Publications

Effects of new combinative antioxidant FeAOX-6 and alpha-tocotrienol on macrophage atherogenesis-related functions

Napolitano M, Avanzi L, Manfredini S, Bravo E.

Vascul Pharmacol. 2007 Jun;46(6):394-405. Epub 2007 Feb 1.

Pivotal role in atherogenesis is played by macrophages, which are early site for lipid accumulation and mediate the inflammatory and immune response in the intima. Epidemiological evidence indicates that natural antioxidants reduce the risk of heart disease, but, so far, supplementation studies have failed to confirm any protective effects of these compounds against cardiovascular disease. This study evaluated the effects of the natural antioxidant alpha-tocotrienol and of the newly designed compound, FeAOX-6, which combines antioxidant structural features of both tocopherols and carotenoids into a single molecule, on macrophage functions involved in foam cell formation. FeAOX-6 or alpha-tocotrienol induce a strong dose-dependent reduction of cholesterol and reduce cholesterol accumulation in human macrophages. The extent of the reduction found with alpha-tocotrienol was greater than that induced by FeAOX-6 and did not correlate with their respective antioxidant capacities. Treatment of HMDM with alpha-tocotrienol or FeAOX-6 enhanced also tumor necrosis factor-alpha secretion. These results are consistent with a reduction in scavenger receptor activity, but we found that antioxidant treatment did not affect cholesterol uptake from modified LDL. The effects on release on pro-inflammatory prostanoid precursors, PGE(2) and cytokine suggest a variety of metabolic responses that are both dependent on antioxidant compounds and macrophages activation status.

 

Tocotrienols potentiate lovastatin-mediated growth suppression in vitro and in vivo

McAnally JA, Gupta J, Sodhani S, Bravo L, Mo H.

Exp Biol Med (Maywood). 2007 Apr;232(4):523-31.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is the rate-limiting enzyme in the mevalonate pathway that provides essential intermediates for the membrane anchorage and biologic functions of growth-related proteins. Contrary to preclinical studies showing the growth-suppressive activity of statins, competitive inhibitors of HMG CoA reductase, clinical application of statins in cancer is precluded by their lack of activity at levels prescribed for the prevention of cardiovascular disease and by their dose-limiting toxicities at high doses. The dysregulated and elevated HMG CoA reductase activity in tumors retains sensitivity to the isoprenoid-mediated posttranscriptional down-regulation, an action that complements the statin-mediated inhibition and may lead to synergistic impact of blends of isoprenoids and lovastatin on tumor HMG CoA reductase activity and consequently tumor growth. d-gamma- and d-delta-tocotrienols, vitamin E isomers containing an isoprenoid moiety, and lovastatin-induced concentration-dependent inhibition of the 48-hr proliferation of murine B16 melanoma cells with IC50 values of 20 +/- 3, 14 +/- 3, and 1.5 +/- 0.4 microM respectively. A blend of lovastatin (1 microM) and d-gamma-tocotrienol (5 microM) totally blocked cell growth, an impact far exceeding the sum of inhibitions induced by lovastatin (12%) and d-gamma-tocotrienol (8%) individually. Synergistic impact of these two agents was also shown in human DU145 prostate carcinoma and human A549 lung carcinoma cells. C57BL6 mice were fed diets supplemented with 12.5 mg lovastatin/kg body weight, 62.5 mg d-delta-tocotrienol/kg body weight, or a blend of both agents for 22 days following B16 cell implantation; only the latter had significantly lower tumor weight than those with no supplementation. Co-administration of isoprenoids that posttranscriptionally down-regulate tumor reductase may lower the effective dose of statins and offer a novel approach to cancer chemo-prevention and/or therapy.

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Triton WR1339, an inhibitor of lipoprotein lipase, decreases vitamin E concentration in some tissues of rats by inhibiting its transport to liver

Abe C, Ikeda S, Uchida T, Yamashita K, Ichikawa T.

J Nutr. 2007 Feb;137(2):345-50.

The aim of this experiment was to clarify the contribution of the alpha-tocopherol transfer activity of lipoprotein lipase (LPL) to vitamin E transport to tissues in vivo. We studied the effect of Triton WR1339, which prevents the catabolism of triacylglycerol-rich lipoproteins by LPL on vitamin E distribution in rats. Vitamin E-deficient rats fed a vitamin E-free diet for 4 wk were injected with Triton WR1339 and administered by oral gavage an emulsion containing 10 mg of alpha-tocopherol, 10 mg of gamma-tocopherol, or 29.5 mg of a tocotrienol mixture with 200 mg of sodium taurocholate, 200 mg of triolein, and 50 mg of albumin. alpha-Tocopherol was detected in the serum and other tissues of the vitamin E-deficient rats, but gamma-tocopherol, alpha- and gamma-tocotrienol were not detected. Triton WR1339 injection elevated (P<0.05) the serum alpha-tocopherol concentration and inhibited (P<0.05) the elevation of alpha-tocopherol concentration in the liver, adrenal gland, and spleen due to the oral administration of alpha-tocopherol. Neither alpha-tocopherol administration nor Triton WR1339 injection affected (P>or=0.05) the alpha-tocopherol concentration in the perirenal adipose tissue, epididymal fat, and soleus muscle despite a high expression of LPL in the adipose tissue and muscle. These data show that alpha-tocopherol transfer activity of LPL in adipose tissue and muscle is not important for alpha-tocopherol transport to the tissue after alpha-tocopherol intake or that the amount transferred is small relative to the tissue concentration. Furthermore, Triton WR1339 injection tended to elevate the serum gamma-tocopherol (P=0.071) and alpha-tocotrienol (P=0.053) concentrations and lowered them (P<0.05) in the liver and adrenal gland of rats administered gamma-tocopherol or alpha-tocotrienol. These data suggest that lipolysis of triacylglycerol-rich chylomicron by LPL is necessary for postprandial vitamin E transport to the liver and subsequent transport to the other tissues.

Gamma-tocotrienol inhibits nuclear factor-kappaB signaling pathway through inhibition of receptor-interacting protein and TAK1 leading to suppression of antiapoptotic gene products and potentiation of apoptosis

Ahn KS, Sethi G, Krishnan K, Aggarwal BB.

J Biol Chem. 2007 Jan 5;282(1):809-20. Epub 2006 Nov 17.

Unlike the tocopherols, the tocotrienols, also members of the vitamin E family, have an unsaturated isoprenoid side chain. In contrast to extensive studies on tocopherol, very little is known about tocotrienol. Because the nuclear factor-kappaB (NF-kappaB) pathway has a central role in tumorigenesis, we investigated the effect of gamma-tocotrienol on the NF-kappaB pathway. Although gamma-tocotrienol completely abolished tumor necrosis factor alpha (TNF)-induced NF-kappaB activation, a similar dose of gamma-tocopherol had no effect. Besides TNF, gamma-tocotrienol also abolished NF-kappaB activation induced by phorbol myristate acetate, okadaic acid, lipopolysaccharide, cigarette smoke, interleukin-1beta, and epidermal growth factor. Constitutive NF-kappaB activation expressed by certain tumor cells was also abrogated by gamma-tocotrienol. Reducing agent had no effect on the gamma-tocotrienol-induced down-regulation of NF-kappaB. Mevalonate reversed the NF-kappaB inhibitory effect of gamma-tocotrienol, indicating the role of hydroxymethylglutaryl-CoA reductase. Gamma-tocotrienol blocked TNF-induced phosphorylation and degradation of IkappaBalpha through the inhibition of IkappaBalpha kinase activation, thus leading to the suppression of the phosphorylation and nuclear translocation of p65. gamma-Tocotrienol also suppressed NF-kappaB-dependent reporter gene transcription induced by TNF, TNFR1, TRADD, TRAF2, TAK1, receptor-interacting protein, NIK, and IkappaBalpha kinase but not that activated by p65. Additionally, the expressions of NF-kappaB-regulated gene products associated with antiapoptosis (IAP1, IAP2, Bcl-xL, Bcl-2, cFLIP, XIAP, Bfl-1/A1, TRAF1, and Survivin), proliferation (cyclin D1, COX2, and c-Myc), invasion (MMP-9 and ICAM-1), and angiogenesis (vascular endothelial growth factor) were down-regulated by gamma-tocotrienol. This correlated with potentiation of apoptosis induced by TNF, paclitaxel, and doxorubicin. Overall, our results demonstrate that gamma-tocotrienol inhibited the NF-kappaB activation pathway, leading to down-regulation of various gene products and potentiation of apoptosis.

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Dose dependent elevation of plasma tocotrienol levels and its effect on arterial compliance, plasma total antioxidant status, and lipid profile in healthy humans supplemented with tocotrienol rich vitamin E

Rasool AH, Yuen KH, Yusoff K, Wong AR, Rahman AR.

J Nutr Sci Vitaminol (Tokyo). 2006 Dec;52(6):473-8.

Tocotrienols are a class of vitamin E reported to be potent antioxidants, besides having the ability to inhibit the HMG-CoA reductase enzyme. This study assessed the effects of 3 doses of tocotrienol-rich vitamin E (TRE) on plasma tocotrienol isomer concentration, arterial compliance, plasma total antioxidant status (TAS), aortic systolic blood pressure (ASBP), serum total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) in healthy males.

Methodology: This randomised, blinded end-point, placebo-controlled clinical trial with a parallel design involved 36 healthy male subjects who took either an oral placebo or TRE at doses of 80, 160 or 320 mg daily for 2 mo. Baseline and end-of-treatment measurements of vitamin E concentration, arterial compliance [assessed by aortic femoral pulse wave velocity (PWV) and augmentation index (AI)], ASBP, plasma TAS, serum TC and LDL-C were taken.

Results: Baseline tocotrienol isomer concentrations were low and not detectable in some subjects. Upon supplementation, all TRE-treated groups showed significant difference from placebo for their change in alpha, gamma and delta tocotrienol concentrations from baseline to end of treatment. There was a linear dose and blood level relationship for all the isomers. There was no significant difference between groups for their change in PWV, AI, plasma TAS, ASBP, TC or LDL-C from baseline to end of treatment. Groups 160 mg (p = 0.024) and 320 mg (p = 0.049) showed significant reductions in their ASBP. Group 320 mg showed a significant 9.2% improvement in TAS.

Conclusion: TRE at doses up to 320 mg daily were well tolerated. Treatment significantly increased alpha, delta, and gamma tocotrienol concentrations but did not significantly affect arterial compliance, plasma TAS, serum TC or LDL-C levels in normal subjects.

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Proposed mechanisms for red palm oil induced cardioprotection in a model of hyperlipidaemia in the rat

Esterhuyse JS, van Rooyen J, Strijdom H, Bester D, du Toit EF.

Prostaglandins Leukot Essent Fatty Acids. 2006 Dec;75(6):375-84.

High-cholesterol diets alter myocardial and vascular NO-cGMP signaling and have been implicated in ischaemic/reperfusion injury. We investigated the effects of dietary red palm oil (RPO) containing fatty acids, carotonoids, tocopherols and tocotrienols on myocardial ischaemic tolerance and NO-cGMP pathway function in the rat. Wistar rats were fed a standard rat chow+/-RPO, or a standard rat chow+cholesterol+/-RPO diet. Myocardial mechanical function and NO-cGMP signaling pathway intermediates were determined before, during and after 25 min ischaemia. RPO-supplementation improved aortic output recovery and increased myocardial ischaemic cGMP concentrations. Simulated ischaemia (hypoxia) increased cardiomyocyte nitric oxide levels in the two RPO supplemented groups, but not in control non-supplemented groups. RPO supplementation also increased hypoxic nitric oxide levels in the control diet fed, but not the cholesterol fed rats. These data suggest that dietary RPO may improve myocardial ischaemic tolerance by increasing bioavailability of NO and improving NO-cGMP signaling in the heart.

Gamma-Tocotrienol inhibits ErbB3-dependent PI3K/Akt mitogenic signalling in neoplastic mammary epithelial cells

Samant GV, Sylvester PW.

Cell Prolif. 2006 Dec;39(6):563-74.

The antiproliferative effects of gamma-tocotrienol are associated with suppression in epidermal growth factor (EGF)-dependent phosphatidylinositol-3-kinase (PI3K)/PI3K-dependent kinase-1 (PDK-1)/Akt mitogenic signalling in neoplastic mammary epithelial cells. Studies were conducted to investigate the direct effects of gamma-tocotrienol treatment on specific components within the PI3K/PDK-1/Akt mitogenic pathway. +SA cells were grown in culture and maintained in serum-free media containing 10 ng/ml EGF as a mitogen. Treatment with 0-8 microm gamma-tocotrienol resulted in a dose-responsive decrease in the +SA cell growth and a corresponding decrease in phospho-Akt (active) levels. However, gamma-tocotrienol treatment had no direct inhibitory effect on Akt or PI3K enzymatic activity, suggesting that the inhibitory effects of gamma-tocotrienol occur upstream of PI3K, possibly at the level of the EGF-receptor (ErbB1). Additional studies were conducted to determine the effects of gamma-tocotrienol on ErbB receptor activation. Results showed that gamma-tocotrienol treatment had little or no effect on ErbB1 or ErbB2 receptor tyrosine phosphorylation, a prerequisite for substrate interaction and signal transduction, but did cause a significant and progressive decrease in the ErbB3 tyrosine phosphorylation. Because ErbB1 or ErbB2 receptors form heterodimers with the ErbB3 receptor, and ErbB3 heterodimers have been shown to be the most potent activators of PI3K, these findings strongly suggest that the antiproliferative effects of gamma-tocotrienol in neoplastic +SA mouse mammary epithelial cells are mediated by a suppression in ErbB3-receptor tyrosine phosphorylation and subsequent reduction in PI3K/PDK-1/Akt mitogenic signalling.

Apoptosis induction by gamma-tocotrienol in human hepatoma Hep3B cells

Sakai M, Okabe M, Tachibana H, Yamada K.

J Nutr Biochem. 2006 Oct;17(10):672-6.

We evaluated the antitumor activity of tocotrienol (T3) on human hepatoma Hep3B cells. At first, we examined the effect of T3 on the proliferation of human hepatoma Hep3B cells and found that gamma-T3 inhibited cell proliferation at lower concentrations and shorter treatment times than alpha-T3. Then, we examined the effect of gamma-T3 apoptosis induction and found that gamma-T3 induced poly (ADP-ribose) polymerase (PARP) cleavage and stimulated a rise in caspase-3 activity. In addition, gamma-T3 stimulated a rise in caspase-8 and caspase-9 activities. We also found that gamma-T3-induced apoptotic cell death was accompanied by up-regulation of Bax and a rise in the fragments of Bid and caspase-8. These data indicate that gamma-T3 induced apoptosis in Hep3B cells and that caspase-8 and caspase-9 were involved in apoptosis induction. Moreover, these results suggest that Bax and Bid regulated apoptosis induction by gamma-T3.

Characterization of the potent neuroprotective properties of the natural vitamin E alpha-tocotrienol

Khanna S, Roy S, Parinandi NL, Maurer M, Sen CK.

J Neurochem. 2006 Sep;98(5):1474-86.

The natural vitamin E tocotrienols possess properties not shared by tocopherols. Nanomolar alpha-tocotrienol, not alpha-tocopherol, is potently neuroprotective. On a concentration basis, this finding represents the most potent of all biological functions exhibited by any natural vitamin E molecule. We sought to dissect the antioxidant-independent and -dependent neuroprotective properties of alpha-tocotrienol by using two different triggers of neurotoxicity, homocysteic acid (HCA) and linoleic acid. Both HCA and linoleic acid caused neurotoxicity with comparable features, such as increased ratio of oxidized to reduced glutathione GSSG/GSH, raised intracellular calcium concentration and compromised mitochondrial membrane potential. Mechanisms underlying HCA-induced neurodegeneration were comparable to those in the path implicated in glutamate-induced neurotoxicity. Inducible activation of c-Src and 12-lipoxygenase (12-Lox) represented early events in that pathway. Overexpression of active c-Src or 12-Lox sensitized cells to HCA-induced death. Nanomolar alpha-tocotrienol was protective. Knock-down of c-Src or 12-Lox attenuated HCA-induced neurotoxicity. Oxidative stress represented a late event in HCA-induced death. The observation that micromolar, but not nanomolar, alpha-tocotrienol functions as an antioxidant was verified in a model involving linoleic acid-induced oxidative stress and cell death. Oral supplementation of alpha-tocotrienol to humans results in a peak plasma concentration of 3 microm. Thus, oral alpha-tocotrienol may be neuroprotective by antioxidant-independent as well as antioxidant-dependent mechanisms.

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Down-regulation of telomerase activity in DLD-1 human colorectal adenocarcinoma cells by tocotrienol.

Eitsuka T, Nakagawa K, Miyazawa T.

Biochem Biophys Res Commun. 2006 Sep 15;348(1):170-5.

As high telomerase activity is detected in most cancer cells, inhibition of telomerase by drug or dietary food components is a new strategy for cancer prevention. Here, we investigated the inhibitory effect of vitamin E, with particular emphasis on tocotrienol (unsaturated vitamin E), on human telomerase in cell-culture study. As results, tocotrienol inhibited telomerase activity of DLD-1 human colorectal adenocarcinoma cells in time- and dose-dependent manner, interestingly, with delta-tocotrienol exhibiting the highest inhibitory activity. Tocotrienol inhibited protein kinase C activity, resulting in down-regulation of c-myc and human telomerase reverse transcriptase (hTERT) expression, thereby reducing telomerase activity. In contrast to tocotrienol, tocopherol showed very weak telomerase inhibition. These results provide novel evidence for the first time indicating that tocotrienol acts as a potent candidate regulator of telomerase and supporting the anti-proliferative function of tocotrienol.