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Pro-apoptotic Mechanisms of Action of a Novel VitaminE Analog (α-TEA) and a Naturally Occurring Form ofVitamin E (δ-Tocotrienol) in MDA-MB-435 Human BreastCancer CellsMing-Chieh Shun , Weiping Yu , Abdul Gapor , Rachel Parsons , Jeffrey Atkinson , Bob G.Sanders & Kimberly KlineVersion of record first published: 18 Nov 2009.

To cite this article: Ming-Chieh Shun , Weiping Yu , Abdul Gapor , Rachel Parsons , Jeffrey Atkinson , Bob G. Sanders &Kimberly Kline (2004): Pro-apoptotic Mechanisms of Action of a Novel Vitamin E Analog (α-TEA) and a Naturally OccurringForm of Vitamin E (δ-Tocotrienol) in MDA-MB-435 Human Breast Cancer Cells, Nutrition and Cancer, 48:1, 95-105

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Pro-apoptotic Mechanisms of Action of a Novel Vitamin E Analog(α-TEA) and a Naturally Occurring Form of Vitamin E

(δ-Tocotrienol) in MDA-MB-435 Human Breast Cancer Cells

Ming-Chieh Shun, Weiping Yu, Abdul Gapor, Rachel Parsons, Jeffrey Atkinson,Bob G. Sanders, and Kimberly Kline

Abstract: Vitamin E derivative, RRR-α-tocopheryl succinate(vitaminEsuccinate,VES), isapotentpro-apoptoticagent, in-ducing apoptosis by restoring both transforming growth fac-tor-β (TGF-β) and Fas (CD95) apoptotic signaling pathwaysthat contribute to the activation of c-Jun N-terminal kinase(JNK)–mediatedapoptosis.Objectivesof thesestudieswere tocharacterize signaling events involved in the pro-apoptoticactions of a naturally occurring form of vitamin E, δ-toco-trienol, and a novel vitamin E analog, α-tocopherol ether ace-tic acid analog [α-TEA; 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxyacetic acid]. Like VES,α-TEA and δ-tocotrienol induced estrogen-nonresponsiveMDA-MB-435 and estrogen-responsive MCF-7 human breastcancercells toundergohigh levelsofapoptosis inaconcentra-tion- and time-dependent fashion. Like VES, the two com-pounds induced either no or lower levels of apoptosis in nor-mal human mammary epithelial cells and immortalized butnontumorigenic human MCF-10A cells. The pro-apoptoticmechanisms triggered by the structurally distinct α-TEA andδ-tocotrienol were identical to those previously reported forVES, that is, α-TEA– and δ-tocotrienol–induced apoptosis in-volved up-regulation of TGF-β receptor II expression andTGF-β-, Fas- and JNK-signaling pathways. These data pro-vide a better understanding of the anticancer actions of a di-etary form of vitamin E (δ-tocotrienol) and a novel non-hydrolyzable vitamin E analog (α-TEA).

Introduction

Vitamin E is a general term used indiscriminately to referto a group of naturally occurring compounds called tocoph-erols and tocotrienols as well as synthetic vitamin E, which isa mixture of eight stereoisomers, only one of which is molec-ularly equivalent to natural RRR-α-tocopherol, as well as ac-etate and succinate ester derivatives of both natural (RRR-)

and synthetic (all-rac or dl) α-tocopherol (1). To date, RRR-δ-tocopherol, α-, γ-, and δ-tocotrienols, RRR-α-toco-pherol–based analogs with the presence of a succinic acidmoiety linked by either an ester [referred to as RRR-α-tocopheryl succinate, vitamin E succinate (VES), or α-to-copheryl hemisuccinate] or an ether bond (referred to asα-tocopheryoxybutyric acid) and β-tocopheryl succinatehave been shown to exhibit antiproliferative properties instudies of tumor cells in culture (2–20). In contrast to the vi-tamin E forms cited previously, natural vitamin E (RRR-α-tocopherol), synthetic vitamin E (all-rac-α-tocopherol),RRR-β-tocopherol, and the acetate derivative of RRR-α-tocopherol are ineffective as inducers of apoptosis in cancercells in vitro (3,5–7,18,20).

In addition to in vitro cell analyses, VES (the hydrolyz-able succinic acid derivative of RRR-α-tocopherol) has beendemonstrated to have antitumor activity in animal xenograftand allograft models when administered i.p., suggesting apossible therapeutic potential (18,21–25). VES has also beenshown to have inhibitory effects on carcinogen (benzo[a]py-rene)-induced forestomach carcinogenesis in mice, suggest-ing potential as an anticarcinogenic, chemopreventive agent(26). Of importance, VES has been shown to be nontoxic tonormal cells in culture and when administered in vivo waswell tolerated and did not exhibit side effects (16,18,22).

Previous studies have identified multiple mechanisms ofaction involved in the antitumor activity induced by VES, in-cluding cell-cycle arrest, differentiation, apoptosis, and an-tiangiogenesis (17,19,24). Cell-cycle arrest and differentia-tion involved the mitogen-activated protein kinase (MAPK),extracellular signal-regulated kinase signaling pathway,up-regulation of cyclin-dependent kinase inhibitor p21(Waf1/Cip1), and inhibition of E2F (27–30). Apoptosis in-volved activation of the transforming growth factor-β (TGF-β) and Fas/CD95 signaling pathways upstream of c-Jun N-terminal kinase (JNK) activation, JNK phosphorylation of

NUTRITION AND CANCER, 48(1), 95–105Copyright © 2004, Lawrence Erlbaum Associates, Inc.

M.-C. Shun and K. Kline are affiliated with the Division of Nutrition/A2703, University of Texas at Austin, Austin, Texas 78712. W. Yu and B. G. Sanders areaffiliated with the School of Biological Sciences/C0900, University of Texas at Austin, Austin, Texas 78712. R. Parsons and J. Atkinson are affiliated with theDepartment of Chemistry, Brock University, St. Catharines, Ontario, Canada L2S 3A1. A. Gapor is affiliated with the Malaysian Palm Oil Board, P.O. Box10620, 50720 Kuala Lumpur, Malaysia.

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the transcription factor c-Jun, JNK-mediated Bax translo-cation to mitochondria, and mitochondrial dysregulation re-sulting in release of cytochrome c, caspase activation, andapoptosis (17,30,31). Some reports suggested the deregula-tion of the protein phosphatase-2A/protein kinase C (PKC)pathway as a contributing factor and mitochondrial and ly-sosomal destabilization as major events in VES-mediatedapoptosis (18,31–34).

Mechanisms of action involved in the growth-inhibitoryactivity induced by the tocotrienols are not entirely defined,but several possibilities have been proposed: 1) suppressionof HMG-CoA reductase (3-hydroxy-3-methylglutaryl coen-zyme A) activity and protein isoprenylation (11,35), 2) in-duction of apoptosis (14), 3) antioxidant effects (36), and 4)blockage of PKC activity (37). Limited animal studies havealso been conducted using tocotrienols administered by i.p.injection or by diet, and these have produced mixed out-comes in different animal models (11,35,38–41).

Based on a desire to develop a clinically useful vitaminE-based chemotherapeutic agent we have produced a novel,nonhydrolyzable ether analog of RRR-α-tocopherol, name-ly, 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chro-man-6-yloxyacetic acid (abbreviated RRR-α-tocopheryloxy-acetic acid or RRR-α-tocopherol ether-linked acetic acid an-alog, referred to as α-TEA; Fig. 1) (42). α-TEA has been

shown to be a potent concentration- and time-dependent in-ducer of apoptosis of murine mammary 66cl.4-GFP tumorcells in vitro and to significantly decrease primary tumor bur-den and lung metastasis of 66cl.4-GFP tumor cells trans-planted into Balb/c mice when administered by liposomalaerosol (42).

The purpose of the studies reported here was to character-ize the apoptotic mechanisms of action of the novel vitamin Eanalog α-TEA as well as the naturally occurring pro-apop-totic vitamin E compound δ-tocotrienol. Similar pro-apop-totic mechanisms of action between VES and α-TEA wereexpected based on structural similarities, but identical mech-anisms of action by δ-tocotrienol were unexpected based onseveral structural differences including number of methylconstituents on the chroman head, presence or absence of afree hydroxyl moiety that is the source of potential antioxi-dant properties at the C-6 on the chroman head, and presenceor absence of an unsaturated side chain (Fig. 1).

Studies reported here show that, like VES, both α-TEAand δ-tocotrienol induced apoptosis of MDA-MB-435 andMCF-7 human breast cancer cells in a concentration- andtime-dependent fashion. Regarding pro-apoptotic mecha-nisms of action, both α-TEA and δ-tocotrienol stimulatedTGF-β-, Fas- and JNK-signaling pathways that are neces-sary, at least in part, for their pro-apoptotic effects. These

96 Nutrition and Cancer 2004

Figure 1. Comparison of the structures of different forms of vitamin E. Structure of the parent compound RRR-α-tocopherol; vitamin E derivativeRRR-α-tocopheryl succinate (VES), possessing an ester-linked succinate moiety attached to C-6 of the chroman head of RRR-α-tocopherol; vitamin E analog(α-TEA) possessing an ether-linked acetic acid moiety attached to the C-6 of the chroman head of RRR-α-tocopherol; and the naturally occurring vitamin Ecompound δ-tocotrienol.

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findings provide important insights into the signaling eventscritical for two structurally distinct vitamin E compounds,α-TEA and δ-tocotrienol, in the induction of apoptosis of hu-man epithelial cancer cells.

Materials and Methods

Vitamin E Compounds

VES (RRR-α-tocopheryl succinate) was purchased fromSigma Chemical Co. (St. Louis, MO). α-TEA was producedby structural modification of RRR-α-tocopherol at the C-6position of the chroman ring by attachment of an acetic acidmoiety via an ether linkage as described previously (42).Methods for bulk production of highly purified α-TEA (99%pure) and testing for anticancer properties have been de-scribed previously (42). 1H-NMR and 13C-NMR analyseswere performed to verify α-TEA chemical composition(U.S. Patent # 6,417,223) (42). δ-Tocotrienol was a gift fromthe Malaysian Palm Oil Board (Kuala Lumpur, Malaysia).Pure δ-tocotrienol was isolated from palm vitamin E (toco-trienol-rich fraction) by silica gel column chromatographyusing hexane as the eluting solvent. Chromatography frac-tions were monitored by high-performance liquid chroma-tography (HPLC) using a Hewlett-Packard HPLC 1100 sys-tem with fluorescence detector (excitation 295 nm, emission325 nm) with a YMC 150 × 6 mm column using 0.5%isopropyl alcohol in hexane as eluting solvent and a 1 ml/minflow rate. The fraction containing δ-tocotrienol was sub-jected to repeated chromatography until a single peak(~100% purity) was obtained. Verification of δ-tocotrienolwas based on HPLC elution profile in comparison with aδ-tocotrienol standard, and structure was verified by NMRanalyses as previously described (43).

Cell Culture and Treatments

MDA-MB-435 human breast cancer cells (originally ob-tained from Dr. Janet E. Price, University of Texas M. D. An-derson Cancer Center, Houston, TX) are an estrogen-re-ceptor negative/estrogen-nonresponsive epithelial cell lineisolated from the pleural effusions of a human with breastcancer (44). The MCF-7 cell line (originally provided by Dr.Suzanne Fuqua, Baylor College of Medicine, Houston, TX)is an estrogen-receptor positive/estrogen-responsive humanbreast cancer cell line (45). Human MCF-10A breast epithe-lial cells (purchased from the American Type Culture Collec-tion, Manassas, VA) are an immortalized, nontumorigeniccell line (46). Human mammary epithelial cells (HMECs)were isolated and purified from normal mammary tissuespecimens obtained from the Cooperative Human TissueNetwork (Birmingham, AL). Cells were maintained and rou-tinely examined to verify absence of Mycoplasma contami-nation as previously described (47).

For experiments, the percentage of fetal bovine serum(FBS, HyClone Laboratories, Logan, UT) was reduced to 2%to reduce the amount of RRR-α-tocopherol in FBS that could

be a confounding factor in these studies and to better modelthe low serum exposure of epithelial tissue in vivo. Exponen-tially growing cells were plated at 5 × 106 cells/T-75 flask forWestern immunoblotting assays or 1.5 × 105 cells per well in12-well plates for apoptosis analyses. Cells were allowed toattach overnight and then were incubated with varying con-centrations of VES or α-TEA [dissolved first in ethanol andthen diluted to desired final concentration containing 0.2%(vol/vol) ethanol] or δ-tocotrienol [dissolved first in dimethylsulfoxide (DMSO) at 100 µg/ml, dissolved at 20 µg/ml inethanol, and then diluted to desired final concentration con-taining 0.04% (vol/vol) DMSO and 0.16% (vol/vol) ethanol].Controls used included 1) vehicle control (VEH, amountsof succinic acid and ethanol equivalent to amounts in VEStreatment), 2) DMSO + ethanol (amounts of DMSO and eth-anol equivalent to amounts in δ-tocotrienol treatment), or 3)ethanol (amount of ethanol equivalent to amount in α-TEAtreatment).

Apoptosis Assays: Nuclear MorphologyAnalyses of 4′,6-Diamidino-2-phenylindole–Stained Cells and Poly(ADP-Ribose)Polymerase Cleavage

MDA-MB-435 cells were treated with VES, α-TEA, orδ-tocotrienol, and apoptosis was measured by morphologicalanalyses of 4′,6-diamidino-2-phenylindole (DAPI)–stainedcells for condensed nuclei and fragmented DNA and byWestern immunoblot analyses of poly(ADP-ribose)polymer-ase (PARP) cleavage as previously described (31,47). Cellsin which the nucleus contained clearly condensed chromatinor cells exhibiting fragmented nuclei were scored asapoptotic. Data are reported as percentage of apoptotic cellsper cell population (that is, number apoptotic cells per totalnumber of cells counted). For each sample, a minimum of500 cells per slide was scored. Data are presented as mean ±SD for three independently performed experiments. Re-agents for morphological analyses of apoptosis were pur-chased from Boehringer Mannheim (Indianapolis, IN).

Western Immunoblot Analyses

Western immunoblot analyses were conducted as previ-ously described (47) to detect PARP and its cleavage frag-ment (antibody sc-7150, Santa Cruz Biotechnology, Inc.,Santa Cruz, CA), phosphorylated c-Jun (sc-822, Santa Cruz),c-Jun (sc-045, Santa Cruz), TGF-β receptor II (sc-400, SantaCruz), GAPDH (rabbit polyclonal antibody to purifiedGAPDH was generated in our laboratory; levels of GAPDHwere used to equalize samples for any variations in loadingwhen conducting densitometric comparisons), phosphory-lated JNK1/2 (V793A, Promega, Madison, WI), and JNK(sc-572, Santa Cruz).

Functional Knock Down of JNK

MDA-MB-435 cells stably transfected with a doxycy-cline-inducible Flag-tagged dominant-negative (DN) mutant

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of JNK1 were used. The Flag-JNK1 (APF) construct was akind gift from Dr. Roger Davis (49). Phosphorylation ofthreonine 183 and tyrosine 185 amino acids in JNK1 is re-quired for kinase activity. These two amino acids have beenreplaced with phenylalanine and alanine in DN-JNK1. Theproduction and characterization of these stably transfectedMDA-MB-435 clones have been described previously (49).Briefly, clone #35 cells were grown in the presence of 2µg/ml doxycycline to induce the expression of DN-JNK1.Cells grown in the absence of doxycycline did not expressDN-JNK1 and served as controls. Cells were cultured in thepresence or absence of doxycycline for 2 d prior to treatmentwith α-TEA (10 or 20 µg/ml) or δ-tocotrienol (5 or 10 µg/ml)for 1 d. Cells were collected and analyzed for percentage ofcells undergoing apoptosis and processed for Western im-munoblot analyses to verify decreased levels of phosphory-lated c-Jun.

Functional Knock Down Assays UsingNeutralizing Antibodies to TGF-β 1or Fas/CD95

Functional knock down assays using neutralizing anti-bodies to TGF-β 1 or Fas/CD95 were conducted as previ-ously described (47,50). Briefly, MDA-MB-435 cells wereallowed to adhere overnight and then treated with varyingconcentrations of α-TEA or δ-tocotrienol plus 0.5 µg/ml ofFas-neutralizing monoclonal antibody (clone ZB4, Immu-notech, Westbrook, ME) or 0.5 µg/ml of irrelevant antibody(mouse IgG, Sigma) as control or 1 µg/ml of TGF-β1–neu-tralizing antibody (R&D Systems, Inc., Minneapolis, MN) or1 µg/ml of irrelevant antibody (chicken IgY, R&D Systems,Inc.) as control. Cells were treated for 1 or 2 d with neutral-izing antibodies to Fas or 1 d with neutralizing antibodiesto TGF-β prior to staining with DAPI as described previous-ly for determination of percentage of cells undergoingapoptosis.

Statistical Analyses

Differences among the treatment groups were evaluatedusing the unpaired t-test with unequal variances. A level of P< 0.05 was regarded as statistically significant.

Results

α-TEA– and δ-Tocotrienol–Induced Apoptosisin MDA-MB-435 Cells in a Concentration-and Time-Dependent Manner

We and others have shown that VES is a potent apoptoticinducer in many human cancer cell lines, including breastcancer (16,50,51). For comparative purposes, we includedVES in these analyses of α-TEA– and δ-tocotrienol–inducedapoptosis. MDA-MB-435 cells treated with 2.5, 5, 10, and 20µg/ml VES, α-TEA, or δ-tocotrienol for 2 d exhibited dose-

dependent apoptosis (Fig. 2A). Untreated, VEH, EtOH, andDMSO controls exhibited background levels of apoptosis.

MDA-MB-435 cells treated with 20 µg/ml VES, 20 µg/mlα-TEA, or 5 µg/ml δ-tocotrienol for 1, 2, and 3 d exhibitedtime-dependent apoptosis (Fig. 2B). Induction of apoptosiswas confirmed by the presence of PARP cleavage followingtreatment with 20 µg/ml α-TEA or δ-tocotrienol for 3, 6, 12,and 24 h (Fig. 2C and D, respectively). The 84-kDa cleavagefragment of PARP was evident at both 12 and 24 h after treat-ment, whereas only intact PARP protein was detected in cellstreated for 3 and 6 h and in the EtOH or DMSO con-trol-treated cells (Fig. 2C and D, respectively).

α-TEA, δ-Tocotrienol, and VES InducedBoth MDA-MB-435 and MCF-7 HumanBreast Cancer Cells to Undergo Apoptosisbut Were Less Effective on MCF-10A Cellsand HMECs

α-TEA, δ-tocotrienol, and VES treatments for 3 d inducedestrogen-responsive MCF-7 and estrogen-nonresponsiveMDA-MB-435 cells to undergo apoptosis in a dose-depend-ent manner, whereas immortalized but nontumorigenic hu-man MCF-10A cells and normal HMECs exhibited no orlower levels of apoptosis (Table 1).

α-TEA and δ-Tocotrienol Increased Levelsof the Phosphorylated Active Forms of JNK1and c-Jun and Increased Levels of c-Jun

Analyses of α-TEA and δ-tocotrienol effects on MAPKsignaling showed that both α-TEA and δ-tocotrienol in-creased levels of the active phosphorylated form of JNK1while having no major effect on the constitutive levels of ex-pression of JNK1. Furthermore, both α-TEA and δ-toco-trienol increased the levels of c-Jun protein as well as the lev-els of its phosphorylated active form (Fig. 3A and B).

When MDA-MB-435 cells were treated with α-TEA (20µg/ml) or δ-tocotrienol (7.5 µg/ml) for 3, 6, 12, and 24 h, ele-vated levels of the phosphorylated form of JNK1 were de-tected by Western immunoblotting analyses (Fig. 3A and B,top panels). Densitometric analyses showed increased levelsof p-JNK1 following treatment with α-TEA (6–24 h) orδ-tocotrienol (3–24 h) when normalized to levels of JNK1 atthe same time point (Fig. 3A and B, top panels). Levels ofphosphorylated c-Jun were increased 6 h after treatment withα-TEA or δ-tocotrienol and remained elevated for 24 h (Fig.3A and B, third panels). Levels of c-Jun protein were also in-creased at 6-, 12-, and 24-h treatment with α-TEA or after 3-to 24-h treatment with δ-tocotrienol (Fig. 3A and B, fourthpanels).

Expression of a DN Mutantof JNK1 Reduced α-TEA–and δ-Tocotrienol–Induced Apoptosis

To address the biological relevance of increased levels ofphosphorylated JNK in α-TEA– and δ-tocotrienol-induced

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Figure 2. VES–, α-TEA–, and δ-tocotrienol–induced MDA-MB-435 human breast cancer cells to undergo apoptosis. MDA-MB-435 cells at 1.5 × 105 cells/ml in12-wellplateswereculturedwith(A)2.5,5,10,and20µg/mlVES,α-TEA,orδ-tocotrienolfor2dor(B)20µg/mlVES,20µg/mlα-TEA,or5µg/mlδ-tocotrienolfor1,2, and 3 d before assessment of apoptosis. Cells were monitored for apoptosis (%) by morphological characterization of nuclei from DAPI-stained cells, as described inMaterialsandMethods.Cell lysates fromMDA-MB-435cellsculturedwith20µg/mlof (C)α-TEAor(D)δ-tocotrienol for3,6,12,or24hwereanalyzedbyWesternimmunoblotting forcleavageofcaspase-3substratePARP.PARPcleavage fragmenthasanMr ofapproximately84,000Daltons (84kDa). (AandB)Apoptoticdataaredepicted as the mean ± SD of three separate experiments. (C and D) Western immunblot data are representative of three independent experiments.

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apoptosis, the effects of a functional knock down of JNKwere assessed using MDA-MB-435 cells (clone #35) stablytransfected with a doxycycline-inducible DN mutant ofJNK1. Cells were cultured with or without 2 µg/ml doxy-cycline for 2 d prior to treatment with 10 or 20 µg/ml α-TEAor 5 or 10 µg/ml δ-tocotrienol for 1 d. Doxycycline inductionof DN-JNK1 significantly reduced levels of α-TEA–inducedapoptosis in comparison with control-uninduced cells (P <0.022 and P < 0.017 for α-TEA treatments of 10 and 20µg/ml, respectively; Fig. 4A). Likewise, doxycycline induc-tion of DN-JNK1 significantly reduced levels of δ-toco-trienol–induced apoptosis in comparison with control-un-induced cells (P < 0.025 and P < 0.014 for δ-tocotrienoltreatments of 5 and 10 µg/ml, respectively; Fig. 4B).

Western immunoblot analyses were conducted on wholecell lysates from cells cultured in the presence or absence ofdoxycycline and treated with α-TEA or δ-tocotrienol. Phos-phorylated c-Jun protein was reduced in α-TEA– and δ-to-cotrienol–treated cells expressing DN-JNK1 (cells culturedwith doxycycline) and compared with identically treatedcells not expressing DN-JNK1 (cells cultured without doxy-cycline; Fig. 4C, top panel).

α-TEA and δ-Tocotrienol IncreasedExpression of TGF-β R-II Protein,Neutralizing Antibodies to TGF-β 1 Blockedα-TEA and δ-Tocotrienol Induced Apoptosis

Western immunblot/ECL analyses of electrophoreticallyseparated whole cell lysates from α-TEA (20 µg/ml)– orδ-tocotrienol (7.5 µg/ml)–treated MDA-MB-435 cells showedthat both compounds increased TGF-β R-II protein levels at3, 6, 12, and 24 h after treatment (Fig. 5A and B).

To determine whether TGF-β signaling was involved inα-TEA– or δ-tocotrienol–induced apoptosis, MDA-MB-435cells were cultured in the presence of neutralizing (blocking)antibody to TGF-β1 or irrelevant antibody control prior to

treatment with α-TEA or δ-tocotrienol. Pretreatment of thecells with TGF-β1–neutralizing antibody significantly re-duced the ability of 10- and 20-µg/ml treatments with α-TEAto induce apoptosis when compared with identically treatedcells pretreated with irrelevant antibody (P < 0.0037 and P <0.0001 for α-TEA treatments at 10 and 20 µg/ml, respec-tively; Fig. 5C) and significantly reduced the ability of δ-to-cotrienol to induce apoptosis when compared with identi-cally treated cells pretreated with irrelevant antibody (P <0.0001 and P < 0.0005 for δ-tocotrienol treatments at 10 and20 µg/ml, respectively; Fig. 5D).

α-TEA– and δ-Tocotrienol–TriggeredApoptosis Involved Fas Signaling

To ascertain if α-TEA– or δ-tocotrienol–induced apop-tosis involved Fas signaling, MDA-MB-435 cells were cul-tured with either α-TEA or δ-tocotrienol + Fas-neutralizingantibody or irrelevant antibody. Fas-neutralizing antibodysignificantly reduced the ability of α-TEA to induce apop-tosis (P < 0.0017 and P < 0.0013 for α-TEA treatments at 10and 20 µg/ml, respectively; Fig. 6A). Likewise, Fas-neutral-izing antibody significantly reduced the ability of δ-toco-trienol to induce apoptosis (P < 0.0044 and P < 0.012 forδ-tocotrienol treatments at 3.75 and 27.5 µg/ml, respectively;Fig. 6B).

Discussion

The studies reported here provide mechanistic insights intohow vitamin E compounds induce apoptosis in human breastcancer cells. α-TEA, a novel nonhydrolyzable, stable analogof RRR-α-tocopherol (acetic acid moiety attached to the C-6phenol of the chroman head of RRR-α-tocopherol via an etherlinkage), and δ-tocotrienol, a naturally occurring form of vita-min E isolated from palm oil, induced apoptosis in both estro-

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Table 1. Comparison of the Ability of VES, α-TEA, and δ-Toc to Induce Apoptosis in Estrogen-Responsive MCF-7,Estrogen-Nonresponsive MDA-MB-435 Human Breast Cancer Cells, Immortalized but Nontumorigenic MCF-10A Cells, andNormal HMECsa

Percentage of Cell Population Undergoing Apoptosisb

Treatment µg/ml MCF-7 MDA-MB-435 MCF-10A HMECs

VES 5 13 ± 2 17 ± 2 2 ± 1 1 ± 110 30 ± 3.5 33 ± 4 8 ± 2 3 ± 120 47 ± 5 53 ± 6 15 ± 2 5 ± 2

α-TEA 5 20 ± 4 26 ± 8 4 ± 1 1 ± 110 51 ± 9 57 ± 7 12 ± 5 4 ± 220 71 ± 6 70 ± 6 23 ± 9 8 ± 3

δ-Toc 1.25 21 ± 5 25 ± 7 3 ± 0.6 1 ± 0.62.5 46 ± 7 51 ± 6 13 ± 7 3 ± 15 64 ± 8 69 ± 8 29 ± 8 12 ± 5

a: Cells were cultured with three concentrations of VES, α-TEA, or δ-tocotrienol for 3 d and scored for apoptosis using morphological criteria of DAPI-stainedcells. δ-Toc, δ-tocotrienol; HMECs, human mammary epithelial cells.

b: Apoptotic data were determined as percentage of apoptotic cells per total number of cells counted.Data are depicted as the mean ± SD of three independent experiments.

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gen-responsive and nonresponsive human breast cancer cellsin culture in a concentration- and time-dependent manner. In-duction of apoptosis was cell type dependent, with both com-pounds inducing high levels of apoptosis in the epithelial de-rived carcinoma cells (MCF-7 and MDA-MB-435) in contrastto no or very low levels of apoptosis in normal HMECs or no or

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Figure 3. Time course of protein expression and activation status of MAPK,JNK, and transcription factor c-Jun after α-TEA or δ-tocotrienol treatment.MDA-MB-435 cells were treated with (A) 20 µg/ml of α-TEA or (B) 7.5µg/ml of δ-tocotrienol or control and harvested at the indicated times. Proteinlevels of JNK and c-Jun as well as active phosphorylated JNK1 (p-JNK1) andc-Jun (p-c-Jun) forms were determined by Western immunoblotting analyses.Densitometric analyses were conducted to determine fold increases of p-JNK1, p-c-Jun, and c-Jun following treatment with α-TEA or δ-tocotrienol.Vehicle-treated controls or, in cases where protein was not detected in the con-trol, the lowest levelofproteinexpressedintreatedcellswasassignedalevelof1for comparative purposes. Densitometric analyses of levels of p-JNK1 werestandardized using JNK-1 levels at the same time points. Densitometric analy-sesof levelsofp-c-Junandc-JunwerestandardizedusingGAPDHlevels.West-ern immunblot data are representative of three independent experiments.

Figure 4. Cells expressing doxycycline-induced DN JNK1 exhibited resistanceto α-TEA– and δ-tocotrienol–induced apoptosis. MDA-MB-435 cell clone #35,which was stably transfected with a tet-on inducible DN JNK1 construct, wastreatedwith2µg/mlofdoxycycline for2d to induceDNJNK1expressionbefore24-h treatment with (A) 10 or 20 µg/ml α-TEA or (B) 5 or 10 µg/ml δ-tocotrienoland calculation of percentage of cells undergoing apoptosis determined by mor-phological analyses of DAPI-stained cells. Apoptotic data are depicted as themean ± SD of three separate experiments. (A and B) *Significant reduction inlevel of apoptosis. (C) Measurement of phosphorylated c-Jun protein levels wasperformed by Western immunoblotting of whole cell lysates 24 h after treatment.Densitometricanalyseswereconductedtodeterminefolddecreaseofp-c-Junfol-lowing treatment with α-TEA or δ-tocotrienol. p-c-Jun levels in cells cultured inthe absence of doxycycline were assigned a level of 1 for comparisons andGAPDH was used to standardize samples. Western immunoblot data are repre-sentative of data generated by three separate experiments.

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lower levels of apoptosis in the immortalized/nontumorigenicMCF-10A cells at the various levels tested. Investigations ofsignaling events in α-TEA– and δ-tocotrienol–induced apop-tosis revealed that both compounds acted in a similar mannerand involved both Fas (CD95) and TGF-β signaling pathwaysas well as prolonged activation of the MAP kinase JNK andprolonged phosphorylation of one of JNK’s downstream tar-gets, the transcription factor c-Jun; thus, both α-TEA and δ-tocotrienol induced apoptosis in human breast cancer cells in amanner similar to VES, a pro-apoptotic derivative of RRR-

102 Nutrition and Cancer 2004

Figure 5. α-TEA– and δ-tocotrienol–induced elevated expression of TGF-βRII– and TGF-β1–neutralizing antibodies blocked α-TEA– and δ-tocotrien-ol–inducedapoptosis.Wholecell lysates fromMDA-MB-435cells treatedfor3,6,12,and24hwith (A)20µg/mlα-TEAor (B)7.5µg/mlδ-tocotrienolwereanalyzed by Western immunoblotting to determine levels of TGF-β RII. (Aand B) Densitometric analyses were conducted for determining fold increaseof TGF-β RII following treatment with α-TEA or δ-tocotrienol. (B) Controllevels of TGF-β RII were assigned a level of 1, and (A) TGF-β RII levels at 3-hposttreatment were assigned a level of 1 for comparative purposes. (A and B)Densitometric analyses of TGF-β RII were standardized using GAPDH lev-els. Western immunblot data are representative of three independent experi-ments. MDA-MB-435 cells were treated with 1 µg/ml of TGF-β1–neutraliz-ing antibody or 1 µg/ml of irrelevant antibody control as well as 10 or 20 µg/ml(C) α-TEA or (D) δ-tocotrienol for 1 d and then examined for percentage ofcell population exhibiting morphological characteristics of apoptosis. Apop-totic data are depicted as the mean ± SD of three separate experiments. (C andD) *Significant reduction in level of apoptosis.

Figure 6. Inhibitory effects of Fas-neutralizing antibody (Ab) on apoptosisinduced by α-TEA or δ-tocotrienol. MDA-MB-435 cells were treated with(A) 10 or 20 µg/ml α-TEA + 0.5 µg/ml neutralizing antibody to Fas for 2 d or(B) 3.75 or 7.5 µg/ml δ-tocotrienol + 0.5 µg/ml neutralizing antibody to Fasfor 1 d and then examined for percentage of cell population exhibiting mor-phological characteristics of apoptosis. Identically treated cells cotreatedwith 0.5 µg/ml irrelevant antibody plus α-TEA or δ-tocotrienol served ascontrols. Apoptotic data are depicted as the mean ± SD of three separate ex-periments. (A and B) *Significant reduction in level of apoptosis.

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α-tocopherol that has been more extensively characterized forits pro-apoptotic mechanisms of action (6,47,49,50,52–54).

Similar pro-apoptotic mechanisms of action betweenVES and α-TEA were expected based on structural similari-ties, but similar mechanisms of action by δ-tocotrienol werenot expected because δ-tocotrienol differs structurally in sev-eral major ways, namely, δ-tocotrienol in comparison withVES and α-TEA has a single rather than three methyl constit-uents on the chroman head, possesses an unsaturated ratherthan a saturated side chain, and, most notably, possesses ahydroxyl moiety that is the source of antioxidant propertiesat the C-6 on the chroman head rather than a free carboxylgroup that has been hypothesized as being critical for theantiproliferative properties of VES (Fig. 1) (7). Thus, in con-trast to the biological activity of vitamin E compounds as de-fined as the ability to support reproduction capacity whentested in a rat gestation-resorption animal model (55), pro-apoptotic properties of vitamin E compounds do not appearto depend on the presence and position of methyl groups onthe aromatic ring of the chroman head. Perhaps the most sur-prising discovery was the ability of δ-tocotrienol to mediateidentical pro-apoptotic mechanisms of action in the absenceof an acid group (succinic acid group in the case of VES oracetic acid group in the case of α-TEA) attached to the C-6phenol of the chroman head. The necessity of an intact VESmolecule and not the free RRR-α-tocopherol or succinic acidcomponents to produce antiproliferative effects was firstdemonstrated by Fariss et al. (7). The importance of the freecarboxyl group on the C-6 of the phenolic ring is also sup-ported by studies that demonstrated that, in contrast to VES,neither RRR-α-tocopherol nor RRR-α-tocopheryl acetateinhibits cell growth or induces apoptosis (14). Thus, the stud-ies reported here appear to defy any easy explanation basedon simple structural comparisons among these vitamin Ecompounds to account for their common pro-apoptotic influ-ences on Fas, TGF-β, and JNK signaling pathways in humanbreast cancer cells.

Knowledge about the precise role of food-derived vitaminE compounds, vitamin E derivatives used in vitamin supple-ments and in food fortification, and novel analogs in cancerprevention and therapy is lacking. One of the many scientificchallenges in the vitamin E field is to identify and better un-derstand the mechanisms of action of specific vitamin Ecompounds in cancer treatment and prevention. Data re-ported here add to our basic understanding of antitumor ac-tions of vitamin E compounds at the cellular level. In sum-mary, data reported here demonstrate that palm oil-derivedδ-tocotrienol and the novel vitamin E analog (α-TEA) are ex-tremely potent pro-apoptotic agents for human breast cancercells yet exhibit little toxicity toward normal HMECs. Fur-thermore, these studies show that cellular events involved inthe pro-apoptotic actions of both δ-tocotrienol and α-TEAare strikingly similar to those previously reported for VES.Similar pro-apoptotic mechanisms of action between VESand α-TEA were expected based on structural similarities,but identical mechanisms of action by δ-tocotrienol were un-expected based on several structural differences. Further

studies are needed to better understand the apoptotic effectsof these forms of vitamin E and to assess their efficacy aschemopreventive and chemotherapeutic agents in vivo.

Acknowledgments and Notes

We thank Dr. Howard Thames for help with statistical analyses (NationalInstitute of Environmental Health Sciences Center Grant ES07784; Univer-sity of Texas, M.D. Anderson, Houston, TX). This work was supported byPublic Health Service Grant CA59739 awarded by the National CancerInstitute, the Foundation for Research (K. Kline and B. G. Sanders), and theNational Institute of Environmental Health Sciences Center Grant ES 07784(K. Kline and B. G. Sanders are members). Address correspondence toKimberly Kline, Division of Nutrition/A2703, University of Texas at Austin,Austin, TX 78712–1097. Phone: (512) 471–8911; FAX: (512) 232–7040;E-mail: k.kline@mail.utexas.edu.

Submitted 10 July 2003; accepted in final form 15 December 2003.

References

1. Kamal-Eldin A and Appelqvist L-A: The chemistry and antioxidantproperties of tocopherols and tocotrienols. Lipids 31, 671–701, 1996.

2. Prasad KN and Edwards-Prasad J: Effects of tocopherol (vitamin E)acid succinate on morphological alterations and growth inhibition inmelanoma cells in culture. Cancer Res 42, 550–554, 1982.

3. Prasad KN and Edwards-Prasad J: Vitamin E and cancer prevention:recent advances and future potentials. J Am Coll Nutr 11, 487–500,1992.

4. Schwartz J and Sklar G: The selective cytotoxic effects of carotenoidsand α-tocopherol on human cancer cell lines in vitro. J Oral Max-illofac Surg 50, 367–373, 1992.

5. Turley JM, Sanders BG, and Kline K: RRR-α-tocopheryl succinatemodulation of human promyelocytic leukemia (HL-60) cell prolifera-tion and differentiation. Nutr Cancer 18, 201–213, 1992.

6. Charpentier A, Groves S, Simmons-Menchaca M, Turley J, Zhao B, etal.: RRR-α-tocopheryl succinate inhibits proliferation and enhancessecretion of transforming growth factor-beta (TGF-β) by human breastcancer cells. Nutr Cancer 19, 225–239, 1993.

7. Fariss MW, Fortuna MB, Everett CK, Smith JD, Trent DF, et al.: Theselective antiproliferative effects of α-tocopheryl hemisuccinate andcholesteryl hemisuccinate on murine leukemia cells result from the ac-tion of the intact compounds. Cancer Res 54, 3346–3351, 1994.

8. Nesaretnam K, Guthrie N, Chambers AF, and Carroll KK: Effect oftocotrienols on the growth of a human breast cancer cell line in culture.Lipids 30, 1139–1143, 1995.

9. Guthrie N, Gapor A, Chambers AF, and Carroll KK: Palm oiltocotrienols and plant flavonoids act synergistically with each otherand with tamoxifen in inhibiting proliferation and growth of estrogenreceptor-negative MDA-MB-435 and -positive MCF-7 human breastcancer cells in culture. Asia Pacific J Clin Nutr 6, 41–45, 1997.

10. Djuric Z, Heilbrun LK, Lababidi S, Everett-Bauer CK, and FarissMW: Growth inhibition of MCF-7 and MCF-10A human breast cellsby α-tocopheryl hemisuccinate, cholesteryl hemisuccinate and theirether analogs. Cancer Lett 111, 133–139, 1997.

11. He L, Mo H, Hadisusilo S, Qureshi AA, and Elson CE: Isoprenoidssuppress the growth of murine B16 melanomas in vitro and in vivo. JNutr 127, 668–674, 1997.

12. Guthrie N, Gapor A, Chambers AF, and Carroll KK: Inhibition of pro-liferation of estrogen receptor-negative MDA-MB-435 and -positiveMCF-7 human breast cancer cells by palm oil tocotrienols andtamoxifen, alone and in combination. J Nutr 127, 544S–548S, 1997.

Vol. 48, No. 1 103

Dow

nloa

ded

by [

Uni

vers

ity o

f St

elle

nbos

ch]

at 1

9:26

21

Apr

il 20

13

13. Nesaretnam K, Stephen R, Dils R, and Darbre P: Tocotrienols inhibitthe growth of human breast cancer cells irrespective of estrogen recep-tor status. Lipids 33, 461–469, 1998.

14. Yu W, Simmons-Menchaca M, Gapor A, Sanders BG, and Kline K: In-duction of apoptosis in human breast cancer cells by tocopherols andtocotrienols. Nutr Cancer 33, 26–32, 1999.

15. McIntyre BS, Briski KP, Gapor A, and Sylvester PW: Antiproliferativeand apoptotic effects of tocopherols and tocotrienols on preneoplasticand neoplastic mouse mammary epithelial cells. Proc Soc Exp BiolMed 224, 292–301, 2000.

16. Neuzil J, Weber T, Gellert N, and Weber C: Selective cancer cell kill-ing by α-tocopheryl succinate. Br J Cancer 84, 87–89, 2000.

17. Kline K, Yu W, and Sanders BG: Vitamin E: mechanisms of action astumor cell growth inhibitors. J Nutr 131, 161S–163S, 2001.

18. Neuzil J, Weber T, Schroder A, Lu M, Ostermann G, et al.: Inductionof cancer cell apoptosis by α-tocopheryl succinate: molecular path-ways and structural requirements. FASEB J 15, 403–415, 2001.

19. Neuzil J, Weber T, Terman A, Weber C, and Brunk UT: Vitamin E ana-logues as inducers of apoptosis: implications for their potential anti-neoplastic role. Redox Rep 6, 143–151, 2001.

20. Yu W, Sanders BG, and Kline K: RRR-α-tocopheryl succinate inhibitsEL4 thymic lymphoma cell growth by inducing apoptosis and DNAsynthesis arrest. Nutr Cancer 27, 92–101, 1997.

21. Malafa MP and Neitzel LT: Vitamin E succinate promotes breast can-cer tumor dormancy. J Surg Res 93, 163–170, 2000.

22. Barnett KT, Fokum FD, and Malafa MP: Vitamin E succinate inhibitscolon cancer liver metastases. J Surg Res 106, 292–298, 2002.

23. Malafa MP, Fokum FD, Mowlavi A, Abusief M, and King M: VitaminE inhibits melanoma growth in mice. Surgery 131, 85–91, 2002.

24. Malafa MP, Fokum FD, Smith L, and Louis A: Inhibition of angio-genesis and promotion of melanoma dormancy by vitamin E suc-cinate. Ann Surg Oncol 9, 1023–1032, 2002.

25. Weber T, Lu M, Andera L, Lahm H, Gellert N, et al.: Vitamin Esuccinate is a potent novel antineoplastic agent with high selectivityand cooperativity with tumor necrosis factor-related apoptosis-induc-ing ligand (Apo2 ligand) in vivo. Clin Cancer Res 8, 863–869, 2002.

26. Wu K, Shan YJ, Zhao Y, Yu JW, and Liu BH: Inhibitory effects ofRRR-alpha-tocopheryl succinate on benzo(a)pyrene (B(a)P)-inducedforestomach carcinogenesis in female mice. World J Gastroenterol 7,60–65, 2001.

27. Turley JM, Ruscetti FW, Kim S-J, Fu T, Gou FV, et al.: Vitamin Esuccinate inhibits proliferation of BT-20 human breast cancer cells: in-creased binding of cyclin A negatively regulates E2F transactivationactivity. Cancer Res 57, 2668–2675, 1997.

28. You H, Yu W, Sanders BG, and Kline K: RRR-α-tocopheryl succinateinduces MDA-MB-435 and MCF-7 human breast cancer cells to un-dergo differentiation. Cell Growth Differ 12, 471–480, 2001.

29. You H, Yu W, Munoz-Medellin D, Brown PH, Sanders BG, et al.: Roleof extracellular signal-regulated kinase pathway in RRR-α-tocopherylsuccinate-induced differentiation of human MDA-MB-435 breast can-cer cells. Mol Carcinogenesis 33, 228–236, 2002.

30. Yu W, Sanders BG, and Kline K: RRR-α-tocopheryl succinate induc-tion of DNA synthesis arrest of human MDA-MB-435 cells involvesTGF-β-independent activation of p21 Waf1/Cip1. Nutr Cancer 43,227–236, 2002.

31. Yu W, Sanders BG, and Kline K: RRR-α-tocopheryl succinate-in-duced apoptosis of human breast cancer cells involves Bax trans-location to mitochondria. Cancer Res 63, 2483–2491, 2003.

32. Neuzil J, Svensson I, Weber T, Weber C, and Brunk UT: α-Tocopherylsuccinate-induced apoptosis in Jurkat T cells involves caspase-3 acti-vation, and both lysosomal and mitochondrial destabilisation. FEBSLett 445, 295–300, 1999.

33. Neuzil J, Zhao M, Ostermann G, Sticha M, Gellert N, et al.: α-To-copheryl succinate, an agent with in vivo anti-tumour activity, inducesapoptosis by causing lysosomal instability. Biochem J 362, 709–715,2002.

34. Weber T, Dalen H, Andera L, Negre-Salvayre A, Auge N, et al.: Mito-chondria play a central role in apoptosis induced by α-tocopheryl

succinate, an agent with antineoplastic activity: comparison with re-ceptor-mediated pro-apoptotic signaling. Biochemisty 42, 4277–4291,2003.

35. Elson CE: Suppression of mevalonate pathway activities by dietaryisoprenoids: protective roles in cancer and cardiovascular disease. JNutr 125, 1666S–1672S, 1995.

36. Serbinova E, Kagan V, Han D, and Packer L: Free radical recycling andintramembrane mobility in the antioxidant properties of α-tocopheroland α-tocotrienol. Free Radic Biol Med 10, 263–275, 1991.

37. Guthrie N, Gapor A, Chambers AF, and Carroll KK: Inhibition of pro-tein kinase C in MDA-MB-435 human breast cancer cells by palm oiltocotrienols. Proc Can Fed Biol Soc 39, Abstract 262, 1996.

38. Theriault A, Chao J-T, Wang Q, Gapor A, and Adeli K: Tocotrienol: areview of its therapeutic potential. Clin Biochem 32, 309–319, 1999.

39. Komiyama K, Iizuka K, Yamaoka M, Watanabe H, Tsuchiya N, et al.:Studies on the biological activity of tocotrienols. Chem Pharm Bull 37,1369–1371, 1989.

40. Gould MN, Haag JD, Kennan WS, Tanner MA, and Elson CE: Acomparison of tocopherol and tocotrienol for the chemoprevention ofchemically induced rat mammary tumors. Am J Clin Nutr 53,1068S–1070S, 1991.

41. Elson CE and Yu SG: The chemoprevention of cancer by mevalonate-derived constituents of fruits and vegetables. J Nutr 124, 607–614,1994.

42. Lawson KA, Anderson K, Menchaca M, Atkinson J, Sun L-Z, et al.:Novel vitamin E analogue decreases syngeneic mouse mammary tu-mor burden and reduces lung metastasis. Mol Cancer Ther 2, 437–444,2003.

43. Gapor A, Leong WL, Ong ASH, Kawada T, Watanabe H, et al.: Pro-duction of high concentration tocopherols and tocotrienols from palmoil by-products. US Patent No. 5,190,618, March 2, 1993.

44. Price JE, Polyzos A, Zhang RD, and Daniel LM: Tumorigenicity andmetastasis of human breast carcinoma cell lines in nude mice. CancerRes 50, 717–721, 1990.

45. Klotz DM, Castles CG, Fuqua SAW, Spriggs LL, and Hill SM: Differ-ential expression of wildtype and variant ER mRNAs by stocks ofMCF-7 breast cancer cells may account for differences in estrogen re-sponsiveness. Biochem Biophys Res Commun 210, 609–615, 1995.

46. Soule HD, Maloney TM, Wolman SR, Peterson WD, Brenz R, et al.:Isolation and characterization of a spontaneously immortalized humanbreast epithelial cell line, MCF-10. Cancer Res 50, 6075–6086, 1990.

47. Yu W, Israel K, Liao QY, Aldaz CM, Sanders BG, et al.: Vitamin Esuccinate (VES) induces Fas sensitivity in human breast cancer cells:role for Mr 43,000 Fas in VES-triggered apoptosis. Cancer Res 59,953–961, 1999.

48. Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, et al.: Pro-inflam-matory cytokines and environmental stress cause p38 mitogen-acti-vated protein kinase activation by dual phosphorylation on tyrosineand threonine. J Biol Chem 270, 7420–7426, 1995.

49. Yu W, Liao QY, Hantash FM, Sanders BG, and Kline K: Activation ofextracellular signal-regulated kinase and c-Jun-NH2-terminal kinasebut not p38 mitogen-activated protein kinases is required for RRR-α-tocopheryl succinate-induced apoptosis of human breast cancer cells.Cancer Res 61, 6569–6576, 2001.

50. Yu W, Heim K, Qian M, Simmons-Menchaca M, Sanders BG, et al.:Evidence for role of transforming growth factor-β in RRR-α-toco-pheryl succinate-induced apoptosis of human MDA-MB-435 breastcancer cells. Nutr Cancer 27, 267–278, 1997.

51. Zhao B, Yu W, Qian M, Simmons-Menchaca M, Brown P, et al.: In-volvement of activator protein-1 (AP-1) in induction of apoptosis byvitamin E succinate in human breast cancer cells. Mol Carcinog 19,180–190, 1997.

52. Turley JM, Fu T, Ruscetti FW, Mikovits JA, Bertolette DC, et al.: Vita-min E succinate induces Fas-mediated apoptosis in estrogen recep-tor-negative human breast cancer cells. Cancer Res 57, 881–890,1997.

53. Yu W, Simmons-Menchaca M, You H, Brown P, Birrer MJ, et al.:RRR-α-tocopheryl succinate induction of prolonged activation of c-jun

104 Nutrition and Cancer 2004

Dow

nloa

ded

by [

Uni

vers

ity o

f St

elle

nbos

ch]

at 1

9:26

21

Apr

il 20

13

amino-terminalkinaseandc-junduring inductionofapoptosis inhumanMDA-MB-435 breast cancer cells. Mol Carcinog 22, 247–257, 1998.

54. Charpentier A, Simmons-Menchaca M, Yu W, Zhao B, Qian M, et al.:RRR-α-tocopheryl succinate enhances TGF-β1, -β2, and -β3 and

TGF-β R-II expression by human MDA-MB-435 breast cancer cells.Nutr Cancer 26, 237–250, 1996.

55. Machlin LJ: Vitamin E. In: Handbook of Vitamins, LJ Machlin (ed).New York: Marcel Dekker, 1984, pp 99–145.

Vol. 48, No. 1 105

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