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Research Article

Omega-3 Fatty Acid Is a Potential Preventive Agent forRecurrent Colon Cancer

Anita Vasudevan1, Yingjie Yu1,2, Sanjeev Banerjee3,4, James Woods1, Lulu Farhana1,2, Sindhu G. Rajendra1,Aamil Patel1, Gregory Dyson3, Edi Levi1,4, Krishna Rao Maddipati3,4,5, Adhip P.N. Majumdar1,2,3, andPratima Nangia-Makker1,2,3

AbstractIncreasing evidence supports the contention that many malignancies, including sporadic colorectal

cancer, are driven by the self-renewing, chemotherapy-resistant cancer stem/stem-like cells (CSC/CSLC),

underscoring the need for improved preventive and therapeutic strategies targeting CSCs/CSLCs.

Omega-3 polyunsaturated fatty acids (w-3 PUFA), have been reported to inhibit the growth of primary

tumors, but their potential as a preventive agent for recurring cancers is unexplored. The primary

objectives of this investigation are (i) to examine whether eicosapentaenoic acid (EPA; one of the w-3PUFA) synergizes with FuOx (5-FUþOxaliplatin), the backbone of colon cancer chemotherapy, and

(ii) whether EPA by itself or in combination with conventional chemotherapy prevents the recurrence of

colon cancer via eliminating/suppressing CSCs/CSLCs. FuOx-resistant (chemoresistant; CR) colon

cancer cells, highly enriched in CSCs, were used for this study. Although EPA alone was effective,

combination of EPA and FuOx was more potent in (i) inhibiting cell growth, colonosphere formation,

and sphere-forming frequency, (ii) increasing sphere disintegration, (iii) suppressing the growth of

SCID mice xenografts of CR colon cancer cells, and (iv) decreasing proinflammatory metabolites in

mice. In addition, EPA þ FuOx caused a reduction in CSC/CSLC population. The growth reduction by

this regimen is the result of increased apoptosis as evidenced by PARP cleavage. Furthermore, increased

pPTEN, decreased pAkt, normalization of b-catenin expression, localization, and transcriptional activity

by EPA suggests a role for the PTEN–Akt axis and Wnt signaling in regulating this process. Our data

suggest that EPA by itself or in combination with FuOx could be an effective preventive strategy for

recurring colorectal cancer. Cancer Prev Res; 7(11); 1138–48. �2014 AACR.

IntroductionCancer stem/stem-like cells (CSC/CSLC), that are self-

renewing undifferentiated cells, are thought to be one of theleading causes of cancer recurrence. In the colon, they areidentified by specific surface epitopes such as CD44,CD166,CD133, andESA (epithelial-specific antigen; refs. 1,2). Like normal stem cells, CSCs/CSLCs grow slowly and aremore likely to survive chemotherapy than other tumor cells(2–5). This is exemplified by the observation that oxalipla-tin treatment of colon cancer boosts the abundance of CSCsby more than 10 times (3). We have also reported that

although exposure of colon cancer HCT-116 or HT-29 cellsto FuOx (5-FUþOxaliplatin) inhibits their growth, thesame treatment leads to enrichment of CSC/CSLC pheno-type (4, 5). These chemoresistant cells show an increasedcolonosphere formation, Wnt–b-catenin signaling, EGFRsignaling, increased expression of miR21, and decreasedmiR145 (6, 7).

Omega 3- and 6-polyunsaturated fatty acids (w-3 and -6PUFA) are substantial components of the diet, comprisingabout 7% to 10% of daily energy intake in U.S. adults(reviewed in ref. 8). A meta-analysis by the World CancerResearch Fund and the American Institute for CancerResearch in 2007 reported that although no definitivecorrelations could be drawn, there was suggestive evidencethat dietary fish (main source ofw-3 PUFAs) intake protectsagainst colorectal cancer risk in humans (9). Additionalsupport came from clinical observations (10, 11), suggest-ing its significance as a chemopreventive agent.

The current investigation examines the potential of w-3PUFA as an effective preventive agent for recurrent colontumors that are reported to be enriched in CSCs/CSLCs.Two main w-3 PUFAs, eicosapentaenoic acid (EPA) anddocosahexaenoic acid (DHA) have been isolated from fish

1Veterans Affairs Medical Center, Wayne State University, Detroit, Michigan.2Department of Internal Medicine,Wayne StateUniversity, Detroit,Michigan.3Karmanos Cancer Institute, Wayne State University, Detroit, Michigan.4Department of Pathology, Wayne State University, Detroit, Michigan.5Lipidomics Core Facility, Wayne State University, Detroit, Michigan.

Corresponding Authors: Adhip P.N. Majumdar, Wayne State University,4646 John R. Street, Detroit, MI 48201. Phone: 313-576-4460; Fax: 313-577-1112; E-mail: [email protected]; and Pratima Nangia-Makker,[email protected].

doi: 10.1158/1940-6207.CAPR-14-0177

�2014 American Association for Cancer Research.

CancerPreventionResearch

Cancer Prev Res; 7(11) November 20141138

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Published OnlineFirst September 5, 2014; DOI: 10.1158/1940-6207.CAPR-14-0177

http://cancerpreventionresearch.aacrjournals.org/

oil. Recent evidence has demonstrated that EPA and DHAreduce inflammation in humans (12, 13) and may haveantineoplastic properties (14–16). Animal studies haverevealed that EPA and, to a lesser extent, DHA reducedVEGF expression andmicrovessel formation (17). Recently,Fan and colleagues (18) demonstrated a stimulatory role ofw-6 PUFA–derived PGE2 on Lgr5þ stem cell population inthe colonic crypts. In contrast, w-3 PUFA derived PGE3 haddiminished ability to support stem cell expansion (18).Hawcroft and colleagues recently showed an inhibition ofliver metastasis in mice that received dietary EPA (19).However, there are no reports on the antineoplastic activityof this PUFA on recurrent colon cancer. The current inves-tigation was undertaken to examine the preventive andtherapeutic potential of EPA alone or when administeredtogether with the conventional chemotherapy on chemo-therapy-resistant colon cancer HT-29 and HCT-116 cells.Herein, we report that EPA alone or in combination withFuOx could be effective in prevention of recurrent coloncancer.

Materials and MethodsCell lines and reagentsHuman colon cancer cells HT-29 and HCT-116 were

obtained from the American Type Culture Collection(ATCC). They were expanded and frozen in aliquots. Freshaliquots were used every 6 to 7 months; therefore, the celllines were not authenticated again. The cells were main-tained in DMEM as reported (5, 20). FuOx-resistant (che-moresistant; CR) cellswere generated as described earlier (5,6, 21) in our laboratory by exposing the cells to 14 conse-cutive cycles of exposure to increasing concentrations of 5-FU and oxaliplatin.Unless otherwise stated, the CR cells were cultured in

medium containing 2� FuOx (50 mmol/L 5-FU and 1.25mmol/L oxaliplatin).

Determination of cell growth and interaction betweenEPA and FuOxCell growth was assessed by mitochondrial-dependent

reduction of 3-(4,5-dimethylthiazol-2yl)-2, 5-diphenylte-trazolium bromide (MTT; Sigma) to formazan as describedpreviously (22). Briefly, the cells (5 � 103) were seeded inquadruplicates onto 96-well culture dishes and subsequent-ly treated with increasing concentrations of EPA and/orFuOx for 48 hours to determine the synergism betweenEPA and FuOx.

Colonosphere formation and disintegrationFormation of colonospheres and their disintegration in

response to EPA and/or FuOx were carried out according toour standard protocols described previously (5, 23).Spheres of size �80 mm [measured by a 100-mm scale(reticule) in the eyepiece] were counted after 14 days.

Extreme limiting dilution analysisExtreme limiting dilution analysis (ELDA) was per-

formed by the method described by Hu and Smyth (24)

with a slight modification (7, 23). Briefly, cells were pre-treated with EPA and/or FuOx for 72 hours, subsequentlyplated at a concentration of 100, 10, and 1 cell per well, andincubated for 8days. The frequencyof sphere formationwasdetermined using ELDA webtool at http://bioinf.wehi.edu.au/software/elda.

Flow cytometryAfter 48-hour incubation in the absence (control) or

presence of EPA and/or FuOx, CR-HT-29 colon cancer cellswere subjected to direct immunofluorescence stainingwith PE-Cy7- or PerCP-Cy5–conjugated anti-human CD44and/or CD166 antibody followed by flow cytometric anal-yses using a FACSDiva (BDBiosciences) at theMICR core ofKarmanos Cancer Institute of Wayne State University asdescribed previously (5). The cells stained with IgG2b(isotype-negative control) served as gating control. Theproportion of CD44þ/CD166þ/low cells was determined onthe basis of fluorescence intensity spectra.

Indirect immunofluorescenceThe cells were seeded at a density of 25,000 per chamber

in an 8-chamber slide. After treatment with EPAþ FuOx for48 hours, the cells were fixed, permeabilized, and processedfor indirect immunofluorescence as described (25) usingappropriate antibodies. Stained cells were observed underan Olympus 1�71 microscope supporting a Hamamatsu1394 ORCA-ERA video camera and the images were storedusing Slidebook Digital Microscopy Software (IntelligentImaging Innovations). For controls, the primary antibodywas omitted.

Western blot analysisWestern blots were performed according to our standard

protocol (26). Briefly, cell lysates containing 25 or 50 mgprotein were separated by SDS-polyacrylamide gel electro-phoresis, transferred onto a polyvinylidene difluoride(PVDF) membrane (Millipore) and subjected to Westernblot analysis with the recommended dilution of primaryand appropriate secondary antibody conjugated to IR Dye680 or IR Dye 800 (Molecular Probes). The membraneswere scanned byOdyssey Infrared Imaging System (LI-CORBiosciences) to locate the respective bands. b-Actin orGAPDH was used as a loading control.

Isolation of RNA and quantitative polymerase chainreaction analysis

Total RNA was extracted from CR cells using the TRIzolreagent (Invitrogen) according to the manufacturer’sinstructions. RNA concentration was measured using aNanoDrop 2000C spectrophotometer.

Quantitative reverse transcription-polymerase chainreaction (qRT-PCR) was performed using the GeneAmpRNA PCR Kit (Applied Biosystems). Briefly, 1 mg of purifiedRNA was reverse transcribed (5, 27). For quantitative PCRamplification, 5 mL of 1:10 diluted cDNA was amplifiedwith SYBR Green Quantitative PCR Master Mix (AppliedBiosystems) using the following PCR primers: CK20

EPA Prevents Colon Cancer Recurrence

www.aacrjournals.org Cancer Prev Res; 7(11) November 2014 1139




forward: 50-TGAAGAGCTGCGAAGTCAGA-30 and reverse:50-GAAGTCCTCAGCAGCCAGTT-30; b-catenin: forward 50-ATACCACCCACTTGGCAGAC-30; reverse 50-GGAAGGTC-TCCTTGGGACTC-30; sequences for stem-cell markers werereported earlier (27). Reactions were carried out in tripli-cates as described previously (5).

TCF/LEF transcriptional activityThe activation of transcription factor TCF/LEF was eval-

uated by using the Cignal TCF/LEF Reporter Assay Kit (SABiosciences) as described (7, 23). The cells were grown to70% to 80% confluence and cotransfected with TCF/LEFreporter constructs using Lipofectamine 2000 transfectionreagent (Invitrogen) according to the manufacturer’sinstructions. After 16 to 24 hours, the cells were trypsinized,seeded into 12wells of a 96-well plate in DMEM containing10%FBS in the presence of EPA and/or FuOx. After 2 days ofincubation, they were collected and analyzed for TCF/LEFactivity using a Dual-Luciferase Assay Kit (Promega Bios-ciences) following the manufacturer’s instructions asdescribed (7, 23). Activity of TCF/LEF was calculated inrelation to positive control.

Tumor growth in SCID miceAll animal experiments were performed according to the

Wayne State University’s Institutional Animal Care andUseCommittee (IACUC) approved protocol #A02-02-13. Ani-mal Welfare Assurance #A3310-01.

Tumors were generated in 4-week-old female SCID mice(Taconic Laboratory) by s.c. injections of 1 � 106 CR HCT-116 or CR HT-29 cells suspended in 100 mL Matrigel oneither side. To study the chemopreventive efficacy of EPA,animals were given EPA (250 mg/kg in sesame oil) by oralgavage 7 days before inoculation of chemoresistance coloncancer cells. The dose of EPA was selected on the basis ofprevious studies (17, 28, 29). To study the therapeuticeffectiveness, EPAwas administered 7days after inoculationof the cells. EPA treatment was continued for 4 weeks everyday for 5 days a week (Monday to Friday). The animals inthis group (control and EPA group) were also injected i.p.with a mixture of 25 mg/kg 5-FU and 2 mg/kg oxaliplatin(FuOx) once a week for 3 weeks. Tumor volumes werecalculated as described previously (20, 23). Mice weremonitored regularly. At the end of treatment period, allanimals were sacrificed, blood was collected immediatelyfrom the heart in a tube containing 50 mL of 80 mmol/LEDTA, centrifuged, and saved at �70�C. The tumors wereharvested and tumor aliquots were frozen for RNA isola-tion, fixed in 10% buffered formalin or immediatelydigested with enzymes for single-cell isolation (23, 27).

Eicosanomic analysisMass spectrometry–based eicosanomic analysis for eico-

sanoids derived from both arachidonic acid and EPA wasperformed on the plasma extracts collected from mice asdescribed earlier (30, 31). Briefly, plasma samples werespiked with a mixture of internal standards (5 ng each ofPGE1-d4, LTB4-d4, and 15-HETE-d8), dilutedwithmethanol

to 15%, and applied to C18 solid phase extraction car-tridges, washed sequentially with 15% methanol in waterand hexane, followed by elution of the eicosanoids withmethanol containing 1% formic acid. The eluates wereevaporated to dryness and reconstituted in HPLC mobilephase for LC/MS analysis.

Eicosanomic analysis was performed by LC/MS usingLuna C18 column (3 mm, 2 � 150 mm; Phenomenex) forHPLC resolution of the eicosanoids and detected byQTRAP5500 mass analyzer (ABSCIEX) using optimizedconditions for each eicosanoid by Multiple Reaction Mon-itoring (MRM) method as described before (31). LC/MSchromatograms were analyzed by MultiQuant (ABSCIEX)for quantitation of each eicosanoid and normalized to theinternal standard signal. Under the standard conditions ofthemethod, the detection limits formost of the eicosanoidswere <2 pg on the column with a signal/ratio of 3.

Statistical analysisUnless otherwise stated, data were expressed as mean �

SD. Where applicable, the results were compared by usingthe unpaired, two-tailed Student t test, as implemented byExcel 2007 (Microsoft). P values smaller than 0.05 wereconsidered statistically significant.

ResultsEPA synergizes with FuOx

The data obtained from synergy analysis of EPA- and/orFuOx-treated CRHT-29 cells revealed that cells treated withthe combined dosage are 6.05 times (P¼ 0.009)more likelyto die than those treated with FuOx alone. This was calcu-latedby the difference in intercepts of the groups in a logisticregressionmodel with data from EPA and FuOx each alone,or in combination (Fig. 1A), assuming a combined slope.The data clearly show synergism between the two.

EPA or EPA þ FuOx treatment inhibits stem-cellcharacteristics in CR cells

To analyze whether EPA and/or FuOx would affect theproperties of colon CSCs/CSLCs, the ability of CR HT-29and CR HCT-116 cells to form colonospheres was exam-ined. ELDAperformedonCRHT-29 cells demonstrated thatthe frequency to form colonosphereswas 139-fold higher incontrol group, compared with 20 mmol/L EPA þ FuOx–treated cells (Fig. 1B).

With respect to formation of colonospheres, EPA alone at10 and 20 mmol/L caused a significant inhibition in CRHCT-116 cells, which was further exacerbated when com-bined with FuOx (Fig. 2A and B). Interestingly, CR HT-29cells appeared to bemore resistant to the EPA at 10 mmol/L,but in the presence of combination treatment, a significantinhibition in colonosphere formation was observed. EPA at20 mmol/L was found to be as effective as the combinatorialtreatment.

In addition, the combined treatment of EPA and FuOxalso induced disintegration of colonospheres, especially inCR HCT-116 cells. We observed that combined treatmentswere significantly more effective than FuOx or EPA alone at

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Cancer Prev Res; 7(11) November 2014 Cancer Prevention Research1140




10 and 20 mmol/L for differentiation and disintegration ofspheres formed by CR HCT-116 cells (Fig. 2C). Interesting-ly, EPA þ FuOx treatment did not affect sphere disintegra-tion anymore than FuOx alone in CRHT-29 cells (Fig. 2D),indicating a cell line specificity.Flow cytometric analysis revealed a reduction in the pro-

portion of CD44þ/CD166low CSC phenotype in CR HT-29cells in response to 10 mmol/L EPA and FuOx, comparedwith the corresponding control (Fig. 3Aa-Af). While theuntreated controls contained 5.12%CD44þ/CD166low phe-notype, FuOx increased theproportionofCD44þ/CD166low

to 7.34%. This increase could be the result of enrichment ofFuOx-resistant phenotype. However, EPA normalized thisphenotype to 5.37%, and the combined treatment furtherreduced it to 2.71%.qPCR analysis of 10 mmol/L EPA and FuOx-treated CR

HCT-116 cells showed a downregulation of stem cell mar-kers CD44, ALDH1, CD133, and b-catenin, and an upre-

gulation of epithelial marker CK20 compared with FuOxalone (Fig. 3B), indicating that EPA þ FuOx affects thecolonic CSC population.

EPA þ FuOx treatment retards tumor growth in SCIDmice

Palpable tumors were formed by 7 to 10 days and grewlinearly in control animals. EPA pretreatment for 7 daysbefore inoculation of CR cells and subsequent continuationreduced the growth, which revealed about 50% reduction atthe end of the 18-day treatment period (Fig. 4A). Thecombination of EPAþ FuOxwas found to bemore effectivethan EPA alone. The tumor size in EPA þ FuOx–treatedgroupwas significantly smaller than the control at 27 and34days (Fig. 4B). CR HT-29 cell xenografts showed a slowgrowth initially, but after 27 days a growth spurt was seen incontrol FuOx-treated mice, whereas the EPA þ FuOx–trea-ted mice did not show any significant tumor growththroughout the 34-day postinjection period (Fig. 4C).Immunohistochemical staining of CR HT-29 xenograftshowed reduced cell proliferation following EPA/FuOxtreatment, as evidenced by low PCNA staining (data notshown).

Single-cell suspensions obtained from the tumors weresubsequently cultured in stem cell medium to examine forcolonosphere formation. The cells, isolated from the EPA/FuOx–treated xenografts formed only a few spheres (Fig.4D), strengthening our observation that EPA/FuOx combi-natorial treatment inhibits the growth of CSCs/CSLCs.

Quantitative real-time PCR performed on RNA isolatedfrom tumor as well as from the CR HT-29 cells showed asignificant increase in CK20 mRNA levels in EPA þ FuOx–treated tumors and cells, indicating an increased number ofdifferentiated cells (Fig. 4E).

The EPA þ FuOx–mediated growth inhibition could bedue to induction of apoptosis asWestern blot analysis of theEPA þ FuOx–treated CR HT-29 cells showed an increasedPARP cleavage (Fig. 5A).

EPA þ FuOx treatment regulates b-catenin activity viaPTEN–Akt axis

To elucidate the regulatory mechanism(s) for EPA andFuOx-mediated inhibition of growth and development ofCSCs in chemoresistant colon cancer cells in vivo and invitro, we examined the localization and transcriptionalactivity of b-catenin, which is known to be activated incolorectal cancer (32) and plays a critical role in maintain-ing the growth and functional properties of colon CSCs (7).Indirect immunofluorescence staining showed b-catenin tobe primarily localized on the cell membrane in normal HT-29 cells (Fig. 5B top, long arrows). In contrast, CR HT-29cells showed nuclear localization of b-catenin in a numberof cells (Fig. 5B middle, short arrow). However, in EPA/FuOx–treated CR HT-29 cells, b-catenin was found to bemainly localized on the cell membrane (Fig. 5B, bottom).Western blot analysis showed a decreased expression ofb-catenin aswell as its target proteins c-myc and cyclinD1 inEPA þ FuOx–treated cells after 24 and 48 hours (Fig. 5C).

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Figure 1. A, dose–response curves for EPA and/or FuOx in CRHT-29 cellsproduced by fixed ratio method demonstrating synergism between EPAand FuOx. A, logistic growth curve was fitted using the R statisticalsoftware (URL http://www.R-project.org/) to the observed data pointswithin each of the three measurement groups: EPA alone, FuOx alone,and the combination. Concentrations of 5-FU/oxaliplatin in relation totheir X value are shown on the x-axis. Each treatment was performedin quadruplicates. B, ELDA of colonosphere forming frequency inresponse to EPA and/or FuOx in CR HT-29 cells.






EPA þ FuOx treatment also caused approximately 40%reduction in the transcriptional activity of TCF/LEF in CRcells (Fig. 5D). These data indicate that EPA þ FuOx treat-ment inhibits the nuclear localization of b-catenin, thuspreventing its transcriptional activity. In addition, dec-reased levels of ATP-binding cassette (ABC) transporterABCG-2 protein, which is involved with drug efflux(3, 33), indicate reduced drug resistance in EPA/FuOx–treated cells (Fig. 5C).

Although the precise mechanism(s) for EPA þ FuOx–mediated changes in b-catenin is not fully understood, wehypothesized that PTEN–Akt signaling may play a role inregulating this process. EGFR, which is known to be acti-vated in colorectal cancer leads to induction of the PTEN–Akt axis (27). In addition, we have reported that miR21-mediated induction of colon CSCs is associated with down-regulation and decreased phosphorylation of PTEN(decreased activation), leading to activation of Akt (34,35). Figure 4E demonstrates a marked increase in pPTENand decreased pAkt levels over the corresponding controlsin response to 20 mmol/L EPAþ FuOx (Fig. 5E). The dose of10 mmol/L EPA þ FuOx also caused reduction in theactivated (phosphorylated) form of Akt (Fig. 5E). The factthat EPA þ FuOx activates PTEN resulting in the decreased

Akt activity suggests that the PTEN–Akt axis is involved inmodulating Wnt–b-catenin signaling.

EPA þ FuOx treatment reduces the levels ofproinflammatory metabolites in SCID mice

Both arachidonic acid (AA) and EPA are metabolized invivo by the cyclooxygenase, lipoxygenase, and epoxygen-ase pathways to prostaglandins, hydroxy fatty acids aswell as leukotrienes, and epoxy fatty acids, respectively.To assess the PUFA metabolic changes in the animalstreated with EPA, we analyzed the plasma eicosanomicprofiles of both treated and untreated animals by LC/MS.The method included all possible metabolites of AA andEPA from all the three enzymatic pathways. Table 1 showsthe detected metabolites of both fatty acids in plasma. Theresults show a lower concentration of AA metabolites inEPA þ FuOx–treated animals compared with controlFuOx-treated mice and a significantly lower concentra-tion of inflammatory mediators, LTB4 (and its metabolite12-Oxo LTB4) and PGE2 (as well as its metabolites, 13,14-dihydro-15-keto PGE2 and bicyclo PGE2) in EPAþ FuOx–treated animals. Interestingly, a majority of EPA metabo-lites shows similar plasma concentrations between thetwo groups, despite the fact that EPA is a significantly

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Figure 2. Changes in formation anddisintegration of colonospheres inresponse to EPA (10 or 20 µmol/L)and or FuOx. ChemoresistantHT-29 or HCT-116 cells wereseeded at a density of 1,000 cellsper well in a 6-well dish. A and B,formation of colonospheres after14 days; C and D, disintegration ofcolonospheres; treatments withvarious regimen were started7 days after seedingB and D, photomicrographs ofrepresentative spheres usingOlympus CKX41 microscopesupporting Olympus DP72 cameraand stored with DP2-BSWsoftware. X400 Spheres �80 mmwere counted. Bars, mean of4–6 readings � SD. �, P < 0.005.

Vasudevan et al.





poorer substrate to the metabolic enzymes comparedwith AA (36).

DiscussionThe main objective of the current investigation was to

study the efficacy of EPA as an inhibitor of recurrent colo-rectal cancer growth and to determine whether EPA incombinationwith FuOxwould bemore effective than eitheragent/regimen alone.The preventive and therapeutic efficacy of a combination

of EPA and DHA or each PUFA alone has been demonstrat-ed in multiple preclinical studies using a variety of rodentmodels of early-stage colorectal cancer (37). These studieshave consistently demonstrated reduction in colorectalcancer incidence (reviewed in ref. 37). Our data demon-strate for the first time that EPA acts synergistically withFuOx to markedly inhibit the growth of chemoresistantcolon cancer cells that form bulk of the recurrent tumor.Although the underlying cause for tumor recurrence is not

fully understood, one of the reasons is thought to be thepresence of CSCs/CSLCs that are resistant to conventionalchemotherapy and retain limitless potential to regenerate(1–3, 38). The resistance of CSCs/CSLCs to therapy hasbeen attributed to a multitude of factors, including inc-reased expression of drug transporters and intracellulardetoxification enzymes, upregulation of antiapoptotic pro-teins, increased efficiency of DNA repair, and alterations incell kinetics (39). As the CR HT-29 and CR HCT-116 cellsexhibit increased stem-like characteristics, as evidenced byincreased colonosphere formation, increaseddrug efflux, anelevated expression of CSC/CSLC markers, higher tumori-genic potential in SCID mice, an increased Wnt–b-cateninand EGFR signaling (4–7), they provide a suitable model tostudy the efficacy of EPA and/or FuOx in inhibiting recur-ring colon cancer.

Our current observation that EPA causes a marked reduc-tion in colonosphere formation by CR HT-29 and CRHCT-116 cells, which is further exacerbated by the combinationof EPA and FuOx, suggests that this regimen not only

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Figure 3. Changes in CSCphenotype and expression of CSCmarkers CR HT-29 cells inresponse to EPA and/or FuOx. A,flow cytometric analysis of CD44þ

and CD166low phenotype (a–e); f,graphical representation of a–e;bars, mean of three readings� SD.�, P < 0.005. B, qRT-PCR showingmRNA levels of CSC anddifferentiation markers normalizedto b-actin. Bars, mean of threereadings � SD. �, P < 0.005.






inhibits proliferation of CSCs, but also their functionalproperties. Furthermore, the fact that the same combinationtreatment also induces disintegration of colonospheres inCRHCT-116 cells suggests that this treatment strategy couldbe used to eliminate/kill colon CSCs that have alreadyextravasated the primary tumor and entered the vascularsystem. In support of this contention, we have observed amarked reduction in the proportion of CD44þ/CD166low

CSC phenotype in EPA/FuOx–treated CR HT-29 cells and areduced expression of stem-cell markers, CD44, ALDH1,CD133, and b-catenin. Yang and colleagues recentlyreported that a combination of EPA andDHA exerts a directantiproliferative andproapoptotic effect on theCSLCsusingSW620 colon cancer cell line and increases their sensitivityto 5-FU (40). Likewise, our data show that although EPA iseffective in inhibiting growth of CSC/CSLC-enriched che-moresistant cells, the combination is even more effective.

An increased expression and activity of Akt has beenreported as an essential factor for cell survival during car-cinogenesis (41). Aberrant Akt activation is the result ofPI3K and PTEN downstream signaling (42), and, in turn,affects various pathways, including inactivation of GSK-3b,resulting in dysregulated canonical Wnt–b-catenin signal-ing (43, 44). Dysregulation of the Wnt–b-catenin pathwayhas been reported to play a pivotal role in the developmentand progression of colorectal cancer. Translocation ofb-catenin to the nucleus activates the transcription ofits target genes like cyclin D1, c-myc, MMP-7, MT1-MMP,

axin-1, etc. (45–47). We have reported that theWnt–b-cate-ninpathwayalsoplays a crucial role in regulating the growthand maintenance of colon CSCs (7). Although the precisemechanism by which EPA þ FuOx inhibits the growth ofchemoresistant colorectal cancer is not known, our currentobservation that EPA/FuOx greatly reduces b-catenin tran-scriptional activity suggests a crucial role for Wnt–b-cateninsignaling in regulating this event. Furthermore, the fact thatEPA þ FuOx activates PTEN resulting in decreased Aktactivity suggests that the PTEN–Akt axis is involved inmodulating Wnt–b-catenin signaling. Additional supportcomes from the observation that the combination of EPAand FuOx also decreases cyclin D1 and c-myc expression,two downstream effector molecules of the Wnt–b-cateninsignaling pathway, that are known to be involved in regu-lating cell proliferation. In addition, the combination ofEPA and FuOxwas found to reduce the expression of ABCG-2 protein, which has been implicated as a key regulator inthe maintenance of stem cells in various human cancer celllines (4, 33, 48) and contributes toward resistance to che-motherapy (3, 33). Downregulation of ABCG-2 in EPA/FuOx–treated cells indicates a reduction in drug effluxcapability resulting in its sensitization to chemotherapy.An increased apoptosis in EPA/FuOx–treated cells as indi-catedby reducedPARP cleavage further confirms the efficacyof combination therapy on viability of CR cells. These datastrongly suggest that EPA/FuOx treatment could be used totarget stem cell–enriched recurrent colorectal cancer.

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Figure 4. Inhibition of CR HCT-116(A and B) or CR HT-29 cells (B)xenografts in EPA and/or FuOx-treated SCID mice. A, EPA wasadministered for 7 days beforeinoculation of 1� 106 CR HCT-116cells and continued for the durationof the experiment. B and C, EPA þFuOx treatments were started 7days after CR cells' inoculation.Each data point representsaverage of eight tumors � SE.�, P > 0.05. D, colonosphereformation in the cells isolated fromCR HCT-116 and CR HT-29xenografts X100. E, qRT-PCR onRNA from tumors of CR HT-29 andCR HCT-116 cells and CR HT-29cells.

Vasudevan et al.





Data generated from our in vivo studies using SCID micexenograft model of colon cancer also support the in vitroobservations. When SCID mice bearing xenografts of CRHCT-116 and CRHT-29 cells were administered with eitherEPA or EPAþ FuOx, the tumor growth was greatly reduced.In fact, no significant increase in growth of xenografts by CRHT-29 was observed following administration of EPA andFuOx. Xenografts formed by CR HCT-116 cells weredecreased by at least 50% following EPA or the combina-torial treatment. The fact that EPA by itself reduced thegrowth indicates its chemopreventive efficacy. This is sup-portedby EPA’s ability to inhibit colonosphere formation invitro, indicating decreased number of CSCs/CSLCs. The factthat these changes were more apparent following combi-nation treatment, also suggests that EPA could be used forpreventive as well as therapeutic purposes.The reduction in tumor growth could be attributed to

decrease in tumor cell proliferation, as evidenced bydecreased PCNA staining in the treated xenograft (data notshown). Furthermore, our observation that the expression

of CK-20, a marker of differentiation, is greatly increased incells isolated from SCID xenografts of chemoresistant cellsfollowing EPA/FuOx treatment strongly suggests that thecurrent combination therapy induces differentiation lead-ing to increased sensitivity to the combination of EPA/FuOxtreatment strategy.

Eicosanomic analysis of plasma fromEPA/FuOx–treatedanimals offers a possibility of modulating inflammatoryresponse in these animals. Both AA and EPA are metabo-lized by the same enzymes to highly physiologically activelipid mediators such as prostaglandins, leukotrienes, andhydroxy and epoxy fatty acids. However, metabolites of AA(an w-6 PUFA) are proinflammatory, whereas thosederived from EPA (an w-3 PUFA) are anti-inflammatoryand participate in active resolution of inflammation (49–51). The eicosanomic profile (Table 1) shows a lowerconcentration of AA metabolites in EPA/FuOx–treatedanimals. Concentration of about 75% of the AA metabo-lites detected was less than 50% in EPA/FuOx–treatedanimals, whereas all EPA metabolites are above 50% or

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Figure5. Inhibition ofWnt/b-cateninsignaling and the PTEN–Akt axis inCR HT-29 cells in response to EPAþ FuOx. A,Western blot analysis ofcleaved PARP in CR HT-29 cellstreated with 10 mmol/L EPA/2�FuOx for 24 and 48 hours. B,indirect immunofluorescencestaining showing membrane (longarrow) and nuclear (short arrow)localization of b-catenin. Counterstaining was done with DAPI (blue)�100. C, Western blot analysis ofCR HT-29 cells treated with 10mmol/L EPA/2� FuOx for 24 and 48hours. 50 mg total protein wasloaded in each lane. b-Actin wasused as loading control. Lines,removal of a band. D,transcriptional activity of TCF/LEFin EPA/FuOx–treated cells. Bars,mean of three readings � SD.�, P < 0.005. E, Western blotanalysis of pPTEN and pAkt in EPAþ FuOx–treated CR HT-29 cells.b-Actin was used as loadingcontrol.






nearly equal between the two groups. Although EPA meta-bolites are expected to be higher in the animals fedwith thefatty acid, it has a 2 to 3 fold metabolic disadvantagecompared with AA (36). Moreover, the apparent suppres-sion of AA metabolites in EPA treatment is not uniformacross all lipid mediators. Although inflammatory media-tors such as LTB4 and PGE2 are lower in EPA-treatedanimals, 5-OxoETE, another neutrophil chemotactic lipidmediator of the 5-lipoxygenase pathway, is similar in bothgroups. It is noteworthy that 5-OxoETE is increased incancer cells under oxidative stress (52). On the other hand,

the anti-inflammatory lipid mediators of the epoxygenasepathway, e.g., 11(12)-EpETE (53), are elevated upon EPAtreatment (Table 1). Although it is difficult to concludeon the limited sample size data presented from thispilot study, the data offer intriguing possibility that EPAtreatment alters the balance of pro- and anti-inflamma-tory lipid mediators toward an improved outcome ofchemotherapy.

In summary, the present data indicate the EPA could be apotential preventive and therapeutic treatment modalityfor recurrent colon cancer, which is known to be highly

Table 1. Concentrations of eicosanoids in control and EPA þ FuOx–treated mouse plasma

Eicosanoid Control (FuOx) EPA þ FuOx % of Control

6-keto PGF1a 48.4 � 11.7 20.4 � 3.8 42TXB2 78.9 � 54.9 17.1 � 0.7 22PGE2 24.0 � 8.4 7.7 � 2.1 3213,14-dihydro-15-keto PGE2 7.9 � 5.2 2.2 � 1.2 27Bicyclo PGE2 21.1 � 18.0 5.0 � 2.8 2413,14-dihydro-15-keto PGF2a 4.6 � 2.5 1.8 � 0.9 3913,14-dihydro-15-keto PGD2 9.4 � 6.9 2.2 � 1.1 23PGJ2 65.4 � 33.2 33.7 � 17.7 525(6)-EpETrE 17.2 � 9.0 5.8 � 1.9 348(9)-EpETrE 17.8 � 9.9 5.0 � 1.0 2811(12)-EpETrE 47.1 � 22.8 14.8 � 3.3 3111,12-DiHETrE 2.8 � 1.4 1.7 � 0.8 6214(15)-EpETrE 19.4 � 16.7 6.4 � 1.4 3314,15-DiHETrE 4.3 � 2.9 3.1 � 1.0 73LTB4 8.4 � 3.5 2.0 � 0.3 2412-Oxo LTB4 4.1 � 1.4 0.1 � 0.1 35-HETE 28.7 � 9.8 14.9 � 2.1 525-oxo ETE 2.7 � 0.6 2.5 � 0.6 908-HETE 33.8 � 14.2 9.8 � 0.5 299-HETE 12.2 � 4.0 4.4 � 0.1 3611-HETE 109.8 � 49.9 33.8 � 6.2 3112-HETE 1629.0 � 855.4 340.4 � 144.8 2112-Oxo ETE 5.1 � 2.5 2.0 � 0.8 3915-HETE 42.5 � 20.2 15.4 � 1.2 3615-Oxo ETE 1.1 � 0.4 0.7 � 0.2 668(9)-EpETE 2.3 � 1.5 1.2 � 0.4 5311(12)-EpETE 1.3 � 1.0 1.4 � 0.1 11014(15)-EpETE 2.2 � 1.7 2.0 � 0.6 9017(18)-EpETE 2.9 � 1.7 1.6 � 0.4 555-HEPE 4.3 � 1.7 3.5 � 0.6 808-HEPE 4.2 � 1.6 2.9 � 0.7 699-HEPE 7.2 � 3.7 4.0 � 2.0 5511-HEPE 5.0 � 2.3 3.1 � 0.4 6112-HEPE 112.1 � 67.9 60.4 � 40.0 5415-HEPE 6.4 � 2.4 4.0 � 0.7 6318-HEPE 3.9 � 1.4 3.2 � 0.6 80

NOTE: All values are ng/mL plasma (mean � SEM; n ¼ 3). Metabolites of EPA are italicized.Abbreviations: DiHETrE, dihydroxyeicosatrienoic acid; EpETrE, epoxyeicosatrienoic acid; EpETE, epoxyeicosatetraenoic acid; ETE,eicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid; HEPE, hydroxyeicosapentaenoic acid; LT, leukotriene; PG, prostaglandin;TX, thromboxane.

Vasudevan et al.





enriched in chemotherapy-resistant CSCs/CSLCs, as evi-denced by the reduction in stem cell characteristics suchas colonosphere formation and increased disintegration,decreased number of CD44þ/CD166low cells, decreasedexpression of stem cell markers, increased CK20 levels, andreduction indrug efflux in vitro and in vivo. These changes areassociated with inhibition of the PTEN–Akt axis leading toreduced Wnt–b-catenin signaling, induction of apoptosis,and reduction in the proinflammation markers.

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

Authors' ContributionsConception anddesign:Y. Yu, E. Levi, A.P.N.Majumdar, P. Nangia-MakkerDevelopment of methodology: Y. Yu, S. Banerjee, K.R. Maddipati,P. Nangia-MakkerAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): A. Vasudevan, S. Banerjee, J. Woods, S.G. Rajen-dra, A. Patel, K.R. Maddipati, P. Nangia-Makker

Analysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): A. Vasudevan, L. Farhana, S.G. Rajendra,G. Dyson, E. Levi, K.R. Maddipati, P. Nangia-MakkerWriting, review, and/or revision of themanuscript: A. Vasudevan, Y. Yu,E. Levi, K.R. Maddipati, A.P.N. Majumdar, P. Nangia-MakkerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): E. Levi, A.P.N. MajumdarStudy supervision: A.P.N. Majumdar, P. Nangia-Makker

Grant SupportThis study was supported by grants from the NIH (AG014343 to A.P.N.

Majumdar) and theDepartment of Veteran Affairs (I101BX001927 to A.P.N.Majumdar). Eicosanomic analysis was supported in part by a grant fromNational Center for Research Resources, NIH (S10RR027926 to K.R.Maddipati).

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.

ReceivedMay 30, 2014; revised August 1, 2014; accepted August 20, 2014;published OnlineFirst September 5, 2014.

References1. Dick JE. Stem cell concepts renew cancer research. Blood 2008;

112:4793–807.2. Jordan CT, Guzman ML, Noble M. Cancer stem cells. N Engl J Med

2006;355:1253–61.3. Dean M, Fojo T, Bates S. Tumour stem cells and drug resistance. Nat

Rev Cancer 2005;5:275–84.4. Kanwar SS, Yu Y, Nautiyal J, Patel BB, Padhye S, Sarkar FH, et al.

Difluorinated-curcumin (CDF): a novel curcumin analog is a potentinhibitor of colon cancer stem-like cells. Pharm Res 2011;28:827–38.

5. Yu Y, Kanwar SS, Patel BB, Nautiyal J, Sarkar FH, Majumdar AP.Elimination of colon cancer stem-like cells by the combination ofcurcumin and FOLFOX. Transl Oncol 2009;2:321–8.

6. Yu Y, Sarkar FH, Majumdar AP. Down-regulation of miR-21induces differentiation of chemoresistant colon cancer cells andenhances susceptibility to therapeutic regimens. Transl Oncol2013;6:180–6.

7. Kanwar SS, Yu Y, Nautiyal J, Patel BB, Majumdar AP. The Wnt/beta-catenin pathway regulates growth andmaintenanceof colonospheres.Mol Cancer 2010;9:212.

8. Azrad M, Turgeon C, Demark-Wahnefried W. Current evidence linkingpolyunsaturated fatty acids with cancer risk and progression. FrontOncol 2013;3:224.

9. Research WCrFAIfC. Food, nutrition, physical activity, and the pre-vention of cancer: a global perspective. Washington DC; 2007.

10. Kim S, Sandler DP, Galanko J, Martin C, Sandler RS. Intake ofpolyunsaturated fatty acids and distal large bowel cancer risk in whitesand African Americans. Am J Epidemiol 2010;171:969–79.

11. Pot GK, Geelen A, van Heijningen EM, Siezen CL, van Kranen HJ,Kampman E. Opposing associations of serum n-3 and n-6 polyunsat-urated fatty acids with colorectal adenoma risk: an endoscopy-basedcase-control study. Int J Cancer 2008;123:1974–7.

12. Ebrahimi M, Ghayour-Mobarhan M, Rezaiean S, Hoseini M, ParizadeSM, Farhoudi F, et al. Omega-3 fatty acid supplements improve thecardiovascular risk profile of subjects with metabolic syndrome,including markers of inflammation and auto-immunity. Acta Cardiol2009;64:321–7.

13. Micallef MA, Garg ML. Anti-inflammatory and cardioprotective effectsof n-3 polyunsaturated fatty acids and plant sterols in hyperlipidemicindividuals. Atherosclerosis 2009;204:476–82.

14. Clarke RG, Lund EK, Latham P, Pinder AC, Johnson IT. Effect ofeicosapentaenoic acid on the proliferation and incidence of apoptosisin the colorectal cell line HT29. Lipids 1999;34:1287–95.

15. Courtney ED,Matthews S, Finlayson C, Di Pierro D, Belluzzi A, Roda E,et al. Eicosapentaenoic acid (EPA) reduces crypt cell proliferation and

increases apoptosis in normal colonic mucosa in subjects with ahistory of colorectal adenomas. Int J Colorectal Dis 2007;22:765–76.

16. Latham P, Lund EK, Johnson IT. Dietary n-3 PUFA increases theapoptotic response to 1,2-dimethylhydrazine, reduces mitosis andsuppresses the induction of carcinogenesis in the rat colon. Carcino-genesis 1999;20:645–50.

17. Calviello G, Di Nicuolo F, Gragnoli S, Piccioni E, Serini S, Maggiano N,et al. n-3 PUFAs reduce VEGF expression in human colon cancer cellsmodulating the COX-2/PGE2 induced ERK-1 and -2 and HIF-1alphainduction pathway. Carcinogenesis 2004;25:2303–10.

18. Fan YY, Davidson LA, Callaway ES, Goldsby JS, Chapkin RS. Differ-ential effects of 2- and 3-series E-prostaglandins on in vitro expansionof Lgr5 þcolonic stem cells. Carcinogenesis 2014;35:606–12.

19. Hawcroft G, Volpato M, Marston G, Ingram N, Perry SL, Cockbain AJ,et al. The omega-3 polyunsaturated fatty acid eicosapentaenoic acidinhibits mouse MC-26 colorectal cancer cell liver metastasis viainhibition of PGE2-dependent cell motility. Br J Pharmacol 2012;166:1724–37.

20. Yu Y, Kanwar SS, Patel BB, Oh PS, Nautiyal J, Sarkar FH, et al.MicroRNA-21 induces stemness by downregulating transforminggrowth factor beta receptor 2 (TGFbetaR2) in colon cancer cells.Carcinogenesis 2012;33:68–76.

21. Nautiyal J, Kanwar SS, Yu Y, Majumdar AP. Combination of dasatiniband curcumin eliminates chemo-resistant colon cancer cells. J MolSignal 2011;6:7.

22. Nangia-Makker P, Tait L, ShekharMP,PalominoE,HoganV,PiechockiMP, et al. Inhibition of breast tumor growth and angiogenesis by amedicinal herb: Ocimum gratissimum. Int J Cancer 2007;121:884–94.

23. Nangia-Makker P, Yu Y, Vasudevan A, Farhana L, Rajendra SG, Levi E,et al. Metformin: a potential therapeutic agent for recurrent coloncancer. PLoS ONE 2014;9:e84369.

24. Hu Y, Smyth GK. ELDA: extreme limiting dilution analysis for compar-ing depleted and enriched populations in stem cell and other assays.J Immunol Methods 2009;347:70–8.

25. Nangia-Makker P, Wang Y, Raz T, Tait L, Balan V, Hogan V, et al.Cleavage of galectin-3 by matrix metalloproteases induces angiogen-esis in breast cancer. Int J Cancer 2010;127:2530–41.

26. Nangia-Makker P, Raz T, Tait L, Shekhar MP, Li H, Balan V, et al.Ocimum gratissimum retards breast cancer growth and progressionand is a natural inhibitor of matrix metalloproteases. Cancer Biol Ther2013;14:417–27.

27. Nautiyal J, Kanwar SS, Majumdar AP. EGFR(s) in aging and carcino-genesis of the gastrointestinal tract. Curr Protein Pept Sci 2010;11:436–50.






28. Uygur R, Aktas C, Tulubas F, Uygur E, Kanter M, Erboga M, et al.Protective effects of fish omega-3 fatty acids on doxorubicin-inducedtesticular apoptosis and oxidative damage in rats. Andrologia 2013Oct 10. [Epub ahead of print].

29. Zugno AI, Chipindo HL, Volpato AM, Budni J, Steckert AV, de OliveiraMB, et al. Omega-3 prevents behavior response and brain oxidativedamage in the ketamine model of schizophrenia. Neuroscience2014;259:223–31.

30. Maddipati KR, Zhou SL. Stability and analysis of eicosanoids anddocosanoids in tissue culture media. Prostaglandins Other Lipid Med-iat 2011;94:59–72.

31. Markworth JF, Vella L, Lingard BS, Tull DL, Rupasinghe TW, SinclairAJ, et al. Human inflammatory and resolving lipid mediator responsesto resistance exercise and ibuprofen treatment. Am J Physiol RegulIntegrative Comp Physiol 2013;305:R1281–96.

32. Fearon ER. Molecular genetics of colorectal cancer. Annu Rev Pathol2011;6:479–507.

33. An Y, Ongkeko WM. ABCG2: the key to chemoresistance in cancerstem cells? Expert Opin Drug Metab Toxicol 2009;5:1529–42.

34. RoyS,Majumdar AP. Signaling in colon cancer stemcells. JMol Signal2012;7:11.

35. Roy S, Yu Y, Padhye SB, Sarkar FH, Majumdar APN. Difluorinated-curcumin (CDF) restores PTEN expression in colon cancer cells bydown-regulating miR-21. PLoS ONE 2013;8:e68543.

36. Wada M, DeLong CJ, Hong YH, Rieke CJ, Song I, Sidhu RS, et al.Enzymes and receptors of prostaglandin pathways with arachidonicacid-derived versus eicosapentaenoic acid-derived substrates andproducts. J Biol Chem 2007;282:22254–66.

37. Cockbain AJ, Toogood GJ, Hull MA. Omega-3 polyunsaturated fattyacids for the treatment and prevention of colorectal cancer. Gut2012;61:135–49.

38. Boman BM, Huang E. Human colon cancer stem cells: a newparadigm in gastrointestinal oncology. J Clin Oncol 2008;26:2828–38.

39. AlisonMR, LinWR, LimSM,Nicholson LJ.Cancer stemcells: in the lineof fire. Cancer Treat Rev 2012;38:589–98.

40. Yang T, Fang S, Zhang HX, Xu LX, Zhang ZQ, Yuan KT, et al. N-3PUFAs have antiproliferative and apoptotic effects on humancolorectal cancer stem-like cells in vitro. J Nutr Biochem 2013;24:744–53.

41. Jiang BH, Liu LZ. PI3K/PTEN signaling in tumorigenesis and angio-genesis. Biochim Biophys Acta 2008;1784:150–8.

42. Testa JR, Bellacosa A. AKT plays a central role in tumorigenesis. ProcNatl Acad Sci U S A 2001;98:10983–5.

43. Kaler P, Godasi BN, Augenlicht L, Klampfer L. The NF-kappaB/AKT-dependent induction of Wnt signaling in colon cancer cells by macro-phages and IL-1beta. Cancer Microenviron 2009 Sep 25. [Epub aheadof print].

44. Vaish V, Sanyal SN. Role of Sulindac andCelecoxib in the regulation ofangiogenesis during the early neoplasm of colon: exploring PI3-K/PTEN/Akt pathway to the canonical Wnt/beta-catenin signaling.Biomed Pharmacother 2012;66:354–67.

45. Kolligs FT, Bommer G, Goke B. Wnt/beta-catenin/tcf signaling: acritical pathway in gastrointestinal tumorigenesis. Digestion 2002;66:131–44.

46. Polakis P. The many ways of Wnt in cancer. Curr Opin Genet Dev2007;17:45–51.

47. WuWK,Wang XJ, Cheng AS, Luo MX, Ng SS, To KF, et al. Dysregula-tion and crosstalk of cellular signaling pathways in colon carcinogen-esis. Crit Rev Oncol Hematol 2013;86:251–77.

48. Katayama R, Koike S, Sato S, Sugimoto Y, Tsuruo T, Fujita N.Dofequidar fumarate sensitizes cancer stem-like side population cellsto chemotherapeutic drugs by inhibiting ABCG2/BCRP-mediateddrug export. Cancer Sci 2009;100:2060–8.

49. Serhan CN, Savill J. Resolution of inflammation: the beginning pro-grams the end. Nat Immunol 2005;6:1191–7.

50. SerhanCN, ChiangN, VanDyke TE. Resolving inflammation: dual anti-inflammatory and pro-resolution lipid mediators. Nat Rev Immunol2008;8:349–61.

51. Serhan CN, Petasis NA. Resolvins and protectins in inflammationresolution. Chem Rev 2011;111:5922–43.

52. Grant GE, Rubino S, Gravel S, Wang X, Patel P, Rokach J, et al.Enhanced formation of 5-oxo-6,8,11,14-eicosatetraenoic acid by can-cer cells in response to oxidative stress, docosahexaenoic acid andneutrophil-derived 5-hydroxy-6,8,11,14-eicosatetraenoic acid. Carci-nogenesis 2011;32:822–28.

53. ZhangG, PanigrahyD,Mahakian LM,Yang J, Liu J-Y, Stephen LeeKS,et al. Epoxy metabolites of docosahexaenoic acid (DHA) inhibit angio-genesis, tumor growth, and metastasis. Proc Natl Acad Sci U S A2013;110:6530–35.


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2014;7:1138-1148. Published OnlineFirst September 5, 2014.Cancer Prev Res Anita Vasudevan, Yingjie Yu, Sanjeev Banerjee, et al. Colon CancerOmega-3 Fatty Acid Is a Potential Preventive Agent for Recurrent

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