08-3028 4242.4249

Mixed Tocopherols Prevent Mammary Tumorigenesis by
γ
Hong Jin Lee, Jihyeung Ju, Shiby Paul, et al. 2009;15:4242-4249. Clin Cancer Res Access the most recent version of this article at: This article cites by 47 articles, 18 of which you can access for free at: This article has been cited by 12 HighWire-hosted articles. Access the articles at: Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications To request permission to re-use all or part of this article, contact the AACR Publications Susceptibility and Prevention Mixed Tocopherols Prevent Mammary Tumorigenesis by InhibitingEstrogen Action and Activating PPAR-;Hong Jin Lee,1Jihyeung Ju,1Shiby Paul,1Jae-Young So,1Andrew DeCastro,1Amanda Smolarek,1Mao-Jung Lee,1Chung S.Yang,1,2 Harold L.Newmark,1,2 and Nanjoo Suh1,2 Purpose: Tocopherols are lipophilic antioxidants present in vegetable oils.Although the antioxi-dant and anticancer activities of a-tocopherol (vitamin E) have been studied for decades, recentintervention studies with a-tocopherol have been negative for protection from cancer in humans.
The tocopherols consist of four isoforms, which are the a, h, g, and y variants, and recent attentionis being given to other isoforms.In the present study, we investigated the inhibitory effect of atocopherol mixture rich in g- and y-tocopherols against mammary tumorigenesis.
Experimental Design: Female Sprague Dawley rats were treated with N-methyl-N-nitrosourea(NMU), and then fed diets containing 0.1%, 0.3 %, or 0.5 % mixed tocopherols rich in g- andy-tocopherols for 9 weeks.Tumor burden and multiplicity were determined, and the levelsof markers of inflammation, proliferation, and apoptosis were evaluated in the serum and inmammary tumors.The regulation of nuclear receptor signaling by tocopherols was studied inmammary tumors and in breast cancer cells.
Results: Dietary administration of 0.1%, 0.3%, or 0.5% mixed tocopherols suppressed mammarytumor growth by 38 %, 50 %, or 80 %, respectively.Tumor multiplicity was also significantlyreduced in all three mixed tocopherol groups.Mixed tocopherols increased the expression ofp21, p27, caspase-3, and peroxisome proliferator activated receptor-g, and inhibited AKT andestrogen signaling in mammary tumors.Our mechanistic study found that g- and y-tocopherols,but not a-tocopherol, activated peroxisome proliferator activated receptor-g and antagonizedestrogen action in breast cancer.
Conclusion: The results suggest that g- and y-tocopherols may be effective agents for theprevention of breast cancer.
Vitamin E is a fat-soluble vitamin essential for humans (1).
United States, a-tocopherol and g-tocopherol are the most The vitamin E family comprises eight lipophilic, naturally common dietary tocopherols due to their high amounts in occurring compounds that include four tocopherols (with a commercially produced vegetable oils such as soybean, corn, saturated phytyl tail) and four tocotrienols (with an unsaturat- and cottonseed (3, 4, 6). Although g-tocopherol is more ed isoprenoid side chain) designated as a, h, g, and y (2). The abundant than a-tocopherol in the human diet, a-tocopherol prevalent tocopherols in foods are the a, g, and y variants, has been considered the classic ‘‘vitamin E'' because it is the natural (RRR) tocopherols differing only in the number and major tocopherol found in plasma and tissues (3), and it has location of the methyl groups in the chromanol ring system superior activity over other tocopherols in the fertility- (Fig. 1). Tocopherols and tocotrienols are phenolic antiox- restoration assay (7, 8).
idants present in many vegetable oils, and all their isomers are Epidemiologic evidence supporting a link between vitamin E known to have strong antioxidant activities (3 – 5). In the and cancer is limited, and the results of a few completed studieswith vitamin E are not consistent (2, 9). Similarly, the results ofstudies with pure a-tocopherol or its more stable acetate ester in Authors'Affiliations: 1Department of Chemical Biology, Ernest Mario School of different cancer models including colon, breast, and prostate, Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey; are also not conclusive (2, 9, 10). A randomized, double-blind, and 2The Cancer Institute of New Jersey, New Brunswick, New Jersey placebo-controlled Alpha-Tocopherol, Beta-Carotene lung can- Received11/19/08; revised 3/13/09; accepted 3/19/09; published OnlineFirst 6/9/09.
Grant support: NIH R03 CA112642, NIH R01CA127645 (N.Suh).
cer prevention study conducted from 1985 to 1993 examined The costs of publication of this article were defrayed in part by the payment of page the effect of a-tocopherol and h-carotene daily supplements charges.This article must therefore be hereby marked advertisement in accordance on the incidence of lung cancer and possibly other cancers (11).
with 18 U.S.C. Section 1734 solely to indicate this fact.
The study found that male smokers receiving 50 mg/day of Note: Current address for Jihyeung Ju: College of Human Ecology, ChungbukNational University, Cheongju, Korea.
synthetic DL-a-tocopherol acetate had a lower prostate cancer Requests for reprints: Nanjoo Suh, Department of Chemical Biology, Ernest incidence and a significant reduction in prostate cancer deaths Mario School of Pharmacy, Rutgers, The State University of New Jersey, 164 when compared with the control group (12).
Frelinghuysen Road, Piscataway, NJ 08854.Phone: 732-445-3400 (x 226); Following the Alpha-Tocopherol, Beta-Carotene cancer Fax: 732-445-0687; E-mail: nsuh@rci.rutgers.edu.
prevention study, two large randomized trials assessing the F 2009 American Association for Cancer Research.
doi:10.1158/1078-0432. CCR-08-3028 effect of vitamin E supplementation on cancer incidence were Clin Cancer Res 2009;15(12) June 15, 200 Mixed Tocopherols and Mammary Tumorigenesis activator, troglitazone (28). Both a- and g-tocopherol have been Translational Relevance shown to activate PPAR-g expression and transactivation incolon cancer cells, and g-tocopherol is a better modulator of Epidemiologic evidence supporting a link between PPAR-g expression than is a-tocopherol (16, 28).
vitamin E (a-tocopherol) and cancer is limited, and the In our earlier study, we showed that dietary administration of results of a few completed human studies with a-tocopherol 0.1% mixed tocopherols inhibited NMU-induced mammary are not conclusive. g-Tocopherol, the most common form tumorigenesis and expression of proliferating cell nuclear of vitamin E in the diet in the United States, has shown antigen in tumor tissues (25). In the present study, we anti-inflammatory and anticancer activity.Our study found determined the efficacy of mixed tocopherols at three different inhibition of mammary tumorigenesis by dietary tocopher- doses on NMU-induced mammary tumorigenesis and also ols rich in g- and/or y-tocopherols.Mechanistic studies investigated the mechanisms of action of tocopherols in showed that the inhibitory activity was associated with mammary tumors and in cultured MCF-7 and T47D human activating peroxisome proliferator activated receptor-g breast cancer cells. We investigated whether mixed tocopherols (PPAR-g) and antagonizing estrogen receptor signaling in inhibit inflammatory markers or regulate nuclear receptor mammary tumors.PPAR-g and estrogen receptor-a are signaling, such as estrogen receptor or PPAR-g, in mammary considered important molecular targets for breast cancer tumors, and whether these actions may contribute to the prevention, and our data showed that g- and y-tocopherols suppression of mammary tumor growth by tocopherols in activate PPAR-g transcription and inhibit estrogen action.
experimental animals. We report here that mixed tocopherols We anticipate that g- and y-tocopherols may be major may inhibit tumor growth through the regulation of nuclear antitumorigenic agents, suggesting that g- and y-tocopher- receptor signaling during mammary tumorigenesis.
ols should be considered for studies in humans.
Materials and Methods established: The Heart Outcomes Prevention Evaluation trial(13) and the Women's Health Study trial (14). These studies Reagents and cell culture.
a-Tocopherol, g-tocopherol, and y-tocoph- found no significant association between cancer incidence and erol were purchased from Sigma. The compounds were dissolved in DMSO.
vitamin E (a-tocopherol) ingestion (13), and there was no The human MCF-7 and T47D breast cancer cell lines were obtained from the overall benefit for prevention of major adverse cardiovascular American Type Culture Collection. The cells were maintained in DMEM/F- events or cancer (14). Interestingly, however, a nested case- 12 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37jC and 5% CO control study with male residents in Washington County, MD, 2, and passed every 3 to 4 d.
Measurement of cell proliferation by [3H]thymidine incorporation.
showed a statistically significant inverse association betweeng MCF-7 and T47D cells were plated at a density of 8,000 cells/well in -tocopherol and the risk of prostate cancer, whereas 24-well plates and treated with compounds in 10% charcoal-stripped a-tocopherol showed no statistically significant benefit (15).
fetal bovine serum/phenol red-free RPMI medium for 3 d. Before g-Tocopherol, the most common form of vitamin E in the U.S.
harvest, the cells were incubated with 1 ACi [3H]thymidine for 3 h at diet and the second most common form in human tissues, has 37jC and were washed with PBS. The cells were precipitated with cold shown anti-inflammatory and anticancer activity in numerous 10% trichloroacetic acid for 10 min and solubilized with 0.5 mL models of colon, breast, and prostate cancer (2, 16 – 19).
solubilization buffer (0.2 M NaOH, 40 Ag/mL salmon sperm DNA) Furthermore, g-tocopherol effectively inhibited cyclooxygenase for 2 h at room temperature. The [3H]thymidine incorporated into the (COX; ref. 20, 21), trapped reactive nitrogen species (22, 23), DNA of MCF-7 and T47D cells was determined using a scintillationspectrometer (Beckman Coulter).
and showed stronger anti-inflammatory activity than a-tocoph- Transient transfection of PPAR-g.
pCMX-mPPAR-g1, PPREx3- erol (21, 23). Our laboratory recently showed cancer-preventive tk-Luc, pCMX-hRXR-a, and pCMV-h-gal vectors were kindly provided activity for mixed tocopherols in animal models of colon and by Dr. David Mangelsdorf (University of Texas Southwestern Medical mammary tumorigenesis (24, 25). We used a readily availablemixed tocopherol preparation (containing over 50% g-tocoph-erol) added at 0.1% to a standardized semipurified laboratorydiet. Administration of the tocopherol-containing diet resultedin a significant inhibition of azoxymethane-induced aberrantcrypt foci in the colon of rats, a recognized early biomarker ofcolon cancer risk (24), and there was also a significant inhibitionof N-methyl-N-nitrosourea (NMU)-induced mammary tumori-genesis (25). In addition, Ju et al. recently reported that themixed tocopherols inhibit colon carcinogenesis by inducingapoptosis, inhibiting inflammation, and reducing the oxidative/nitrosative stress (26).
Tocopherols have been shown to bind to the estrogen receptor and to work as antagonists of estrogen signaling (27). This led usto study the mechanism of inhibition of mammary tumorigen-esis by each tocopherol isoform. In addition to the antiestrogenicaction of tocopherols, they may activate a nuclear receptor,peroxisome proliferator activated receptor-g (PPAR-g), due totheir structural similarity to the thiazolidinedione PPAR-g Fig. 1. Structures of tocopherol variants, a-, h-, g-, and y-tocopherol.
Clin Cancer Res 2009;15(12) June 15, 2009 Susceptibility and Prevention Table 1. Analysis of tocopherol levels in the serum of Sprague-Dawley rats fed a control diet or mixedtocopherol diets AIN-93M control diet 0.1% mixed tocopherols in AIN-93M diet 24.0 F 9.2 (1.2-fold) 1.14 F 0.74b(7.6-fold) 0.59 F 0.41b (19.7-fold) 0.3% mixed tocopherols in AIN-93M diet 29.4 F 7.8 (1.4-fold) 2.87 F 0.73x (19.1-fold) 1.69 F 0.44x (56.3-fold) 0.5% mixed tocopherols in AIN-93M diet 27.7 F 4.3 (1.3-fold) 3.53 F 1.03x (23.5-fold) 2.42 F 0.47x (80.7-fold) *All rats (21 F 1 d old; n = 12 per group) were given an i.p. injection of 50mg NMU per kilogram body weight 1 wk before starting the feeding ofmixed tocopherols. Rats were fed a control or mixed tocopherol – containing diets for 9 wk. Serum samples were collected at autopsy andanalyzed for the levels of retinol, a-tocopherol, g-tocopherol, and y-tocopherol.
cThe data are expressed as the mean F SE (n = 6).
bSignificantly different from control by the Student's t test, P < 0.01.
x Significantly different from control by the Student's t test, P < 0.001.
Center, Dallas, TX) and previously reported (29). For the transient weight). One week after NMU injection, rats were fed AIN-93M control transfection of PPAR-g, PPRE-luc, and RXR-a vectors, DNAs were mixed diet or AIN-93M diets containing mixed tocopherols (0.1, 0.3, or 0.5% with FuGene6 Transfection Reagent (Invitrogen), and this mixture was of the diet). Tumors were palpated weekly. Nine weeks after NMU directly added to MCF-7 and T47D cells in DMEM/F12 medium injection, the rats were sacrificed and the tumors were weighed and without serum. After incubation with the mixture for 4 h, the cells were counted at autopsy. The average tumor burden was the sum of tumor treated with the compounds for an additional 36 h in 10% charcoal- weights in the group per number of rats in the group. The average stripped fetal bovine serum/phenol red free RPMI medium. Luciferase tumor multiplicity was the sum of the number of tumors in each group activity was measured with a luminometer (Turner Biosystems) and per number of rats per group. All animal studies were done in normalized by h-galactosidase activity.
accordance with an institutionally approved protocol.
Diets for animal experiment.
The mixed tocopherols were supplied Analysis of tocopherol levels in rat serum.
Rat serum was collected at by the Cognis Corporation, and contained 57% g-tocopherol, 24% autopsy, and serum tocopherol levels (a-, y-, or g-tocopherol) were y-tocopherol, 13% a-tocopherol, and an insignificant amount of measured by a method modified from a previously described procedure h-tocopherol (about 0.5%). Semipurified modified AIN-93M diet was (30). In brief, fat-soluble vitamins were extracted from 150 AL of obtained from Research Diets Laboratory and used as the control diet.
plasma with ethanol and hexane, and then dissolved in a mixture of The test diet was prepared by adding 0.1%, 0.3%, or 0.5% of mixed ethanol and acetonitrile. A high performance liquid chromatography tocopherols to AIN-93M diet. The diets were stored in sealed containers at (HPLC) system was developed using a Supelcosil LC18 column, 5 Am 4jC, and the food cups were replenished with fresh diets twice weekly.
(4.6  150 mm) with ethanol:acetonitrile (45:55) as the mobile phase.
Animals and experimental procedure.
Female Sprague-Dawley rats A Waters 490 multiwavelength detector (Waters-Millipore) was used to were obtained from Taconic Farms. Rats (21 F 1 d old) were treated detect absorbance at 292 nm (a-, g-, y-tocopherol) and 325 nm with a single i.p. injection of the carcinogen NMU (50 mg/kg body (retinol). Reference samples of pure a-, g-, and y-tocopherol as well as Fig. 2. Mixed tocopherols (MT) inhibitmammary tumor growth in a dose-dependentmanner.All rats (21 F1 d old) were given ani. p. injection of 50 mg NMU per kilogrambody weight 1wk before starting the feedingof 0.1%, 0.3%, and 0.5% mixedtocopherols.Rats were fed with control ormixed tocopherols containing diet for 9 wk.
Tumor volume and body weight weremeasured weekly.The data are expressed asmean F S.E. (n = 12). A, average body weightat autopsy was not affected up to 0.5% mixedtocopherols in the diet. B, mixed tocopherolssignificantly inhibited the mammary tumorgrowth in a dose-dependent manner.
C, average tumor burden in the groups fedmixed tocopherols (0.1%, 0.3%, and 0.5% inthe diet) was reduced by 37.9%, 50.0%, and80.3%, respectively. D, tumor multiplicity ofthe group fed 0.1%, 0.3%, and 0.5% mixedtocopherols in the diet was reduced by39.6%, 47.9%, and 62.5% respectively.
Significant difference between control andmixed tocopherols fed groups wasdetermined by Student's t-test (# P = 0. 05,*P < 0.05, **P < 0.01, *** P < 0.001).
Clin Cancer Res 2009;15(12) June 15, 200 Mixed Tocopherols and Mammary Tumorigenesis Table 2. Serum levels of PGE2, LTB4, and 8-isoprostane from Sprague-Dawley rats fed control diet or mixedtocopherols diet 8-Isoprostane (pg/mL)c AIN-93M control diet 0.1% mixed tocopherols in AIN-93M diet 8,898.7 F 1676.0714.7 F 47.9 0.3% mixed tocopherols in AIN-93M diet 700.9 F 49.0248.4 F 40.6 0.5% mixed tocopherols in AIN-93M diet *All rats (21 F 1 d old, n = 12 per group) were given an i.p. injection of 50mg NMU per kilogram body weight 1 wk before starting the feeding ofmixed tocopherols. Rats were fed with control or mixed tocopherol containing diet for 9 weeks. Serum samples were collected at autopsy andanalyzed for the levels of PGE2, LTB4 and 8-isoprostane.
cThe data are expressed as mean F S.E. (n = 6). There is no significant difference among groups by statistical analysis.
retinol were obtained from the Centers for Disease Control and Inc.) and PPAR-g (1:200; Santa Cruz Biotechnology). The slides were Prevention. The structures of a-, h-, g-, and y-tocopherol are shown in incubated with biotinylated secondary antibody, and then with avidin/ biotinylated peroxidase complex for 30 min at room temperature Histopathologic analyses and immunohistochemistry.
(Vector Labs). The slides were then incubated with 3¶-diaminobenz- tumors from each group were harvested at autopsy and fixed in 10% amine substrate, and the sections were counterstained with Modified formalin for 24 h. They were paraffin-embedded, and cut into 4-Am- Harris Hematoxylin. The images were taken randomly using a Zeiss thick tissue sections. Individual tumors were evaluated histopatholog- AxioCam HRc camera fitted to a Zeiss Axioskope 2 Plus microscope.
ically in H&E-stained tumor sections. For immunohistochemistry, the Western blot analysis.
Tumor samples were homogenized in radio- slides were incubated overnight at room temperature with antibody immunoprecipitation assay buffer (10 mmol/L Tris-HCl, 5 mmol/L against cleaved caspase-3 (1:200 diluted; Cell Signaling Technology EDTA, 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, Fig. 3. Mixed tocopherols (MT) regulate the markers of apoptosis and cell proliferation, as well as the expression of nuclear receptors, PPAR-g and estrogenreceptor-a, in NMU-induced mammary tumors. A, mammary tumor tissues were stained with H&E and immunostained with cleaved caspase-3 and PPAR-g antibodies.
Representative sections in mammary tumor tissues from the control group or mixed tocopherols fed groups (0.1, 0.3, and 0.5%) are shown. B, mRNA level of PPAR-g andestrogen receptor-a in tumors from the control group and those from mixed tocopherols fed groups (0.1, 0.3, and 0.5%) were determined by quantitative PCR. Fivedifferent tumors from each group were analyzed and the average value of mRNA expression levels was shown.Significant difference was determined by Student's t-test(*P < 0.05, **P < 0.01, *** P < 0.001). C, the expression levels of protein markers in mammary tumors were determined by Western blot analysis.Three tumor tissues from eachgroup were randomly selected and pooled for protein analysis.The antibodies against p21, p27, cleaved caspase-3, and PARP were used to probe for the indicatedproteins.The levels of estrogen receptor-a, p-Akt, Akt, and COX-2 were also determined as described in Materials and Methods.
Clin Cancer Res 2009;15(12) June 15, 2009


Susceptibility and Prevention Fig. 4. Mixed tocopherols (MT), g-tocopherol (g-T), and y-tocopherol (y-T) exert antiestrogenic activity and activate PPAR-g in estrogen receptor positive human breastcancer cells. A, inhibition of estrogen-induced growth of MCF-7 andT47D by mixed tocopherols and individual tocopherol isoforms.MCF-7 andT47D cells were treated withtocopherols (10, 30, 60, and 100 Amol/L) in10% charcoal stripped fetal bovine serum/phenol red free RPMI medium for 3 d, together with17-h-estradiol (10 pmol/L), and the cellproliferation was compared using [3H]thymidine uptake assay.Statistical significance, *P < 0.05, **P < 0.01, ***P < 0.001. B, PPAR-g transactivation by mixed tocopherols andindividual tocopherol isoforms.MCF-7 andT47D cells were transfected with DNA vectors (50 ng PPAR-g, 40 ng PPRE-Luc, 50 ng, RXR-a, and 10 ng h-gal in 24-well plates) for4 h in serum-free media.Then, cells were treated with compounds (50 and 100 Amol/L in MCF-7 cells and 20, 50, and100 Amol/L inT47D cells) for an additional 36 h in10%charcoal stripped fetal bovine serum in phenol red free RPMI.Luciferase activity was measured with a luminometer and normalized by h-galactosidase activity.Statisticalsignificance, *P < 0.05, **P < 0.01, ***P < 0.001. C, the effect of protein expression by mixed tocopherols (100 Amol/L) in the presence of17-h-estradiol (10 pmol/L) in MCF-7 cells.
After the incubation of 17-h-estradiol and mixed tocopherols for 24 h, the protein levels of estrogen receptor-a, p-Akt, Akt, and h-actin were determined.
0.1% sodium dodecyl sulfate, 0.1 mmol/L Na3VO4, 1% phenylmethyl- PGE2, LTB4, and 8-isoprostane were determined using enzyme sulfonylfluoride, 1% aprotinin and 0.1% leupeptin) using a Dounce immunoassay kits (Cayman Chemical).
homogenizer (Wheaton), and the protein extracts were electrophoresed Statistical significance was evaluated using in 4% to 15% gradient gels (Biorad) and transferred to a polyvinylidene Student's t-test.
difluoride membrane (PALL). The primary antibodies against poly ADPribose polymerase (PARP), cleaved PARP, cleaved caspase-3, phospho-Akt, Akt (Cell Signaling Technology Inc.), p21, p27, estrogen receptor-a, and COX-2 (Santa Cruz Biotechnology), actin (Sigma), and secondaryantibodies (Santa Cruz Biotechnology) were used.
The serum levels of g-tocopherol and d-tocopherol are Quantitative reverse transcription-PCR analysis.
significantly increased by the administration of mixed tocopherols have been previously reported (31). Labeled primers, including in rats. We used AIN-93M control diet, 0.1%, 0.3%, or 0.5% glyceraldehyde-3-phosphate dehydrogenase, estrogen receptor-a, and mixed tocopherols in AIN-93M diet throughout the 9-week study.
PPAR-g, were obtained from Applied Biosystems.
To determine the bioavailability of the diet tocopherol Enzyme immunoassay.
Procedures for prostaglandin E2 (PGE2) in experimental animals, we collected blood samples at and leukotriene B4 (LTB4) enzyme immunoassay have been reported autopsy and determined the serum levels of retinol, a-, g-, and previously (26). For the determination of PGE2, LTB4, and y-tocopherol (Table 1). Serum levels of retinol in rats fed with 8-isoprostane levels, the tissue homogenates or serum samples 0.1%, 0.3%, or 0.5% mixed tocopherols were comparable were mixed with ethyl acetate, vortexed for 30 min, and thencentrifuged at 10,000  g for 20 min (Sorvall RT 6000B). The with those in rats fed with the control diet. However, rats fed organic layer was collected and dried using a Speed Vacuum Evaporator with 0.1%, 0.3%, or 0.5% mixed tocopherols had much higher (VWRInternational, Inc.). The dried samples were then reconstituted levels of g- and y-tocopherols than the control group (Table 1).
in enzyme immunoassay buffer (Cayman Chemical), and levels of Rats fed with 0.1%, 0.3%, or 0.5% mixed tocopherols had the Clin Cancer Res 2009;15(12) June 15, 200 Mixed Tocopherols and Mammary Tumorigenesis levels of g-tocopherol increased by 7.6-, 19.1-, or 23.5-fold, receptor-a were also affected by mixed tocopherols. As shown respectively. The level of y-tocopherol was barely detectable in the in Fig. 3B, there was a significant induction of PPAR-g mRNA control group fed the AIN-93M diet, but rats fed with 0.1%, 0.3%, level but a decrease of estrogen receptor-a mRNA level. In or 0.5% mixed tocopherols had the levels of y-tocopherol Western blot analysis, we found that dietary administration of increased by 19.7-, 56.3-, or 80.7-fold, respectively. Mixed mixed tocopherols up-regulated the cyclin-dependent kinase tocopherols contained only a negligible amount of h-tocopherol, inhibitors p21 and p27, and increased the level of apoptosis and the serum level of h-tocopherol was not detectable.
markers, cleaved PARP and cleaved caspase-3, in mammary Mixed tocopherols inhibit NMU-induced mammary tumor tumors (Fig. 3C). Mixed tocopherols also down-regulated the growth and multiplicity without affecting the body weight. The protein levels of estrogen receptor-a and phospho-Akt in body weight of rats fed the experimental diet containing 0.1%, mammary tumor tissues (Fig. 3C). However, the expression of 0.3%, or 0.5% mixed tocopherols was not significantly different total Akt and COX-2 protein in NMU-induced mammary from that of rats fed the control diet throughout the 9-week tumor tissues was not significantly changed by treatment with experimental period (Fig. 2A). Starting from five weeks after the mixed tocopherols (Fig. 3C).
carcinogen injection, mammary tumors became palpable, and g- and d-Tocopherols inhibit estradiol-induced cell proliferation the volume of mammary tumors was measured weekly (Fig. 2B).
in estrogen receptor – positive human breast cancer cells. We next Mammary tumors continued to grow in the control group, investigated whether the tumor inhibitory effect of mixed whereas the tumor growth was inhibited in a dose-dependent tocopherols is due to the regulation of estrogen action in the fashion in groups fed with three different doses of mixed breast. Because we found a significant inhibitory effect of mixed tocopherols (Fig. 2B). As shown in Fig. 2C and D, the tumor tocopherols in an estrogen receptor – positive animal model of burden and tumor multiplicity at autopsy were also significantly breast cancer in rats, we chose the estrogen receptor – positive reduced by the administration of mixed tocopherols. The human breast cancer cell lines MCF-7 and T47D for further percentage inhibition of the average tumor burden per rat for analysis in vitro (32). We used 10% charcoal-stripped fetal the groups fed 0.1%, 0.3%, or 0.5% mixed tocopherols was 38%, bovine serum/phenol red free RPMI medium to determine the 50%, or 80%, respectively (Fig. 2C). The average tumor possible antiestrogenic action of the compounds. As shown in multiplicity from the control group was 4.8 F 0.6 tumors per Fig. 4A, a-tocopherol (10, 30, 60, and 100 Amol/L final rat, whereas the average tumor multiplicity from the groups fed concentration) did not significantly inhibit the growth of 0.1%, 0.3%, or 0.5% mixed tocopherols was 2.9 F 0.6, 2.5 F 0.4, MCF-7 and T47D cells. Mixed tocopherols, g-tocopherol and or 1.8 F 0.4 tumors per rat, respectively, which translated into y-tocopherol inhibited the estrogen-induced cell proliferation reductions by 40%, 48%, or 63%, respectively (Fig. 2D).
in MCF-7 cells. y-Tocopherol markedly inhibited estrogen- Treatment with mixed tocopherols does not affect the levels induced cell proliferation in both MCF-7 and T47D cells in a of markers of inflammation and oxidation in the serum. Because g-tocopherol is known to inhibit the activity of the COX g- and d-Tocopherols, but not a-tocopherol, enhance the enzyme (20, 21), we next determined whether inflammatory transactivation of PPAR-g in estrogen receptor – positive human markers are modulated by tocopherols in our animal model of breast cancer cells. PPAR-g, which belongs to the nuclear mammary tumorigenesis. Pro-inflammatory eicosanoids, PGE2 receptor family, is known to be important for the inhibition of and LTB4, are the enzymatic products of the arachidonic acid experimental breast cancer (33 – 35). Because we found that pathway, COX and lipooxygenase-5, respectively. Different mixed tocopherols inhibited mammary tumorigenesis and from a previous report in the azoxymethane/dextran sulfate increased the expression of PPAR-g mRNA and protein in sodium – induced colon cancer model in mice (26), the serum mammary tumors, we investigated which individual isoforms levels of PGE2 and LTB4 in the control group were not activate PPAR-g transcription in MCF-7 and T47D breast cancer significantly different from those in the groups fed with mixed cells. As shown in Fig. 4B, mixed tocopherols activated PPAR-g tocopherols (Table 2). The plasma level of 8-isoprostane, a transcription in MCF-7 and T47D cells. Among the tocopherol marker of oxidative stress, was also measured to determine the isoforms tested, a-tocopherol did not significantly activate antioxidant activity of the tocopherols during mammary PPAR-g transcription, whereas g-tocopherol, and more strongly carcinogenesis. As shown in Table 2, the plasma level of y-tocopherol, activated PPAR-g transcription in MCF-7 and 8-isoprostane in the control group was also similar to that in T47D cells (Fig. 4B).
the mixed tocopherol – fed groups.
Mixed tocopherols inhibit Akt phosphorylation and estrogen Mixed tocopherols regulate PPAR-g and estrogen receptor-a receptor-a expression in MCF-7 human breast cancer cells. To nuclear receptor signaling as well as cell proliferation and determine whether the mixed tocopherols regulate the same apoptosis in mammary tumors. To determine which factors molecular targets in vivo and in vitro, we tested the expression may contribute to the inhibition of mammary tumorigenesis by levels of estrogen receptor-a and phospho-Akt in the presence tocopherols, we next analyzed the mammary tumors for of 17-h-estradiol in MCF-7 human breast cancer cells. As shown various markers. Using H&E staining, we found that all tumors in Fig. 4C, mixed tocopherols down-regulated the expression of evaluated were adenocarcinomas, either papillary, cribriform, estrogen receptor-a and inhibited the level of phospho-Akt or tubular adenocarcinoma type. The tumor grade among the without affecting total Akt.
control group and mixed tocopherol-treated groups was notdifferent (Fig. 3A). However, immunohistochemical analyses determined that administration of mixed tocopherols in-creased the expression of an apoptosis marker, cleaved Tocopherols are phenolic antioxidants present in a variety caspase-3, and a nuclear receptor, PPAR-g, in mammary of vegetable oils, and the biological effects of the classic tumors (Fig. 3A). The mRNA levels of PPAR-g and estrogren vitamin E (a-tocopherol) in cancer prevention have been Clin Cancer Res 2009;15(12) June 15, 2009 Susceptibility and Prevention investigated over many decades (3 – 5). However, the potential breast cancer cells (Fig. 4B). Because estrogen receptor-a has benefits of a-tocopherol in the prevention of cancer in been shown to bind to PPARresponse element and repress preclinical and clinical studies were not conclusive (2, 9, 10, transactivation of the PPAR-g (45), it is likely that activation of 36). Knowledge of the effect of other key tocopherol isomers PPAR-g may mediate the antiestrogenic action of tocopherols, on breast cancer formation is very limited, although recent especially for g- and y-tocopherols.
studies suggest that g- and y-tocopherols have more potent Bonofiglio et al. reported that the PPAR-g and estrogen anti-inflammatory and antioxidant properties than a-tocoph- receptor-a pathways have an opposite effect on the regulation of erol (2, 16 – 21, 23).
the PI3K/Akt transduction (45). Recently, the PPAR-g agonist Because g-tocopherol as a pure isoform is not easily available rosiglitazone was reported to inhibit the Akt phosphorylation for studying long-term dietary administration, we investigated induced by insulin-like growth factor-1 in human adrenocorti- the cancer chemopreventive activity of dietary mixed tocopherols cal carcinoma cells (46), suggesting a possible cross-talk containing 57% g-tocopherol and 24% y-tocopherol in the between the PPAR-g and Akt signaling pathways. Interestingly, NMU-induced mammary tumor model in rats. Oral administra- g-tocotrienol has been shown to inhibit the proliferation of tion of 500 mg of mixed tocopherols (containing 60% g- mammary epithelial cells by regulating ErbB3 phosphorylation tocopherol) daily to human subjects (37) resulted in levels of and eventually reducing Akt signaling (47, 48). We found that serum g-tocopherol similar to what was observed with a 0.1% mixed tocopherols down-regulated the phosphorylation of Akt mixed tocopherol diet in rats (Table 1). The dose of mixed in both tumor tissues and MCF-7 human breast cancer cells tocopherols used in humans (500 mg/day) is approximately (Figs. 3C and 4C), which may contribute to the strong induction 0.1% of a 2,000 kcal daily diet, so a rough correlation of of apoptosis in tumor tissues. Further studies are necessary to supplemented serum levels in humans and in rats in the present determine whether there is interaction between activation of study seems reasonable. We found that mixed tocopherols PPAR-g and regulation of Akt signaling by mixed tocopherols.
significantly suppressed mammary tumor growth, tumor burden, Ju et al. recently reported that mixed tocopherols inhibit and tumor multiplicity in a dose-dependent fashion without inflammation and thus prevent colon carcinogenesis in azoxy- affecting body weight (Fig. 2A to D). We further determined that methane/dextran sulfate sodium – treated CF-1 mice (26). In administration of mixed tocopherols induced apoptosis, breast cancer, there was a significant association between a high inhibited cell proliferation, and regulated the nuclear receptors, expression level of COX-2 and a high risk of ductal carcinoma PPAR-g and estrogen receptor-a, in mammary tumors (Fig. 3).
in situ recurrence among women with ductal carcinoma in situ Estrogen receptor is one of the most important markers in a case-control study (44). In addition, treatment with a COX- involved in human breast carcinogenesis. Administration of 2 inhibitor together with a PPAR-g agonist significantly delayed selective estrogen receptor modulators such as tamoxifen and mammary tumorigenesis in the C3(1)-SV40 tumor antigen raloxifene have significantly reduced the risk of estrogen mouse model (49). These studies suggest that inflammation receptor – positive breast cancer (38, 39). In our study, we may play a role in breast cancer. However, in our NMU-induced found that mixed tocopherols down-regulated the expression of breast cancer model, the serum levels of PGE2 and LTB4 in estrogen receptor-a in mammary tumor tissues (Fig. 3B and C) Sprague Dawley rats were not changed by NMU treatment (data and in estrogen receptor – positive MCF-7 human breast cancer not shown) compared with saline-treated groups, and mixed cells (Fig. 4C). Among the tocopherol isoforms tested, g- tocopherols did not affect their levels in the serum (Table 2) or tocopherol and more strikingly y-tocopherol significantly in mammary tumors (data not shown). Furthermore, the inhibited estradiol-induced growth of estrogen receptor – protein expression level of COX-2 in mammary tumors was not positive human breast cancer cells (Fig. 4A). a-Tocopherol is changed by administration of the mixed tocopherols (Fig. 3C).
tri-methylated at the 5-, 7-, and 8-positions of the chromanol Taken together, our data indicate that inflammation may not be ring, whereas g-tocopherol is dimethylated at the 7- and a key factor in NMU-induced mammary tumorigenesis.
8-positions and y-tocopherol is monomethylated at the 8- PPAR-g and estrogen receptor-a are considered important position (Fig. 1). The difference in the chromanol ring structure molecular targets for cancer chemoprevention, and our data may, in part, contribute to different antiestrogenic activities of showed that mixed tocopherols regulate estrogen receptor-a each isoform. Our studies suggest that y- and/or g-tocopherols and PPAR-g nuclear receptor signaling which may, in part, may inhibit estrogen receptor – positive tumor growth by contribute to their preventive effects in mammary tumorigen- altering the cellular response to estrogen.
esis. In conclusion, mixed tocopherols, particularly g- and/or Interestingly, the tocopherols may regulate another nuclear y-tocopherols, are safe and effective agents for the prevention of receptor, PPAR-g, due to the structural similarity to a known breast cancer in animals, suggesting that g- and y-tocopherols PPAR-g activator, troglitazone (28). The ligands for PPAR-g have should be considered for studies in humans.
shown growth inhibitory effects on different tumor cell typessuch as colon (40, 41), lung (42), and breast cancer (33, 43), andnuclear expression of PPAR-g was reported to be associated with Disclosure of Potential Conflicts of Interest a lower risk of recurrence of women's breast ductal carcinomain situ (44). Therefore, PPAR-g has been considered as a No potential conflicts of interest were disclosed.
molecular target for cancer chemoprevention (33, 34). Here,we showed that mixed tocopherols markedly induced the mRNAand protein expression of PPAR-g in mammary tumors (Fig. 3A and B). In addition, g-, y-, and mixed tocopherols (but not We thank Maria Hyra and Lamberto R.Navoa of the Animal Facility in the a-tocopherol) significantly increased the transactivation of Department of Chemical Biology for their technical assistance in taking care of the PPAR-g in estrogen receptor – positive MCF-7 and T47D human animals, and Dr.Allan Conney for helpful advice on our work.
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Differential response to a selective cannabinoid receptorantagonist (SR141716: rimonabant) in female mice from linesselectively bred for high voluntary wheel-running behaviourBrooke K. Keeneya, David A. Raichlenb, Thomas H. Meeka,Rashmi S. Wijeratnea, Kevin M. Middletona,*, Gregory L. Gerdemanb,wand Theodore Garland Jra Exercise is a naturally rewarding behaviour in human

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