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Dietary Administration of the Proapoptotic Vitamin E Analogue -Tocopheryloxyacetic Acid Inhibits Metastatic Murine Breast Cancer

Departments of ¹ Immunobiology and ² Chemistry and ³ College of Pharmacy, Arizona Cancer Center, BIO5 Institute for Collaborative Bioresearch, Department of Chemistry, University of Arizona, Tucson, Arizona

Requests for reprints: Emmanuel T. Akporiaye, Department of Immunobiology, University of Arizona, 1501 North Campbell Avenue, Tucson, AZ 85724. Phone: 520-626-6859; Fax: 520-626-2100; E-mail: akporiay{at}email.arizona.edu.

	Abstract

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The ability of the vitamin E (RRR--tocopherol) derivatives -tocopheryl succinate (-TOS) and -tocopheryloxyacetic acid (-TEA) to suppress tumor growth in preclinical animal models has recently led to increased interest in their potential use for treating human cancer. To make the use of these vitamin E analogues more clinically relevant, we compared the antitumor efficacy of orally and i.p. delivered forms of -TEA and -TOS against a murine mammary cancer (4T1) that bears resemblance to human breast cancer because of its poor immunogenicity and high metastatic potential. In cell culture studies, we showed that both compounds inhibited tumor colony formation and induced apoptotic death of tumor cells. To avoid solubility concerns associated with the hydrophobicity of -TEA and -TOS, we used the vesiculated forms of -TEA (V-TEA) and -TOS (V-TOS) for the in vivo tumor studies. Both compounds inhibited the growth of preestablished 4T1 tumors when given i.p. However, when given by oral gavage, only the esterase-resistant V-TEA was able to suppress primary tumor growth and reduce lung metastasis. To make this approach more translatable to the clinic, -TEA was incorporated into the diet and fed to tumor-bearing mice. We report here for the first time that dietary -TEA delivery significantly inhibited primary tumor growth and dramatically reduced spontaneous metastatic spread to the lung in prophylactic and therapeutic settings. This study suggests that dietary -TEA could prove useful as a relatively easy and effective modality for treating metastatic breast cancer. (Cancer Res 2006; 66(19): 9374-8)

	Introduction

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Chemotherapy, which is a standard form of adjuvant cancer therapy, is plagued by undesirable adverse side effects, including toxicity to normal cells and suppression of the immune system. These drawbacks have fueled efforts to develop novel anticancer agents that are toxic to tumor cells while sparing normal host cells. In this respect, the antitumor properties of the vitamin E [-tocopherol (-TOH)] derivatives -tocopheryl succinate (-TOS) and -tocopheryloxyacetic acid (-TEA) have recently sparked interest (1) because they have been shown to exhibit selective toxicity toward tumor cells (1–5) and to suppress tumor growth in various rodent and human xenograft tumor models (2, 3, 5–9). -TOS and -TEA are semisynthetic derivatives of vitamin E that structurally share the phytyl tail and the chroman head with vitamin E (Fig. 1A ). -TOS differs from vitamin E in that the hydroxyl group at the number 6 carbon of the phenolic ring of the chroman head has been replaced by a succinic acid residue (Fig. 1B; ref. 4). Because this succinate group is linked to the chroman head by an ester bond, -TOS is sensitive to hydrolytic cleavage by intestinal esterases (5). In contrast, -TEA has an acetic acid moiety attached via a nonhydrolyzable ether bond to the chroman head at this position (Fig. 1C; ref. 3). However, translation of -TOS and -TEA chemotherapy to the clinic has been hampered by the hydrophobic nature of both compounds. To improve the bioavailability of -TOS, Jizomoto et al. (10) developed a vesiculated formulation of -TOS (V-TOS) that is formed by spontaneous self-assembly on addition of sodium hydroxide, rendering it more readily soluble in aqueous solvents. V-TOS generated by this method is effective at inhibiting tumor growth and prolonging survival of B16 melanoma and 4T1 mammary tumor-bearing mice (11, 12). The susceptibility of -TOS to hydrolytic cleavage by intestinal esterases makes it unsuitable for oral delivery because only the intact molecule has antitumor activity (13). In contrast, -TEA is resistant to esterase cleavage and has been shown to exhibit antitumor activity when given by oral gavage to tumor-bearing mice, whereas -TOS was ineffective in this setting (3, 8). In this report, we evaluated the antitumor effect of V-TOS and the vesiculated form of -TEA (V-TEA) delivered by i.p. injection or oral gavage against a highly metastatic murine breast cancer cell line (4T1). With an eye to moving -TEA chemotherapy to the clinic, we incorporated -TEA into mouse chow to prevent and treat spontaneous metastatic breast cancer. We report here for the first time that dietary -TEA significantly inhibited primary tumor growth and dramatically reduced lung metastases before and after primary tumor establishment. This study shows that dietary administration of -TEA is a relatively easy and effective treatment modality that could have translational potential for treatment of metastatic breast cancer.

View larger version (10K): [in this window] [in a new window]   Figure 1. Structure of vitamin E (-TOH; A) and the vitamin E derivatives -TOS (B) and -TEA (C). -TOS possesses an ester-linked succinate moiety attached to carbon number 6 of the chroman head, making it susceptible to hydrolysis and cleavage by esterases. This is in contrast to -TEA that possesses an ether-linked acetic acid moiety attached to the carbon number 6 of the chroman head.

	Materials and Methods

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Tumor cells and cell culture. The 4T1 cell tumor cell line is a variant of 410.4, a tumor subline that was isolated from a spontaneous mammary tumor in a BALB/cfC3H mouse. 4T1 is a poorly immunogenic and highly metastatic murine mammary tumor model that spontaneously metastasizes to the liver, lungs, bone marrow, and brain (15, 16), a characteristic that is shared with human breast cancers. The tumor cells were maintained in complete Iscove's modified Dulbecco's medium (IMDM; JRH Biosciences, Lenexa, KS) containing 100 units/mL penicillin, 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA), 0.75 µg/mL fungizone (Gemini Bio-Products, Woodland, CA), and 10% fetal bovine serum (Gemini Bio-Products).

Assessment of tumor cell viability, clonogenic potential, and apoptotic cell death. For the in vitro cell viability assays, 2 x 10⁵ 4T1 tumor cells per well were allowed to adhere in six-well tissue plates overnight in triplicate and then treated with V-TEA or V-TOS. After 24 hours, nonadherent and adherent cells were collected (200 x g, 5 minutes) and cell number and viability were determined by trypan blue dye exclusion (12). For the clonogenicity assay, 10², 10³, 10⁴, and 10⁵ viable cells (trypan blue negative) from each treatment group were plated in triplicate in 100-mm dishes and incubated (7% CO₂, 37°C) for 10 days in complete IMDM. The resulting colonies were fixed with methanol, stained with Giemsa, and counted. The surviving cell fraction was then determined as described previously (8, 9) using the following formula: surviving fraction = [no. of colonies counted at a given concentration of V-TEA or V-TOS / no. of cells plated at that concentration] / [no. of control colonies counted (PBS) / no. of control cells plated (PBS)].

Determination of poly (ADP-ribose) polymerase cleavage. 4T1 tumor cells (2 x 10⁶) were allowed to adhere overnight in 100-mm tissue culture plates (Sarstedt, Newton, NC) and then treated with V-TEA or V-TOS for 4, 8, 12, or 16 hours after which nonadherent and adherent cells were collected and lysed using Complete Lysis-M Buffer containing protease inhibitors (Roche, Indianapolis, IN) and homogenized by forcing through a 21-gauge needle and placing on ice for 10 minutes. Supernatant was recovered (14,000 x g, 4°C, 15 minutes), and 20 µg of protein per sample were resolved by 10% SDS-PAGE and analyzed using a poly (ADP-ribose) polymerase (PARP)-specific antibody (Cell Signaling Technology, Beverly, MA). To control for equal loading, the membranes were stripped using Restore Western Blot Stripping Buffer (Pierce, Rockford, IL) and reprobed using a glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific antibody (Chemicon, Temecula, CA).

Animal studies. Six- to eight-week-old female BALB/c mice (National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD) were housed at the University of Arizona Animal Facilities (Tucson, AZ) in accordance with the Principles of Animal Care (NIH publication no. 85-23). -TEA was incorporated into the AIN93G diet by Harlan Teklad (Madison, WI) at a concentration of 3.3 g -TEA/kg. Tumor growth was monitored by measuring the tumor length and width with calipers and calculating the tumor volume [V = (L x W²) / 2]. Visible metastatic lung nodules were enumerated by staining with India ink and Fekete's solution (15).

Statistical analysis. Statistical significance of differences among data sets of treatment groups was assessed either by Student's t test, where applicable, or by one-way ANOVA, including Tukey-Kramer post-tests for multiple comparisons. To compare tumor growth rates, growth curves were transformed to linearity, and linear regression analysis was used to determine slopes that were then compared by Student's t test. All analyses were done using the Prism software (GraphPad, San Diego, CA). Ps 0.05 were considered indicative of significant differences between data sets.

	Results and Discussion

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Tumoricidal properties of V-TEA and V-TOS. We compared the growth-inhibitory and tumoricidal properties of V-TEA and V-TOS on the murine breast cancer cell line 4T1. Exposure to V-TEA and V-TOS for 24 hours killed 4T1 tumor cells in a dose-dependent manner with similar efficacy (Fig. 2A ). Treatment of cells with 20 µg/mL of V-TEA or V-TOS caused 67% cell death, which increased to 99% and 100% when treated with 40 and 80 µg/mL of the drug, respectively. In addition, tumor cells that survived the V-TEA or V-TOS treatment were significantly impaired in their ability to proliferate and form colonies in a dose-dependent manner (Fig. 2B).

View larger version (19K): [in this window] [in a new window]   Figure 2. A, tumoricidal effect of V-TEA and V-TOS. 4T1 tumor cells were allowed to adhere overnight in six-well tissue culture plates in triplicate. The cells were then treated with none (PBS), 5, 10, 20, 40, or 80 µg/mL of V-TEA or V-TOS (in PBS). After a 24-hour exposure, nonadherent and adherent cells were collected and cell number and viability were determined by trypan blue dye exclusion. Data are representative of two independent experiments. Points, mean of triplicate samples; bars, SE. B, clonogenic potential of surviving cells following V-TEA or V-TOS treatment. 4T1 tumor cells were allowed to adhere overnight in six-well tissue culture plates. The cells were then treated with none (PBS), 20, 40, or 60 µg/mL of V-TEA or V-TOS. Nonadherent and adherent cells were collected and cell number and viability were determined by trypan blue dye exclusion 24 hours later. Subsequently, 102, 103, 104,and 105 viable cells from each treatment group were plated in triplicate in 100-mm tissue culture dishes and incubated (7% CO2, 37°C) for 10 days in complete culture medium. The resulting colonies were fixed and Giemsa stained. Colonies were counted, and the surviving cell fraction was determined using the following formula: surviving fraction = [no. of colonies counted at a given concentration of V-TEA or V-TOS / no. of cells plated at that concentration] / [no. of control colonies counted (PBS) / no. of control cells plated (PBS)]. Points, mean of triplicate samples; bars, SE. C, induction of tumor cell apoptosis by V-TEA and V-TOS. 4T1 cells were allowed to adhere overnight in tissue culture plates. The cells were then treated with none (PBS) or 40 µg/mL of V-TEA or V-TOS for 4, 8, 12, or 16 hours after which nonadherent and adherent cells were collected and lysed using lysis buffer containing protease inhibitors. Proteins (20 µg/sample) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked using TBS (Tris-buffered saline, 0.05% Tween 20, 5% nonfat dry milk) and stained with a PARP-specific antibody. Full-length PARP, 116 kDa; cleaved PARP, 89 kDa. To control for equal loading, the membranes were stripped and reprobed using a GAPDH-specific antibody.

Oral administration of V-TEA inhibits tumor growth in vivo. Having shown that V-TEA and V-TOS kill tumor cells in vitro, we compared their efficacy as a single treatment modality when given by i.p. injection or oral gavage to control the growth of established 4T1 tumors. For this purpose 5 x 10⁴ viable 4T1 tumor cells were injected orthotopically into the mammary fat pad of BALB/c mice. When tumors became palpable (30 mm³), mice received three i.p. injections of 4 mg of either V-TEA or V-TOS every 4 days as described previously (12). I.p. injection of both V-TEA and V-TOS significantly reduced the tumor growth rate (P = 0.002 and 0.005, respectively), resulting in over 2-fold lower average tumor volumes in the V-TEA-treated (400 ± 129 mm³; P < 0.001) and V-TOS-treated (406 ± 109 mm³; P < 0.001) groups compared with PBS-treated mice (987 ± 229 mm³; Fig. 3A ). When given by daily oral gavage, the esterase-resistant V-TEA significantly inhibited the tumor growth rate (P = 0.017) and reduced the average tumor volume 2-fold to 274 ± 53 mm³ (P < 0.001) compared with PBS-treated mice (699 ± 66 mm³). In contrast, V-TOS given by oral gavage had no effect on tumor progression as tumors grew to a final average tumor volume of 723 ± 67 mm³ in this group (Fig. 3B). Increasing the oral gavage dose of V-TEA to 8 mg did not result in further tumor growth inhibition (data not shown). These findings corroborate studies by others in which liposomal -TEA or -TOS dissolved in peanut oil and ethanol given by oral gavage was shown to suppress lung metastasis of the 66cl murine breast cancer line (3, 8). In addition to its effect on established primary tumors, V-TEA given by oral gavage caused a 2-fold reduction in the average number of spontaneously arising, visible lung metastases (75 ± 5; P < 0.001) compared with V-TOS (152 ± 22) and PBS (151 ± 28; Fig. 3C).

View larger version (22K): [in this window] [in a new window]   Figure 3. A, effect of i.p. injection of V-TEA and V-TOS on tumor growth. BALB/c mice were injected with 5 x 104 4T1 tumor cells into the right mammary fat pad. On tumor establishment (30 mm3, day 14 after tumor cell injection), mice received PBS () or 4 mg (in 200 µL) of V-TEA () or V-TOS () by i.p. injections () on days 14, 18, and 22. Points, mean tumor volumes or the tumor volumes of individual mice and average (—) on day 30 after tumor injection; bars, SE. B, effect of oral delivery of V-TEA and V-TOS by gavage on tumor growth and metastases formation. BALB/c mice were injected with 5 x 104 4T1 tumor cells into the right mammary fat pad. On tumor establishment (, 30 mm3, day 14 after tumor cell injection), mice received PBS () or 4 mg (in 200 µL) of V-TEA () or V-TOS () orally by daily gavage (days 14-30). Points, mean tumor volumes or the tumor volumes of individual mice and average (—) on day 30 after tumor injection; bars, SE. The values in (C) represent the number of lung metastases of individual mice and average (—) on day 30. Asterisks, significant differences between the groups.

Delivery of -TEA in the diet significantly suppresses the growth of established 4T1 mammary tumors.

View larger version (18K): [in this window] [in a new window]   Figure 4. Effect of dietary delivery of -TEA on primary tumor growth and metastases formation. BALB/c mice were injected with 5 x 104 4T1 tumor cells in the right mammary fat pad. A, mice received -TEA diet (5.5 mg -TEA per mouse daily) either from the day of tumor cell injection (, prevention) throughout the study or were switched to the -TEA diet (, established) after tumors became palpable (, 30 mm3, day 11 after tumor cell injection). Untreated mice () received control diet throughout the study. Points, mean tumor volumes or the tumor volumes of individual mice and average (—) on day 29 after tumor injection; bars, SE. The values in (B) represent the number of lung metastases of individual mice and average (—) on day 29. Asterisks, significant differences between the groups.

	Acknowledgments

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Deborah Bradley-Dunlop and Matthew Rausch for technical assistance.

	Footnotes

 
⁴ Unpublished observation.

Received 7/ 3/06. Revised 7/20/06. Accepted 8/21/06.

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This Article Abstract Full Text (PDF) Correction (v67,p1877) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hahn, T. Articles by Akporiaye, E. T. Search for Related Content PubMed PubMed Citation Articles by Hahn, T. Articles by Akporiaye, E. T.