-tables has potential either alone or in combination withcapecitabine in the treatment of CRC.Whether examined incell culture or in animal model, we found that ursolic acidalone inhibited the growth of CRC and enhanced theactivity of capecitabine. When examined for the mecha-nism, we found that ursolic acid inhibited several transcrip-tion factors such as NF-kB, STAT3, and b-catenin activationand various biomarkers linked to survival, proliferation,and metastasis. We also observed that ursolic acid poten-tiated capecitabine-induced cell death.This is the first report to show that ursolic acid can

suppress CRC growth and enhances the effect of capecita-bine in an orthotopic mouse model. Whether ursolic acid

affects the concentration of capecitabine or its analog is notknown at present. However, ursolic acid is known to inhibitcytochrome P450 1A2 activities in human livermicrosomes(39). Although one study reported that ursolic acidenhanced the postirradiation responses to gamma radiationand decreased undesirable radiation damage to hemato-poietic tissue after radiotherapy (9), this study did notdescribe the mechanism of action. In our study, the inhi-bition of tumor growth by ursolic acid alone or in combi-nation with capecitabine seemed to be because of thesuppression of multiple biomarkers. Ursolic acid inhibitedproliferative proteins, cyclin D1 and Ki-67, indicating oneof the probable mechanisms of the antitumorigenic poten-tial of ursolic acid. EGFR overexpression has also beenshown in some of the CRC tumor cells (40), which wasdownregulated by ursolic acid in the present study. Previ-ously Shan and colleagues (41) showed that ursolic acidsuppressed the phosphorylation of EGFR, ERK1/2, p38MAPK, and JNK in colorectal cancer cells, which was cor-related with its growth inhibitory effect.

We also found that ursolic acid alone can inhibit CRCmetastasis to different organs and also suppress CRC-induced ascites. The inhibition of metastasis could be dueto the suppression ofMMP-9 andVEGF.Ursolic acid indeeddecreased angiogenesis, as indicated by inhibition of CD31,a marker for microvessel density, and VEGF. It has beenreported earlier that ursolic acid inhibited tumor-associatedcapillary formation by reducing VEGF, and proinflamma-tory cytokines in C57BL/6 mice induced by highly meta-static B16F-10 melanoma cells (42). Ursolic acid can also

Figure 6. A, ursolic acid enhancesthe effect of capecitabine against theexpression of MMP-9, and VEGF inCRC samples analyzed byimmunohistochemistry. Samplesfrom 3 animals in each group wereanalyzed, and representative dataare shown. Percentages indicatepositive staining for the givenbiomarker. B, Western blot analysisof proteins involved in invasion,angiogenesis, and metastasis(ICAM-1, VEGF and MMP-9). C,ursolic acid induced the expressionof tumor suppressor p53 and p21.Tissue extracts were subjected toWestern blotting using respectiveantibodies. Samples from 3 animalsin each group were analyzed, andrepresentative data are shown.






downregulate MMP-9, which is in agreement with previousreports (18). Inhibition of metastasis by ursolic acid mayalso be due to CXCR4, a chemokine receptor closely linkedto metastasis and which has been reported to be down-regulated by ursolic acid (43).

Our finding that ursolic acid downregulated expressionsof MMP-9 and ICAM-1, which are involved in tumorinvasion, were significantly suppressed in tumor tissuesfrom ursolic acid -treated mice. It has been shown thatMMP-9 and ICAM-1 are overexpressed in patients with CRC(44, 45), and it has also been observed that negativeMMP-9expression levels correlate with longer survival time inpatients with CRC (44). Finally, our results showed that,inCRC, ursolic acid inhibited several antiapoptotic proteinsregulated by NF-kB, including survivin, Bcl-2, Bcl-xL, andcIAP-1. These results suggest that ursolic acid enhances theapoptotic effect of capecitabine by inhibiting antiapoptoticproteins in CRC cells.

Overall, our results suggest that ursolic acid has signif-icant potential for the treatment of CRC, and it canfurther enhance the effects of capecitabine by inhibitingNF-kB and associated biomarkers that are involved inproliferation, angiogenesis, invasion, and metastasis. Onthe basis of these results, further studies are required inpatients to explore the potential of ursolic acid as ananticancer agent.

Disclosure of Potential Conflicts of InterestDr. B.B. Aggarwal is the Ransom Horne, Jr, Professor of Cancer Research.

No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: S. Prasad, S. Reuter, R. Kannappan, S. Krishnan, S.Guha, Bharat B. AggarwalDevelopment of methodology: S. Prasad, V.R. Yadav, S. Reuter, Bharat B.AggarwalAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): V.R. Yadav, P. Diagaradjane, Bharat B. AggarwalAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): C. WeiWriting, review, and/or revision of the manuscript: S. Prasad, V. Bala-dandayuthapani, S. Guha, Bharat B. AggarwalAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S. Guha, Bharat B. AggarwalStudy supervision: Bharat B. AggarwalHelped to perform experiments: B. SungProvided technical help in tumor implantation, bioluminscence imag-ing, tumor collection, and ingeneral animal handlingduring the in vivoexperiment: A.A. DeorukhkarOrganized the imaging schedule and helped in the data acquisition ofin vivo bioluminescence imaging: P. Diagaradjane

AcknowledgmentsThe authors thank Michael Worley and the Department of Scientific

Publications for carefully editing the manuscript, and Dr. Juri G. Gelovani,Department of Experimental Diagnostic Imaging, for providing biolumi-nescence imaging system (IVIS) 200 facility.

Grant SupportThis work was supported by a grant from Center for Targeted Therapy of

MD Anderson Cancer Center. Veera Baladandayuthapani’s and CaimiaoWei’s work is supported in part by the NI H through MD Anderson CancerCenter’s Support Grant CA016672.

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 to indicatethis fact.

Received October 31, 2011; revised July 3, 2012; accepted July 17, 2012;published OnlineFirst July 25, 2012.

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2012;18:4942-4953. Published OnlineFirst July 25, 2012.Clin Cancer Res Sahdeo Prasad, Vivek R. Yadav, Bokyung Sung, et al. Cell Signaling Pathways: Chemosensitization with CapecitabineCancer in an Orthotopic Nude Mouse Model by Targeting Multiple Ursolic Acid Inhibits Growth and Metastasis of Human Colorectal

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