Dose-response impact of various tocotrienols on serum lipid parameters in 5-week-old female chickens.

The cholesterol-suppressive action of the tocotrienol-rich-fraction (TRF) of palm oil may be due to the effect of its constituent tocotrienols on beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase activity. The tocotrienols, modulate HMG-CoA reductase activity via a post-transcriptional mechanism. As a consequence small doses (5-200 ppm) of TRF-supplemented diets fed to experimental animals lower serum cholesterol levels. These findings led us to evaluate the safety and efficacy of large supplements of TRF and its constituents. Diets supplemented with 50, 100, 250, 500, 1000, or 2000 ppm of TRF, alpha-tocopherol, alpha-tocotrienol, gamma-tocotrienol, or 6-tocotrienol were fed to chickens for 4 wk. There were no differences between groups or within groups in weight gain, or in feed consumption at the termination of the feeding period. Supplemental TRF produced a dose-response (50-2000 ppm) lowering of serum total and LDL cholesterol levels of 22% and 52% (P < 0.05), respectively, compared with the control group. alpha-Tocopherol did not affect total or LDL-cholesterol levels. Supplemental alpha-tocotrienol within the 50-500 ppm range produced a dose-response lowering of total (17%) and LDL (33%) cholesterol levels. The more potent gamma and delta isomers yielded dose-response (50-2,000 ppm) reductions of serum total (32%) and LDL (66%) cholesterol levels. HDL cholesterol levels were minimally impacted by the tocotrienols; as a result, the HDL/LDL cholesterol ratios were markedly improved (123-150%) by the supplements. Serum triglyceride levels were significantly lower in sera of pullets receiving the higher supplements. The safe dose of various tocotrienols for human consumption might be 200-1000 mg/d based on this study.

Evidence for the preventive effect of the polyunsaturated phytol side chain in tocotrienols on 17beta-estradiol epoxidation.

BACKGROUND: We found that 17beta-estradiol (E2) could be activated by epoxidation to bind DNA and to inhibit nuclear RNA synthesis. Vitamin E compounds are powerful antioxidants and chain-breaking free radical scavengers. The chromanol ring in Vitamin E is believed to be involved in these reactions. METHODS: Here, we examined the preventive effect of alpha-tocopherol, alpha-, gamma- and delta-tocotrienols on E2 activation. RESULTS: We found that when any one of these Vitamin E compounds was mixed with E2 for epoxidation by the epoxide-forming oxidant dimethyldioxirane (DMDO), alpha-tocopherol was the least effective as compared with the tocotrienols against the formation of E2 epoxide as reflected by the loss of the ability of E2 to inhibit nuclear RNA synthesis. This conclusion was further confirmed by the binding studies of [3H] labeled E2 to DNA using either DMDO or liver microsomes activation system. CONCLUSIONS: Since the chromanol ring is shared by both tocopherols and tocotrienols and the only difference between these two subgroups of Vitamin E is the phytol side chain, we conclude that the polyunsaturated phytol group in tocotrienols plays a key preventive role in E2 epoxidation. This is the first report showing that the polyunsaturated phytol side chain in tocotrienols is involved in an antioxidative activity and it may also have a preventive effect against the E2 epoxide induced breast cancer carcinogenesis at the initiation.

Pro-apoptotic mechanisms of action of a novel vitamin E analog (alpha-TEA) and a naturally occurring form of vitamin E (delta-tocotrienol) in MDA-MB-435 human breast cancer cells.

Vitamin E derivative, RRR-alpha-tocopheryl succinate (vitamin E succinate, VES), is a potent pro-apoptotic agent, inducing apoptosis by restoring both transforming growth factor-beta (TGF-beta) and Fas (CD95) apoptotic signaling pathways that contribute to the activation of c-Jun N-terminal kinase (JNK)-mediated apoptosis. Objectives of these studies were to characterize signaling events involved in the pro-apoptotic actions of a naturally occurring form of vitamin E, delta-tocotrienol, and a novel vitamin E analog, alpha-tocopherol ether acetic acid analog [alpha-TEA; 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxyacetic acid]. Like VES, alpha-TEA and delta-tocotrienol induced estrogen-nonresponsive MDA-MB-435 and estrogen-responsive MCF-7 human breast cancer cells to undergo high levels of apoptosis in a concentration- and time-dependent fashion. Like VES, the two compounds induced either no or lower levels of apoptosis in normal human mammary epithelial cells and immortalized but nontumorigenic human MCF-10A cells. The pro-apoptotic mechanisms triggered by the structurally distinct alpha-TEA and delta-tocotrienol were identical to those previously reported for VES, that is, alpha-TEA- and delta-tocotrienol-induced apoptosis involved up-regulation of TGF-beta receptor II expression and TGF-beta-, Fas- and JNK-signaling pathways. These data provide a better understanding of the anticancer actions of a dietary form of vitamin E (delta-tocotrienol) and a novel nonhydrolyzable vitamin E analog (alpha-TEA).

Role of caspase-8 activation in mediating vitamin E-induced apoptosis in murine mammary cancer cells.

The vitamin E family of compounds is divided into two subgroups, tocopherols and tocotrienols. However, tocotrienols display more potent apoptotic activity in mammary cancer cells. Although the mechanism(s) mediating tocotrienol-induced apoptosis is presently unknown, apoptosis is carried out by activation of initiator caspases (caspase-8 or -9) that subsequently activate effector caspases (caspase-3, -6, or -7). Studies were conducted to determine whether tocotrienol-induced apoptosis is mediated by activation of the caspase-8 and/or caspase-9 pathway. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained on serum-free media. Treatment with tocotrienol-rich-fraction of palm oil (TRF) and g-tocotrienol, but not a-tocopherol, induced a dose-dependent decrease in +SA cell viability. TRF- and g-tocotrienol-induced cell death resulted from apoptosis, as determined by DNA fragmentation and positive TUNEL assay staining. Additional studies showed that treatment with 50 mM TRF or 20 mM g-tocotrienol increased intracellular activity and levels of processed caspase-8 and -3 but not caspase-9. Furthermore, treatment with specific caspase-8 or -3 inhibitors, but not caspase-9 inhibitor, completely blocked the tocotrienol-induced apoptosis in +SA cells. These findings demonstrate that tocotrienol-induced apoptosis in +SA mammary cancer cells is mediated through activation of the caspase-8 signaling pathway and is independent of caspase-9 activation.

Tocotrienol is the most effective vitamin E for reducing endothelial expression of adhesion molecules and adhesion to monocytes.

Alpha-tocopherol and its esterified derivatives have been shown to be effective in reducing monocytic-endothelial cell adhesion. However, the effect of alpha-tocotrienol (alpha-T3) has not been characterized. In the present study, using human umbilical vein endothelial cells (HUVEC) as the model system, we examined the relative inhibitory effects of alpha-T3 and other vitamin E derivatives on cell surface adhesion molecule expression under TNF-alpha stimulation. Using enzyme-linked immunosorbent assay, we demonstrated that alpha-T3 markedly inhibited the surface expression of vascular cell adhesion molecule-1 in TNF-alpha activated HUVEC in a dose- and time-dependent manner. The optimal inhibition was observed at 25 micromol/l alpha-T3 within 24 h (77+/-5%) without cytotoxicity. In addition, the surface expression of intercellular adhesion molecule-1 and E-selectin were also reduced by 40+/-7 and 42+/-5%, respectively. In order to further evaluate the effects of alpha-T3 on the vascular endothelium, we investigated the ability of monocytes to adhere to endothelial cells. Interestingly, a 63+/-3% decrease in monocytic cell adherence was observed. Compared to alpha-tocopherol and alpha-tocopheryl succinate, alpha-T3 displayed a more profound inhibitory effect on adhesion molecule expression and monocytic cell adherence. This inhibitory action by alpha-T3 on TNF-alpha-induced monocyte adhesion was shown to be NF-kappaB dependent and was interestingly reversed with co-incubation with farnesol and geranylgeraniol, suggesting a role for prenylated proteins in the regulation of adhesion molecule expression. In summary, the above results suggest that alpha-T3 is a potent and effective agent in the reduction of cellular adhesion molecule expression and monocytic cell adherence.

Inhibitory effect of delta-tocotrienol, a HMG CoA reductase inhibitor, on monocyte-endothelial cell adhesion.

We have previously shown that alpha-tocotrienol (alpha-T3), a vitamin E analogue and HMG CoA reductase (HMGR) inhibitor, markedly inhibited monocyte-endothelial cell adhesion, a process that was reversed with the addition of mevalonate intermediates involved in protein prenylation. Since delta-T3 and gamma-T3 possess greater HMGR inhibition than alpha-T3, we postulated that these analogues might have a greater effect on protein prenylation, and thus on monocyte adhesion and endothelial adhesion molecule expression in comparison to alpha-T3. Hence, we pursued to investigate the effect of various analogues of tocotrienol (alpha, gamma, delta) on monocytic cell adhesion and expression of adhesion molecules using a human umbilical vein endothelial cell-line, EA.hy926, as the model system. Relative to alpha-T3, delta-T3 displayed a more profound inhibitory effect on monocytic cell adherence using a 15 micromol/L concentration within 24 h (delta: 42 +/- 5%; alpha: 26 +/- 8% vs. control). This inhibitory action was reversed by co-incubation with farnesol and geranylgeraniol, suggesting a role for prenylated proteins in the regulation of monocyte adhesion. To further evaluate the effect of tocotrienols on the vascular endothelium, we measured the surface expression of adhesion molecules. Compared to alpha-T3, delta-T3 markedly inhibited the expression of VCAM-1 (delta: 57 +/- 6%; alpha: 37 +/- 10% vs. control) and E-selection (delta: 36 +/- 3%; alpha: 18 +/- 6% vs. control) in TNF-alpha activated endothelial cells. The above result suggests that delta-T3 is a potent and effective agent for the reduction of cellular adhesion molecule expression and monocytic cell adherence.