Orally active alpha-tocopheryloxyacetic acid suppresses tumor growth and multiplicity of spontaneous murine breast cancer

Abstract

We recently demonstrated the antitumor efficacy of orally administered alpha-tocopheryloxyacetic acid (alpha-TEA), a redox silent and nonhydrolyzable derivative of naturally occurring vitamin E. In order to move alpha-TEA closer to the clinic to benefit patients with breast cancer, the present study had two goals. First, to determine the minimal effective treatment dose; and second, to test the efficacy of dietary administration of alpha-TEA in the clinically relevant MMTV-PyMT mouse model of spontaneous breast cancer that more closely resembles human disease. The minimal effective dose of alpha-TEA was evaluated in the transplantable 4T1 tumor model and we show a dose-dependent decrease of primary tumor growth and reduction of metastatic spread to the lung. Six-week-old MMTV-PyMT mice were treated with oral alpha-TEA for 9 weeks, with no apparent signs of drug toxicity. The alpha-TEA treatment delayed tumor development and significantly slowed tumor progression, resulting in a 6-fold reduction of the average cumulative tumor size. In addition, oral alpha-TEA caused an 80% reduction in spontaneous metastases. In situ analysis of tumor tissue identified apoptosis as an important mechanism of alpha-TEA-mediated tumor suppression in addition to inhibition of tumor cell proliferation. This study shows, for the first time, the ability of orally administered alpha-TEA to delay tumor onset and to inhibit the progression and metastatic spread of a clinically relevant model of spontaneous breast cancer. Our finding of the high efficacy in this tumor model highlights the translational potential of oral alpha-TEA therapy.

Figure 1. Effect of dietary delivery of α-TEA on primary tumor growth

BALB/c mice were injected with 4T1 mammary tumor cells in the right mammary fat pad (day 0). When tumors became palpable (,~17 mm², day 10 post-tumor cell injection), mice received α-TEA in the diet for 18 days. Untreated mice received control diet throughout the study. (A) The values represent the mean tumor areas ± SEM of 9 mice per group. 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 t-test. (B) The values represent tumor areas of individual mice on day 28 post-tumor injection. Boxed numbers are mean tumor areas ± SEM. Differences of the mean tumor areas were determined by ANOVA including Tukey-Kramer post tests for multiple comparisons. (C) Effect of dietary delivery of α-TEA on tumor spread. BALB/c mice with implanted 4T1 mammary tumors from the above study were sacrificed on day 28 post-tumor cell injection. To determine the number of pulmonary metastases, lungs were inflated with India ink and removed, and the surface lung metastases were counted. Boxed numbers are mean number of lung metastases ± SEM. Differences of the mean number of lung metastases were determined by ANOVA including Tukey-Kramer post tests for multiple comparisons.

Figure 2. Determination of α-TEA serum and tissue levels

(A) Tumor-bearing mice received diets containing indicated amounts of α-TEA for 18 days (day 10 to day 28 post-tumor injection). Subsequently, serum was isolated and α-TEA levels of 4 to 5 individual mice per group were analyzed by HPLC/MSD. (B) Tumor-bearing mice received the 0.3% or the 0.1% α-TEA diet for 8 days (day 18 post-tumor injection). Tumor tissues and sera were isolated, and α-TEA levels of three individual mice per group were analyzed by HPLC/MSD. Boxed numbers are corresponding average serum levels ± SEM.

Figure 3. Effect of dietary delivery of α-TEA on the growth of spontaneously developing mammary tumors

MMTV-PyMT mice (10 mice) received α-TEA in the diet (0.1%) starting at 6 weeks of age (day 42) until 15 weeks of age (day 105) when mice were sacrificed. Untreated mice (9 mice) received control diet throughout the study. (A) The values represent the cumulative tumor areas per mouse. Tumor growth curves were determined by non-linear regression analysis and statistically evaluated for differences by f-test. (B) The values represent tumor multiplicity calculated as: Tumor multiplicity = [# tumors per group] / [# of mice per group]. (C) Effect of dietary α -TEA on tumor spread. Tumor-bearing MMTV-PyMT mice from the above study were sacrificed on day 105. To determine the number of pulmonary metastases, lungs were inflated with India ink and the surface lung metastases were counted. Boxed numbers are the mean numbers of lung metastases per group ± SEM. Differences were evaluated by Mann-Whitney test.

Figure 4. Effect of α-TEA treatment on tumorigenesis in MMTV-PyMT mice

MMTV-PyMT mice received α-TEA in the diet (0.1% diet) starting at 6 weeks of age for 4 weeks when mice were sacrificed. Mammary glands were dissected and either whole mounts or tissue sections were prepared. To prepare whole mounts, the tissues were dried flat on a microscope slide, fixed and stained with carmine red (20x magnification). Tissue sections were prepared from formalin fixed, paraffin embedded tissues and stained with H&E (100x magnification).

Figure 5. α-TEA increases apoptosis and inhibits cell proliferation in MMTV-PyMT tumors

After 4 weeks (day 70) or 9 weeks (day 105) of α-TEA treatment (0.1% diet), tumor sections were examined for cell proliferation by Ki-67 stain and apoptosis by TUNEL assay. Data are depicted as average number of Ki-67 or TUNEL-positive cells in fifteen microscopic fields per tumor sample (Ki-67: 400x magnification; apoptosis: 200x magnification). Differences were evaluated by t-test.