Cancer Research -Tocotrienol Inhibits Pancreatic Tumors and … · Therapeutics, Targets, and...

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Therapeutics, Targets, and Chemical Biology γ-Tocotrienol Inhibits Pancreatic Tumors and Sensitizes Them to Gemcitabine Treatment by Modulating the Inflammatory Microenvironment Ajaikumar B. Kunnumakkara 1 , Bokyung Sung 1 , Jayaraj Ravindran 1 , Parmeswaran Diagaradjane 2 , Amit Deorukhkar 2 , Sanjit Dey 1 , Cemile Koca 1 , Vivek R. Yadav 1 , Zhimin Tong 4 , Juri G. Gelovani 3 , Sushovan Guha 4 , Sunil Krishnan 2 , and Bharat B. Aggarwal 1 Abstract Pancreatic cancers generally respond poorly to chemotherapy, prompting a need to identify agents that could sensitize tumors to treatment. In this study, we investigated the response of human pancreatic cells to γ-tocotrienol (γ-T3), a novel, unsaturated form of vitamin E found in palm oil and rice bran oil, to determine whether it could potentiate the effects of gemcitabine, a standard of care in clinical treatment of pancreatic cancer. γ-T3 inhibited the in vitro proliferation of pancreatic cancer cell lines with variable p53 status and potentiated gemcitabine-induced apoptosis. These effects correlated with an inhibition of NF-κB activation by γ-T3 and a suppression of key cellular regulators including cyclin D1, c-Myc, cyclooxygenase-2 (COX-2), Bcl-2, cellular inhibitor of apoptosis protein, survivin, vascular endothelial growth factor (VEGF), ICAM-1, and CXCR4. In an orthotopic nude mouse model of human pancreatic cancer, p.o. administration of γ-T3 inhib- ited tumor growth and enhanced the antitumor properties of gemcitabine. Immunohistochemical analysis indicated a correlation between tumor growth inhibition and reduced expression of Ki-67, COX-2, matrix metalloproteinase-9 (MMP-9), NF-κB p65, and VEGF in the tissue. Combination treatment also downregu- lated NF-κB activity along with the NF-κBregulated gene products, such as cyclin D1, c-Myc, VEGF, MMP-9, and CXCR4. Consistent with an enhancement of tumor apoptosis, caspase activation was observed in tumor tissues. Overall, our findings suggest that γ-T3 can inhibit the growth of human pancreatic tumors and sen- sitize them to gemcitabine by suppressing NF-κBmediated inflammatory pathways linked to tumorigenesis. Cancer Res; 70(21); 8695705. ©2010 AACR. Introduction The National Cancer Institute estimated that 18,770 men and 18,030 women in the United States would die of pancre- atic cancer in 2010 (1). The only agents approved by the U.S. Food and Drug Administration to treat pancreatic cancer are gemcitabine and erlotinib, both of which produce responses in <10% of patients and are associated with multiple adverse events and drug resistance. Therefore, the need for novel strategies to treat this lethal disease is imperative. The transcription factor NF-κB has been linked with cell pro- liferation, invasion, angiogenesis, metastasis, antiapoptosis, and chemoresistance in multiple tumors (2, 3). In addition, numerous lines of evidence suggest that NF-κB plays a major role in the growth and chemoresistance of pancreatic cancer. NF-κB is constitutively active in pancreatic cancer cells (4) but not in immortalized, nontumorigenic pancreatic ductal epithelial cells (5). Thus, NF-κB activation has been reported in animal models of pancreatic cancer (6) and in human pan- creatic cancer tissue (4). NF-κB promotes pancreatic cancer growth via antiapoptosis (4, 7) and mediates the induction of mitogenic gene products, such as c-Myc and cyclin D1 (8), the latter of which is overexpressed in human pancreatic cancer tissue and inversely correlated with patient survival (9). Additionally, NF-κB enhances the angiogenic potential of pancreatic cancer cells via increased expression of proan- giogenic factors, including vascular endothelial growth factor (VEGF; ref. 10), whereas other NF-κBregulated gene pro- ducts promote the migration and invasion of pancreatic can- cer cells (11). Finally, NF-κB plays a pivotal role in promoting gemcitabine resistance in pancreatic cancer (3). Together, this evidence implicates NF-κB in pancreatic cancer and sug- gests that agents that block NF-κB activation could reduce chemoresistance of the cells to gemcitabine and perhaps be used in combination with gemcitabine as a novel thera- peutic regimen for pancreatic cancer. Authors' Affiliations: Departments of 1 Experimental Therapeutics, 2 Radiation Oncology, 3 Experimental Diagnostic Imaging, and 4 Gastrointestinal Medicine and Nutrition, University of Texas M.D. Anderson Cancer Center, Houston, Texas Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Bharat B. Aggarwal, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 143, Houston, TX 77030. Phone: 713-794-1817; Fax: 713-745-6339; E-mail: aggarwal@ mdanderson.org. doi: 10.1158/0008-5472.CAN-10-2318 ©2010 American Association for Cancer Research. Cancer Research www.aacrjournals.org 8695 on July 14, 2021. © 2010 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst September 23, 2010; DOI: 10.1158/0008-5472.CAN-10-2318 on July 14, 2021. © 2010 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst September 23, 2010; DOI: 10.1158/0008-5472.CAN-10-2318 on July 14, 2021. © 2010 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst September 23, 2010; DOI: 10.1158/0008-5472.CAN-10-2318

Transcript of Cancer Research -Tocotrienol Inhibits Pancreatic Tumors and … · Therapeutics, Targets, and...

Page 1: Cancer Research -Tocotrienol Inhibits Pancreatic Tumors and … · Therapeutics, Targets, and Chemical Biology γ-Tocotrienol Inhibits Pancreatic Tumors and Sensitizes Them to Gemcitabine

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creatic cancers generally respond poorly to chemotherapy, prompting a need to identify agents thatsensitize tumors to treatment. In this study, we investigated the response of human pancreatic cells totrienol (γ-T3), a novel, unsaturated form of vitamin E found in palm oil and rice bran oil, to determineer it could potentiate the effects of gemcitabine, a standard of care in clinical treatment of pancreatic. γ-T3 inhibited the in vitro proliferation of pancreatic cancer cell lines with variable p53 status andiated gemcitabine-induced apoptosis. These effects correlated with an inhibition of NF-κB activation3 and a suppression of key cellular regulators including cyclin D1, c-Myc, cyclooxygenase-2 (COX-2),cellular inhibitor of apoptosis protein, survivin, vascular endothelial growth factor (VEGF), ICAM-1, and4. In an orthotopic nude mouse model of human pancreatic cancer, p.o. administration of γ-T3 inhib-mor growth and enhanced the antitumor properties of gemcitabine. Immunohistochemical analysisted a correlation between tumor growth inhibition and reduced expression of Ki-67, COX-2, matrixoproteinase-9 (MMP-9), NF-κB p65, and VEGF in the tissue. Combination treatment also downregu-F-κB activity along with the NF-κB–regulated gene products, such as cyclin D1, c-Myc, VEGF, MMP-9,

XCR4. Consistent with an enhancement of tumor apoptosis, caspase activation was observed in tumor. Overall, our findings suggest that γ-T3 can inhibit the growth of human pancreatic tumors and sen-

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sitize them to gemcitabine by suppressing NF-κB–mediated inflammatory pathways linked to tumorigenesis.Cancer Res; 70(21); 8695–705. ©2010 AACR.

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National Cancer Institute estimated that 18,770 men,030 women in the United States would die of pancre-ncer in 2010 (1). The only agents approved by the U.S.nd Drug Administration to treat pancreatic cancer aretabine and erlotinib, both of which produce responses% of patients and are associated with multiple adverseand drug resistance. Therefore, the need for novelies to treat this lethal disease is imperative.

tion factorNF-κBhas been linkedwith cell pro-ion, angiogenesis, metastasis, antiapoptosis,

the lacance(9). Adof pangiogen(VEGFductscer cegemcithis evgestschembe usepeutic

ons: Departments of 1Experimental Therapeutics,logy, 3Experimental Diagnostic Imaging, andMedicine and Nutrition, University of Texas M.D.enter, Houston, Texas

ry data for this article are available at Cancer Researchrres.aacrjournals.org/).

thor: Bharat B. Aggarwal, University of Texas M.D.enter, 1515 Holcombe Boulevard, Box 143, Houston,13-794-1817; Fax: 713-745-6339; E-mail: aggarwal@

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hemoresistance in multiple tumors (2, 3). In addition,rous lines of evidence suggest that NF-κB plays a majorthe growth and chemoresistance of pancreatic cancer.is constitutively active in pancreatic cancer cells (4)t in immortalized, nontumorigenic pancreatic ductallial cells (5). Thus, NF-κB activation has been reportedal models of pancreatic cancer (6) and in human pan-

c cancer tissue (4). NF-κB promotes pancreatic cancerh via antiapoptosis (4, 7) and mediates the induction ofenic gene products, such as c-Myc and cyclin D1 (8),tter of which is overexpressed in human pancreaticr tissue and inversely correlated with patient survivalditionally, NF-κB enhances the angiogenic potentialcreatic cancer cells via increased expression of proan-ic factors, including vascular endothelial growth factor; ref. 10), whereas other NF-κB–regulated gene pro-promote the migration and invasion of pancreatic can-lls (11). Finally, NF-κB plays a pivotal role in promotingtabine resistance in pancreatic cancer (3). Together,idence implicates NF-κB in pancreatic cancer and sug-that agents that block NF-κB activation could reduceoresistance of the cells to gemcitabine and perhaps

d in combination with gemcitabine as a novel thera-regimen for pancreatic cancer.

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Vitaered ivitamidentif(13). A(overnols.moreexhibinstanthat γboth ccancerregulawith sTocoteratiobreastreportstatindietarinosit(21). Mangiofactorcycloostudieprogrestudieeratiowhenthe paγ-T3to doccell ncleaveThe

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min E, as represented by tocopherols, was first discov-n 1922 (12). Tocotrienols, unsaturated derivatives ofin E found in rice, barley, oats, and palm, were firstied in 1965 and first found to prevent cancer in 1989lthough tocopherols have been studied extensively30,000 citations), very little is known about tocotrie-Limited studies have revealed that tocotrienols arepotent antioxidant agents than tocopherols (14) andit activities superior to those of tocopherols. Force, studies from our laboratory and others have shown-tocotrienol (γ-T3), but not tocopherol, can inhibitonstitutive and inducible NF-κB activation in variouscell lines (15, 16). This activity correlated with down-tion of the NF-κB–regulated gene products associatedurvival, proliferation, invasion, and angiogenesis (16).rienols have been shown to suppress the in vitro prolif-n of a wide variety of tumor cells, including human, colon, prostate, and pancreatic (12, 17). γ-T3 has beened to potentiate the effect of gefitinib, celecoxib, ands against various human cancers (12, 18–20). Thisy agent can also inhibit ErbB3-dependent phospho-ide 3-kinase/Akt mitogenic signaling in cancer cellsoreover, tocotrienols have been shown to inhibit

genesis (22, 23) by suppressing hypoxia-inducible-1α (24) and to inhibit proinflammatory markers andxygenase-2 (COX-2) expression in vitro (25). Others have shown that γ-T3 inhibits human cancer cellssion by downregulating cyclin D1 and E (26). Recents have shown that tocotrienols can suppress the prolif-n of various human pancreatic cancer cells (17) and,given p.o. to mice, significant levels accumulate inncreatic tissues. Another recent study indicate thatcan sensitize androgen-independent prostate canceretaxel in vivo through downregulation of proliferatinguclear antigen, Ki-67 and Id1, and upregulation ofd caspase-3 and poly(ADP-ribose) polymerase (27).possibility that dietary agents can potentiate theof chemotherapeutic agents such as gemcitabine is

ractive strategy. In the present study, we investigateder γ-T3 could sensitize human pancreatic tumors totabine in vitro and in an orthotopic mouse model.owed that γ-T3 inhibited the in vitro proliferation ofs pancreatic cancer cells, enhanced gemcitabine-d apoptosis, and potentiated the antitumor activitycitabine against orthotopically implanted human

eatic tumors through the downregulation of NF-κBF-κB–regulated gene products.

rials and Methods

ials3 (Fig. 1A) was kindly supplied by Davos Life Sciencepore). The following polyclonal antibodies againstellular adhesion molecule-1 (ICAM-1), cyclin D1, matrixoproteinase 9 (MMP-9), survivin, cellular inhibitor ofosis protein-1 (cIAP-1), procaspase-3, procaspase-9,

, c-Myc, Bcl-2, and Bcl-xL were obtained from SantaBiotechnology. The polyclonal antibody against p65

Thethe M

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nizing the epitope within the NH2 terminal domainan NF-κB) was obtained from Abcam. Antiobodies

t VEGF and Ki-67 were purchased from Thermo Fisherific. The liquid DAB+ substrate chromogen system–adish peroxidase (HRP) used for immunocytochemis-s obtained from Dako. Penicillin, streptomycin, RPMIand fetal bovine serum (FBS) were obtained from Invi-. Tris, glycine, NaCl, SDS, and bovine serum albuminwere obtained from Sigma Chemical. Gemcitabinear; kindly supplied by Eli Lilly) was stored at 4°C andved in sterile PBS on the day of use.

inespancreatic cancer cell lines BxPC-3, MIA PaCa-2, and-1 were obtained from American Type Culture Collec-Panc-96 was a kind gift from Dr. C. Logsdon (Univer-

f Texas M.D. Anderson Cancer Center). All cell linescultured in RPMI 1640 supplemented with 10% FBS,its/mL penicillin, and 100 μg/mL streptomycin. The-mentioned cell lines were procured >6 months agoave not been tested recently for authentication in ourtory.

eration assay

dependent experiments.

1. γ-T3 inhibits proliferation in pancreatic cancer cells in vitro.ture of γ-T3. B, MTT assay results showed dose-dependentssion of cell proliferation in all four pancreatic cancer cell lines

effect of γ-T3 on cell proliferation was determined byTT uptake method as described previously (28). The

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cells (2a 96-wA MT2 houdimetovernimeasuscann

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,000 per well) were incubated with γ-T3 in triplicate inell plate and then incubated for 2, 4, or 6 days at 37°C.T solution was added to each well and incubated forrs at 37°C. An extraction buffer (20% SDS and 50%hylformamide) was added, and the cells were incubatedght at 37°C. The absorbance of the cell suspension wasred at 570 nm with an MRX Revelation 96-well multi-er (Dynex Technologies).

tosis assayinvestigate whether γ-T3 could potentiate the apo-effects of gemcitabine in pancreatic cancer cells,ed a LIVE/DEAD cell viability assay kit (Invitrogen),is used to determine intracellular esterase activitylasma membrane integrity. This assay uses calcein,anionic, green fluorescent dye that is retained withinlls, and a red fluorescent ethidium homodimer dyean enter cells through damaged membranes ando nucleic acids but is excluded by the intact plasmaranes of live cells (29). Briefly, cells (5,000 per well)ncubated in chamber slides, pretreated with γ-T3 forrs, and treated with gemcitabine for 24 hours. Cellsthen stained with the assay reagents for 30 minutesm temperature. Cell viability was determined underrescence microscope by counting live (green) and(red) cells.

alse athymic nu/nu mice (4 weeks old) were obtainedhe breeding colony of the Department of Experimentalion Oncology at University of Texas M.D. Andersonr Center. The animals were housed in standard plexi-ages (five per cage) in a room maintained at constantrature and humidity and in a 12-hour light/12-hourycle. Their diet consisted of regular autoclave chowater ad libitum. None of the mice exhibited anys, and all were tested pathogen free. Before initiatingperiment, we acclimatized all of the mice to a pul-d diet for 3 days. Our experimental protocol wased and approved by the Institutional Animal Carese Committee at University of Texas M.D. Andersonr Center.

topic implantation of MIA PaCa-2 cellsPaCa-2 cells were stably transfected with luciferase asusly described for PANC-1 cells (30). The MIA PaCa-2ere orthotopically implanted as described previouslyriefly, luciferase-transfected MIA PaCa-2 cells wereted from subconfluent cultures after a brief exposure5% trypsin and 0.2% EDTA. Trypsinization wasd with medium containing 10% FBS. The cells wered once in serum-free medium and resuspended inOnly suspensions consisting of single cells, withviability, were used for the injections. After micenesthetized with ketamine-xylazine solution, a smalln was made in the left abdominal flank, and MIA

2 cells (1 × 106) in 100 μL PBS were injected intobcapsular region of the pancreas with a 27-gauge

utes.stored

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and a calibrated push button–controlled dispensing(Hamilton Syringe Co.). A cotton swab was held

usly for 1 minute over the site of injection to prevente. The abdominal wound was closed in one layeround clips (Braintree Scientific, Inc.).

imental protocolweek after implantation, the mice were randomizede following treatment groups (n = 6 per group) basedbioluminescence first measured with an in vivo imag-stem (IVIS 200, Xenogen Corp.): (a) untreated controloil, 100 μL daily), (b) tocotrienol (400 mg/kg once daily(c) gemcitabine alone (25 mg/kg twice weekly by i.p.on), and (d) combination (tocotrienol, 400 mg/kg oncep.o., and gemcitabine, 25 mg/kg twice weekly by i.p.on). Tumor volumes were monitored weekly with theinescence IVIS, which includes a cryogenic coolingnd a data acquisition computer running Living Imagere (Xenogen Corp.). Before imaging, the animals wereetized in an acrylic chamber with 2.5% isoflurane/airre and injected i.p. with 40 mg/mL D-luciferin potassiumPBS at a dose of 150 mg/kg body weight. After

nutes of incubation with luciferin, each mouse wasin a right lateral decubitus position and a digital

ale animal image was acquired, followed by the acqui-and overlay of a pseudocolor image representing thel distribution of detected photons emerging from ac-ciferase within the animal. Signal intensity was quan-as the sum of all detected photons within the region ofst per second. The mice were subjected to imaging on, 7, 17, 24, and 29 of treatment. Therapy was contin-r 4 weeks, and the animals were euthanized 1 weekPrimary tumors in the pancreas were excised, andal tumor volume was measured as V = 2/3πr3, wheree mean of the three dimensions (length, width, and). Half of the tumor tissue was fixed in formalin andded in paraffin for immunohistochemistry and rou-&E staining. The other half was snap frozen in liquiden and stored at −80°C. H&E staining confirmed thece of tumor(s) in each pancreas.

ration of nuclear extract from tumor samplescreatic tumor tissues (75–100 mg/mouse) from micecontrol and treatment groups were minced and in-d on ice for 30 minutes in 0.5 mL of ice-cold buffermmol/L HEPES (pH 7.9), 1.5 mmol/L KCl, 10 mmol/L, 0.5 mmol/L DTT, and 0.5 mmol/L phenylmethylsul-fluoride (PMSF)]. The minced tissue was homoge-using a Dounce homogenizer and centrifuged at× g at 4°C for 10 minutes. The resulting nuclear pel-

as suspended in 0.2 mL of buffer B [20 mmol/LS (pH 7.9), 25% glycerol, 1.5 mmol/L MgCl2,mol/L NaCl, 0.5 mmol/L DTT, 0.2 mmol/L EDTA,mol/L PMSF, and 2 μg/mL leupeptin] and incubatedfor 2 hours with intermittent mixing. The suspen-as then centrifuged at 16,000 × g at 4°C for 30 min-

The supernatant (nuclear extract) was collected andat −70°C until used as previously described (28).

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n concentration was measured by the Bradford assaySA as the standard.

activation in pancreatic cancer cells andr samplesassess NF-κB activation, we isolated nuclei from pan-cancer cell lines and tumor samples and carried outphoretic mobility shift assays (EMSA) essentially asbed previously (29). Briefly, nuclear extracts preparedancreatic cancer cells (1 × 106/mL) and tumor samplesncubated with 32P-end-labeled 45-mer double-strandedoligonucleotide (4 μg of protein with 16 fmol of DNA)the HIV long terminal repeat (5′-TTGTTACAAGG-TTCCGCTGGGGACTTTCCAGGGGGAGGCGTGG-3′;line indicates NF-κB–binding sites) for 15 minutes atThe resulting DNA-protein complex was separatedree oligonucleotide on 6.6% native polyacrylamide gels.dried gels were visualized, and radioactive bands wereitated with a PhosphorImager and ImageQuant soft-GE Healthcare).

nolocalization of NF-κB p65, VEGF, and COX-2 inr samplesnuclear localization of p65 and the expression of COX-2EGF were examined via an immunohistochemicald described previously (28). Briefly, pancreatic cancersamples were embedded in paraffin and fixed withrmaldehyde. After being washed in PBS, the slideslocked with protein block solution (Dako) for 20 min-nd then incubated overnight with rabbit polyclonaluman p65, mouse monoclonal anti-human VEGF,nti-COX-2 antibodies (1:100, 1:50, and 1:75, respect-After incubation with the antibodies, the slides wered and then incubated with biotinylated link universalrum followed by HRP-streptavidin detection with thekit (Dako). The slides were rinsed, and color was de-d with 3,3′-diaminobenzidine hydrochloride used as aogen. Finally, sections were rinsed in distilled water,erstained with Mayer's hematoxylin, and mountedDPX mounting medium (Sigma) for evaluation.es were captured with a Photometrics CoolSNAP CFcamera (Nikon) and MetaMorph software versionUniversal Imaging).

immunohistochemistrymalin-fixed, paraffin-embedded sections (5 μm) wered with anti-Ki-67 (rabbit monoclonal clone SP6) anti-as described previously (29). Results were expressedpercentage of Ki-67+ cells ± SEM per 40× magnifica-total of ten 40× fields were examined and counted

hree tumors each of the treatment groups.

rn blot analysiscreatic tumor tissues (75–100 mg/mouse) from micecontrol and treatment groups were minced and in-d on ice for 30 minutes in 0.5 mL of ice-cold whole-

sate buffer (10% NP40, 5 mol/L NaCl, 1 mol/L HEPES,l/L EGTA, 0.5 mol/L EDTA, 0.1 mol/L PMSF, 0.2 mol/L

Weof hu

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orthovanadate, 1 mol/L NaF, 2 μg/mL aprotinin,μg/mL leupeptin). The minced tissue was homoge-with a Dounce homogenizer and centrifuged at× g at 4°C for 10 minutes. The proteins were thennated by SDS-PAGE, electrotransferred to nitrocellu-embranes, blotted with each antibody, and detectedhanced chemiluminescence reagents (GE Healthcare).

performance liquid chromatography analysisextraction of γ-T3 was performed according to thed described earlier (31). Frozen tissue (100 mg) wasd, and 400 μL of ethanol were added and homogenizedseconds. Next, 500 μL of water were added and homog-for 15 seconds. And then, 500 μL of hexane wereand homogenized for 15 seconds. The samples werefuged at 5,000 rpm for 5 minutes at 4°C. The organicwas evaporated to dryness under purified air at 40°C.ellets were reconstituted with mobile phase and esti-by high-performance liquid chromatography (HPLC;s separation module Alliance 2695, Waters). Silica-C18 column and mobile phase that was a mixture ofitrile/methanol/acetic acid (10%) in a ratio of 45:45:10,tively, were used for separation. Analyses were run at aate of 0.4 mL/min with the detector operating at wave-of 291 nm. Tissues from vehicle-fed mice are set asls that reflect the baseline.

ticsitro experiments were repeated twice, and statisticalis was performed. The values were initially subjected-way ANOVA, which revealed significant differencesen groups, and then later compared between groupsn unpaired Student's t test, which revealed significantnces between two sample means. In vivo experimentsone as at least three independent assays. The valuesinitially subjected to one-way ANOVA and then laterred among groups with an unpaired Student's t test.

lts

objective of this study was to (a) determine whethercan inhibit the growth of human pancreatic cancerb) determine whether γ-T3 can enhance the antitumorof gemcitabine in vitro, (c) determine whether γ-T3

otentiate the effects of a chemotherapeutic agentin models of human pancreatic cancer, and (d) inves-the mechanism by which γ-T3 enhances the effects ofemotherapeutic agent. We used four different humaneatic cancer cell lines (AsPC-1, BxPC-3, MIA PaCa-2,ANC-1) of different origin for this investigation. Thesees differ with respect to K-Ras and p53 mutations. Toor tumor growth in vivo, we used noninvasive imagingluciferase-transfected MIA PaCa-2 cell line.

inhibits the proliferation of pancreatic cancern vitro

examined whether γ-T3 could inhibit the proliferationman pancreatic cancer cell lines AsPC-1, PANC-1,

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3, and MIA PaCa-2, all of which exhibit K-Ras andutations, except BxPC-3, which harbors wild-type. We treated these cell lines with different doses ofor various time periods and determined the inhibitionliferation by examining mitochondrial activity via theptake method. As Fig. 1B shows, γ-T3 suppressed theration of all four pancreatic cancer cell lines in aand time-dependent manner, irrespective of thec background (Fig. 1B). Proliferation of pancreaticr cells was almost completely suppressed with aol/L dose.

sensitizes human pancreatic cancer cells totabinecitabine is a standard treatment for patients with

eatic cancer. To determine whether γ-T3 could en-the apoptotic effects of gemcitabine in these cell lines,rformed LIVE/DEAD assay that measured the esterasey. At a dose at which γ-T3 (10 μmol/L) and gemcita-200 nmol/L) alone had minimally toxic effects, the two

er were highly effective for inducing apoptosis in pan-cancer cell lines in vitro (Fig. 2). No significant differ-

Likelinked

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was observed between the cell lines with respect toensitivity to the combination.

inhibits constitutive NF-κB activation andins associated with inflammation, proliferation,ion, and angiogenesis in pancreatic cancer cellsause NF-κB has been linked with both proliferationemoresistance, we next examined whether γ-T3 couldt constitutive NF-κB activation in MIA PaCa-2 cellOur results showed that γ-T3 inhibited constitutiveactivation (Fig. 3A).next investigated the effects of γ-T3 on the constitu-pression of antiapoptotic proteins Bcl-2, cIAP-1, andin. The expression of all these proteins was down-ted by γ-T3 in a dose-dependent manner (Fig. 3B).we examined whether γ-T3 could modulate the

tutive expression of proliferative proteins cyclin D1,, and COX-2, we found that γ-T3 suppressed thesion of all these proteins in a dose-dependent man-ig. 3C).

2. γ-T3 potentiates thetic effects of gemcitabine intic cancer cells in vitro.AD assay results indicated3 potentiates gemcitabineinduced cytotoxicity.tages, proportions oftic pancreatic cancer cells.e representative of two

wise, γ-T3 downregulated the expression of proteinsto invasion, adhesion, and angiogenesis-MMP-9,

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ICAM(Fig. 3

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mine tplanteThe dositizatLuc

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-1, VEGF, and CXCR4-in a dose-dependent mannerD).

potentiates the antitumor activity of gemcitabineorthotopic pancreatic tumor model in nude miceed on our in vitro results, we designed studies to deter-he effects of γ-T3 on gemcitabine in orthotopically im-d human pancreatic tumors in nude mice (Fig. 4A).se of γ-T3 used has been used previously for radiosen-ion of tumors in mice (32).iferase-transfected MIA PaCa-2 cells were implanted inils of the pancreas in nude mice. Based on the IVISg, 1 week later, the mice were randomized into fours. The treatment was started 1 week after tumor cellntation and continued per experimental protocol forks. The animals were euthanized 6 weeks after tumorjection and 5 weeks from the date of treatmentA). To determine tumor development and the effects3 and gemcitabine, we assessed the tumors with theinescence IVIS on days 7, 17, 24, and 29 after the starttment. The bioluminescence imaging results (Fig. 4B

present two independent experiments.

) indicated a gradual increase in tumor volume inntrol group compared with the three treatment groups.

(MMPFig. 6

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aging results showed that the tumor volume in thenation group was significantly lower than in the groupd with tocotrienol or gemcitabine alone as well as inhicle-treated control group (P < 0.001 versus gemcita-< 0.001 versus control). On the 42nd day, we eutha-

the mice and measured the tumor volume with Vernierrs. The results were in concordance with those frominescence imaging and showed that the combinationed the tumor volume more than in the other threes (P < 0.001 versus control; Fig. 4D).next examined the expression of the cell prolifera-arker Ki-67 in tumor tissues from the four groups.

esults in Fig. 5A show that γ-T3 in gemcitabine sig-tly downregulated the expression of Ki-67 in tumors compared with the control group (P < 0.05 versusl). The results also showed that γ-T3 alone signifi-suppressed the expression of Ki-67 (P < 0.05 versusl; Fig. 5B).

inhibits NF-κB activation and NF-κB–regulatedproducts in orthotopic pancreatic tumorsnext investigated whether the effects of γ-T3 on tu-rowth in mice were associated with the inhibition ofactivation. The results of immunohistochemical anal-owed that γ-T3 alone completely suppressed NF-κBtion in orthotopically grown human pancreatic tumores (Fig. 5B). γ-T3 also further suppressed NF-κB acti-in gemcitabine-treated tissues. Immunohistochemicalis further showed that γ-T3 suppressed the expres-f COX-2 (Fig. 5B, second panel), MMP-9 (Fig. 5B, third, and VEGF (Fig. 5B, bottom) in human pancreatictissues, and this effect was enhanced in tissues trea-

ith gemcitabine.

potentiates the effects of gemcitabine inregulating the expression of NF-κB–regulatedproductsalso examined NF-κB activation in tumor tissues by. The results showed that γ-T3 alone abolished thetutive activation of NF-κB in the pancreatic tumor, and this down-modulation was maintained in tissueed from animals treated with gemcitabine (Fig. 6A).EMSA results confirmed those obtained from immu-ochemical analysis. NF-κB is known to regulate thesion of proteins involved in proliferation: cyclin D1,, and COX-2. The expression of all these proteins wasto be maximally downregulated in pancreatic tissueed from animals treated with both agents together6B). We also examined the expression of survivalns Bcl-xL, cIAP-1, and Bcl-2 by Western blot. Maxi-downregulation was noted when both agents weretogether (Fig. 6C). When activation of caspase-9 andse-3 were examined, maximum activation was alsowhen both agents were used together (Fig. 6C).sults also showed that γ-T3 and gemcitabine togethersed the expression of proteins linked to metastasis

3. γ-T3 inhibits constitutive activation of NF-κB and NF-κB–d gene products in vitro. A, MIA PaCa-2 (1 × 106) cells werewith γ-T3 (10 and 50 μmol/L) for 4 h; nuclear extracts wered and then assayed for NF-κB activation by EMSA. B-D, Westernalysis showed that γ-T3 inhibited constitutive expression ofregulated gene products that regulate antiapoptosis (B),ation (C), and metastasis and angiogenesis (D) in pancreaticcells. The MIA PaCa-2 (1 × 106) cells were treated with γ-T350 μmol/L) for 24 h. Whole-cell lysates were prepared and

d for NF-κB–regulated gene products by Western blotting.

-9), invasion (ICAM-1), and angiogenesis (VEGF;D). Thus, γ-T3 in combination with gemcitabine

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inhibitcreatic

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Figureeffect othe groin nudeexperimMateriawas givdaily) aweekly)(400 mwas givi.p., twiwas givdaily) ai.p., twiB, bioluorthototumorsC, measecondvariousimaging(n = 6).D, tumomeasurexperimcalipersthe formColumn

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ed the expression of various gene products in pan-tumors from mice.

can be detected in pancreatic tumor tissueetermine whether γ-T3 is directly detectable in pancre-sue when given p.o., we extracted tissues and analyzedby HPLC for γ-T3. Purified γ-T3 was used as a control.o used tissues from untreated animals as a control. Thein Fig. 6E show a significant presence of γ-T3 in human

eatic tumor tissue (94.12 ng/g tissue) compared with

ted control. γ-T3 estimation by HPLC was linear in this(see Supplementary Fig. S1).

variou

s, mean; bars, SE.

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ssion

creatic cancer is the most lethal type of cancer, withimated 5-year survival rate of around 4% despite thereatment available today. Agents that are nontoxicet potentiate the effects of chemotherapy are highlyble. In the current study, we investigated whetheran analogue of vitamin E, could enhance the anti-activity of gemcitabine against human pancreatic

r. We found that γ-T3 inhibited the proliferation of

s pancreatic cancer cell lines in vitro, potentiated

gemcitabine-induced apoptosis, and inhibited constitutively

4. γ-T3 potentiates thef gemcitabine in blockingwth of pancreatic cancermice. A, schematic ofental protocol described inls and Methods. Group Ien olive oil (100 μL, p.o.,nd PBS (100 μL, i.p., twice, group II was given γ-T3g/kg, p.o., daily), group IIIen gemcitabine (25 mg/kg,ce weekly), and group IVen γ-T3 (400 mg/kg, p.o.,nd gemcitabine (25 mg/kg,ce weekly; n = 6).minescence IVIS images ofpically implanted pancreaticin live, anesthetized mice.surements of photons perdepicting tumor volume attime points using live IVISat the indicated timesPoints, mean; bars, SEM.r volumes in miceed on the last day of theent at autopsy with Vernierand calculated usingula V = 2/3πr3 (n = 6).

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activegivenin esttumorthe effin nudWe

ious pfromcouldand Bto cell

the mof celland CationOur

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ureemliferκBancunliferntifdescthodthemmF-κesance. Pitivemarmalslyzea are shown.

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NF-κB and NF-κB–regulated gene products. Whenalone, γ-T3 significantly inhibited the growth of PaCaablished orthotopically grown human pancreatics in nude mice. In addition, γ-T3 further potentiatedects of gemcitabine against human pancreatic tumorse mice.found that γ-T3 can suppress the proliferation of var-ancreatic cancer cell lines. Our results agree with thoseHussein and colleagues (17) that showed that δ-T3suppress the proliferation of Panc-1, MIA PaCa-2,

xPC-3 cells. This suppression of proliferation was duecycle arrest at G1. However, they did not investigate

and tpartia

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echanism in detail (17). We found that downregulationproliferative gene products such as cyclin D1, c-Myc,OX-2 could account for the suppression of cell prolifer-by γ-T3.study is the first report to suggest that γ-T3 cance the apoptotic effect of gemcitabine in variouseatic cancer cell lines in vitro. Previously, it has beened that γ-T3 can enhance the effect of gefitinib, doc-, celecoxi, and statins against various human cancers, 27). Although different cancer has its own property

Figof gproNF-in pimmproquaasMeasB, iof Ngenin pmicposbioanianadat

herapeutic strategy, the synerglly confirmed.

010 American Association for

5. γ-T3 enhances the effectcitabine against tumor cellation and the expression ofand NF-κB–regulated genesreatic cancer. A, left,ohistochemical analysis ofation maker Ki-67; right,ication of Ki-67+ cells,ribed in Materials ands. Values are representedmean ± SEM of triplicate.unohistochemical analysisB and NF-κB–regulatedCOX-2, MMP-9, and VEGFreatic tumor tissues fromercentages indicatestaining for the given

ker. Samples from threein each group wered, and representative

istic action of γ-T3 is

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Wethe docIAP-1expreslinkedactivaresultfor cu

showeimplic(HMGinhibiferativγ-T3

Figureeffect oexpressregulatepancreaA, detebindingsampleNF-κBanalysisgemcitaexpressproliferD1, c-Mblot anand gethe expcaspasBcl-xLD, Westhat γ-TinhibitemetastaVEGF,pancreafrom thwere andata arγ-T3 byHPLC cmobile

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found that this effect may be mediated throughwnregulation of cell survival proteins such as Bcl-2,and survivin by γ-T3. Moreover, constitutive NF-κBsed in human pancreatic cancer cell lines has beenwith chemoresistance (3). We found that this NF-κBtion in cell lines was also suppressed by γ-T3. These

s are consistent with those previously reportedrcumin (29) and resveratrol (33). Hussein and Mo

lovastductas

phase.

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d that mevalonate reversed the effect of δ-T3, thusating the role of f3-hydroxy-f3-methylglutaryl CoA-CoA) in action of δ-T3. Furthermore, lovastatin, antor of HMG-CoA reductase, potentiated the antiproli-e effect of δ-T3 (17, 34). These results suggest thatcan be used in combination with gemcitabine and

atin, as γ-T3 is known to also inhibit HMG-CoA re-e (35–37). Similarly, we have shown previously that

6. γ-T3 enhances thef gemcitabine against theion of NF-κB and NF-κB–d gene products intic cancer samples.ction of NF-κB by DNAin orthotopic tumor tissues showed the inhibition ofby γ-T3. B, Western blotshowed that γ-T3 andbine together inhibited theion of NF-κB–dependentative gene products cyclinyc, and COX-2. C, Westernalysis showed that γ-T3mcitabine together inhibitedression of caspase-3,e-9, Bcl-2, cIAP-1, andin pancreatic tumor tissues.tern blot analysis showed3 and gemcitabine togetherd the expression oftic and angiogenic genesMMP-9, and CXCR4 intic tumor tissues. Samplesree animals in each groupalyzed, and representativee shown. E, uptake ofpancreatic tissue in micehromatogram of γ-T3

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tatins and γ-T3 are potent inhibitors of NF-κB acti-(16, 38).found for the first time that p.o. administration of γ-T3inhibited the growth of human pancreatic tumorsexamined in vivo in an orthotopic nude mice model.r growth was inhibited by almost 50% by 400 mg/kglone, and this level of inhibition was comparable withduced by gemcitabine alone. γ-T3 was very well toler-y the animals. When the two agents were used in com-on, they were found to be much more effective.γ-T3 exhibited its effects in vivowere also investigated

eral ways. First, analysis of Ki-67 revealed that γ-T3ed its proliferation in tumor tissues. Likewise, analysis-κB in pancreatic tumors showed that γ-T3 aloneed constitutive activation of NF-κB. γ-T3 significantlyegulated the expression of proinflammatory marker, suppressed the expression of invasion biomarker9, and inhibited the angiogenic biomarker VEGF inssues. All of these effects were further enhanced bytabine. The synergistic interaction between the twofor down-modulation of various biomarkers was moreent by Western blot analysis than by immunohisto-cal analysis. All these data indicate for the first timeechanisms by which γ-T3 could exhibit its effectst human pancreatic cancer.also found a significant presence of intact γ-T3 in theatic tumor tissue (94.12 ng/g). These results agree withreported by Husain and colleagues (39) in a previousof δ-T3. They showed that δ-T3, when given p.o. atg/kg to mice, has a peak plasma concentration ofol/L at 2 hours, plasma T1/2 of 3.5 hours. Six-week feed-δ-T3 gave a tissue level of 41 nmol/L/g tissue; levels werees higher in pancreas than in tumor or liver tissue (39).

min E consists of four different isoforms of tocopher- Rece

n pancreatic cancer is associated with decreased postoperativevival. Oncology 1998;55:363–9.ng HQ, Abbruzzese JL, Lin E, et al. NF-κB activity blockade

imInt

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that anticancer activities are limited to only γ ands. Why α and β forms of tocotrienols exhibit mini-nticancer activities is not understood at present. Dif-ial cellular uptake, membrane insertion, bioavailabilty,tioxidant or anti-inflammatory potential may accountese differences.rall, our results show for the first time that γ-T3 po-tes the antitumor activity of gemcitabine by inhibit-F-κB and NF-κB–regulated gene products, leading tohibition of proliferation, angiogenesis, and invasion.ver, further studies are necessary to confirm our find-n patients with pancreatic cancer. Considering thatienols, although structurally homologous to toco-ls, exhibit distinct molecular targets, they merit fur-xploration.

osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

owledgments

thank Markeda Wade for carefully proof reading the manuscriptoviding valuable comments. Dr. Aggarwal is the Ransom Horne,essor of Cancer Research.

Support

ysian Palm Oil Board, NIH core grant CA-16 672, NIH programgrant NIH CA-124787-01A2, and Center for Targeted Therapy ofderson Cancer Center.costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.

ived 06/28/2010; revised 08/18/2010; accepted 09/06/2010; published

d of tocotrienols (12). Among the tocotrienols, it OnlineFirst 09/23/2010.

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18. Baaptini

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Editor's Note

Editor's Note: g-Tocotrienol InhibitsPancreatic Tumors and Sensitizes Them toGemcitabine Treatment by Modulating theInflammatory Microenvironment

The editors are publishing this note to alert readers to a concern about this article (1).It appears that the same image was used to represent b-actin Western blot bands inFig. 3C and D and another image was used to represent b-actin Western blot bandsin Fig. 6C and D. Following an institutional review, the original research recordsrelated to the figures did not sufficiently address these concerns. Therefore, theinstitution was not able to determine the relation of the images to the experimentsdescribed.

Reference1. Kunnumakkara AB, Sung B, Ravindran J, Diagaradjane P, Deorukhkar A, Dey S, et al. g-Tocotrienol

inhibits pancreatic tumors and sensitizes them to gemcitabine treatment by modulating theinflammatory microenvironment. Cancer Res 2010;70:8695–705.

Published online September 4, 2018.doi: 10.1158/0008-5472.CAN-18-1826�2018 American Association for Cancer Research.

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2010;70:8695-8705. Published OnlineFirst September 23, 2010.Cancer Res   Ajaikumar B. Kunnumakkara, Bokyung Sung, Jayaraj Ravindran, et al.   Microenvironmentto Gemcitabine Treatment by Modulating the Inflammatory -Tocotrienol Inhibits Pancreatic Tumors and Sensitizes Themγ

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