First Evidence That G-Tocotrienol Inhibits the Growth of Human

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

Clinical Cancer Cancer Therapy: Preclinical Research

First Evidence That g-Tocotrienol Inhibits the Growth of Human Gastric Cancer and Chemosensitizes It to Capecitabine in a Xenograft Mouse Model through the Modulation 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

Abstract Purpose: Because of poor prognosis and development of resistance against chemotherapeutic drugs, the existing treatment modalities 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 the NF-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-tocotrienol alone (1 mg/kg body weight, 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 compared with vehicle control, g-tocotrienol also suppressed the NF-kB activation and the expression of cyclin D1, COX-2, intercellular adhesion molecule-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.

Introduction accounting for more than 600,000 deaths annually world- Gastric cancer remains one of the most common malig- wide (1, 2). Chemotherapy constitutes an important treat- nancies and the second leading cause of cancer mortality ment regimen for gastric cancers besides surgical resection (3). Unfortunately, only few patients experience complete pathologic response to chemotherapeutic drugs like cape- Authors' Afﬁliations: 1Department of Pharmacology, Yong Loo Lin School citabine, mainly because of their resistance to chemother- of Medicine, 2Cancer Science Institute of Singapore, National University of apy (4). Hence, novel approaches to enhance the effects of 3 4 Singapore; Davos Life Science Pte Ltd; Genome Institute of Singapore; chemotherapeutic drugs and reduce their resistance are and 5Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore; and 6School of Anatomy, Physiology and Human Biology, The imperative. University of Western Australia, Crawley, Perth, Western Australia Various lines of evidence suggest that the activation of Note: K.A. Manu and M.K. Shanmugam contributed equally to this work. master transcription factor NF-kB and its regulated gene Corresponding Authors: Gautam Sethi, Department of Pharmacology, products play a pivotal role in growth, metastasis, and Yong Loo Lin School of Medicine, Cancer Science Institute of Singapore, chemoresistance of gastric cancer. First, NF-kB is constitu- National University of Singapore, 10 Medical Drive, Singapore 117456. tively activated in human gastric cancer tissue and is asso- Phone: 65-65163267; Fax: 65-68737690; E-mail: [email protected] and Patrick Tan, Cancer and Stem Cell Biology, Duke-NUS Graduate Medical ciated with tumor progression (5). Second, it promotes School, Singapore 169857. Phone: 65-65161783; Fax: 65-62212402; gastric cancer growth by inhibiting apoptosis (6). Third, E-mail: [email protected] NF-kB mediates the induction of mitogenic gene products doi: 10.1158/1078-0432.CCR-11-2470 such as cyclin D1, which is overexpressed in human gastric 2012 American Association for Cancer Research. cancer tissue and is inversely correlated with poor prognosis

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(17), phosphoinositide 3-kinase (PI3K)/Akt (30), NF-kB Translational Relevance (13), STAT3 (20), telomerase (31), PPAR-g (32), hypoxia- Despite advances in earlier detection and therapy for inducible factor-1a (33), b-catenin (23), EGF (24), and gastric cancer, it still remains the second leading cause of inhibitor of differentiation family proteins (34) have been cancer death worldwide, killing more than 600,000 implicated. people annually worldwide. Existing drugs lack efficacy Although g-tocotrienol has been found to suppress pro- and yet are highly toxic. For example, although capeci- liferation, to inhibit invasion/migration, and induce apo- tabine is used routinely in the treatment of gastric cancer, ptosis in human gastric cancer SGC-7901 cells (28–31), but development of resistance to this treatment in the its potential to act as a chemosensitizing agent in gastric patients is one of the major problems. Thus agents which cancer cell lines and xenograft models has never been can overcome the resistance and can enhance the effect explored before (16–19). Thus, in the present study, we of capecitabine are urgently needed. Through in vitro and investigated whether g-tocotrienol could sensitize human in vivo experiments, we show for the first time that gastric cancer to capecitabine in vitro and in a xenograft g-tocotrienol, a vitamin E analogue, is one such agent mouse model. Our observations indicate for the first time that can reduce the resistance and can potentiate the that g-tocotrienol can inhibit the proliferation of various effect of capecitabine against gastric cancer. Because gastric cancer cells, enhanced capecitabine-induced apopto- g-tocotrienol is already in clinical trials, the present study sis, and potentiated the antitumor activity of capecitabine in may form the basis of novel therapeutic options for the human xenograft gastric cancer model through the modu- treatment of patients with gastric cancer. lation of NF-kB and NF-kB–regulated gene products.

Materials and Methods Reagents and poor survival (7). Fourth, NF-kB plays an important g-Tocotrienol with purity more than 97% was obtained role in regulating the CXC motif receptor 4 (CXCR4; ref. 8) from Davos Life Science. MTT, Tris base, glycine, NaCl, SDS, and COX-2 (9) that are associated with gastric cancer bovine serum albumin (BSA), b-actin antibody, and corn metastasis. Finally, it is well established that Helicobacter oil were purchased from Sigma-Aldrich. g-Tocotrienol was pylori, a well-known risk factor for gastric carcinoma, is a dissolved in dimethylsulfoxide as a 10 mmol/L stock solu- potent activator of NF-kB in gastric epithelial cells (10). tion and stored at 4 C for in vitro and in corn oil for in vivo Moreover, NF-kB pathway is also responsible for the experiments, respectively. Further dilution was done in cell increased generation of several cell adhesion molecules culture medium. RPMI-1640 media, FBS, 0.4% trypan blue including intercellular adhesion molecule-1 (ICAM-1), vital stain, and antibiotic–antimycotic mixture were whose expression is significantly correlated with an increase obtained from Invitrogen. Antibodies against p65, matrix in H. pylori–induced gastritis (11). Together, these findings metalloproteinase-9 (MMP-9), Bcl-2, Bcl-xL, COX-2, ICAM- implicate the involvement of NF-kB pathway in gastric 1, cyclin D1, survivin, Mcl-1, VEGF, and XIAP were obtained cancer and thus the agents that can modulate NF-kB and from Santa Cruz Biotechnology. CXCR4 antibody was NF-kB–regulated gene products have enormous potential obtained from Abcam. CD31 antibody was purchased from for the treatment of gastric cancer. Cell Signaling Technology. Ki-67 antibody was purchased One such agent derived from natural sources that can from BD Pharmingen, Inc. Goat anti-rabbit horseradish have a great potential for gastric cancer prevention and peroxidase (HRP) conjugate and goat anti-mouse HRP were treatment is vitamin E constituent, g-tocotrienol derived purchased from Invitrogen. Capecitabine was obtained from palm oil and rice bran. Vitamin E family of com- from Duheng International Trading Company Ltd. and pounds primarily consists of 4 tocotrienols and 4 tocopher- dissolved in sterile PBS on the day of use. ols (12). However, while tocopherols had been intensively studied for their health benefits, many novel benefits of Cell lines tocotrienols are only beginning to be brought to light by The gastric cancer cell lines SNU-5 and SNU-16 were research in the last decade (13, 14). For instance, g-toco- obtained from the American Type Culture Collection. trienol has been reported to suppress the proliferation of a MKN45 cells were obtained from JCRB (Japanese Collec- wide variety of tumor cells (15), including gastric (16–19), tion of Research Bioresources), Japan. All the gastric cancer hepatocellular carcinoma (20), melanoma (21), breast cell lines were cultured in RPMI-1640 media supplemented (22), colorectal (23), and prostate (24). In vivo mice studies with 10% FBS, 100 units/mL penicillin, and 100 mg/mL have shown that g-tocotrienol can suppress the growth of streptomycin. breast tumor (25), prostate (26), lung cancer and melanoma (27) and also inhibit the growth of liver and pancreatic Western blotting cancer either alone or in combination with chemothera- For detection of various proteins, gastric cancer cells (2 peutic drugs and radiation (28, 29). How g-tocotrienol 106 per mL) were treated with g-tocotrienol for different mediates its anticancer effects is not completely understood, time intervals. The cells were then washed and extracted but the roles of various signaling cascades/kinases/tran- by incubation for 30 minutes on ice in 0.05 mL buffer scription factors such as mitogen-activated protein kinases containing 20 mmol/L HEPES, pH 7.4, 2 mmol/L EDTA,

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250 mmol/L NaCl, 0.1% NP-40, 2 mg/mL leupeptin, 2 mg/ cutaneously in the right flank with SNU-5 cells (3 106 mL aprotinin, 1 mmol/L phenylmethylsulfonylfluoride cells/100 mL saline). When tumors have reached 0.25 cm in (PMSF), 0.5 mg/mL benzamidine, 1 mmol/L DTT, and 1 diameter, the mice were randomized into the following mmol/L sodium vanadate. The lysate was centrifuged, and treatment groups (n ¼ 5 per group): (i) untreated control the supernatant was collected. Whole-cell extract protein (corn oil, 100 mL daily); (ii) tocotrienol [1 mg/kg body (30 mg) was resolved on 12% SDS-PAGE, electrotransferred weight, suspended in corn oil, intraperitoneal (i.p.) onto a nitrocellulose membrane, blotted with antibodies injection] 3 times/wk; (iii) capecitabine alone (60 mg/kg against survivin, Bcl-2, Bcl-xL, cyclin D1, VEGF, procaspase- bodyweight, suspended in corn oil, twice weekly by gavage); 3, and PARP and then detected by chemiluminescence and (iv) combination (tocotrienol, 1 mg/kg body weight, (ECL; GE Healthcare). suspended in corn oil, i.p. injection) 3 times/wk and capecitabine (60 mg/kg body weight, suspended in corn oil, Cell proliferation MTT assay twice weekly by gavage). Therapy was continued for 4 The effect of g-tocotrienol on cell proliferation was deter- weeks, and the animals were euthanized 1-week later. mined by the MTT uptake method as described previously Primary tumors were excised and the final tumor volume (20). The cells (5,000 per well) were incubated with g-toco- was measured as V ¼ 4/3pr3, where r is the mean radius of trienol in triplicate in a 96-well plate and then incubated for the 3 dimensions (length, width, and depth). Half of the indicated time points at 37C. An MTT solution was added tumor tissue was fixed in formalin and embedded in par- to each well and incubated for 2 hours at 37C. A lysis buffer affin for immunohistochemistry and routine hematoxylin (20% SDS and 50% dimethylformamide) was added, and and eosin (H&E) staining. The other half was snap frozen in the cells were incubated overnight at 37C. The absorbance liquid nitrogen and stored at 80C. of the cell suspension was measured at 570 nm by Tecan plate reader. Immunohistochemical analysis of gastric tumor samples LIVE/DEAD assay Solid tumors from control and various treatment groups To investigate whether g-tocotrienol could potentiate the were fixed with 10% phosphate-buffered formalin, pro- apoptotic effects of capecitabine in gastric cancer cells, we cessed, and embedded in paraffin. Sections were cut and used a LIVE/DEAD Cell Viability Assay Kit (Invitrogen), deparaffinized in xylene and dehydrated in graded alcohol which is used to determine intracellular esterase activity and and finally hydrated in water. Antigen retrieval was carried plasma membrane integrity. This assay uses calcein, a poly- out by boiling the slide in 10 mmol/L sodium citrate (pH anionic, green fluorescent dye that is retained within live 6.0) for 30 minutes. Immunohistochemistry was carried out cells, and a red fluorescent ethidium homodimer dye that following manufacturer instructions (DAKO LSAB kit). can enter cells through damaged membranes and bind to Briefly, endogenous peroxidases were quenched with 3% nucleic acids but is excluded by the intact plasma mem- hydrogen peroxide. Nonspecific binding was blocked by branes of live cells (20). Briefly, gastric cancer cells (5,000 incubation in the blocking reagent in the LSAB kit (Dako) per well) were incubated in chamber slides, pretreated with according to the manufacturer’s instructions. Sections were g-tocotrienol for 4 hours, and treated with capecitabine for incubated overnight with primary antibodies as follows: 24 hours. Cells were then stained with the assay reagents for anti-p65, anti-COX-2, anti-VEGF, anti-MMP-9, anti-Ki-67, 30 minutes at room temperature. Cell viability was deter- and anti-CD31 (each at 1:100 dilutions). Slides were sub- mined under a fluorescence microscope by counting live sequently washed several times in TBS with 0.1% Tween 20 (green) and dead (red) cells. and were incubated with biotinylated linker for 30 minutes, followed by incubation with streptavidin conjugate provid- Flow cytometric analysis ed in LSAB kit according to the manufacturer’s instructions. To determine the effect on the cell cycle, cells were Immunoreactive species were detected with 3,30-diamino- exposed to combination of g-tocotrienol for 4 hours and benzidine (DAB) as a substrate. Sections were counter- treated with capecitabine for 24 hours. Thereafter cells were stained with the Gill hematoxylin and mounted under glass washed, fixed with 70% ethanol, and incubated for 30 cover slips. Images were taken with an Olympus BX51 minutes at 37C with 0.1% RNase A in PBS. Cells were microscope (magnification, 20). Positive cells (brown) then washed again, resuspended, and stained in PBS con- were quantitated with the ImagePro plus 6.0 software taining 25 mg/mL propidium iodide (PI) for 30 minutes at package (Media Cybernetics, Inc.). room temperature. Cell distribution across the cell cycle was analyzed with a CyAn ADP flow cytometer (Dako Preparation of nuclear extract from gastric tumor Cytomation). samples Gastric tumor tissues (100 mg/mouse) from mice in the Gastric tumor model control and treatment groups were minced and incubated All procedures involving animals were reviewed and on ice for 30 minutes in 0.5 mL of ice-cold buffer A [10 approved by NUS Institutional Animal Care and Use Com- mmol/L HEPES (pH 7.9), 1.5 mmol/L KCl, 10 mmol/L mittee. Six-week-old athymic nu/nu female mice (Animal MgCl2, 0.5 mmol/L dithiothreitol (DTT), and 0.5 mmol/L Resource Centre, Western Australia) were implanted sub- PMSF]. The minced tissue was homogenized with a Dounce

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homogenizer and centrifuged at 16,000 g at 4C for was also examined. We used flow cytometric analysis and an 10 minutes. The resulting nuclear pellet was suspended in esterase staining assay (LIVE/DEAD assay) to establish 0.2 mL of buffer B [20 mmol/L HEPES (pH 7.9), 25% whether g-tocotrienol can potentiate the apoptosis induced glycerol, 1.5 mmol/L MgCl2, 420 mmol/L NaCl, 0.5 by capecitabine. As shown in Fig. 1C and D, the dose of mmol/L DTT, 0.2 mmol/L EDTA, 0.5 mmol/L PMSF, and g-tocotrienol (10 mmol/L) or capecitabine (10 mmol/L) that 2 mg/mL leupeptin] and incubated on ice for 2 hours had minimum effect on apoptosis alone produced enhance- with intermittent mixing. The suspension was then centri- ment of apoptosis when used in combination. fuged at 16,000 g at 4C for 30 minutes. The supernatant (nuclear extract) was collected and storedat70Cuntilused g-Tocotrienol inhibits constitutive and capecitabine- as previously described (35). Protein concentration was induced NF-kB activation in gastric cancer cells measured by the Bradford assay with BSA as the standard. We next examined that how g-tocotrienol potentiates the effects of capecitabine in gastric cancer cells. NF-kB has been Measurement of NF-kB activation in gastric cancer cells shown to be constitutively expressed in gastric cancer and and tumor samples mediates resistance to apoptosis (5, 36, 37). Whether To determine NF-kB activation, we conducted DNA- g-tocotrienol induces downregulation of constitutive NF- binding assay by TransAM NF-kB p65 transcription factor kB activation in SNU-16 and SNU-5 cells was examined by assay kit (Active Motif) according to the manufacturer’s using an ELISA-based DNA-binding assay. Results show that instructions and as described previously (20). Briefly, nucle- the treatment with g-tocotrienol inhibited NF-kB expres- ar extracts from g-tocotrienol–treated gastric cancer cell sion in a dose-dependent manner (Fig. 2A and B). We lines and tumor tissues were incubated in a 96-well plate further observed that chemotherapeutic drug capecitabine coated with oligonucleotide containing the NF-kB consen- was also able to further induce NF-kB activation in a dose- 0 0 sus–binding sequence 5 -GGGACTTTCC-3 . Bound NF-kB dependent manner in MKN45 cells, with maximum acti- was then detected by a specific primary antibody. An HRP- vation observed at 25 mmol/L (Fig. 2C). Interestingly, conjugated secondary antibody was then applied to detect g-tocotrienol was also found to suppress capecitabine- the bound primary antibody and provided the basis for induced NF-kB activation in a dose-dependent manner in colorimetric quantification. The enzymatic product was MKN45 cells (Fig. 2D), thereby indicating that it is a potent measured at 450 nm with a microplate reader (Tecan modulator of both constitutive and inducible NF-kB acti- Systems). Specificity of this assay was tested by the addition vation in gastric cancer cells. Whether g-tocotrienol can also of wild-type or mutated NF-kB consensus oligonucleotide modulate the expression of various NF-kB–regulated gene in the competitive or mutated competitive control wells products was also examined. We found that the g-tocotrie- before the addition of the nuclear extracts. nol suppressed the constitutive expression of antiproliferative (cyclin D1), antiapoptotic (Bcl-2), invasive/metastatic Statistical analysis (ICAM-1, MMP-9, CXCR4), and angiogenic (VEGF) protein Statistical analysis was conducted by the Student t test and expression in a time-dependent manner in SNU16 cells one way ANOVA. A P value of less than 0.05 was considered (Fig. 2E). g-Tocotrienol also induced the cleavage of PARP statistically significant. in SNU-16 cells (Fig. 2E). On the basis of these observations in vitro, we decided to study the effect of g-tocotrienol and Results capecitabine either alone or in combination in an in vivo The purpose of this study was to determine whether gastric cancer xenograft model. g-tocotrienol, a component of vitamin E (with chemical structure shown in Fig. 1A) might have a role in the g-Tocotrienol potentiates the antitumor effects of treatment of gastric cancer either alone or in combination capecitabine in a xenograft gastric cancer model in with capecitabine and if so, through what mechanism(s). nude mice For this, we used 4 different well-characterized human We examined the therapeutic potential of g-tocotrienol gastric cancer cell lines. To facilitate the monitoring of and capecitabine either alone or in combination on the tumor growth in mice, one of these cell lines, SNU-5 was growth of subcutaneously implanted human gastric cancer subcutaneously injected and used in the xenograft trans- cells in nude mice. The experimental protocol is depicted plant model in mice. in Fig. 3A. SNU-5 cells were implanted subcutaneously in the right flank of nude mice. When tumors have reached g-Tocotrienol inhibits the proliferation and 0.25 cm in diameter after a week, the mice were randomized potentiates the effect of capecitabine in gastric cancer into 4 groups and started the treatment as per the experi- cells in vitro mental protocol. The treatment was continued for 4 weeks We first investigated the effect of g-tocotrienol on the and animals were sacrificed after 5 weeks. We found that proliferation of 3 different gastric cancer cell lines. g-Toco- g-tocotrienol alone when given at 1 mg/kg body weight trienol inhibited the growth of all 3 human gastric cancer significantly inhibited the growth of the tumor (P < 0.001 cells (SNU-5, MKN45, and SNU-16) in a dose- and time- when compared with control; Fig. 3B and C). Capecitabine dependent manner (Fig. 1B). Whether g-tocotrienol can alone was also very effective (P < 0.001 when compared potentiate the effect of capecitabine against these 3 cell lines with control; P > 0.05 when compared with g-tocotrienol

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AB γ-Toco μ SNU-5 SNU-16 MKN45 ( mol/L) 1 H 0 0.8 HO 10 CH CH CH3 3 3 0.6 25 H2C O 0.4 CH3 CH3 50 CH3 0.2 0 0 24 48 72 0 24 48 72 0 24 48 72 γ -Tocotrienol Time (h)

C Control γ-Toco Cape γ-Toco + Cape

Figure 1. g-Tocotrienol inhibits the

SNU-5 growth and proliferation, Sub G0–G1 1.24% 8.8% 15.5% 41.16% potentiates the apoptotic effects 37.5% 36.3% G0–G1 60.98% 57.38% of capecitabine in gastric cancer S-phase 15.46% 14.53% 18.1% 5.4% cells in vitro. A, structure of 28.87% 17.13% G2–M 22.32% 19.29% g-tocotrienol. B, MTT assay results showed dose-dependent suppression of cell proliferation in all 3 gastric cancer cell lines treated

SNU-16 with g-tocotrienol. Points, mean of Sub G0–G1 4.14% 7.86% 11.16% 43.25% triplicate; bars, SE. Data are a representative of 2 independent G0–G1 45.02% 42.04% 38.21% 39.02% S-phase 15.04% 15.75% 15.81% 8.79% experiments. C and D, ﬂow G2–M 35.02% 30.97% 29.4% 9.38% cytometric analysis and LIVE/ DEAD assay results indicate that g-tocotrienol (g-Toco, 10 mmol/L) potentiates capecitabine (Cape, MKN45 10 mmol/L)-induced apoptosis in Sub G0–G1 5.78% 8.28% 13.04% 48.81% gastric cancer cells. Data indicated G0–G1 55.81% 46.48% 52.79% 46.84% as percentage proportions of S-phase 15.45% 13.46% 13.3% 3.7% apoptotic gastric cancer cells for G2–M 22.96% 29.46% 20.3% 0.82% LIVE/DEAD assay. Values are mean SE of triplicate. Data are a Control γ-Toco Cape γ-Toco + Cape D representative of 2 independent experiments. SNU-5

3.8 ± 2.5 9.7 ± 3.1 11.8 ± 2.6 41.8 ± 4.3 SNU-16 Apoptosis %

3 ± 1.2 7.1 ± 2.5 13.1 ± 2.2 42.3 ± 5.9 MKN45 5.7 ± 1.7 8.3 ± 2.3 15.1 ± 3.1 47.7 ± 6.1

alone group); and the combination of the 2 agents were density. Whether g-tocotrienol and capecitabine modu- more effective in reducing the tumor burden. The tumor late these markers, was examined. Figure 4A shows that volume in the combination of g-tocotrienol and capecita- both g-tocotrienol (P < 0.05) and capecitabine (P < 0.05) bine group was significantly lower than g-tocotrienol alone alone significantly downregulated the expression of group (P < 0.001) or capecitabine alone group (P < 0.001) Ki-67 in gastric cancer SNU-5 tissue and the combination on day 35 (Fig. 3C and D). of the 2 was most effective (P < 0.001). Similarly when examined for CD31, we found that both agents signi- g-Tocotrienol inhibits CD31 and Ki-67 expression in ficantly reduced the CD31 expression as compared gastric tumor tissues with control group and 2 together were most effective While Ki-67–positive index is used as a marker for cell (P < 0.001 when compared with capecitabine alone; proliferation, the CD31 index is a marker for microvessel Fig. 4B).

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ABC 0.4 0.4 SNU-16 0.4 MKN45 * Figure 2. A and B, DNA-binding SNU-5 assay results showing that 0.35 * 0.3 0.3 g-tocotrienol suppresses the 0.3 * * constitutive activation of NF-kBin * SNU-16 and SNU-5 cells in a dose- 0.25 dependent manner. SNU-16 and 0.2 * 0.2 * 0.2 SNU-5 (1 106) cells were treated with g-tocotrienol (10, 25, and 50 * * 0.15 mmol/L) for 4 hours, and nuclear 0.1 0.1 extracts were prepared and assayed 0.1 Optical density (450 nm) Optical density (450 nm) for NF-kB activation by ELISA-linked 0.05 DNA-binding assay. C, MKN45 0 6 0 0 (1 10 ) cells were treated with 0 102550 01025 50 01020 25 capecitabine (10, 20, and 25 mmol/L) for 4 hours, and nuclear extracts γ-Tocotrienol (μmol/L) γ-Tocotrienol (μmol/L) Capecitabine (μmol/L) were prepared and assayed for NF- DE kB activation by ELISA-linked DNA- γ-Tocotrienol binding assay. D, MKN45 (1 106) cells were pretreated with 0 3 6 12 24 Time (h) g-tocotrienol (10, 25, and 50 mmol/L) Cyclin D1 for 4 hours, stimulated with 0.4 capecitabine 25 mmol/L for 4 hours, Bcl-2 and then the nuclear extracts were prepared and assayed for NF-kB MMP-9 activation by ELISA-linked DNA- 0.3 * binding assay. E, g-tocotrienol ICAM-1 suppressed the constitutive * expression of gene products CXCR4 0.2 involved in proliferation, metastasis, * and antiapoptosis in gastric cancer β-Actin cells. SNU-16 (1 106) cells were treated with 50 mmol/L g-tocotrienol * 0.1 VEGF

for the indicated time points, and Optical density (450 nm) Western blot analysis was β-Actin conducted as described in Materials and Methods. , P < 0.05. 0 μ –– 25 25 25 25 (Cape mol/L) PARP – 50 – 2510 50 (γ-Toco μmol/L) β-Actin

g-Tocotrienol inhibited the constitutive NF-kB was examined by Western blot analysis. We found that expression and NF-kB–regulated gene products in treatment with combination of g-tocotrienol and capecita- gastric tumor tissues bine was effective in downregulating the overexpression of We also evaluated the effect of g-tocotrienol and cape- various gene products regulated by NF-kB (Fig. 5B) and also citabine on NF-kBlevelsingastrictumortissue.Figure involved in various aspects of gastric cancer growth, surviv- 5A, left shows that g-tocotrienol either alone or in com- al, invasion, and metastasis. bination with capecitabine was quite effective in suppres- Whether modulation of nuclear NF-kB, COX-2, VEGF, sing the constitutive expression of NF-kBingastriccancer and MMP-9 can also be detected by immunohistochem- tissue. Capecitabine alone hadnosigniﬁcanteffecton ical methods was also examined. As shown in Figure 6, constitutive NF-kBactivationingastrictissue.AWestern these gene products were signiﬁcantly downregulated in blot analysis for p65 in extracts from tumor samples gastric tumor samples treated with g-tocotrienol in com- showed that g-tocotrienol alone inhibited NF-kB (p65) bination with capecitabine. The downregulation was activation (Fig. 5A, right). more impressive with either g-tocotrienol or capecitabine NF-kB is known to regulate the expression of number of alone. The immunohistochemical analysis results further proteins, including those involved in proliferation (cyclin supports the data obtained from Western blotting. These D1, COX-2), invasion/metastasis (ICAM-1, MMP-9), and results collectively indicate that g-tocotrienol suppresses survival (Bcl-xL, survivin, and XIAP; ref. 38). Whether the activation of NF-kB thereby inhibiting the expression g-tocotrienol and capecitabine can modulate the expression of genes involved in proliferation, survival, invasion, and of these NF-kB–regulated gene products in tumor tissues angiogenesis.

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A A *** D-7 D-0 D-28 D-35 Ki-67 immunohistochemistry 250 Vehicle 200 *** γ-Tocotrienol (1 mg/kg b.wt, i.p. thrice/wk 150 Capecitabine-60 mg/kg, twice/wk-gavage Vehicle γ-Toco

γ-Tocotrienol + Capecitabine 100

50 Tumor Randomization Treatment Sacrifice injection stop animals cells of Ki-67–positive No. B Cape γ-Toco + Cape 0 Cape γ -Toco γ -Toco Vehicle B + Cape CD31 immunohistochemistry *** Vehicle γ-Toco 500

400

300 Vehicle γ-Toco *** Cape γ-Toco + Cape 200 CD*** 2.5 100 2.5 ) 3 ) γ cells of CD31-positive No. 3 Cape -Toco + Cape Vehicle control 2 2 0 Capecitabine 1.5 1.5 γ-Tocotrienol Cape γ -Toco γ -Toco Vehicle γ-Toco + Cape 1 *** + Cape 1 0.5 0.5 Tumor volume (cm volume Tumor

Tumor volume (cm volume Tumor 0 Figure 4. g-Tocotrienol enhances the effect of capecitabine against 0 0 1 4 7 10 13 16 19 22 25 28 tumor cell proliferation and angiogenesis in gastric cancer. A, left,

Cape immunohistochemical analysis of proliferation marker Ki-67 indicates the γ -Toco Days Vehicle inhibition of gastric cancer cell proliferation in g-tocotrienol either alone or in combination with capecitabine-treated groups of animals. Right, þ -Toco + Cape γ -Toco quantification of Ki-67 cells as described in Materials and Methods. Columns, mean of triplicate; bars, SE. , P < 0.001. Left, immunohistochemical analysis of CD31 for microvessel density in gastric Figure 3. g-Tocotrienol potentiates the effect of capecitabine to inhibit the cancer tumors indicates the inhibition of angiogenesis by either growth of gastric cancer in nude mice. A, schematic representation of g-tocotrienol alone and in combination with capecitabine. B, right, experimental protocol described in Materials and Methods. Group I was quantification of CD31þ microvessel density as described in Materials per os given corn oil (100 mL, , daily), group II was given g-tocotrienol and Methods. Columns, mean of triplicate; bars, SE. , P < 0.001. [1 mg/kg body weight (b wt.), i.p. 3 times/wk], group III was given capecitabine (60 mg/kg body weight, twice weekly by gavage), and group IV was given g-tocotrienol (1 mg/kg body weight, i.p. 3 times/wk) and cancer cell lines, potentiated capecitabine-induced apopto- capecitabine (60 mg/kg body weight, twice weekly by gavage). B, sis, and inhibited constitutively active and inducible NF-kB necropsy photographs of mice bearing subcutaneously implanted gastric tumors. C, tumor volumes in mice measured during the course of activation as well as NF-kB–regulated gene products. We experiment and calculated using the formula V ¼ 4/3pr3, , P < 0.001. D, also found that in a xenograft mouse model g-tocotrienol tumor volumes in mice measured on the last day of the experiment at effectively suppressed the growth of gastric cancer alone and 3 autopsy with Vernier calipers and calculated using the formula V ¼ 4/3pr also when used in combination with capecitabine. (n ¼ 5). Columns, mean; bars, SE. , P < 0.001. We first observed that g-tocotrienol can suppress the proliferation of various gastric cancer cell lines in a dose- and time-dependent manner. Our results are in part agree- Discussion ment with those of Sun and colleagues (17) who reported Despite the major improvements in diagnosis and treat- that g-tocotrienol could suppress the proliferation of gastric ment regimens, gastric cancer remains one of the most lethal adenocarcinoma SGC-7901 cells. The inhibitory effects of cancers, with less than 20% of patients surviving up to 5 g-tocotrienol were correlated with the DNA damage and years. Thus, novel agents that are nontoxic, efficacious, and cell-cycle arrest at G0–G1 phase, although the detailed can significantly enhance the effects of existing chemother- molecular mechanism(s) were not elucidated. We found apeutic drugs are urgently needed. The aim of the present that g-tocotrienol caused downregulation of cell prolifer- study was to investigate whether g-tocotrienol, a compo- ative gene products such as cyclin D1, which may explain its nent of vitamin E, could enhance the antitumor activity of potent antiproliferative effects in gastric cancer. capecitabine against human gastric cancer. We found that Specifically, we also found for the first time that g-toco- g-tocotrienol suppressed the proliferation of various gastric trienol when used in combination with capecitabine, is

2226 Clin Cancer Res; 18(8) April 15, 2012 Clinical Cancer Research

g-Tocotrienol Enhances the Effect of Capecitabine in Gastric Cancer

p65 COX-2 VEGF MMP-9 A *** 0.35 *** 0.3 Vehicle – + – + γ-Toco 0.25 ± ± ± ± * – – + + Cape 82 0.92 78 13.5 79 12.1 82 15.7 0.2 p65 0.15 β -Actin γ -Toco 0.1 13 ± 1.3 35 ± 8.1 25 ± 7.8 12 ± 1.9 0.05 Optical density (450 nm) 0 Cape

Cape 45 ± 13.1 41 ± 8.9 51 ± 14.5 69 ± 13.2 γ -Toco Vehicle -Toco + Cape γ -Toco

-Toco + Cape γ -Toco ± ± ± ± B – + – + γ-Toco 7 0.9 11 1.4 9 1.9 9.1 1.4 – – + + Cape

Cyclin D1 Figure 6. Immunohistochemical analysis of nuclear p65, COX-2, VEGF, COX-2 and MMP-9 showed the inhibition of NF-kB, COX-2, VEGF, and MMP-9 MMP-9 by either g-tocotrienol alone or in combination with capecitabine. Percentage, positive staining for the given biomarker. ICAM-1 β-Actin itory effects on invasion, metastasis, and angiogenesis Bcl-xL (18, 41). Survivin We found for the first time that the intraperitoneal XIAP administration of g-tocotrienol alone inhibited the growth β-Actin of human gastric tumors when examined in vivo in a xenograft nude mice model. Tumor growth was inhibited by more than 50% on treatment with g-tocotrienol and Figure 5. g-Tocotrienol enhances the effect of capecitabine against the capecitabine alone, respectively. Also, when the 2 agents – expression of NF-kBandNF-kB regulated gene products in gastric were used in combination, they were found to be much cancer tissue samples. A, left, detection of NF-kBbyDNA-binding assay in tumor tissue samples showed the significant inhibition of NF- more effective and potent. When examined for the mech- kB by combination. , P < 0.001. Right, Western blot analysis anism by which g-tocotrienol manifests its effects in the showed the inhibition of NF-kB(p65)byg-tocotrienol in whole-cell mice against gastric cancer, we found that the proliferation extracts from animal tissue. B, Western blotting showing that marker Ki-67 as well as microvessel density indicator CD31 combination of g-tocotrienol and capecitabine inhibit the expression of NF-kB–dependent gene products cyclin D1, COX-2, MMP-9, was downregulated by g-tocotrienol. Further investigation ICAM-1, Bcl-xL, survivin, and XIAP in gastric tumor tissues. Samples also revealed the downregulation of NF-kB and NF-kB– from 3 mice in each group were analyzed and representative data regulated cyclin D1, COX-2, survivin, Bcl-xL, XIAP, ICAM-1, are shown. MMP-9, and VEGF. All of these effects were further enhanced by capecitabine. g-Tocotrienol has been used in combination therapy with several chemotherapeutic highly effective in inducing apoptosis in gastric cancer cell agents/targeted therapies such as statins in breast and colo- lines. This is very intriguing because although g-tocotrienol rectal cancers (42, 43), celecoxib in breast cancer (44), has been previously reported to induce apoptosis in SGC- gemcitabine in pancreatic cancer (29), EGFR inhibitors 7901 cells (16, 17), its effect in combination with chemo- erlotinib and gefitinib in breast cancer (45, 46), but so far therapeutic agents like capecitabine has never been inves- its effects on gastric cancer mice models either alone or in tigated before in gastric cancer. We observed that this effect combination has never been investigated before. Our results may be mediated because of the downregulation of cell overall suggest for the first time that g-tocotrienol has survival proteins such as Bcl-2 in gastric cancer. Interest- significant potential for the treatment of gastric cancer and ingly, we also observed for the first time that both consti- its effects can be further enhanced by capecitabine. A num- tutive and capecitabine-induced NF-kB activation was sup- ber of clinical trials with tocotrienols in patients with pressed by g-tocotrienol in gastric cancer cells. These results pancreatic, prostate, and breast cancer are already in prog- are consistent with those previously reported with other ress, and based on our results, well-designed clinical studies dietary agents like curcumin (39) and phenethyl isothio- are required for potential translation of our preclinical cyanate (40). Also, g-tocotrienol was found to downregu- findings also in patients with gastric cancer. late the expression of various invasive, metastatic, and angiogenic gene products (ICAM-1, MMP-9, CXCR4, and Disclosure of Potential Conflicts of Interest VEGF) which may account for its recently reported inhib- No potential conflicts of interest were disclosed.

www.aacrjournals.org Clin Cancer Res; 18(8) April 15, 2012 2227

Manu et al.

Grant Support The costs of publication of this article were defrayed in part by the This work was supported by grants from NUS Academic Research Fund payment of page charges. This article must therefore be hereby marked (grant R-184-000-170-112) and National Medical Research Council of advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Singapore (grants R-184-000-211-213 and R-184-000-201-275) to G. Sethi. this fact. A.P. Kumar was supported by grants from the National Medical Research Council of Singapore (grant R-713-000-124-213) and Cancer Science Insti- tute of Singapore, Experimental Therapeutics I Program (grant R-713-001- Received September 26, 2011; revised January 18, 2012; accepted February 011-271). 8, 2012; published OnlineFirst February 20, 2012.

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First Evidence That γ-Tocotrienol Inhibits the Growth of Human Gastric Cancer and Chemosensitizes It to Capecitabine in a Xenograft Mouse Model through the Modulation of NF-κB Pathway

Kanjoormana A. Manu, Muthu K. Shanmugam, Lalitha Ramachandran, et al.

Clin Cancer Res 2012;18:2220-2229. Published OnlineFirst February 20, 2012.

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