Vitamin E Analog α -TEA, Methylseleninic Acid, and Trans-Resveratrol in Combination Synergistically Inhibit Human Breast Cancer Cell Growth

Abstract

Alpha-tocopherol ether-linked acetic acid analog [2,5,7,8-tetramethyl-2R-(4R, 8R-12-trimethyltridecyl) chroman-6-yloxyacetic acid (α -TEA)] is a novel form of vitamin E effective at killing cancer cells but not normal cells. α -TEA alone and together with methylseleninic acid (MSA) and trans-resveratrol (t-RES) were investigated for ability to induce apoptosis, DNA synthesis arrest, and cellular differentiation and inhibit colony formation in human MDA-MB-435-F-L breast cancer cells in culture. The 3 agents alone were effective in inhibiting cell growth by each of the 4 different assays, and 3-way combination treatments synergistically inhibited cell proliferation in each assay in comparison to individual treatments. Furthermore, combinations of α -TEA, t-RES, and MSA significantly enhanced levels of apoptosis in human breast (MDA-MB-231, MCF7, and T47D) and prostate (LnCaP, PC-3, and DU-145) cancer cell lines as well as in immortalized but nontumorigenic MCF10A cells but not primary cultures of human mammary epithelial cells. Western immunoblotting confirmed the induction of apoptosis in that the 3 agents induced poly(adenosine diphosphate-ribose) polymerase cleavage, with earlier detection and more complete cleavage seen in the combination treatment. Mechanistic studies showed combination treatments to inhibit cell proliferation via downregulation of cyclin D1 and induce apoptosis via activation of caspases 8 and 9 and downregulation of prosurvival proteins FLIP and survivin. In summary, the combination of α -TEA, MSA, and t-RES is more effective than single treatments for inhibiting cell proliferation, inducing cellular differentiation, and inducing cell death by apoptosis in human cancer cells in culture.

Acknowledgments

This work was supported by Public Health Service Grant CA59739 awarded by the National Cancer Institute, the Foundation for Research (K. Kline & B. G. Sanders), the National Institute of Environmental Health Sciences Center Grant ES 07784 (K. Kline and B. G. Sanders are members), and Toxicology Training Grant T32 ES 07247 (predoctoral support for R. M. Snyder).

Notes

^a Cells were treated with Alpha-tocopherol ether-linked acetic acid analog [2,5,7,8-tetramethyl-2R-(4R, 8R-12-trimethyltridecyl) chroman-6-yloxyacetic acid] (α -TEA), methylseleninic acid (MSA), trans-resveratrol (t-RES), or a combination and analyzed after 3 days treatment. Treatments are given in μ M concentrations. Data reflect an average of 3 experiments +/− SD. Percentages have been rounded to the nearest integer. Numbers in italics and parentheses represent fold increase of combination over individual treatment. Abbreviations are as follows: LnCAP, lymph node carcinoma of the prostate; VEH, vehicle.

∗P < 0.05 compared to the individual treatments for that cell line. Data was analyzed using a 1-way analysis of variance followed by a Tukey post hoc test. Align values in TB on ones

^b Data represent synergistic effect as calculated by CalcuSyn.

^c Data represent additive effect as calculated by CalcuSyn.

^a Data are representative of 3 experiments. Abbreviations are as follows: α-TEA, alpha-tocopherol ether-linked acetic acid analog [2,5,7,8-tetramethyl-2R-(4R, 8R-12-trimethyltridecyl) chroman-6-yloxyacetic acid]; t-RES, trans-resveratrol; MSA, methylseleninic acid; CI, combination index; ED50, effective dose 50. The ED₅₀ values were 4.73, 13.2, and 6.9 μ M for α -TEA, t-RES, and MSA, respectively. These values were treated as a 2:2:2 ratio. Other ratios were made with respect to the ED₅₀ values.

^b Data represent synergistic effect as calculated by CalcuSyn.

^c Data represent additive effect as calculated by Calcusyn.