Oxidative stress plays a pivotal role in the pathogenesis of several chronic diseases and antioxidants may represent potential tools for the prevention of these diseases. Here, we investigated the antioxidant efficiency of different tocotrienol isoforms (alpha-, delta-, gamma-tocotrienols), and that of FeAox-6, a novel synthetic compound which combines, by a stable covalent bond, the chroman head of vitamin E and a polyisoprenyl sequence of four conjugated double bonds into a single molecule. The antioxidant efficiency was evaluated as the ability of the compounds to inhibit lipid peroxidation, reactive oxygen species (ROS) production, heat shock protein (hsp) expression in rat liver microsomal membranes as well as in RAT-1 immortalized fibroblasts challenged with different free radical sources, including 2,2′-azobis(2-amidinopropane) (AAPH), tert-butyl hydroperoxide (tert-BOOH) and H2O2. Our results show that individual tocotrienols display different antioxidant potencies. Irrespective of the prooxidant used, the order of effectiveness was:delta-tocotrienol > gamma-tocotrienol = alpha-tocotrienol in both isolated membranes and intact cells. This is presumably due to the decreased methylation of delta-tocotrienol chromane ring, which allows the molecule to be more easily incorporated into cell membranes. Moreover, we found that FeAox-6 showed an antioxidant potency greater than that of delta-tocotrienol. Such an efficiency seems to depend on the concomitant presence of a chromane ring and a phytyl chain in the molecule, which because of four conjugated double bonds, may induce a greater mobility and a more uniform distribution within cell membrane. In view of these results, FeAox-6 represents a new potential preventive agent in chronic diseases in which oxidative stress plays a pathogenic role.
Publications
Tocotrienol-rich fraction of palm oil induces cell cycle arrest and apoptosis selectively in human prostate cancer cells
Srivastava JK, Gupta S.
Biochem Biophys Res Commun. 2006 Jul 28;346(2):447-53. Epub 2006 Jun 2.
One of the requisite of cancer chemopreventive agent is elimination of damaged or malignant cells through cell cycle inhibition or induction of apoptosis without affecting normal cells. In this study, employing normal human prostate epithelial cells (PrEC), virally transformed normal human prostate epithelial cells (PZ-HPV-7), and human prostate cancer cells (LNCaP, DU145, and PC-3), we evaluated the growth-inhibitory and apoptotic effects of tocotrienol-rich fraction (TRF) extracted from palm oil. TRF treatment to PrEC and PZ-HPV-7 resulted in almost identical growth-inhibitory responses of low magnitude. In sharp contrast, TRF treatment resulted in significant decreases in cell viability and colony formation in all three prostate cancer cell lines. The IC(50) values after 24h TRF treatment in LNCaP, PC-3, and DU145 cells were in the order 16.5, 17.5, and 22.0 microg/ml. TRF treatment resulted in significant apoptosis in all the cell lines as evident from (i) DNA fragmentation, (ii) fluorescence microscopy, and (iii) cell death detection ELISA, whereas the PrEC and PZ-HPV-7 cells did not undergo apoptosis, but showed modestly decreased cell viability only at a high dose of 80 microg/ml. In cell cycle analysis, TRF (10-40 microg/ml) resulted in a dose-dependent G0/G1 phase arrest and sub G1 accumulation in all three cancer cell lines but not in PZ-HPV-7 cells. These results suggest that the palm oil derivative TRF is capable of selectively inhibiting cellular proliferation and accelerating apoptotic events in prostate cancer cells. TRF offers significant promise as a chemopreventive and/or therapeutic agent against prostate cancer.
A rice bran oil diet increases LDL-receptor and HMG-CoA reductase mRNA expressions and insulin sensitivity in rats with streptozotocin/nicotinamide-induced type 2 diabetes
Chen CW, Cheng HH.
J Nutr. 2006 Jun;136(6):1472-6.
A rice bran oil (RBO) diet can reduce plasma lipids; this was attributed to the specific components, gamma-oryzanol and gamma-tocotrienol, which individually were shown to be hypocholesterolemic; however, the mechanism of their effects on diabetic hyperlipidemia and the development of diabetes is not known. Rats with streptozotocin/nicotinamide-induced type 2 diabetes were divided into control, RO10, and RO15 groups, and fed cholesterol-free diets containing 0, 10, and 15 g RBO with 0, 352, and 528 g gamma-oryzanol and 0, 6.0 and 9.0 mg gamma-tocotrienol/100 g diet for 4 wk. Diabetic rats fed the RBO diet had greater insulin sensitivity (P = 0.02) than rats fed the control diet. Diabetic rats fed the RBO diet also had lower plasma triglyceride (P = 0.003), LDL cholesterol (P = 0.028), and hepatic triglyceride concentrations (P = 0.04), as well as greater fecal neutral sterol and bile acid excretion than those fed the control diet. After 4 wk, there was an approximately 100% (P < 0.001) increase in the abundance of hepatic cholesterol 7alpha-hydroxylase, an 89% (P < 0.001) increase in the hepatic LDL-receptor, and a 50% (P < 0.001) increase in hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA in rats fed the RBO diet compared with those fed the control diet. These findings support the conclusion that a rice bran oil-containing diet can significantly suppress hyperlipidemic and hyperinsulinemic responses in diabetic rats. The high contents of gamma-oryzanol and gamma-tocotrienol in RBO can lead to increased fecal neutral sterol and bile acid excretion, via upregulation of cholesterol synthesis and catabolism.
Molecular mechanism of antioxidant synergism of tocotrienols and carotenoids in palm oil
Schroeder MT, Becker EM, Skibsted LH.
J Agric Food Chem. 2006 May 3;54(9):3445-53.
During repeated deep-fat frying of potato slices at 163 degrees C in yellow or red palm olein of comparable fatty acid profiles, the oxidative stability (peroxide value and anisidine value) of the palm oleins was similar, and in yellow palm olein, the rate of antioxidant depletion decreased in the order gamma-T3 > alpha-T3 > delta-T3 (T3, tocotrienol). In red palm olein, which had a total tocopherol/tocotrienol content of 1260 vs 940 ppm in yellow palm olein and a corresponding longer induction period in the Rancimat stability test at 120 degrees C, only depletion of gamma-T3 was significant among the phenols during frying and slower as compared to that in yellow palm olein. The carotenes in the red palm olein were depleted linearly with the number of fryings, apparently yielding an overall protection of the phenols. In antioxidant-depleted palm olein and in phospholipid liposomes with added increasing concentrations of phenols, gamma-T3 was found to be a better antioxidant than alpha-T3. alpha-T3 and alpha-T (T, tocopherol) had a similar antioxidant effect in antioxidant-depleted palm olein in the Rancimat stability test, while in the liposomes the ordering as determined by induction period for the formation of conjugated dienes was gamma-T3 > alpha-T3 > alpha-T. The addition of 100-1000 ppm beta-carotene to antioxidant-depleted palm olein or liposomes (lycopene also tested) did not provide any protection against oxidation. In the liposomes, synergistic interactions were observed between beta-carotene or lycopene and alpha-T, alpha-T3, or gamma-T3 for carotene/phenol ratios of 1:10 and 1:2 but not for 1:1. In chloroform, carotenes were regenerated by tocopherols/tocotrienols from carotene radicals generated by laser flash photolysis as shown by transient absorption spectroscopy, suggesting that carotenes rather than phenols are the primary substrate for lipid-derived radicals in red palm olein, in effect depleting carotenes prior to phenols during frying. Regeneration of carotenes by the phenols also explains the synergism in liposomes. In the laser flash photolysis experiments, gamma-T3 was also found to be faster in regenerating carotenes than alpha-T3 and alpha-T.
Dose-response impact of various tocotrienols on serum lipid parameters in 5-week-old female chickens
Yu SG, Thomas AM, Gapor A, Tan B, Qureshi N, Qureshi AA.
Lipids. 2006 May;41(5):453-61.
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.
Synthesis and study of the cancer cell growth inhibitory properties of alpha-, gamma-tocopheryl and gamma-tocotrienyl 2-phenylselenyl succinates
Vraka PS, Drouza C, Rikkou MP, Odysseos AD, Keramidas AD.
Bioorg Med Chem. 2006 Apr 15;14(8):2684-96. Epub 2005 Dec 27.
Vitamin E succinate selenium-conjugated molecules were synthesized and their apoptogenic properties were evaluated. 4-Methyl-2-phenylselenyl succinate (4) was prepared by the reaction of sodium benzeneselenolate with 2-bromosuccinic anhydrite in methanol solution. The methyl ester was converted to the acid (5) by hydrolysis with aqueous hydrochloric acid. Reaction of the 2-phenylselenyl succinic anhydrite (6) with alpha-tocopherol (1a), gamma-tocopherol (1c), and gamma-tocotrienol (2c) in acidic conditions gave the respective esters. The free radical scavenging properties of alpha-tocopheryl-2-phenylselenyl succinate (7), gamma-tocopheryl-2-phenylselenyl succinate (8), and gamma-tocotrienyl-2-phenylselenyl succinate (9) were evaluated in comparison with those of alpha-tocopheryl succinate (10), gamma-tocopheryl succinate (11), and gamma-tocotrienyl succinate (12), respectively, and the free tocopherols and gamma-tocotrienol. Compounds 7-9 induced a statistically significant decrease in prostate cancer cell viability compared to 10-12, respectively, or 5, exhibiting features of apoptotic cell death and associated with caspase-3 activation. These data show that structural modifications of vitamin E components by 5 enhance their apoptogenic properties in cancer cells.
Comparative effects of alpha-tocopherol and gamma-tocotrienol against hydrogen peroxide induced apoptosis on primary-cultured astrocytes
Mazlan M, Sue Mian T, Mat Top G, Zurinah Wan Ngah W.
J Neurol Sci. 2006 Apr 15;243(1-2):5-12.
Oxidative stress is thought to be one of the factors that cause neurodegeneration and that this can be inhibited by antioxidants. Since astrocytes support the survival of central nervous system (CNS) neurons, we compared the effect of alpha-tocopherol and gamma-tocotrienol in minimizing the cytotoxic damage induced by H2O2, a pro-oxidant. Primary astrocyte cultures were pretreated with either alpha-tocopherol or gamma-tocotrienol for 1 h before incubation with 100 microM H2O2 for 24 h. Cell viability was then assessed using the MTS assay while apoptosis was determined using a commercial ELISA kit as well as by fluorescent staining of live and apoptotic cells. The uptake of alpha-tocopherol and gamma-tocotrienol by astrocytes were also determined using HPLC. Results showed that gamma-tocotrienol is toxic at concentrations >200 microM but protects against H2O2 induced cell loss and apoptosis in a dose dependent manner up to 100 microM. alpha-Tocopherol was not cytotoxic in the concentration range tested (up to 750 microM), reduced apoptosis to the same degree as that of gamma-tocotrienol but was less effective in maintaining the viable cell number. Since the uptake of alpha-tocopherol and gamma-tocotrienol by astrocytes is similar, this may reflect the roles of these 2 vitamin E subfamilies in inhibiting apoptosis and stimulating proliferation in astrocytes.
Preferential radiation sensitization of prostate cancer in nude mice by nutraceutical antioxidant gamma-tocotrienol
Kumar KS, Raghavan M, Hieber K, Ege C, Mog S, Parra N, Hildabrand A, Singh V, Srinivasan V, Toles R, Karikari P, Petrovics G, Seed T, Srivastava S, Papas A.
Life Sci. 2006 Mar 27;78(18):2099-104. Epub 2006 Jan 18.
Gamma-tocotrienol (GT) is a member of the vitamin E family. Our preliminary studies indicated that it protected mice from lethal irradiation, so we hypothesized that GT might be a radiation sensitizing agent for tumors. To test this, we induced prostate tumors by injecting PC3 cells into nude BALB/c mice. When the tumors were about 5 mm in diameter, mice were injected subcutaneously with 400 mg/kg gamma-tocotrienol and irradiated 24 h later at the site of the tumor with a dose of 12 Gy (60)Cobalt. Tumor size was monitored for 24 days after radiation. Tumor tissues as well as normal tissues like rectum, kidney, and liver were monitored for lipid peroxidation on day 4 and day 24 after radiation. The results indicated that the size of the tumors was reduced by almost 40%, but only in GT-treated and irradiated mice. In unstimulated and Fe-stimulated lipid peroxidation groups, lipid peroxidation in the tumors from irradiated mice increased to 135% and 150%, respectively, four days after irradiation and 33% and 66% in the same groups, respectively, 24 days after irradiation. In general, lipid peroxidation in the rectum did not increase in GT-treated and irradiated mice, although there was a slight increase in Fe-stimulated lipid peroxidation (29%) four days after irradiation. Unexpectedly, the kidneys were as equally sensitized to lipid peroxidation as the tumors. Liver tissue was protected in the short-term from radiation-induced lipid peroxidation. These studies indicate that the radiotherapy efficacy of prostate cancer can be increased with GT and a pro-oxidant if the kidneys can be shielded.
Tocotrienol inhibits proliferation of human Tenon’s fibroblasts in vitro: A comparative study with vitamin E forms and mitomycin C
Meyenberg A, Goldblum D, Zingg JM, Azzi A, Nesaretnam K, Kilchenmann M, Frueh BE.
Graefes Arch Clin Exp Ophthalmol. 2005 Dec;243(12):1263-71. Epub 2005 May 21.
PURPOSE: To evaluate the potential of the vitamin E compound alpha-tocotrienol as antifibrotic agent in vitro.
METHODS: Using human Tenon’s capsule fibroblast cultures, the antiproliferative and cytotoxic effects of the different vitamin E forms alpha-tocopherol, alpha-tocopheryl acetate, alpha-tocopheryl succinate and alpha-tocotrienol were compared with those of mitomycin C. To mimic subconjunctival and regular oral application in vivo, exposure time of serum-stimulated and serum-restimulated fibroblasts (SF and RF, respectively) to vitamin E forms was set at 6 days. Cultures were only exposed for 5 min to mitomycin C due to its known acute toxicity and to mimic the short-time intraoperative administration. Proliferation (expressed as % of control) was determined by DNA content quantification on days 2, 4 and 6, whereas cytotoxicity was assessed by cell morphology and glucose 6-phosphate dehydrogenase (G6PD) release after 24 h.
RESULTS: alpha-Tocopherol and alpha-tocopheryl acetate stimulated growth of SF, but not RF. Reduction of fibroblast content by alpha-tocopheryl succinate was accompanied by increased G6PD release and necrosis. Contrary to alpha-tocopheryl succinate, 50 microM or repeatedly 20 microM of alpha-tocotrienol significantly inhibited proliferation without causing cellular toxicity (maximal effect: 46.8%). RF were more sensitive to this effect than SF. Mitomycin C 100-400 microg/ml showed a stronger antiproliferative effect than alpha-tocotrienol (maximal effect: 13.8%). Morphologic characteristics of apoptosis were more commonly found under treatment with mitomycin C.
CONCLUSIONS: Of the vitamin E forms tested, only alpha-tocotrienol significantly inhibited growth at non-toxic concentrations. In this in vitro study, antiproliferative effects of mitomycin C were stronger than those of alpha-tocotrienol.
Tumor suppressive effects of tocotrienol in vivo and in vitro
Wada S, Satomi Y, Murakoshi M, Noguchi N, Yoshikawa T, Nishino H.
Cancer Lett. 2005 Nov 18;229(2):181-91. Epub 2005 Aug 10.
Tocotrienols have been reported to have higher biological activities than tocopherols. We investigated the antitumor effect of tocotrienols both in vivo and in vitro. Oral administration of tocotrienols resulted in significant suppression of liver and lung carcinogenesis in mice. In human hepatocellular carcinoma HepG2 cells, delta-tocotrienol exerted more significant antiproliferative effect than alpha-, beta-, and gamma-tocotrienols. delta-Tocotrienol induced apoptosis, and also tended to induce S phase arrest. On the other hand, gene expression analysis showed that delta-tocotrienol increased CYP1A1 gene, a phase I enzyme. Although further study will be necessary to investigate possible adverse effect, the data obtained in present study suggest that tocotrienols could be promising agents for cancer prevention.