First Evidence That g-Tocotrienol Inhibits the Growth of ... · in H. pylori–induced gastritis (11). Together, these ﬁndings implicate the involvement of NF-kB pathway in gastric

Cancer Therapy: Preclinical

First Evidence That g-Tocotrienol Inhibits the Growth ofHuman Gastric Cancer and Chemosensitizes It toCapecitabine in a Xenograft Mouse Model through theModulation of NF-kB Pathway

Kanjoormana A. Manu1, Muthu K. Shanmugam1, Lalitha Ramachandran1, Feng Li1, Chee Wai Fong3,Alan Prem Kumar1,2,6, Patrick Tan2,4,5, and Gautam Sethi1,2

AbstractPurpose: Because of poor prognosis and development of resistance against chemotherapeutic drugs, the

existing treatmentmodalities for gastric cancer are ineffective. Hence, novel agents that are safe and effective

are urgently needed. Whether g-tocotrienol can sensitize gastric cancer to capecitabine in vitro and in a

xenograft mouse model was investigated.

Experimental Design: The effect of g-tocotrienol on proliferation of gastric cancer cell lines was

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

binding assay, and gene expression by Western blotting. The effect of g-tocotrienol on the growth and

chemosensitization was also examined in subcutaneously implanted tumors in nude mice.

Results: g-Tocotrienol inhibited the proliferation of various gastric cancer cell lines, potentiated the

apoptotic effects of capecitabine, inhibited the constitutive activation of NF-kB, and suppressed theNF-kB–regulated expression of COX-2, cyclin D1, Bcl-2, CXCR4, VEGF, and matrix metalloproteinase-9 (MMP-9).

In a xenograft model of human gastric cancer in nude mice, we found that administration of g-tocotrienolalone (1mg/kg bodyweight, intraperitoneally 3 times/wk) significantly suppressed the growth of the tumor

and this effect was further enhanced by capecitabine. Both the markers of proliferation index Ki-67 and for

microvessel density CD31 were downregulated in tumor tissue by the combination of capecitabine and

g-tocotrienol. As comparedwith vehicle control, g-tocotrienol also suppressed theNF-kB activation and the

expression of cyclin D1, COX-2, intercellular adhesionmolecule-1 (ICAM-1), MMP-9, survivin, Bcl-xL, and

XIAP.

Conclusions: Overall our results show that g-tocotrienol can potentiate the effects of capecitabine

through suppression of NF-kB–regulated markers of proliferation, invasion, angiogenesis, and metastasis.

Clin Cancer Res; 18(8); 2220–9. �2012 AACR.

IntroductionGastric cancer remains one of the most common malig-

nancies and the second leading cause of cancer mortality

accounting for more than 600,000 deaths annually world-wide (1, 2). Chemotherapy constitutes an important treat-ment regimen for gastric cancers besides surgical resection(3). Unfortunately, only few patients experience completepathologic response to chemotherapeutic drugs like cape-citabine, mainly because of their resistance to chemother-apy (4). Hence, novel approaches to enhance the effects ofchemotherapeutic drugs and reduce their resistance areimperative.

Various lines of evidence suggest that the activation ofmaster transcription factor NF-kB and its regulated geneproducts play a pivotal role in growth, metastasis, andchemoresistance of gastric cancer. First, NF-kB is constitu-tively activated in human gastric cancer tissue and is asso-ciated with tumor progression (5). Second, it promotesgastric cancer growth by inhibiting apoptosis (6). Third,NF-kB mediates the induction of mitogenic gene productssuch as cyclin D1, which is overexpressed in human gastriccancer tissue and is inversely correlatedwith poor prognosis

Authors' Affiliations: 1Department of Pharmacology, Yong Loo Lin Schoolof Medicine, 2Cancer Science Institute of Singapore, National University ofSingapore; 3Davos Life Science Pte Ltd; 4Genome Institute of Singapore;and 5Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School,Singapore; and 6School of Anatomy, Physiology and Human Biology, TheUniversity of Western Australia, Crawley, Perth, Western Australia

Note: K.A. Manu and M.K. Shanmugam contributed equally to this work.

Corresponding Authors: Gautam Sethi, Department of Pharmacology,Yong Loo Lin School of Medicine, Cancer Science Institute of Singapore,National University of Singapore, 10 Medical Drive, Singapore 117456.Phone: 65-65163267; Fax: 65-68737690; E-mail: [email protected] andPatrick Tan, Cancer and Stem Cell Biology, Duke-NUS Graduate MedicalSchool, Singapore 169857. Phone: 65-65161783; Fax: 65-62212402;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-2470

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(8) April 15, 20122220

on February 24, 2020. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 20, 2012; DOI: 10.1158/1078-0432.CCR-11-2470

http://clincancerres.aacrjournals.org/

and poor survival (7). Fourth, NF-kB plays an importantrole in regulating the CXC motif receptor 4 (CXCR4; ref. 8)and COX-2 (9) that are associated with gastric cancermetastasis. Finally, it is well established that Helicobacterpylori, a well-known risk factor for gastric carcinoma, is apotent activator of NF-kB in gastric epithelial cells (10).Moreover, NF-kB pathway is also responsible for theincreased generation of several cell adhesion moleculesincluding intercellular adhesion molecule-1 (ICAM-1),whose expression is significantly correlatedwith an increasein H. pylori–induced gastritis (11). Together, these findingsimplicate the involvement of NF-kB pathway in gastriccancer and thus the agents that can modulate NF-kB andNF-kB–regulated gene products have enormous potentialfor the treatment of gastric cancer.One such agent derived from natural sources that can

have a great potential for gastric cancer prevention andtreatment is vitamin E constituent, g-tocotrienol derivedfrom palm oil and rice bran. Vitamin E family of com-pounds primarily consists of 4 tocotrienols and 4 tocopher-ols (12). However, while tocopherols had been intensivelystudied for their health benefits, many novel benefits oftocotrienols are only beginning to be brought to light byresearch in the last decade (13, 14). For instance, g-toco-trienol has been reported to suppress the proliferation of awide variety of tumor cells (15), including gastric (16–19),hepatocellular carcinoma (20), melanoma (21), breast(22), colorectal (23), and prostate (24). In vivomice studieshave shown that g-tocotrienol can suppress the growth ofbreast tumor (25), prostate (26), lung cancer and melano-ma (27) and also inhibit the growth of liver and pancreaticcancer either alone or in combination with chemothera-peutic drugs and radiation (28, 29). How g-tocotrienolmediates its anticancer effects is not completely understood,but the roles of various signaling cascades/kinases/tran-scription factors such as mitogen-activated protein kinases

(17), phosphoinositide 3-kinase (PI3K)/Akt (30), NF-kB(13), STAT3 (20), telomerase (31), PPAR-g (32), hypoxia-inducible factor-1a (33), b-catenin (23), EGF (24), andinhibitor of differentiation family proteins (34) have beenimplicated.

Although g-tocotrienol has been found to suppress pro-liferation, to inhibit invasion/migration, and induce apo-ptosis in human gastric cancer SGC-7901 cells (28–31), butits potential to act as a chemosensitizing agent in gastriccancer cell lines and xenograft models has never beenexplored before (16–19). Thus, in the present study, weinvestigated whether g-tocotrienol could sensitize humangastric cancer to capecitabine in vitro and in a xenograftmouse model. Our observations indicate for the first timethat g-tocotrienol can inhibit the proliferation of variousgastric cancer cells, enhanced capecitabine-induced apopto-sis, andpotentiated the antitumor activity of capecitabine inhuman xenograft gastric cancer model through the modu-lation of NF-kB and NF-kB–regulated gene products.

Materials and MethodsReagents

g-Tocotrienol with purity more than 97% was obtainedfromDavos Life Science.MTT, Tris base, glycine, NaCl, SDS,bovine serum albumin (BSA), b-actin antibody, and cornoil were purchased from Sigma-Aldrich. g-Tocotrienol wasdissolved in dimethylsulfoxide as a 10 mmol/L stock solu-tion and stored at 4�C for in vitro and in corn oil for in vivoexperiments, respectively. Further dilution was done in cellculture medium. RPMI-1640 media, FBS, 0.4% trypan bluevital stain, and antibiotic–antimycotic mixture wereobtained from Invitrogen. Antibodies against p65, matrixmetalloproteinase-9 (MMP-9), Bcl-2, Bcl-xL, COX-2, ICAM-1, cyclinD1, survivin,Mcl-1, VEGF, andXIAPwere obtainedfrom Santa Cruz Biotechnology. CXCR4 antibody wasobtained fromAbcam. CD31 antibody was purchased fromCell Signaling Technology. Ki-67 antibody was purchasedfrom BD Pharmingen, Inc. Goat anti-rabbit horseradishperoxidase (HRP) conjugate and goat anti-mouseHRPwerepurchased from Invitrogen. Capecitabine was obtainedfrom Duheng International Trading Company Ltd. anddissolved in sterile PBS on the day of use.

Cell linesThe gastric cancer cell lines SNU-5 and SNU-16 were

obtained from the American Type Culture Collection.MKN45 cells were obtained from JCRB (Japanese Collec-tion of Research Bioresources), Japan. All the gastric cancercell lines were cultured in RPMI-1640media supplementedwith 10% FBS, 100 units/mL penicillin, and 100 mg/mLstreptomycin.

Western blottingFor detection of various proteins, gastric cancer cells (2�

106 per mL) were treated with g-tocotrienol for differenttime intervals. The cells were then washed and extractedby incubation for 30 minutes on ice in 0.05 mL buffercontaining 20 mmol/L HEPES, pH 7.4, 2 mmol/L EDTA,

Translational RelevanceDespite advances in earlier detection and therapy for

gastric cancer, it still remains the second leading cause ofcancer death worldwide, killing more than 600,000people annually worldwide. Existing drugs lack efficacyand yet are highly toxic. For example, although capeci-tabine is used routinely in the treatment of gastric cancer,development of resistance to this treatment in thepatients is one of themajor problems. Thus agentswhichcan overcome the resistance and can enhance the effectof capecitabine are urgently needed. Through in vitro andin vivo experiments, we show for the first time thatg-tocotrienol, a vitamin E analogue, is one such agentthat can reduce the resistance and can potentiate theeffect of capecitabine against gastric cancer. Becauseg-tocotrienol is already in clinical trials, the present studymay form the basis of novel therapeutic options for thetreatment of patients with gastric cancer.

g-Tocotrienol Enhances the Effect of Capecitabine in Gastric Cancer

www.aacrjournals.org Clin Cancer Res; 18(8) April 15, 2012 2221




250 mmol/L NaCl, 0.1% NP-40, 2 mg/mL leupeptin, 2 mg/mL aprotinin, 1 mmol/L phenylmethylsulfonylfluoride(PMSF), 0.5 mg/mL benzamidine, 1 mmol/L DTT, and 1mmol/L sodium vanadate. The lysate was centrifuged, andthe supernatant was collected. Whole-cell extract protein(30 mg) was resolved on 12% SDS-PAGE, electrotransferredonto a nitrocellulose membrane, blotted with antibodiesagainst survivin, Bcl-2, Bcl-xL, cyclin D1, VEGF, procaspase-3, and PARP and then detected by chemiluminescence(ECL; GE Healthcare).

Cell proliferation MTT assayThe effect of g-tocotrienol on cell proliferation was deter-

mined by the MTT uptake method as described previously(20). The cells (5,000 per well) were incubated with g-toco-trienol in triplicate in a 96-well plate and then incubated forindicated time points at 37�C. An MTT solution was addedto eachwell and incubated for 2 hours at 37�C. A lysis buffer(20% SDS and 50% dimethylformamide) was added, andthe cells were incubated overnight at 37�C. The absorbanceof the cell suspension was measured at 570 nm by Tecanplate reader.

LIVE/DEAD assayTo investigate whether g-tocotrienol could potentiate the

apoptotic effects of capecitabine in gastric cancer cells, weused a LIVE/DEAD Cell Viability Assay Kit (Invitrogen),which is used to determine intracellular esterase activity andplasma membrane integrity. This assay uses calcein, a poly-anionic, green fluorescent dye that is retained within livecells, and a red fluorescent ethidium homodimer dye thatcan enter cells through damaged membranes and bind tonucleic acids but is excluded by the intact plasma mem-branes of live cells (20). Briefly, gastric cancer cells (5,000per well) were incubated in chamber slides, pretreated withg-tocotrienol for 4 hours, and treated with capecitabine for24 hours. Cells were then stainedwith the assay reagents for30 minutes at room temperature. Cell viability was deter-mined under a fluorescence microscope by counting live(green) and dead (red) cells.

Flow cytometric analysisTo determine the effect on the cell cycle, cells were

exposed to combination of g-tocotrienol for 4 hours andtreated with capecitabine for 24 hours. Thereafter cells werewashed, fixed with 70% ethanol, and incubated for 30minutes at 37�C with 0.1% RNase A in PBS. Cells werethen washed again, resuspended, and stained in PBS con-taining 25 mg/mL propidium iodide (PI) for 30 minutes atroom temperature. Cell distribution across the cell cyclewasanalyzed with a CyAn ADP flow cytometer (DakoCytomation).

Gastric tumor modelAll procedures involving animals were reviewed and

approved by NUS Institutional Animal Care and Use Com-mittee. Six-week-old athymic nu/nu female mice (AnimalResource Centre, Western Australia) were implanted sub-

cutaneously in the right flank with SNU-5 cells (3 � 106

cells/100 mL saline). When tumors have reached 0.25 cm indiameter, the mice were randomized into the followingtreatment groups (n ¼ 5 per group): (i) untreated control(corn oil, 100 mL daily); (ii) tocotrienol [1 mg/kg bodyweight, suspended in corn oil, intraperitoneal (i.p.)injection] 3 times/wk; (iii) capecitabine alone (60 mg/kgbodyweight, suspended in cornoil, twiceweekly by gavage);and (iv) combination (tocotrienol, 1 mg/kg body weight,suspended in corn oil, i.p. injection) 3 times/wk and cape-citabine (60 mg/kg body weight, suspended in corn oil,twice weekly by gavage). Therapy was continued for 4weeks, and the animals were euthanized 1-week later.Primary tumors were excised and the final tumor volumewas measured as V ¼ 4/3pr3, where r is the mean radius ofthe 3 dimensions (length, width, and depth). Half of thetumor tissue was fixed in formalin and embedded in par-affin for immunohistochemistry and routine hematoxylinand eosin (H&E) staining. The other half was snap frozen inliquid nitrogen and stored at �80�C.

Immunohistochemical analysis of gastric tumorsamples

Solid tumors from control and various treatment groupswere fixed with 10% phosphate-buffered formalin, pro-cessed, and embedded in paraffin. Sections were cut anddeparaffinized in xylene and dehydrated in graded alcoholand finally hydrated in water. Antigen retrieval was carriedout by boiling the slide in 10 mmol/L sodium citrate (pH6.0) for 30minutes. Immunohistochemistrywas carriedoutfollowing manufacturer instructions (DAKO LSAB kit).Briefly, endogenous peroxidases were quenched with 3%hydrogen peroxide. Nonspecific binding was blocked byincubation in the blocking reagent in the LSAB kit (Dako)according to the manufacturer’s instructions. Sections wereincubated overnight with primary antibodies as follows:anti-p65, anti-COX-2, anti-VEGF, anti-MMP-9, anti-Ki-67,and anti-CD31 (each at 1:100 dilutions). Slides were sub-sequently washed several times in TBS with 0.1% Tween 20andwere incubatedwith biotinylated linker for 30minutes,followed by incubation with streptavidin conjugate provid-ed in LSAB kit according to the manufacturer’s instructions.Immunoreactive species were detected with 3,30-diamino-benzidine (DAB) as a substrate. Sections were counter-stained with the Gill hematoxylin andmounted under glasscover slips. Images were taken with an Olympus BX51microscope (magnification, �20). Positive cells (brown)were quantitated with the ImagePro plus 6.0 softwarepackage (Media Cybernetics, Inc.).

Preparation of nuclear extract from gastric tumorsamples

Gastric tumor tissues (100 mg/mouse) from mice in thecontrol and treatment groups were minced and incubatedon ice for 30 minutes in 0.5 mL of ice-cold buffer A [10mmol/L HEPES (pH 7.9), 1.5 mmol/L KCl, 10 mmol/LMgCl2, 0.5 mmol/L dithiothreitol (DTT), and 0.5 mmol/LPMSF]. Theminced tissue was homogenized with aDounce

Manu et al.

Clin Cancer Res; 18(8) April 15, 2012 Clinical Cancer Research2222




homogenizer and centrifuged at 16,000 � g at 4�C for10 minutes. The resulting nuclear pellet was suspended in0.2 mL of buffer B [20 mmol/L HEPES (pH 7.9), 25%glycerol, 1.5 mmol/L MgCl2, 420 mmol/L NaCl, 0.5mmol/L DTT, 0.2 mmol/L EDTA, 0.5 mmol/L PMSF, and2 mg/mL leupeptin] and incubated on ice for 2 hourswith intermittent mixing. The suspension was then centri-fuged at 16,000 � g at 4�C for 30 minutes. The supernatant(nuclear extract)was collectedand storedat�70�Cuntilusedas previously described (35). Protein concentration wasmeasured by the Bradford assay with BSA as the standard.

Measurement of NF-kB activation in gastric cancer cellsand tumor samplesTo determine NF-kB activation, we conducted DNA-

binding assay by TransAM NF-kB p65 transcription factorassay kit (Active Motif) according to the manufacturer’sinstructions and as described previously (20). Briefly, nucle-ar extracts from g-tocotrienol–treated gastric cancer celllines and tumor tissues were incubated in a 96-well platecoated with oligonucleotide containing the NF-kB consen-sus–binding sequence 50-GGGACTTTCC-30. Bound NF-kBwas then detected by a specific primary antibody. An HRP-conjugated secondary antibody was then applied to detectthe bound primary antibody and provided the basis forcolorimetric quantification. The enzymatic product wasmeasured at 450 nm with a microplate reader (TecanSystems). Specificity of this assay was tested by the additionof wild-type or mutated NF-kB consensus oligonucleotidein the competitive or mutated competitive control wellsbefore the addition of the nuclear extracts.

Statistical analysisStatistical analysiswas conducted by the Student t test and

oneway ANOVA. A P value of less than 0.05 was consideredstatistically significant.

ResultsThe purpose of this study was to determine whether

g-tocotrienol, a component of vitamin E (with chemicalstructure shown in Fig. 1A) might have a role in thetreatment of gastric cancer either alone or in combinationwith capecitabine and if so, through what mechanism(s).For this, we used 4 different well-characterized humangastric cancer cell lines. To facilitate the monitoring oftumor growth in mice, one of these cell lines, SNU-5 wassubcutaneously injected and used in the xenograft trans-plant model in mice.

g-Tocotrienol inhibits the proliferation andpotentiates the effect of capecitabine in gastric cancercells in vitroWe first investigated the effect of g-tocotrienol on the

proliferation of 3 different gastric cancer cell lines. g-Toco-trienol inhibited the growth of all 3 human gastric cancercells (SNU-5, MKN45, and SNU-16) in a dose- and time-dependent manner (Fig. 1B). Whether g-tocotrienol canpotentiate the effect of capecitabine against these 3 cell lines

was also examined.Weusedflow cytometric analysis and anesterase staining assay (LIVE/DEAD assay) to establishwhether g-tocotrienol can potentiate the apoptosis inducedby capecitabine. As shown in Fig. 1C and D, the dose ofg-tocotrienol (10 mmol/L) or capecitabine (10 mmol/L) thathadminimumeffect on apoptosis alone produced enhance-ment of apoptosis when used in combination.

g-Tocotrienol inhibits constitutive and capecitabine-induced NF-kB activation in gastric cancer cells

We next examined that how g-tocotrienol potentiates theeffects of capecitabine in gastric cancer cells.NF-kBhas beenshown to be constitutively expressed in gastric cancer andmediates resistance to apoptosis (5, 36, 37). Whetherg-tocotrienol induces downregulation of constitutive NF-kB activation in SNU-16 and SNU-5 cells was examined byusing anELISA-basedDNA-binding assay. Results show thatthe treatment with g-tocotrienol inhibited NF-kB expres-sion in a dose-dependent manner (Fig. 2A and B). Wefurther observed that chemotherapeutic drug capecitabinewas also able to further induce NF-kB activation in a dose-dependent manner in MKN45 cells, with maximum acti-vation observed at 25 mmol/L (Fig. 2C). Interestingly,g-tocotrienol was also found to suppress capecitabine-induced NF-kB activation in a dose-dependent manner inMKN45 cells (Fig. 2D), thereby indicating that it is a potentmodulator of both constitutive and inducible NF-kB acti-vation in gastric cancer cells.Whether g-tocotrienol can alsomodulate the expression of various NF-kB–regulated geneproducts was also examined. We found that the g-tocotrie-nol suppressed the constitutive expression of antiprolifera-tive (cyclin D1), antiapoptotic (Bcl-2), invasive/metastatic(ICAM-1,MMP-9, CXCR4), and angiogenic (VEGF) proteinexpression in a time-dependent manner in SNU16 cells(Fig. 2E). g-Tocotrienol also induced the cleavage of PARPin SNU-16 cells (Fig. 2E). On the basis of these observationsin vitro, we decided to study the effect of g-tocotrienol andcapecitabine either alone or in combination in an in vivogastric cancer xenograft model.

g-Tocotrienol potentiates the antitumor effects ofcapecitabine in a xenograft gastric cancer model innude mice

We examined the therapeutic potential of g-tocotrienoland capecitabine either alone or in combination on thegrowth of subcutaneously implanted human gastric cancercells in nude mice. The experimental protocol is depictedin Fig. 3A. SNU-5 cells were implanted subcutaneously inthe right flank of nude mice. When tumors have reached0.25 cm in diameter after aweek, themicewere randomizedinto 4 groups and started the treatment as per the experi-mental protocol. The treatment was continued for 4 weeksand animals were sacrificed after 5 weeks. We found thatg-tocotrienol alone when given at 1 mg/kg body weightsignificantly inhibited the growth of the tumor (P < 0.001when compared with control; Fig. 3B and C). Capecitabinealone was also very effective (P < 0.001 when comparedwith control; P > 0.05 when compared with g-tocotrienol






alone group); and the combination of the 2 agents weremore effective in reducing the tumor burden. The tumorvolume in the combination of g-tocotrienol and capecita-bine group was significantly lower than g-tocotrienol alonegroup (P < 0.001) or capecitabine alone group (P < 0.001)on day 35 (Fig. 3C and D).

g-Tocotrienol inhibits CD31 and Ki-67 expression ingastric tumor tissues

While Ki-67–positive index is used as a marker for cellproliferation, the CD31 index is a marker for microvessel

density. Whether g-tocotrienol and capecitabine modu-late these markers, was examined. Figure 4A shows thatboth g-tocotrienol (P < 0.05) and capecitabine (P < 0.05)alone significantly downregulated the expression ofKi-67 in gastric cancer SNU-5 tissue and the combinationof the 2 was most effective (P < 0.001). Similarly whenexamined for CD31, we found that both agents signi-ficantly reduced the CD31 expression as comparedwith control group and 2 together were most effective(P < 0.001 when compared with capecitabine alone;Fig. 4B).

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Figure 1. g-Tocotrienol inhibits thegrowth and proliferation,potentiates the apoptotic effectsof capecitabine in gastric cancercells in vitro. A, structure ofg-tocotrienol. B, MTT assay resultsshowed dose-dependentsuppression of cell proliferation inall 3 gastric cancer cell lines treatedwith g-tocotrienol. Points, mean oftriplicate; bars, SE. Data are arepresentative of 2 independentexperiments. C and D, flowcytometric analysis and LIVE/DEAD assay results indicate thatg-tocotrienol (g-Toco, 10 mmol/L)potentiates capecitabine (Cape,10 mmol/L)-induced apoptosis ingastric cancer cells. Data indicatedas percentage proportions ofapoptotic gastric cancer cells forLIVE/DEAD assay. Values aremean � SE of triplicate. Data are arepresentative of 2 independentexperiments.

Manu et al.





g-Tocotrienol inhibited the constitutive NF-kBexpression and NF-kB–regulated gene products ingastric tumor tissuesWe also evaluated the effect of g-tocotrienol and cape-

citabine on NF-kB levels in gastric tumor tissue. Figure5A, left shows that g-tocotrienol either alone or in com-bination with capecitabine was quite effective in suppres-sing the constitutive expression of NF-kB in gastric cancertissue. Capecitabine alone had no significant effect onconstitutive NF-kB activation in gastric tissue. A Westernblot analysis for p65 in extracts from tumor samplesshowed that g-tocotrienol alone inhibited NF-kB (p65)activation (Fig. 5A, right).NF-kB is known to regulate the expression of number of

proteins, including those involved in proliferation (cyclinD1, COX-2), invasion/metastasis (ICAM-1, MMP-9), andsurvival (Bcl-xL, survivin, and XIAP; ref. 38). Whetherg-tocotrienol and capecitabine canmodulate the expressionof these NF-kB–regulated gene products in tumor tissues

was examined by Western blot analysis. We found thattreatment with combination of g-tocotrienol and capecita-bine was effective in downregulating the overexpression ofvarious gene products regulated byNF-kB (Fig. 5B) and alsoinvolved in various aspects of gastric cancer growth, surviv-al, invasion, and metastasis.

Whether modulation of nuclear NF-kB, COX-2, VEGF,and MMP-9 can also be detected by immunohistochem-ical methods was also examined. As shown in Figure 6,these gene products were significantly downregulated ingastric tumor samples treated with g-tocotrienol in com-bination with capecitabine. The downregulation wasmore impressive with either g-tocotrienol or capecitabinealone. The immunohistochemical analysis results furthersupports the data obtained from Western blotting. Theseresults collectively indicate that g-tocotrienol suppressesthe activation of NF-kB thereby inhibiting the expressionof genes involved in proliferation, survival, invasion, andangiogenesis.

Figure 2. A and B, DNA-bindingassay results showing thatg-tocotrienol suppresses theconstitutive activation of NF-kB inSNU-16 and SNU-5 cells in a dose-dependent manner. SNU-16 andSNU-5 (1 � 106) cells were treatedwith g-tocotrienol (10, 25, and 50mmol/L) for 4 hours, and nuclearextracts were prepared and assayedfor NF-kB activation by ELISA-linkedDNA-binding assay. C, MKN45(1 � 106) cells were treated withcapecitabine (10, 20, and 25 mmol/L)for 4 hours, and nuclear extractswere prepared and assayed for NF-kB activation by ELISA-linked DNA-binding assay. D, MKN45 (1 � 106)cells were pretreated withg-tocotrienol (10, 25, and 50 mmol/L)for 4 hours, stimulated withcapecitabine 25 mmol/L for 4 hours,and then the nuclear extracts wereprepared and assayed for NF-kBactivation by ELISA-linked DNA-binding assay. E, g-tocotrienolsuppressed the constitutiveexpression of gene productsinvolved in proliferation, metastasis,and antiapoptosis in gastric cancercells. SNU-16 (1 � 106) cells weretreated with 50 mmol/L g-tocotrienolfor the indicated time points, andWestern blot analysis wasconducted as described in Materialsand Methods. �, P < 0.05.

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DiscussionDespite the major improvements in diagnosis and treat-

ment regimens, gastric cancer remainsoneof themost lethalcancers, with less than 20% of patients surviving up to 5years. Thus, novel agents that are nontoxic, efficacious, andcan significantly enhance the effects of existing chemother-apeutic drugs are urgently needed. The aim of the presentstudy was to investigate whether g-tocotrienol, a compo-nent of vitamin E, could enhance the antitumor activity ofcapecitabine against human gastric cancer. We found thatg-tocotrienol suppressed the proliferation of various gastric

cancer cell lines, potentiated capecitabine-induced apopto-sis, and inhibited constitutively active and inducible NF-kBactivation as well as NF-kB–regulated gene products. Wealso found that in a xenograft mouse model g-tocotrienoleffectively suppressed the growth of gastric cancer alone andalso when used in combination with capecitabine.

We first observed that g-tocotrienol can suppress theproliferation of various gastric cancer cell lines in a dose-and time-dependent manner. Our results are in part agree-ment with those of Sun and colleagues (17) who reportedthat g-tocotrienol could suppress the proliferation of gastricadenocarcinoma SGC-7901 cells. The inhibitory effects ofg-tocotrienol were correlated with the DNA damage andcell-cycle arrest at G0–G1 phase, although the detailedmolecular mechanism(s) were not elucidated. We foundthat g-tocotrienol caused downregulation of cell prolifer-ative gene products such as cyclinD1, whichmay explain itspotent antiproliferative effects in gastric cancer.

Specifically, we also found for the first time that g-toco-trienol when used in combination with capecitabine, is

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Figure 3. g-Tocotrienol potentiates the effect of capecitabine to inhibit thegrowth of gastric cancer in nude mice. A, schematic representation ofexperimental protocol described in Materials and Methods. Group I wasgiven corn oil (100 mL, per os, daily), group II was given g-tocotrienol[1 mg/kg body weight (b wt.), i.p. 3 times/wk], group III was givencapecitabine (60mg/kgbodyweight, twiceweekly by gavage), and groupIV was given g-tocotrienol (1 mg/kg body weight, i.p. 3 times/wk) andcapecitabine (60 mg/kg body weight, twice weekly by gavage). B,necropsy photographs of mice bearing subcutaneously implantedgastric tumors. C, tumor volumes in mice measured during the course ofexperiment and calculated using the formula V¼ 4/3pr3, ��,P < 0.001. D,tumor volumes in mice measured on the last day of the experiment atautopsywith Vernier calipers and calculated using the formula V¼ 4/3pr3

(n ¼ 5). Columns, mean; bars, SE. ��, P < 0.001.

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Figure 4. g-Tocotrienol enhances the effect of capecitabine againsttumor cell proliferation and angiogenesis in gastric cancer. A, left,immunohistochemical analysis of proliferationmarker Ki-67 indicates theinhibition of gastric cancer cell proliferation in g-tocotrienol either alone orin combination with capecitabine-treated groups of animals. Right,quantification of Ki-67þ cells as described in Materials and Methods.Columns, mean of triplicate; bars, SE. ��, P < 0.001. Left,immunohistochemical analysis of CD31 formicrovessel density in gastriccancer tumors indicates the inhibition of angiogenesis by eitherg-tocotrienol alone and in combination with capecitabine. B, right,quantification of CD31þ microvessel density as described in Materialsand Methods. Columns, mean of triplicate; bars, SE. ��, P < 0.001.

Manu et al.





highly effective in inducing apoptosis in gastric cancer celllines. This is very intriguing because although g-tocotrienolhas been previously reported to induce apoptosis in SGC-7901 cells (16, 17), its effect in combination with chemo-therapeutic agents like capecitabine has never been inves-tigated before in gastric cancer. We observed that this effectmay be mediated because of the downregulation of cellsurvival proteins such as Bcl-2 in gastric cancer. Interest-ingly, we also observed for the first time that both consti-tutive and capecitabine-induced NF-kB activation was sup-pressed by g-tocotrienol in gastric cancer cells. These resultsare consistent with those previously reported with otherdietary agents like curcumin (39) and phenethyl isothio-cyanate (40). Also, g-tocotrienol was found to downregu-late the expression of various invasive, metastatic, andangiogenic gene products (ICAM-1, MMP-9, CXCR4, andVEGF) which may account for its recently reported inhib-

itory effects on invasion, metastasis, and angiogenesis(18, 41).

We found for the first time that the intraperitonealadministration of g-tocotrienol alone inhibited the growthof human gastric tumors when examined in vivo in axenograft nude mice model. Tumor growth was inhibitedby more than 50% on treatment with g-tocotrienol andcapecitabine alone, respectively. Also, when the 2 agentswere used in combination, they were found to be muchmore effective and potent. When examined for the mech-anism by which g-tocotrienol manifests its effects in themice against gastric cancer, we found that the proliferationmarker Ki-67 as well as microvessel density indicator CD31was downregulated by g-tocotrienol. Further investigationalso revealed the downregulation of NF-kB and NF-kB–regulated cyclinD1,COX-2, survivin, Bcl-xL, XIAP, ICAM-1,MMP-9, and VEGF. All of these effects were furtherenhanced by capecitabine. g-Tocotrienol has been used incombination therapy with several chemotherapeuticagents/targeted therapies such as statins in breast and colo-rectal cancers (42, 43), celecoxib in breast cancer (44),gemcitabine in pancreatic cancer (29), EGFR inhibitorserlotinib and gefitinib in breast cancer (45, 46), but so farits effects on gastric cancer mice models either alone or incombinationhasnever been investigatedbefore.Our resultsoverall suggest for the first time that g-tocotrienol hassignificant potential for the treatment of gastric cancer andits effects can be further enhanced by capecitabine. A num-ber of clinical trials with tocotrienols in patients withpancreatic, prostate, and breast cancer are already in prog-ress, and based on our results, well-designed clinical studiesare required for potential translation of our preclinicalfindings also in patients with gastric cancer.

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

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Figure 5. g-Tocotrienol enhances the effect of capecitabine against theexpression of NF-kB and NF-kB–regulated gene products in gastriccancer tissue samples. A, left, detection of NF-kB by DNA-bindingassay in tumor tissue samples showed the significant inhibition of NF-kB by combination. ��, P < 0.001. Right, Western blot analysisshowed the inhibition of NF-kB (p65) by g-tocotrienol in whole-cellextracts from animal tissue. B, Western blotting showing thatcombination of g-tocotrienol and capecitabine inhibit the expressionof NF-kB–dependent gene products cyclin D1, COX-2, MMP-9,ICAM-1, Bcl-xL, survivin, and XIAP in gastric tumor tissues. Samplesfrom 3 mice in each group were analyzed and representative dataare shown.

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Figure 6. Immunohistochemical analysis of nuclear p65, COX-2, VEGF,and MMP-9 showed the inhibition of NF-kB, COX-2, VEGF, and MMP-9by either g-tocotrienol alone or in combination with capecitabine.Percentage, positive staining for the given biomarker.






Grant SupportThis work was supported by grants from NUS Academic Research Fund

(grant R-184-000-170-112) and National Medical Research Council ofSingapore (grants R-184-000-211-213 and R-184-000-201-275) to G. Sethi.A.P. Kumar was supported by grants from the National Medical ResearchCouncil of Singapore (grant R-713-000-124-213) and Cancer Science Insti-tute of Singapore, Experimental Therapeutics I Program (grant R-713-001-011-271).

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 September 26, 2011; revised January 18, 2012; accepted February8, 2012; published OnlineFirst February 20, 2012.

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2012;18:2220-2229. Published OnlineFirst February 20, 2012.Clin Cancer Res Kanjoormana A. Manu, Muthu K. Shanmugam, Lalitha Ramachandran, et al.

B PathwayκXenograft Mouse Model through the Modulation of NF-Gastric Cancer and Chemosensitizes It to Capecitabine in a

-Tocotrienol Inhibits the Growth of HumanγFirst Evidence That

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First Evidence That g-Tocotrienol Inhibits the Growth of ... · in H. pylori–induced gastritis (11). Together, these ﬁndings implicate the involvement of NF-kB pathway in gastric