Anticancer actions of natural and synthetic vitamin E forms: RRR-alpha-tocopherol blocks the anticancer actions of gamma-tocopherol

Abstract

Two naturally occurring dietary sources of vitamin E (i.e. RRR-alpha-tocopherol (alphaT) and RRR-gamma-tocopherol (gammaT)), the manufactured synthetic form of vitamin E, all-racemic-alpha-tocopherol (all-rac-alphaT), as well as a potent antitumor analog of vitamin E, RRR-alpha-tocopherol ether-linked acetic acid analog (alpha-TEA), were assessed for anticancer actions. Data showed that gammaT, all-rac-alphaT, and alpha-TEA but not alphaT or alphaT+gammaT significantly inhibited tumor burden of human MDA-MB-231 cells in nude mice. Immunohistochemical analyses of tumor tissue showed that all-rac-alphaT and alpha-TEA increased apoptosis and decreased proliferation in tumor cells while gammaT was associated with increased tumor cell apoptosis only. In vitro data showed alpha-TEA and gammaT but not all-rac-alphaT or alphaT to inhibit colony formation and induce apoptosis. Anticancer actions of alpha-TEA and gammaT involved death receptor 5 protein upregulation, Survivin protein downregulation and poly (ADP-ribose) polymerase cleavage, all of which were blocked by co-treatment with alphaT. In summary, both gammaT and alpha-TEA exhibited promising anticancer properties in vivo and in vitro, whereas all-rac-alphaT exhibited promising anticancer properties in vivo only. Importantly, alphaT not only failed to exhibit anticancer properties but it also reduced anticancer actions of gammaT in vivo and gammaT and alpha-TEA in vitro.

Figure 1

Evaluation of tumor growth. Supplemented diets were initiated at day 0 which was 7 days after tumor cell injections when all tumors were approximately the same size (i.e. 25 mm³). Tumor volumes (mm³) were determined at two-day intervals and are depicted as mean ± S.E. *designates significant difference from control group (p < 0.05).

Figure 2

Assessment of biomarkers of antitumor action. Measures of apoptosis (TUNEL) and proliferation (Ki67) were detected by immunohistochemical analyses of tumor tissue. A and C are photo-picture of representative of TUNEL (A) and Ki67 (B), respectively. B and D are presented as mean ± S.E. of 5 samples in each group. * designates significant difference from control group (p < 0.05).

Figure 3

Colony formation assays. Cells were treated with a range of concentrations of different vitamin E compounds for 10 days. A. photo-picture. B. Colonies are expressed as cell survival (%), which was calculated as number of colonies in treatment/number of colonies in control × 100%. Data are mean ± SD of at least two separate experiments.

Figure 4

Evaluation of pro-apoptotic properties. Cells were treated with vitamin E compounds at the indicated concentrations for 5 days. (A) Quantification of percent apoptotic cells by FACS analyses of annexin V-PE positive cells treated with different doses of different vitamin E compounds. * Significant difference between control and treatments p<0.05, ** significant difference between higher dose and lower dose p<0.05. B. Quantification of percent apoptotic cells by FACS analyses of annexin V-PE positive cells treated with 40 μM γT and 10 μM α-TEA alone or plus 10 μM αT. The data are presented as mean ± S.D of at least three independent experiments.

Figure 5

Assessment of biomarkers of apoptosis. PARP cleavage and pro-apoptotic DR5 and anti-apoptotic survivin protein expression were detected by western blot analyses using the same samples in Fig 4A. Dose-response expression of the markers treated with γT and α-TEA. B. Comparison of the marker expression in different vitamin E compounds. C. The effect of αT on γT and α-TEA-induced apoptotic biomarker expression. Data are representative of two independent experiments.